U.S. patent application number 12/096594 was filed with the patent office on 2009-09-03 for detection of fetal cells from maternal blood.
This patent application is currently assigned to FCMB APS. Invention is credited to Britta Christensen, Sten Kolvraa, Palle Schelde, Morten Draeby Sorensen.
Application Number | 20090220933 12/096594 |
Document ID | / |
Family ID | 37776097 |
Filed Date | 2009-09-03 |
United States Patent
Application |
20090220933 |
Kind Code |
A1 |
Kolvraa; Sten ; et
al. |
September 3, 2009 |
DETECTION OF FETAL CELLS FROM MATERNAL BLOOD
Abstract
The present application relates to methods for identification of
foetal cells and generation and isolation of binding members
recognising foetal cells. Said methods may further be used for
other purposes relating to characterisation of biological samples
and biological antigens. The methods are characterised by the
applicability in situations where the interesting objects are
present in a limited amount, or where the interesting objects are
intermixed with other material, thus the methods are suitable for
use in situations where the ratio of the interesting material
compared to other material is low. The application discloses
methods for use of detecting foetal cells and method of
generating/isolating binding members towards antigenic material of
low abundancy.
Inventors: |
Kolvraa; Sten; (Skoedstrup,
DK) ; Christensen; Britta; (Birkeroed, DK) ;
Schelde; Palle; (Hoejbjerg, DK) ; Sorensen; Morten
Draeby; (Aarhus, DK) |
Correspondence
Address: |
DAVIS WRIGHT TREMAINE LLP - San Francisco
505 MONTGOMERY STREET, SUITE 800
SAN FRANCISCO
CA
94111
US
|
Assignee: |
FCMB APS
Vejle
DK
|
Family ID: |
37776097 |
Appl. No.: |
12/096594 |
Filed: |
December 7, 2006 |
PCT Filed: |
December 7, 2006 |
PCT NO: |
PCT/DK2006/000693 |
371 Date: |
February 2, 2009 |
Current U.S.
Class: |
435/2 ;
435/366 |
Current CPC
Class: |
C12Q 1/6806 20130101;
G01N 33/566 20130101; C12Q 1/6879 20130101; C12Q 1/6841 20130101;
C12N 15/1037 20130101; C12Q 2600/156 20130101; C12N 5/0081
20130101 |
Class at
Publication: |
435/2 ;
435/366 |
International
Class: |
A01N 1/02 20060101
A01N001/02; C12N 5/08 20060101 C12N005/08 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 8, 2005 |
DK |
PA 2005 01745 |
Claims
1. A method of detecting a fetal cell in a maternal blood sample
comprising the following steps of: a. providing a maternal blood
sample, b. fixing and permealizing nucleated cells present in said
blood sample while maintaining cell morphology, maintaining protein
content of the cells and ensuring accessibility for a hybridization
probe. c. adding a hybridization probe, and d. detecting at least
one fetal cell.
2. The method according to claim 1, comprising a pre-fixation
step.
3. The method according to claim 2, wherein the pre-fixation step
comprises PFA treatment.
4. The method according to claim 3, wherein the pre-fixation step
comprises incubation of cells in a solution comprising 0.1-4%
PFA.
5. The method according to claim 1, wherein the fixation and
permeabilization step comprises methanol and acetone treatment.
6. The method according to claim 1, comprising a re-fixation
step.
7. The method according to claim 6, wherein the re-fixation step
comprises PFA treatment.
8. The method according to claim 7, wherein the re-fixation step
comprises incubation of cells in a solution comprising 1-5%
PFA.
9. The method according to claim 1, comprising a dehydration steps
in ethanol.
10. The method according to claim 1, comprising an enrichment
step.
11. The method according to claim 10, wherein the enrichment step
does not discriminate between different nucleated cell types.
12. The method according to claim 11, wherein the enrichment step
comprises a step of erythrocyte lysis.
13. The method according to claim 12, wherein erythrocyte lysis is
obtained by NH.sub.4Cl mediated lysis.
14. The method according to claim 1, wherein the fetal cell is
detected by selective labelling of DNA in the fetal cell.
15. The method according to claim 14, wherein cell type specific
DNA is selectively labelled by a hybridization technique.
16. The method according to claim 1, wherein the fetal cell is
detected by the presence of cell type specific epigenetic
characteristics.
17. The method according to claim 1, wherein the fetal cell is
detected by selective labelling of cell type specific RNA in the
fetal cell.
18. The method according to claim 17, wherein cell type specific
RNA is selective labelled by a hybridization technique.
19. The method according to claim 18, wherein the cell type
specific RNA is an mRNA.
20. The method according to claim 1, wherein the fetal cell is
detected by selective labelling of at least one cell type specific
protein.
21. The method according to claim 1, wherein the fetal cell is
detected by morphological characteristics.
22. The method according to claim 1, wherein the detected fetal
cell is a male fetal cell.
23. The method according to claim 22, wherein the male fetal cell
is detected by reverse colour color FISH.
24. The method according to claim 1, wherein the at least one fetal
cell is detected using an automated scanner system.
25. A fetal cell identified by the method according to claim 1.
26. A binding member specifically recognizing the fetal cell
according to claim 25.
27. A fetal cell antigen recognized by the binding member according
to claim 26.
28. A method of isolating binding members recognizing target
antigenic material of limited availability comprising the steps: a.
identifying the target antigenic material, b. contacting said
target antigenic material with a binding member generating system
and c. isolating binding member(s) recognizing the target antigenic
material.
29.-46. (canceled)
47. A fetal cell antigen recognized by a binding member isolated
using the method of claim 28.
48. (canceled)
49. An assay method comprising the steps of: a. providing a
maternal blood sample, b. selectively labelling at least one fetal
cell by labelling of a fetal cell antigen according to claim 47 in
said blood sample.
50. An assay method comprising the steps of: a. providing a
maternal blood sample, b. selectively labelling at least one fetal
cell by use of a binding member isolated using the method of claim
28 in said blood sample.
51.-64. (canceled)
Description
[0001] All patent and non-patent references cited in the
application, or in the present application, are also hereby
incorporated by reference in their entirety.
FIELD OF INVENTION
[0002] The present invention relates to methods for detection and
characterisation of cells, tissue or target antigenic material of
very limited availability. The analysis of rare cells, such as
foetal cells in a maternal blood sample or a micometastatic cell,
is complicated by the low abundance of such cells and because the
biological sample often will comprise a majority of other cells or
tissue material besides the rare cell of interest. The possibility
to analyse rare cells and other biological material of limited
availability will permit the development of new diagnostic methods
with a less invasive character.
BACKGROUND OF INVENTION
[0003] The examination of foetal cells for early detection of
foetal diseases and genetic abnormalities is carried out in
connection with many pregnancies, in particular when the maternal
age is high (35 years or above) or where genetic diseases are known
in the family. Foetal cells may be obtained by amniocentesis, the
removal of amniotic fluid from the amniotic cavity within the
amniotic sac or by chorion biopsy, where biopsies are taken from
the placenta, so-called invasive sampling.
[0004] Prenatal aneuploidy screening employs either standard
chromosome analysis or FISH analysis using specific DNA probes for
the elucidation of numerical aberrations of chromosomes, particular
chromosomes 13, 18, 21, X and Y in the foetus.
[0005] Due to the invasiveness of the methods described above and
the risk of abortion associated herewith, it would be
advantageously to perform foetal diagnosis by a non-invasive
procedure, such as for example by use of a maternal blood
sample.
[0006] During pregnancy a variety of cell types of foetal origin
cross the placenta and circulate within maternal peripheral blood.
The feasibility of using foetal cells in the maternal circulation
for diagnostic purposes has been hindered by the fact that foetal
cells are present in maternal blood in only very limited numbers,
reported numbers have been from one foetal cell per
10.sup.5-10.sup.8 nucleated maternal cells or 1-10 foetal cells per
ml maternal blood.
[0007] In order to use foetal cell present in maternal blood for
diagnostic purposes, methods suitable for isolation and/or
identification of foetal cells are required.
[0008] Most foetal cells cannot be distinguished from maternal
cells on the basis of morphology alone, thus alternative methods of
identification of foetal cells have been investigated.
[0009] Foetal cells present in maternal blood include erythroblast,
foetal leukocytes and trophoblast cells. Recently, foetal cells
with stem cell properties have been indicated to be present in
pregnant women and described as pregnancy-associated progenitor
cells (PAPCs) and foetal mesenchymal stem cells (Khosrotehrani and
Bianchi, 2005)
[0010] As described above different types of foetal cells, such as
nucleated erythrocytes have be identified in maternal blood samples
but so far, the efficiency of detecting these cells is very low.
This may be due to the low number of foetal cells present in a
maternal blood sample and/or due to the method employed for
detection of said foetal cells.
[0011] Due to the very limited number of foetal cells in maternal
blood separation or enrichment of the maternal blood sample with
respect to the foetal cells is often conducted by for example
negative selection, i.e. removal of maternal cells.
[0012] Maternal cells may be removed by density gradient
centrifugation, by removing maternal cells with an antibody to a
cell surface antigen or alternatively by lyses of maternal
erythrocytes, optionally combined with immunologic methods for
removing the maternal cells.
[0013] Alternatively foetal cells are separated from maternal cells
by using flow cytometirc methods or alternatively by a new
technique, in which, a two-step enrichment procedure is performed
to isolate trophoblast cells, consisting of a density gradient
centrifugation and negative immunoaffinity isolation using Magnetic
Activated Cell Sorter (MACS) Separation techniques and monoclonal
antibodies or ligands specific to foetal cells.
[0014] It is however, a problem that due to the
enrichment/separation procedures some of the foetal cells may also
be removed leading to even fewer foetal cells in the blood sample
to be analysed. Particular antibody selection methods may lead to a
loss of foetal cells not expressing the antigen at a sufficient
level.
[0015] Most methods are thus biased by the selection criteria of
the enrichment/separation step. Two methods for unbiased
quantification of total amount of foetal cells are available. Both
methods are based on detection of Y-chromosome sequences. One is
quantitative PCR, which is rather imprecise and the other is FISH
staining of Y-chromosome in nuclei followed by counting. The
enrichment step in the latter is based on initial carnoid fixation
of whole blood, which dissolves all cell membranes thereby lysing
the erythrocytes and removing the cytoplasm and many nucleoproteins
of the nucleated blood cells. This treatment thus results in a
nuclear pellet, which can be smeared onto slides. These slides are
ideal for FISH-analysis, since the removal of so many proteins
makes the nuclear Y-chromosome sequences easy assessable for the
Y-chromosome specific probe. Both the PCR and FISH based techniques
have been used to quantify total number of foetal cells and the
most reliable reports all come to similar results, namely about two
foetal cells pr. ml full blood. The problem with this
quantification is however, that neither of the two methods allows
identification or characterisation of the foetal cell disclosed.
For the PCR-method due to only specific Y-chromosomes sequences
being measured, and for the FISH-method due to the carnoid fixation
removing all cytoplasm.
[0016] Immunocytochemical methods for detecting erythroblast,
lymphoblast and trophoblast have been developed and blood from
pregnant women investigated. In general very varying results have
been obtained, some very compatible with the previous mentioned
cell unspecific estimates. If however the foetal origin of a
candidate cell is established without doubt (double and independent
verifications) the general experience is, that the number of the
three candidate cell types mentioned are one to two orders of
magnitude lower than what was found by the cell unspecific methods.
Thus the maternal blood may comprise foetal cells which are none of
these three proposed cell types.
[0017] In general the methods described so far are laborious and
complex and thus not suitable for routine analysis.
[0018] Thus improved methods for identification of foetal cells are
required in order to utilise foetal cells circulating in pregnant
women for non-invasive pre-natal diagnosis.
[0019] Usable tools in elucidating the identity of foetal cells are
binding members such as antibodies or antibody fragments or
alternative binding molecules capable of specifically recognising
said foetal cells.
[0020] In general antibodies are generated by immunization of
animals, preferably rodents. This usually requires a substantial
amount of the antigenic material for sequential immunisation and/or
several rounds of selection. These methods are thus not suitable
when only a small amount of the antigenic material is available and
when the antigenic material comprises multiple independent
antigens. It is further normally accepted that a high purity of the
target antigenic material is required to obtain suitable antibodies
using these methods.
[0021] In some situations a binding member towards a minor fraction
of molecules present in a sample is desired. That may be such as a
specific cell type in a sample comprising multiple cell types. As
purification of this minor fraction of molecules for which a
binding member is thought is not fisiable or otherwise undesired,
alternative methods are required for generation of binding members
towards target antigenic material present in a low ratio compared
to the amount of non target antigenic material in a sample.
[0022] As described above the abundance of foetal cells in maternal
blood is very limited and only available in small amounts and the
ratio of foetal cells compared to maternal cells in maternal blood
is less than 1/1000. In addition foetal cells are not easily
purified from the maternal blood sample, as a substantially amount
of the foetal cells may be lost during purification, thus
conventional methods of generating antibodies are not efficient for
the purpose of generating antibodies for recognition of foetal
cells.
[0023] As described herein, the applicant has developed a method of
identifying foetal cells furthermore said method allow the
generation of binding members recognising said foetal cells. Hereby
tools suitable for the development of prenatal diagnostic methods
are made available.
[0024] It is further foreseen that the method of generating binding
members may have a general application for generation of binding
members recognising target antigenic material of limited
availability or for the generation of binding members recognising
target antigenic material present intermixed with non-target
antigenic material in a sample.
SUMMARY OF THE INVENTION
[0025] It is an object of the present invention to provide for a
method for detecting foetal cells in a maternal blood sample. The
method developed by the inventors is cell type unspecific and
enables detection of chromosomes, conservation of cell morphology
and immunoassaying of cell type specific proteins.
[0026] Previous methods used for the identification of foetal cells
in maternal blood samples were generally aimed at identifying
foetal cells of a particular type by including an immunologic
selection step selecting specific cell types expected to be present
in the maternal blood sample. Foetal cells as trophoblasts,
erythroblast (nucleated erythocytes) and foetal leukocytes have
been identified using such procedures. The method according to the
present invention does not discriminate between different nucleated
cell types and thus enables detection of foetal cells of various
cell types. The identified cells may thus be any type of foetal
nucleated blood cells, such as, but not limited to, leucocytes,
stem cells (such as hematopoietic stem cells or mesenchymal stem
cells) or placenta derived cells, such as trophoblast cells. The
method according to the invention has further provided a method
allowing detection of foetal cells of new cell types as the method
is not limited to foetal cells of one or more particular cell
types. Foetal cells of any cell type may be identified using the
method described herein including foetal cells of cell types
previously not seen, which may be such as a primitive stem
cell.
[0027] The method described herein is based on the ability to
analyse a large number of cells. As a consequence thereof, the need
for strong enrichments procedures have disappeared and the risk of
loosing foetal cell present in the maternal blood sample is
minimized. The method includes applying a mild fixation and
permeabilisation step, which at the same time does not prevent
accessibility for a hybridisation probes. The method according to
the invention conserve protein content and cell morphology, the
subcellular compartments of the cells, particular the cytoplasm and
the nucleus.
[0028] An aspect of the present invention relates to a method of
detecting a foetal cell in a maternal blood sample comprising the
following steps; [0029] a. providing a maternal blood sample,
[0030] b. fixing and permealizing nucleated cells present in said
sample while; [0031] maintaining cell morphology, [0032]
maintaining protein content of the cells and [0033] ensuring
accessibility for a hybridisation probe and [0034] c. adding a
hybridisation probe
[0035] d. detecting at least one foetal cell.
[0036] Any suitable type of fixation and permeabilisation method
may be used, such as for example methanol and/or acetone
treatment.
[0037] The method may further comprise a pre-fixation step, whereby
any fragile foetal cells are maintained, which preferably comprise
a PFA treatment, most preferably the cells are pre-fixed in a
solution comprising 0.1-4% PFA.
[0038] Following a re-fixation step may be used to provide further
stability and sensibility of the sample during the hybridisation
procedures. Such re-fixation step may comprise PFA treatment, such
as incubation of the cells in a solution comprising 0, 1-5% PFA.
Following the samples are conveniently dehydrated in ethanol.
[0039] Thus in an embodiment the invention relates to a method of
detecting a foetal cell in a maternal blood sample comprising the
following steps; [0040] a. providing a maternal blood sample,
[0041] b. pre-fixing [0042] c. fixing and permealizing nucleated
cells present in said sample while; [0043] maintaining cell
morphology, [0044] maintaining protein content of the cells and
[0045] ensuring accessibility for a hybridisation probe [0046] d.
re-fixing [0047] e. adding a hybridisation probe and [0048] f.
detecting at least one foetal cell.
[0049] The number of maternal cells largely exceeds the number of
foetal cells present in a maternal blood sample, thus it may be
useful to include a step of enrichment whereby maternal cells are
removed from the sample to be analysed. The enrichment step may be
performed at any suitable time point during the procedure, most
suitable as an initial step. In order not to remove any foetal
cells it is preferred that the enrichment step does not
discriminate between different foetal nucleated cell types. A large
fraction of the maternal cells in the blood sample is comprised by
erythrocytes. Several methods of removing erythrocytes is known,
and most convenient is erythrocyte lysis, which may be achieved by
NH.sub.4 Cl mediated lysis.
[0050] As the method of detection of at least one foetal cell
according to the invention includes a fixation and permeabilization
step which ensures accessibility for a hybridization probe, the
foetal cell may following be detected using a hybridisation
technique, which may be used for detecting DNA or RNA within the
nucleus and/or cytoplasm of the cells. Hybridization may be used
for detection of chromosomal DNA or RNA molecules, such as mRNA.
The present invention may for example be used for the detection of
male foetal cells by used of FISH, which may be confirmed by
reverse colour FISH.
[0051] In a further embodiment the methods of detection of at least
one foetal cell according to the invention may include examination
of morphological or epigenetic characteristics for identification
of at least on foetal cell.
[0052] The method described further enables detection of a rare
foetal cell, such as a cell present in a ratio of less than 1 to
10.sup.3 maternal nucleated cells. Thus the method provided herein
enables examination of a large number of cells, which may be
performed using an automated scanner system.
[0053] The method of the invention provides a novel approach to the
analysis of a maternal blood samples and thus provides a new method
for identification of foetal cells, and it follows that foetal
cells identified using this method may be of a cell type different
from foetal cells previously identified.
[0054] An aspect of the invention relates to a foetal cell
identified by the method according to the invention.
[0055] The at least one foetal cell identified by the method
according to the invention thus provides a new tool for generating
foetal cell specific binding members, identification of foetal cell
specific antigens and following the use of binding members and
antigens in assays methods, for use in such as pre-natal
diagnosis.
[0056] The at least one foetal cell identified by the method
described herein are useful for generating foetal cell specific
binding members, which may be obtained by any suitable method such
as, but not limited to, phage display.
[0057] The present invention in an aspect relates to a method of
isolating binding member, such as, but not limited to, antibodies
or antibody fragments recognising antigenic material of limited
availability, such as cells of a specific cell type or a small
sub-populations of cells, such as foetal cells in a maternal blood
sample.
[0058] It is envisioned that this method has a general application
for the generation of binding members recognizing target antigenic
material which is only available in very limited amounts, Antigenic
material of limited availability may be such as a single cell
either in a suspension of other cells of a different cell type or a
specific cell in a heterogeneous tissue, e.g. a micrometastatic
cell, or e.g. a single cell of a tumor. The method may further be
applied to antigens present in a tissue sample, such antigens
present in the extracellular matrix, Limited availability, further
relates to the rareness of the antigenic material, i.e. the low
ratio of the amount of target antigenic material compared to the
amount of non target antigenic material, such as a ratio of less
than 1 target cell to 10.sup.3 non-target cells.
[0059] An aspect of the invention relates to a method of isolating
a binding member recognising target antigenic material of limited
availability, comprising the steps: [0060] a. identifying the
target antigenic material, [0061] b. contacting said antigenic
material with a binding member generating system. [0062] c.
isolating binding members recognising the antigenic material.
[0063] In an embodiment the target antigenic material may be
limited to only one cell.
[0064] Herein limited availability may refer to the rareness of the
target antigenic material.
[0065] In an embodiment the ratio of the target antigenic material
compared to non-target antigenic material is less than 1/1000. In a
preferred embodiment the ratio of the target antigenic material
compared to non-target antigenic material is less than
1/10,000.
[0066] In a preferred embodiment the binding member generating
system allows linking of the phenotype and the genotype of binding
members generated by the system.
[0067] The method according to the invention may further comprise a
step of minimizing isolation of binding members recognising
non-antigenic material, as may be obtained by protection of the
antigenic material and any genetic information encoding the binding
member, bound thereto, from agents or treatment capable of
rendering the genetic material non-replicable, such as UV
irradiation.
[0068] The method according to the invention is useful for
generation of binding members recognizing target antigenic material
of limited availability, such as foetal cell as identified by the
method described herein with out prior purification.
[0069] In an embodiment the method is for generating foetal cell
specific binding members such as recombinant antibody fragments
generated by use of phage display.
[0070] In addition the method of generating binding members
according to the invention is not dependent on the specific
identity of the antigen, e.g. isolated binding members may
recognize different antigens available on/in the target antigenic
materiel, e.g. the cell or tissue. The method further allows the
generated binding members to be retested on the original target
antigenic material whereby specificity may be confirmed.
[0071] Binding members obtained by the method according to the
invention may subsequently be used for the identification of
antigens recognized by the binding member.
[0072] An aspect of the invention relates to binding members
specifically recognising foetal cells such as foetal cells
identified by the method described herein. Such binding members may
be used to identify foetal cell antigens, which may further be used
for generating alternative binding members recognising said foetal
cell antigen.
[0073] Binding members specifically recognising said foetal cell
antigen, may be used in an assay method. Such methods may have
various purposes, such as isolation of foetal cells from a maternal
blood sample or for pre-natal diagnosis using a maternal blood
sample.
[0074] An aspect of the invention relates to an assay method
comprising the following steps; [0075] a. providing a maternal
blood sample, [0076] b. selectively labelling at least one foetal
cell by labelling of a foetal cell antigen according to the
invention in said blood sample.
[0077] An aspect of the invention relates to an assay method
comprising the following steps; [0078] a. providing a maternal
blood sample, [0079] b. selectively labelling at least one foetal
cell by use of a binding member according to the invention, in said
blood sample.
[0080] Selectively labelling may be performed using an
immunodetection technique, preferably by use of a binding member
according to the invention.
[0081] The assay method may further comprise any of the steps
described herein in relation to the method of detecting at least
one foetal cell according to the invention.
[0082] Said selectively labelled foetal cell may be isolated from
the sample, by used of any suitable method such as FACS analysis
and MACS analysis.
[0083] In further embodiments said selectively labelled foetal cell
may be subject to in situ chromosomal/DNA analysis, for the purpose
of gender determination or for detection of chromosomal
abnormality. This is preferably performed by a DNA hybridisation
technique. Examples of such techniques includes, but is not limited
to fluorescent in situ hybridization (FISH). primed in situ
labelling (PRINS), quantitative FISH (Q-FISH) and
multicolor-banding (MCB). Alternatively gender and/or chromosomal
abnormality may be determined by a PCR technique.
[0084] Analysis of a maternal blood sample may be performed at any
time during pregnancy, preferably at 4-24 weeks of gestation and
more preferably at 6-15 weeks of gestation.
[0085] Isolated foetal cells may further be isolated in order to
obtain a foetal cell population, such as a stem cell population,
which may be used for treatment.
DESCRIPTION OF DRAWINGS
[0086] FIG. 1. Detection of one male foetal cell in a maternal
blood sample by use of FISH.
[0087] FIG. 2. Shielding using a puck.
[0088] FIG. 3. Shielding using a shadow stick.
[0089] FIG. 4. Illustration of UV reflection.
[0090] FIG. 5. Prevention of UV reflection using a cylinder.
[0091] FIG. 6. Graphic illustration of ELISA assay tests of 10
selected antibody phage clones. A shows the binding to K564 cells,
B and C show the control experiments using lymphocytes coated
plates and blank plates, respectively.
[0092] FIG. 7. The binding to K562 cells relative to the binding to
lymphocytes.
DETAILED DESCRIPTION OF THE INVENTION
Method of Detecting a Foetal Cell
[0093] The present invention relates to a method of detecting a
foetal cell in a maternal blood sample.
[0094] In order to detect foetal cells in the sample, the applicant
have developed a suitable method for detection foetal cells in a
sample, such a method may include FISH analysis or immunostaining.
Detection of the Y chromosome may for example be used, for the
detection of foetal cells from male embryos. Using a protocol as
describe herein the inventors have detected foetal cells in
maternal blood samples.
[0095] The steps of the method according to the invention is
performed under mild condition suitable for maintaining the cell
morphology i.e., the cytoplasm and nucleus of the cells, in
particular the cytoplasm of foetal cells which may be more fragile
than nucleated maternal cells. Maintaining the morphology of the
cells requires that the cytoplasm and the nucleus may be clearly
visualised in a microscopy following the fixation and
permeabilization step. It is further preferred than the protein
content is maintained by said fixation and permeabilization step,
i.e. that cellular proteins expressed by the cell and in particular
surface proteins expressed by the cells is conserved an available
for recognition by a binding member, such as an antibody following
fixation and permeabilization of said cell.
[0096] The protocol for detection of at least one foetal cell
according to the invention enables detection of foetal cells, which
based on morphological characters appears to be of a different cell
type than foetal cells previously recognised in maternal blood.
[0097] Particularly, a male foetal cell was found to be of a
different type than previously identified nucleated foetal cells,
such as nucleated erythrocytes (erythoblasts), syncytiotrophoblasts
and leukocytes described above. Thus the results suggest that a
foetal cell of a previously unrecognised cell type is detected
using the method according to the invention.
[0098] The detected foetal cell may be discriminated from cells
previously detected in maternal blood samples by the morphology,
extra- or intra-cellular markers. In an embodiment the foetal cell
is discriminated from previously detected foetal cells by a
enlarged cytoplasm and/or an irregular or elongated nucleus.
[0099] In an embodiment the invention relates to a method of
detecting a foetal cell in a maternal blood sample comprising the
following steps: [0100] a. providing a maternal blood sample,
[0101] b. fixing and permealizing nucleated cells present in said
sample while [0102] maintaining cell morphology, [0103] maintaining
protein content of the cells and ensuring accessibility for a
hybridisation probe [0104] c. adding a hybridisation probe and
[0105] d. detecting at least one foetal cell.
[0106] Foetal cells may be distinguished from maternal cells by the
specific recognition of a foetal cell antigen, such as by staining
with a labelled antibody to a protein selectively produced by
foetal cells. Alternatively foetal cells may be distinguished from
maternal cells by the specific recognition of DNA or RNA encoding a
protein selectively or substantially selectively produced by foetal
cells. The foetal cell may further be distinguished from the
maternal cells by epigenetic characteristics, such as telomere
length or methylation status. Alternatively, morphological
characteristics may be used to distinguish foetal cells from
maternally derived cells.
[0107] Detection of at least one foetal cell may include [0108]
detecting DNA in a foetal cell by hybridisation technique or [0109]
detecting RNA, particular mRNA, in a foetal cell by hybridisation
technique or [0110] detection of foetal cell antigen in/on a foetal
cell.
[0111] The method steps described herein may further be used in
combination with the assay method based on the foetal cell, foetal
cell specific antigen and foetal cell specific antigen binding
member described herein below.
Maternal Blood Sample
[0112] It is desirable to obtain as large a maternal blood sample
as possible in order to increase the total number of foetal cells.
However, due to practical problems the sample must be within
certain limits. Accordingly, the size of the maternal blood sample
is preferably in the range of 0.5 to 40 ml, such as in the range of
1 to 30 ml, such as from 2 to 20 ml or 3 to 10 ml.
[0113] The ratio of nucleated foetal cells in a maternal blood
sample is a described in the background section very low, maybe as
low as 1/10.sup.7, or even lower. The method according to the
invention enables detection of at least one foetal cell in a
maternal blood sample wherein the ratio of foetal cells to
nucleated maternal cells is less than 1/100, such as preferably
1/1000 or such as more preferably 1/10.000. In specific embodiments
the ratio of foetal cells to nucleated maternal cells is less than
1/50.000 or preferably less than 1/100.000, such as more preferred
less than 1/10.sup.8, even more preferably less than 1/10.sup.7 or
most preferably 1/10.sup.8.
[0114] Also, according to the invention the sample may be diluted
or concentrated at anytime during the method of identification of
the foetal cells (to facilitate the identification of the foetal
cells). The sample may be diluted at least 1.5 times, such as
twice, more preferred at least three times, such as five times by
adding isotonic buffers, such as saline solutions, phosphate
buffered saline solutions, PBS, and/or suitable growth media, such
as basal media, and tissues growth media. The sample volume may be
decreased to less than 80%, such as 70, or 60 or 50% of the
original sample volume, or even preferable to less than 40%, such
as 25% of the original sample volume.
[0115] The maternal blood sample provided is preferably obtained
from a pregnant woman between 5-24 or 6-20 weeks of gestation, more
preferably between 7-16, or 8-12 weeks of gestation.
Sedimentation
[0116] The cells present in the blood sample may be concentrated by
sedimentation, where the majority of cells present in the sample is
allowed to sediment. The blood sample may prior to sedimentation be
diluted in a suitable solution, such as 0.15 M NaCl.
[0117] The sedimentation may continue until sedimentation has
occurred, such as for at least 5 hours, or over night (see example
1).
[0118] Preferably the sample is allowed to sediment at a
temperature below room temperature, such as at a temperature of
less than 15.degree. C., such as less than 10.degree. C. or
8.degree. C. or 6.degree. C., preferably at a temperature of
2-8.degree. C. or around 4.degree. C.
[0119] A minor population of cells with a low density may not
sediment and may be isolated by mild pre-fixation as described
below, such as in 0.5% paraformaldehyde followed by centrifugation
as described in example 1.
Enrichment
[0120] According to the invention enrichment may be performed to
increase in the number of foetal cells relative to the number of
maternal cells and/or an increase in the number of foetal cells
relative to the current sample volume.
[0121] Methods of enrichment can include but is not limited to,
density gradient centrifugation, FACS, MACS, whereby negative or
positive selection of different cells may be accomplished. The
enrichment procedure may involve multiple steps such as lyses of
maternal cells followed by any suitable separation technique.
[0122] A preferred method of enrichment is lysis of erythrocytes
such as NH.sub.4Cl mediated lysis, which allows selective lysis of
erythrocytes leaving nucleated cells intact. Methods of NH.sub.4 Cl
mediated erythrocyte lysis is know by a person skilled in the art.
A method of erythrocyte lysis is described in the example 1 herein.
Preferably a concentration of 0.1-0.2 mM NH.sub.4Cl is used, such
as 0.14-0.18 mM NH.sub.4Cl more preferably mM 0.15-0.17 NH.sub.4
Cl.
Pre-Fixation
[0123] The inventors have found that an improved result is achieved
when cells are prefixed prior to mounting of the cells on a slide
for further processing. Thus the application provides a method of
detecting foetal cells in a maternal blood sample comprising a step
of pre-fixation, such pre-fixation may involve a mild
paraformaldehyde treatment, such as incubation of cells in a
solution comprising 0-5% PFA, 0.1-4% PFA, preferably 0.2-1% or
0.2-0.7% PFA, most preferably 0.4-0.6 or about 0.5% PFA.
Pre-fixation may be performed for such as at least 1 minute, 2
minutes, 4 minutes or 6 minutes, or such as 10 minutes. In specific
embodiments pre-fixation may be performed for up to 24 hours,
although preferably for less than 12 hours, such as 8, 4 or 2
hours, more preferably less than 1 hour, or less than 30 minutes,
such as 20 or 15 minute, most preferably for less than 10 minutes,
such as for about, 8, 6, 4 or 2 minutes.
Detection
[0124] According to the method of the invention the foetal cell may
be detected using various suitable techniques. In order to detect
the foetal cell the foetal cells are selectively labelled. The
selective labelling of the foetal cells may be carried out by any
suitable method as described herein distinguishing foetal cells
from maternal cells by the specific recognition of a foetal cell
antigen or a protein selectively produced by foetal cells or by the
specific recognition of DNA or RNA encoding a protein selectively
or substantially selectively produced by foetal cells.
Alternatively foetal cells may be detected based on epigenetic
characteristics as described above.
Hybridisation Techniques
[0125] Accordingly, it is an object of the present invention to
provide for the selective labelling of foetal blood cells in the
maternal blood sample based on a hybridisation technique, Male
foetal cells may be detected using a probe to male specific mRNA or
male specific chromosomal DNA. Alternatively, probes recognising
m-RNA selectively expressed by foetal cells may be used.
[0126] Foetal cell specific RNA, generally messenger RNA (mRNA)
sequences may be used as foetal cell markers. The presence of such
mRNA indicates that the gene for the foetal protein is being
transcribed and expressed. The probes used to identify foetal cells
in a sample containing foetal and maternal cells include nucleic
acid molecules, which comprise the nucleotide sequence
complementary to the nucleotide sequence of the RNA molecule
encoding a specific protein. Foetal cells contain distinct mRNAs or
RNA species that do not occur in other cell types. The detection of
these RNAs, can serve to identify cells of foetal or embryonic
origin.
[0127] As described below foetal cell antigens and mRNA encoding
these antigens may be detected in a foetal cell although such
methods have so far not resulted in satisfactory results, which may
be due to the lack of suitable foetal cell markers.
Probe
[0128] According to the invention the probe may be any type of
probe known in the art for detection of RNA or DNA molecules.
Conventional probes know by a person skilled in the art comprise,
RNA and DNA probes synthesised from nucleotides of deoxynucleotide,
respectively using a commercial synthesiser. Probes may be
comprised of the natural nucleotide bases or known analogues of the
natural nucleotide bases. It is further contemplated that the
probes may be oligomers comprising one or more nucleotide analogs
including peptide nucleic acids and other synthetic molecules
capable of Watson Crick-base pairing.
[0129] For detection of chromosomal DNA Flourescence in Situ
Hybridization (FISH) is frequently employed. One procedure for
performing X-Y FISH is described in example 2. Male specific
chromosomal DNA or RNA may be detected by similar means. As an
initial procedure the method of detection a foetal cell according
to the invention was aimed at identifying male cells present in the
maternal blood sample, whereby identification of a foetal cell is
achieved.
Automated Scanning
[0130] Since the preparation of cell according to the invention
involves only limited enrichment of foetal cells and embodiment of
the invention relates to the detection of the very rare foetal
cells by automated scanning using commercial scanners at high
speed. These scanners are in principle fluorescence microscopes
with a moving stage and a camera. The whole slide is photographed
in sections and each picture analysed for presence of foetal cell
specific signals i.e. Y-chromosome signals. Specialized software
that can precisely define a foetal cell as such under the
preparative conditions employed is used. The parameters developed
in the software are size and intensity of signal and shape, density
and size of nucleus.
Identification of Male Foetal Cell
[0131] In an embodiment of the invention a male foetal cells is
detected using a chromosome Y specific probe. Such labelling
methods are well know to persons skilled in the art and are thus
only briefly described below. A male foetal cell may further be
detected by HLA typing for the paternal isotypes.
[0132] Detection of male foetal cells has been used as an example
herein as any male cell detected in a maternal blood sample is
considered to be of foetal origin.
[0133] The method described herein (example 1 and 2) was employed
for the identification of a male foetal cell (FIG. 1). The method
is based on a method of fixation traditionally used for conserving
proteins, which at the same time insures that FISH probes have
access to the nucleus. The pre-fixation is followed by additional
fixation and permeabilization steps. By use of this method,
followed by reverse colour FISH, a male foetal cell was identified
in a maternal blood sample from a woman pregnant with a male
foetus. The method of fixation conserving cellular proteins, allows
further analysis of the detected cells and thus the identification
of cell type specific proteins as is described herein. This method
of identification of foetal cells may thus lead to the
identification of novel foetal cell types. FIG. 1 shows one male
foetal cell identified by the method according to the invention
(for protocol see example 2). FIG. 1A shows a field of vision
wherein one spectrum green labelled cell is identified, while the
remaining nucleated cells are labelled with spectrum orange. FIGS.
1B and 1C show enlargements of the area containing the male foetal
cell from the first and second hybridization, respectively. The
labelling with spectrum green and spectrum orange has switched from
B to C, confirming that the cell is a true foetal cell. The FISH
labelling of the male foetal cell is indicated by an arrow.
[0134] The cell is characterized by an irregular and/or elongated
nucleus and a large cytoplasm. Such morphology is not consistent
with the morphology of foetal cell previously identified in
maternal blood samples and thus a new foetal cell type may have
been identified, for example a stem cell.
[0135] The new approach described herein for detection of at least
one foetal cell, has enable identification of a foetal cell with
characteristics different from previously identified foetal cells,
indicating that a new type of foetal cell is identified. Such new
foetal cell is useful for the generation of new foetal cell
specific antibodies and identification of new foetal cell
antigens.
[0136] It is furthermore possible to identify foetal cell specific
morphological characteristics by characterisation of said
identified foetal cell,
Foetal Cell Antigen
[0137] Foetal cell specific antigens are proteins or other
macromolecules, including modifications of said protein or
macromolecules selectively produced by foetal cells. Proteins which
are foetal cell specific antigens are e.g. embryonic hemoglobin,
such as z globin chains. Foetal cell specific antigens may be used
for detection of foetal cells. Detection of male specific antigen
may likewise be employed for the detection of male foetal
cells.
[0138] Surface antigens are suitable for enrichment procedures by
enabling labelling of foetal cells using conventional methods,
whereby foetal cells may be isolated using FACS, Cellular antigens
may likewise be used in an enrichment procedure although improved
methods of using FAGS may be required.
[0139] In a preferred embodiment a foetal cell antigen is a surface
antigen.
[0140] The present invention provides a method of identification of
a foetal cell with in a sample of maternal blood comprising a
majority of non-foetal cells. The identified foetal cell is
according to the invention used for identification of new foetal
antigens or foetal cell markers. Screening of binding members for
reactivity towards the identified foetal cells may lead to the
identification of new foetal cell specific binding members and
antigens as described herein below and in example 3 and 4.
Binding Members
[0141] A binding member according to the invention is any type of
molecule, such as the molecules defined here below, capable of
binding to a ligand, i.e. the target antigenic material or antigen
as described herein.
[0142] The binding member may be a single moiety, e.g., a
polypeptide or protein, or it may include two or more moieties,
e.g., a pair of polypeptides such as a pair of single chain
antibody domains. Methods of generating antibodies are well know to
person skilled in the art, by immunisation strategies for the
generation of monoclonal or polyclonal antibodies or in vitro
methods for generating alternative binding members. Polyclonal
antibodies may be such as sheep, goat, rabbit or rat polyclonal
antibody. In addition any suitable molecule capable of high
affinity binding may be used including antibody fragments such as
single chain antibodies (scFv), particularly, Fab and scFv
antibodies which may be obtained by phage-display (see below) or
single domain antibodies (VHH) or chimeric antibodies. The binding
member may be derived from a naturally occurring protein or
polypeptide; it may be designed de novo, or it may be selected from
a library. For example, the binding member may be or be derived
from an antibody, a single chain antibody (scFv), a single domain
antibody (VHH), a lipocalin, a single chain MHC molecule, an
Anticalin.TM. (Pieris), an Affibody.TM., a nanobody (Ablynx) or a
Trinectin.TM. (Phylos). Thus methods of generating binding members
of various types are well known in the art.
Antibodies
[0143] A binding member may according to the invention be an
antibody, such as any suitable antibody known in the art including
other immunologically active fragments of antibodies or single
chain antibodies. Antibody molecules are typically Y-shaped
molecules whose basic unit consist of four polypeptides, two
identical heavy chains and two identical light chains, which are
covalently linked together by disulfide bonds. Each of these chains
is folded in discrete domains. The C-terminal regions of both heavy
and light chains are conserved in sequence and are called the
constant regions, also known as C-domains. The N-terminal regions,
also known as V-domains, are variable in sequence and are
responsible for the antibody specificity. The antibody specifically
recognizes and binds to an antigen mainly through six short
complementarity-determining regions located in their V-domains.
Antibody Fragments
[0144] In one embodiment of the invention the binding member is a
fragment of an antibody, preferably an antigen binding fragment or
a variable region. Examples of anti-body fragments useful with the
present invention include Fab, Fab', F(ab')) and Fv fragments.
Papain digestion of antibodies produces two identical antigen
binding fragments, called the Fab fragment, each with a single
antigen binding site, and a residual "Fc" fragment, so-called for
its ability to crystallize readily. Pepsin treatment yields an
F(ab').sub.2 fragment that has two antigen binding fragments which
are capable of cross-linking antigen, and a residual other fragment
(which is termed pFc').
[0145] Additional fragments can include diabodies, linear
antibodies, single-chain antibody molecules, and multispecific
antibodies formed from antibody fragments.
[0146] The antibody fragments Fab, Fv and scFv differ from whole
antibodies in that the antibody fragments carry only a single
antigen-binding site. Recombinant fragments with two binding sites
have been made in several ways, for example, by chemical
cross-linking of cysteine residues introduced at the C-terminus of
the VH of an Fv (Cumber et al., 1992), or at the C-terminus of the
VL of an scFv (Pack and Pluckthun, 1992), or through the hinge
cysteine residues of Fab's (Carter et al., 1992).
[0147] Preferred antibody fragments retain some or essential all
the ability of an antibody to selectively binding with its antigen
or receptor. Some preferred fragments are defined as follows:
[0148] Fab is the fragment that contains a monovalent
antigen-binding fragment of an anti-body molecule. A Fab fragment
can be produced by digestion of whole antibody with the enzyme
papain to yield an intact light chain and a portion of one heavy
chain.
[0149] Fab' is the fragment of an antibody molecule and can be
obtained by treating whole antibody with pepsin, followed by
reduction, to yield an intact light chain and a portion of the
heavy chain, Two Fab' fragments are obtained per antibody molecule.
Fab' fragments differ from Fab fragments by the addition of a few
residues at the carboxyl terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
[0150] (Fab').sub.2 is the fragment of an antibody that can be
obtained by treating whole anti-body with the enzyme pepsin without
subsequent reduction. F(ab').sub.2 is a dimer of two Fab' fragments
held together by two disulfide bonds.
[0151] Fv is the minimum antibody fragment that contains a complete
antigen recognition and binding site. This region consists of a
dimer of one heavy and one light chain variable domain in a tight,
non-covalent association (V.sub.H-V.sub.L dimer). It is in this
configuration that the three CDRs of each variable domain interact
to define an antigen binding site on the surface of the
V.sub.H-V.sub.L dimer. Collectively, the six CDRs confer antigen
binding specificity to the antibody. However, even a single
variable domain (or half of an Fv comprising only three CDRs
specific for an antigen) has the ability to recognize and bind
antigen, although at a lower affinity than the entire binding
site.
[0152] In one embodiment of the present invention the antibody is a
single chain antibody ("SCA"), defined as a genetically engineered
molecule containing the variable region of the light chain, the
variable region of the heavy chain, linked by a suitable
poly-peptide linker as a genetically fused single chain molecule.
Such single chain anti-bodies are also referred to as "single-chain
Fv" or "sFv" antibody fragments. Generally, the Fv polypeptide
further comprises a polypeptide linker between the V.sub.H and
V.sub.L domains that enables the sFv to form the desired structure
for antigen binding.
[0153] The antibody fragments according to the invention may be
produced in any suitable manner known to the person skilled in the
art. Several microbial expression systems have already been
developed for producing active antibody fragments, e.g. the
production of Fab in various hosts, such as E. coli, yeast, and the
filamentous fungus Trichoderma reesei are known in the art. The
recombinant protein yields in these alternative systems can be
relatively high (1-2 g/l for Fab secreted to the periplasmic space
of E. coli in high cell density fermentation or at a lower level,
e.g. about 0.1 mg/l for Fab in yeast in fermenters, and 150 mg/l
for a fusion protein CBHI-Fab and 1 mg/l for Fab in Trichoderma in
fermenters and such production is very cheap compared to whole
antibody production in mammalian cells (hybridoma, myeloma,
CHO).
[0154] The fragments can be produced as Fab's or as Fv's, but
additionally it has been shown that a VH and a VL can be
genetically linked in either order by a flexible polypeptide
linker, which combination is known as an scFv.
Natural Single Domain Antibodies
[0155] Heavy-chain antibodies (HCAbs) are naturally produced by
camelids (camels, dromedaries and llamas). HCAbs are homodimers of
heavy chains only, devoid of light chains and the first constant
domain (Hamers-Casterman et al., 1993). The possibility to immunise
these animals allows for the cloning, selection and production of
an antigen binding unit consisting of a single-domain only.
Furthermore these minimal-sized antigen binding fragments are well
expressed in bacteria, interact with the antigen with high affinity
and are very stable.
[0156] New or Nurse Shark Antigen Receptor (NAR) protein exists as
a dimer of two heavy chains with no associated light chains. Each
chain is composed of one variable (V) and five constant domains.
The NAR proteins constitute a single immunoglobulin variable-like
domain (Greenberg et al) which is much lighter than an antibody
molecule.
[0157] According to the invention natural single domain antibodies
may be considered an antibody fragment. The proteins may be
produced and purified by any suitable method know by a person
skilled in the art as described above.
[0158] In a further embodiment the binding member is active
fragments of antibodies selected from Fab, Fab', F(ab).sub.2, Fv,
HCAbs and NARs.
Non-Immonoglobulin Binding Members
[0159] In one preferred embodiment, the present invention relates
to binding members derived from a naturally occurring protein or
polypeptide; said protein or polypeptide may for example be
designed de novo, or may be selected from a library. The binding
member may be a single moiety, e.g., a polypeptide or protein
domain, or it may include two or more moieties, e.g., a pair of
polypeptides such as a pair polypeptides. The binding member may
for example, but exclusively, be a lipocalin, a single chain MHC
molecule, an Anticalin.TM. (Pieris), an Affibody.TM., or a
Trinectin.TM. (Phylos), Nanobodies (Ablynx). The binding member may
be selected or designed by recombinant methods known by people well
known in the art.
[0160] In another embodiment of the invention the binding member is
an affibody, such as any suitable affibody known in the art, in
particular antibodies as defined herein, such as affibodies or
immonologically fragments of affibodies, Affibodies are selected in
vitro, from an affibody library constructed by combinatorial
variation of the IgG binding domain of Protein A. Protein A is a
surface protein from the bacterium Staphylococcus aureus. The
biding domain consists of 58 residue, where of 13 are randomized to
generate Affibody.RTM. libraries Thus, the size of an affibody is
considerably less than of an antibody (www.affibody.com).
Method of Generating Binding Members
[0161] As described above foetal cells, obtained from a maternal
blood sample, are of very limited availability and are not usable
for immunization or other standard methods of generating binding
members. The applicants have developed a method that allows
generation of binding members recognising target antigenic material
of limited availability and purity. The limited availability of the
target antigen material may be due to the target antigen material
being present in a sample intermixed with non-target antigenic
material, in situations where purification of the target antigen
material is not desirable or feasible alternative methods of
providing specific binding members are required. The method
described herein is thus not limited to the isolation and
generation of binding members recognising foetal cells, but applies
equally to other target antigen material of limited
availability.
[0162] An aspect of the invention relates to a method of isolating
binding members recognizing target antigenic material of limited
availability comprises the steps: [0163] a. identifying the target
antigenic material, [0164] b. contacting said target antigenic
material with a binding member generating system and [0165] c.
isolating binding member(s) recognising the target antigenic
material,
Target Antigenic Material
[0166] The target antigenic material may be any type of material
for which specific binding members are thought. The method allows
selection of binding members to non purified antigens or
non-homogenouse antigens, the target antigenic material may be such
as a mixture of antigens, cells or other material comprising
multiple antigens.
[0167] In an embodiment the ratio of the amount of target antigen
material compared to the amount of non-target antigen material is
less than 1/100, such as less 1/5000, such as less 1/10.000 or
preferably less than 1/50.000 or more preferably less than
1/100,000, such as less than 1/10.sup.6, more preferably less than
1/10.sup.7 or most preferably 1/10.sup.8.
[0168] The target antigen ratio,
R TA = target antigen material non - target antigen material .
##EQU00001##
[0169] In an embodiment T.sub.TA is less than 1/10.sup.2, or
preferably less than 1/10.sup.3, or more preferably less than
1/10.sup.5, even further preferred less than 1/10.sup.6, more
preferably less than 1/10.sup.7 or most preferably 1/10.sup.8.
[0170] Depending on the type of antigen material the amounts may be
calculated in numbers, weight or other suitable units.
[0171] In an embodiment the target antigenic material is a sample
of cells, such as cells of a specific cell type for which binding
members are thought. In an embodiment the target antigenic material
may be limited to only one cell in a sample, where such a cell may
be present either in suspension or fixed to a slide intermixed with
other cells,
[0172] In an embodiment the target antigenic material is present on
a slide, such as cells fixed on a slide. Said cell may be located
on a slide in between other cells. Location of the cell may be
available using micro-dissection/micro manipulation systems.
[0173] In a preferred embodiment the target antigenic material is
one or more foetal cell(s), such as foetal cells identified
according to the method described herein.
[0174] In a preferred embodiment the target antigenic material is a
foetal cell identified as described herein. In a preferred
embodiment the foetal cells is such as a male foetal cell
identified by reverse colour FISH and automated scanning.
[0175] In an embodiment the target antigenic material is
extracellular matrix.
[0176] In an embodiment the binding member is selected from the
group of antibodies, antibody fragments and non-immune binding
members.
[0177] In a specific embodiment the binding member is an antibody.
In a further embodiment the binding member is an antibody fragment.
In a subsequent embodiment the binding member is a non-immune
binding member.
Binding Member Generating System
[0178] The target antigenic material is contacted with any suitable
binding member generating system known in the art.
[0179] The binding member generating system preferably allows a
linkage between the genotype of the binding member and the
phenotype of the binding member. Such linkage allows convenient
characterisation of the sequence of an identified binding member.
Such binding member generating system known in the art, include but
are not limited to, ribosome display (Hanes, J et al and Lipovsek,
D et al), mRNA display (reviewed in Gold, L (2001), DNA display
(including cis display (Odegrip R et al. 2004)), yeast display
(Boder, E T., et al. 1997), bacterial display (Francisco, J. A. et
al. 1993 and Georgiu G et al 1997), retroviral display (Ager S,
1996, Buckholtz et al. 1998) etc. and phage display as described
here below. The linkage of the genotype and phenotype provides easy
aces to the genetic information related to any identified binding
member, whereby the region of specificity may be easily
characterised.
[0180] The linkage between the phenotype and the genotype permit
manipulation of the genetic material encoding isolated binding
members.
[0181] In an embodiment the binding member generating system allows
linkage between the genotype and the phenotype of binding members
generated by the system
[0182] In a preferred embodiment the invention relates to a method
of isolating binding members recognizing target antigenic material
of limited availability comprising the steps: [0183] a. identifying
the target antigenic material, [0184] b. contacting said target
antigenic material with a binding member generating system which
allows linkage between the genotype and the phenotype of generated
binding members and [0185] c. isolating binding member(s)
recognising the target antigenic material.
[0186] In a preferred embodiment the binding member generating
system is based on phages expressing suitable binding members, such
as antibody fragments on there surface as described here below.
[0187] Alternative methods such as compartmentalisation of genotype
and phenotype may also be applied if the target antigenic material
is analysed in suspension.
Binding Members Generated by Phage Display
[0188] Phage display technology (Smith et al. 1985, Winter et al,
1994 and Griffiths et al., 1994) is a useful system for generating
binding member according to the invention.
[0189] Phage-displayed antibody technology enables generation of
high-affinity binding sites without the constraint imposed by
classical method for generating either polyclonal or monoclonal
antibody. Phage libraries that are successfully used to generate
desired binding sites are constructed from the genetic material
obtained from human or other eukaryotic species, Various types of
library formats are known to the person skilled in the art, such as
but not limited to immunised libraries, naive libraries,
semisynthetic libraries, single scaffold or single domain
libraries. These libraries can for example, but not limited to, be
generated from the light-chain and heavy-chain IgM-V-gene pools of
B cells of non-immunized healthy donors, which are isolated from
peripheral blood lymphocytes, bone marrow, or spleen cells. The
antibody can also be engineered with in-built features that suit
various downstream applications. The whole antibodies cannot be
functionally expressed in bacteria, thus only the antibody
fragments that contain the binding regions are displayed on the
surface of the bacteriophage. It has been shown that both Fab and
single-chain Fv (scFv) can be expressed on the surface of M13
without apparent loss of the antibody's specificity and affinity.
Most of the phage libraries that have been constructed, display the
antibody fragment on the surface of the phage minor coat proteins
(pIII), Antibodies with an affinity in the 1-200 nM range are
routinely selected. The affinity of the antibodies isolated from
phage library can be further improved by various methods of
affinity maturation.
[0190] In a preferred embodiment the binding member is an antibody
fragment generated by phage display.
Isolation of Binding Members Recognising the Target Antigenic
Material
[0191] Following the initial contacting of the target antigenic
material and the binding member generating system, binding members
associated with the target antigenic material are isolated and
binding members recognising the target antigenic material are
thereby obtained. Due to the linkage between the gene encoding the
binding member and the binding member, the genetic information (the
sequence) of the binding member is obtained at the same time.
Binding members may be subjected to further rounds of selection in
order to obtained binding members with high specificity. Steps of
protein evolution/maturation may further be included in order to
obtain binding members with a high affinity.
[0192] If the target antigenic material is not isolated from
non-target antigenic material, the isolation of specific binding
members may be more challenging as binding members associated with
non-target antigenic material are preferably removed, inactivated
and/or eliminated before isolating binding members bound to the
target antigenic material.
[0193] By removing, inactivating and/or eliminating binding members
associated with non-target antigenic material a higher proportion
of antigen specific binding members specifically recognising the
target antigenic material, compared to binding members recognising
the non-target antigenic material is obtained.
[0194] The method of the present invention may further comprise a
step of minimizing the number of binding members retrieved
recognizing any non-target antigenic material present adjacent to
the target antigenic material. This step serves to narrow down the
number of binding members recognising non-target antigenic material
which may be such as proteins or other macromolecules which are
expressed at identical level in target antigenic material and in
non-target antigenic material. In an embodiment the binding member
generating system binding non-target antigenic material is
inactivated.
[0195] As described above the binding activity and the genetic
information of a binding member is preferably linked in the binding
member generating system. The genetic material encoding the binding
members may thus be compromised by introducing damage or
crosslinks, where by the genetic information is rendered
non-replicable. Such damage or crosslinks can be generated by
various means known to a person skilled in the art, such as by UV
irradiation.
[0196] In a preferred embodiment the minimizing step comprise UV
irradiation.
[0197] It is evident that target antigenic material, including
associated binding members must be protected from agents damaging
the genetic information, such as UV irradiation. In an embodiment
the target antigenic material and associated binding members is
protected from UV irradiation by shielding. This is particularly
useful when the binding members are linked to genetic/sequence
information responsible for further replication or amplification of
the binding member. During UV irradiation the target antigenic
material is protected from UV irradiation by shielding the selected
area. Binding members bound to the target antigenic material may be
retrieved with out contamination of binding members bound to
material outside the protected region and these binding members may
then be amplificated by suitable methods.
[0198] The shield may be any suitable particle or item capable of
preventing UV light from reaching the selected region. Suitable
shields may be shadow stick, gold plate or gold puck, which is to
be position above the selected region during UV light exposure
(FIG. 2 and FIG. 3).
[0199] The size of the shield may be such as 50, .mu.m, 80 .mu.m,
100 .mu.m, 120 .mu.m, 150 .mu.m or 200 .mu.m depending on the size
of the selected region, preferably between 60 .mu.m and 200 .mu.m,
more preferably between 70-120 .mu.m.
[0200] In order to optimise the selection of binding members
recognising the target antigenic material in the selected region,
reflection of the UV light may be minimized, to avoid inactivation
of phages bound in the selected region. The problem of reflection
is illustrated in FIG. 4.
[0201] UV reflection may be limited by reducing the light with
non-vertical angel, by placing the shield close to the selected
area, such as with in 5 mm, 3 mm, 2 mm and 1 mm. Reflection may
further be decrease by a black back colour of the slide, or by
grinding of the back surface. Alternatively a cylindrical wall may
be placed around the selected area to prevent non-vertical UV light
to reach the selected area from the side (FIG. 5).
Isolation of Binding Members
[0202] The step(s) of isolating binding members are dependent on
the binding member generating system applied, and are thus selected
accordingly.
[0203] In a particular embodiment, as described in example 8,
binding members are generated by use of phages. Phages, expressing
binding members, binding the target antigenic material may be
isolated by eluting the phages from the selected area by use of any
suitable method including such as by use of trypsin or in low pH
buffer followed by neutralisation such as by use of mM Glycine, pH
2.2 and Tris-HCl for neutralization.
[0204] Following, the binding members are subcloned into a suitable
vector for large scale production of the binding members. For
binding members generated by used of phages this may conveniently
be performed without the binding members being displayed on phage
particles. Likewise for other methods, the availability of the
genetic information may ease subsequent steps of selection.
[0205] The specificity of isolated binding members may then be
tested against target anti-genic material and non target antigenic
material. Cell type specific binding members may be tested against
cells of the same type and cells of a different type whereby cell
type specific binding members may be identified.
[0206] In order to obtain foetal cell specific binding members the
isolated binding members must be screen for reactivity towards
non-foetal cells, such as adult blood cells particularly maternal
blood cells.
[0207] Binding members recognizing antigens present at similar or
higher levels on adult blood cells, compared to foetal cells are to
be excluded. This can be performed by staining a slide containing a
mixed population of cells (such as the one use initial for
selection of the binding members) or in an ELISA assay. The
antibodies may be tested in ELISA against different cell types. As
many different cultures as possible may be used, preferably
including embryonic, foetal and stem cells cultures. Any cell line
or cell lines similar or related to the identified foetal cell
based on morphology may be preferred. Identification of a pattern
of recognition may serve to validate the specificity of the
isolated antibodies or antibody fragments
[0208] In example 8, generation of phage antibody fragments to K562
cells is described. The specificity of the antibody fragments is
evaluated by comparing the binding activity towards K562 cells to
the binding activity towards lymphocytes (FIG. 6.9). The data shows
that the method of the present invention is useful for generation
of cell type specific antibody fragments, based on a very limited
amount of target antigenic material, e.g. few cells on a slide. The
graphs shows examples of an antibody fragment (S2.1A8) with
approximately 20 fold higher activity and an antibody fragment
(S3.1A9) with approximately 48 fold higher activity at a dilution
of 1:200, towards K562 cells than towards lymphocytes. As seen
phage display is particular useful for generation of cell type
specific antibody fragments.
[0209] According to the invention foetal cell specific binding
member may be generated using identified foetal cells as antigen in
the method described herein and in example 3 and 8.
[0210] The identified binding members such as scFv antibody
fragments may be used for staining of foetal blood samples whereby
the specificity and localization of the antigen is tested. The
method described herein further allows retesting on the identified
antibodies on the slide by re-hybridization procedures.
[0211] The binding members preferably bind specifically to a foetal
cell antigen present on/in a foetal cell. As the method of
identification of foetal cell and following the method of isolating
binding members as described herein, are cell type unspecific.
antibodies towards foetal cell antigens identified using these
methods are cell type non-specific.
[0212] Accordingly, an aspect of the present invention relates to
foetal cell specific binding members isolated by its binding
capability to a foetal cell, such as a foetal cell identified as
described herein. Binding members isolated by phage display are
further preferred.
Identification of New Foetal Cell Specific Antigens
[0213] In order to obtain binding members specific for the foetal
cell identified according to the method described herein the
binding members are screened for cross reactivity with other cells
and antigens. The specificity of the antibody is tested against a
panel of previously known foetal cell markers to confirm the
identification of binding members recognising a previously unknown
foetal cell antigen/marker. Due to the initial detection method
which does now discriminate between different cell types, the
identified binding members may recognise a plurality of foetal cell
types, even foetal cell types not previously known.
[0214] The binding members are screened against adult blood cells
and/or previously know foetal cell makers, thus the binding members
do preferably not bind foetal cell antigens such as embryonic
hemoglobin, such as .epsilon. globin chains and zeta globin chains,
and foetal hemoglobin, such as gamma globin chains and trophoblast
specific antigens.
[0215] When specificity of the antibody is confirmed, the identity
of the foetal cell marker/antigen recognised by the identified
foetal cell specific antibody fragments may be determined, Antigens
bound/recognised by the selected antibody may be identified by
several methods (see example 5) know by the skilled person such as
screening of c-DNA expression libraries or proteins purification
procedures, examples of such methods include, but is not limited
to, immunoprecipitation or 2D-PAGE followed by mass
spectroscopy.
[0216] The identified foetal cell antigen may then be used to
select further binding members specifically recognising said
antigen, which may be used for labelling of foetal cells in
maternal blood samples.
[0217] An aspect of the present invention relates to binding
members, such as antibodies or antibody fragments recognising
foetal cell antigens identified by the method described herein,
such as by characterisation of antigens recognised by antibody
fragments isolated by bio-panning of a phage display library on a
detected foetal cell. Following such binding members, antibody or
antibody fragments may be used for labelling of foetal cells.
[0218] The labelling as described below may be carried out by use
of any specific binding member. Particular by use of an antibody
selected from monoclonal and polyclonal antibodies or phage display
generated antibody fragments. The binding members may be unlabelled
antibodies, biotin labelled antibodies, fluorochrome labelled
anti-bodies or both fluorochrome and biotin labelled antibodies
recognising any foetal specific antigen as identified by the method
of the present invention.
Fluorochrome
[0219] The fluorochrome is selected to be excited in the
wave-length area of the detection means, and furthermore in
suitable combination with an optional second labelling. In
particular the fluorochromes may be selected from FITC
(fluorescein-isofluocyanate) or TRITC (Rhodanine
Tetramethyl-isofluocyanate) having excitation at 495 nm and 520-530
nm, respectively. Further fluorochromes which may be used are
listed in table 1.
TABLE-US-00001 TABLE 1 Wave length of excitation and emission of
various flourochroms. Fluorochome Ex (nm) Em (nm) MW Notes Reactive
and conjugated probes Hydroxycoumarin 325 386 331 Succinimidyl
ester Aminocoumarin 350 445 330 Succinimidyl ester Methoxycoumarin
360 410 317 Succinimidyl ester Cascade Blue 375; 400 423 596
Hydrazide Lucifer yellow 425 528 NBD 466 539 294 NBD-X
R-Phycoerythrin (PE) 480; 565 578 240 PE-Cy5 conjugates 480; 565;
650 670 aka Cychrome, R670, Tri- Color, Quantum Red PE-Cy7
conjugates 480; 565; 743 767 APC-Cy7 conjugates 650; 755 767
PharRed Red 613 480; 565 613 PE-Texas Red Fluorescein 495 519 389
FITC; pH sensitive FluorX 494 520 587 (AP Biotech) BODIPY-FL 503
512 Tetramethylrhodamine 550 560-608 444 TRITC Tetramethylrhodamine
547 530-560 isothiocyanate X-Rhodamine 570 576 548 XRITC Lissamine
Rhodamine B 570 590 PerCP 490 675 Peridinin chlorphyll protein
Texas Red 589 (603) 615 625 Sulfonyl chloride Allophycocyanin (APC)
650 660 104 TruRed 490, 675 695 PerCP-Cy5.5 conjugate Alexa Fluor
dyes (Molecular Probes) Alexa Fluor 350 346 445 410 Alexa Fluor 430
430 545 701 Alexa Fluor 488 494 517 643 Alexa Fluor 532 530 555 724
Alexa Fluor 546 556 573 1079 Alexa Fluor 555 556 573 1250 Alexa
Fluor 568 578 603 792 Alexa Fluor 594 590 617 820 Alexa Fluor 633
621 639 1200 Alexa Fluor 647 650 668 1250 Alexa Fluor 660 663 690
1100 Alexa Fluor 680 679 702 1150 Alexa Fluor 700 696 719 Alexa
Fluor 750 752 779 Spectrum dyes (Vysis) SpectrumOrange 559 588
SpectrumGreen1 497 524 SpectrumGreen.sup.2 509 538 SpectrumAqua 433
480 SpectrumBlue 400 450 SpectrumGold 530 555 SpectrumRed 592 612
SpectrumFRed (far red) 655 675 Cy Dyes (AP Biotech) Cy2 489 506 714
Cy3 (512); 550 570; (615) 767 Cy3.5 581 596; (640) 1102 Cy5 (625);
650 670 792 Cy5.5 675 694 1128 Cy7 743 767 818
[0220] Unlabelled binding members may be used as known in the art,
by use of a second labelling step with e.g. a secondary antibody
against the unlabelled binding members, said antibody being
labelled as discussed above, such as fluorochrome labelled. By this
two-step it may be possible to enhance the signals from the foetal
cells. Further detection steps may be included by using indirect
labelling as described here below.
Labelling
[0221] In one embodiment of the invention a binding member (such as
an antibody) or a synthetic probe is directly labelled, by having
fluorochromes covalently attached thereto. The binding of such
probes or binding member to the target in the cell may be observed
under a microscope as a bright fluorescence or may be detected by a
fluorimetric apparatus,
[0222] Instead of direct labelling or in addition to the direct
labelling in another embodiment the probes or binding members are
indirectly labelled with biotin or enzymes for example, such as
alkaline phosphatase or peroxidase. Biotin may be detected using
suitable streptavidin/avidin molecules know to people skilled in
the art. These complexes may comprise fluorochroms or suitable
enzymes.
[0223] By use of a combination of labelling methods it is possible
to enhance the signals from the foetal cells, thereby facilitating
the identification thereof.
[0224] In order to enhance the probability and/or selectivity of
identifying the foetal cells over the background of maternal cells,
two or more selective labellings may be performed. The two or more
labellings may be a combination of any of the labellings used for
single labelling described above. Accordingly, the combined
labelling may be carried out by the use of two or more different
hybridisation probes, such as a combination of a DNA probe and a
PNA probe for hybridisation with the same foetal RNA or more
preferred with different foetal RNAs. Also, two or more different
DNA probes (or PNA probes, or similar probes capable of specific
hybridisation) may be used for hybridisation with different foetal
RNAs. Likewise a combination of different binding members may be
used, either with specificity for the same foetal antigen or with
specificity for different antigens. In further embodiments
labelling with a combination of nucleotide probes and binding
members may be performed.
Use of a Binding Member According to the Invention
[0225] The identification of a new foetal cell using the method
according to the invention have opened the possibility to use
binding members, such as binding members identified as described
herein for various application. Such binding members may be useful
in several assay method such as for the purpose of pre-natal
diagnosis or for the purpose of isolating a population of foetal
cells. Such binding members may be used as described above for
selectively labelling of foetal cells which may be processed
further depending on the purpose of the assay.
[0226] In a further aspect the invention relates to an assay method
comprising the steps of; [0227] a. providing a maternal blood
sample and [0228] b. selectively labelling at least one foetal cell
by labelling of a foetal cell antigen according to the invention or
[0229] a. providing a maternal blood sample and [0230] b.
selectively labelling at least one foetal cell by use of a binding
member according to the invention, [0231] in said blood sample.
[0232] An embodiment of the invention relates to an assay method
for identifying at least on foetal cell by use of a binding member
as described herein, recognising a foetal cell type specific
protein of morphological characteristics as described herein, Said
at least on foetal cell may for example be selectively labelled
using an immunodetection technique by use of a binding member,
particular a binding member specifically recognizing the foetal
cell identified by the method of the invention.
[0233] The invention further relates to an assay as described here
above, comprising one or more steps of labelling foetal cell as
described herein for specific purposes. The assay method may
include steps of labelling foetal cell antigens of mRNA of foetal
cell antigens such as the previously known foetal cell antigens
described in the section related thereto.
Isolation of Foetal Cells
[0234] Selective labelling of foetal cells using the binding member
according to the invention may in a further embodiment be used for
selection of foetal cells recognized by the binding member. By use
of suitable techniques known in the art, the selectively labelled
cells may be isolated from the cell sample and processed further.
The isolated foetal cells may be used for pre-natal diagnosis or
for specific application related to the cell type isolated (see
below) in further embodiment the assay may be for determination of
gender or chromosomal abnormalities.
Pre-Natal Gender Determination
[0235] The findings according to the present invention may further
be used for determination of gender of the foetus, either by use of
male specific probes or by employing antigen binding members
identified by the method described herein for the detection of
foetal cells, followed by suitable methods for determination of
gender known to a person skilled in the art.
Prenatal Diagnosis of Chromosomal Abnormality
[0236] In parallel to determination of gender, the invention
further relates to methods for determination of chromosomal
abnormalities by detection of foetal cells based on antigens or
binding member recognising said foetal cell antigens isolated or
identified based on the present invention. Such methods of
determination of chromosomal abnormalities relates to the detection
of such as aneuploidy, translocation, unbalanced translocation,
rearrangement, subtelomeric rearrangement, unbalance chromosomal
rearrangement, unbalance subtelomeric rearrangement, deletion,
inversions, unbalanced inversions, duplication and telomere
instability and or shortening. The chromosomal abnormality may
further be such as single nucleotide substitution, micro deletion,
micro-insertion, short deletions, short insertion, multi-nucleotide
changes, DNA methylation and/or loss of imprint. (LOI)
[0237] In a preferred embodiment chromosomal aneuploidy is a
complete and/or partial trisomy. Such as trisomy 21, trisomy 18,
trisomy 13, trisomy 16 and/or XXX and other sex chromosome
abnormalities. Alternatively the aneuploidy is a complete and/or
partial monosomy, such as monosomy X, monosomy 21, monosomy 22,
monosomy 16 and/or monosomy 15.
[0238] DNA hybridisation techniques may be used for determination
of gender or determination of chromosomal abnormalities. Techniques
known in the art includes methods such as fluorescent in situ
hybridization (FISH), primed in situ labelling (PRINS),
quantitative FISH (Q-FISH) and multicolor-banding (MCB).
Fluorescense in situ hybridization (FISH) makes use of molecular
probes labelled as described above with e.g. a fluorescence. A
probe corresponding to a gene or DNA sequence is used and shows a
signal under a microscope at a specific locus on a chromosome. The
FISH technique may be applied to interphase cells and may confirm
the presence of an euploid or an aneuploid of chromosomes X, Y, 13,
15, 18, 21. FISH is useful for identifying abnormal numbers of
chromosomes such as trisomies and monosomies and may, when probes
are available for specific regions of chromosomes, be used to
determine if deletions, translocations, or duplications are
present.
[0239] As an alternative to the above mentioned hybridisation
techniques PCR methods may be used for determining chromosomal
abnormalities. PCR methods according to the invention includes
suitable method known in the art, capable of detecting
abnormalities as trisomies etc. as described above. PCR methods may
further be employed for determination of minor abnormalities, such
as small deletions of mutation in specific genes. Quantitative
fluorescent PCR (QF-PCR) is an example of such methods suitable for
detection of for example trisomy 13, 18, 21, triploidies, double
trisomies as well as X and Y aneuploidies (V. Cirigliano, 2004). By
the design of suitable primers for minor but non the less severe
chromosomal abnormalities PCR methods may be used for determination
of disease such as for example Cystic Fibrosis which is often
caused by a 3 bp deletion in the Cystic Fibrosis Gene leading to a
protein which lacks a critical phenylalanine amino acid.
Foetal Stem Cell
[0240] The foetal cells may as described above may be a stem cells.
Stem cells come in different varieties, relating to when and where
they are produced during development, and how versatile they are.
The foetal stem cells detected may be of any type, such as
embryonic, or somatic, being pluripotent or multipotent.
Use of Stem Cells.
[0241] By applying the technology described herein, foetal stem
cells may be isolated from a maternal blood samples by use of a
binding member, antibody or antibody fragment recognising said
foetal cell antigen according to the invention. Stem cells can
produce more stem cells and they can be used to generate
specialized cell types such as nerve, blood or liver cells.
Depending on the types of stem cells isolated the cells may have
varying application in the development of cells of specific cell
types or tissue. Pluripotent stem cells may give rise to any cell
type whereas multipotent stem cells may give rise to a more limited
number of cell types. For example, blood-forming (haematopoietic)
stem cells may be capable of forming all types of blood cells,
whereas mesenchymal stem cells are capable of forming mesenchymal
cells.
[0242] Stem cells, especially pluripotent stem cells may be used
for treatment of a variety of disease. Pluripotent stem cells are
traditionally embryonic stem cells, which due to ethical
considerations are of limited availability. The possibility of
using stem cells isolated from a maternal blood sample is an
attractive alternative. Pluripotent stem cells may be used for
treatment of a plurality of diseases for which conventional methods
does not provide suitable treatment.
DETAILED DESCRIPTION OF THE DRAWINGS
[0243] FIG. 1
Detection of One Male Foetal Cell.
[0244] A slide comprising maternal blood cells prepared as in
example 1, is hybridized with DXZ1 and DYZ1 probes labelled with
spectrum green and spectrum orange as described in example 2. FIG.
1B shows the area of the male foetal cell after the first
hybridisation wherein the spectrum orange label is indicated with
an arrow. Following stripping and re-hybridisation with reversed
labelling the labelling has switched confirming the male identity
of the cell. The green labelling of the cell is indicated by the
arrow on FIG. 10. FIG. 1A shows an enlarged area of the slide after
the second labelling again the male cell is indicated by an
arrow.
[0245] FIG. 2
Shielding of Target Antigenic Material (Cells) from UV Light.
[0246] The figure shows a graphical view of a slide covered with
cells, including a cell, for antibody selection. The slides are
hybridized with phages and following "the cell" is covered with a
puck protecting the cell, and the associated phages from damage
during UV irradiation.
[0247] FIG. 3
Shielding of Target Antigenic Material (Cells) from UV Light Using
a Shadow Stick.
[0248] The figure shows a situation as in FIG. 2, wherein the
shielding is performed by a shadow stick.
[0249] FIG. 4
Reflection of UV Light.
[0250] The figure shows a drawing illustrating the problem of
reflection of UV light, Non-vertical UV light may indirectly reach
"the cell" and thus reduce the number of viable phages that can be
eluted from the cell.
[0251] FIG. 5
Shielding of Target Antigenic Material (Cells) from Reflected UV
Light Using a Cylinder.
[0252] A cylinder is placed around "the cell" to minimised the
amount of reflected UV light.
[0253] FIG. 6
Graphic Illustration of ELISA Assay Results
[0254] Elisa data, obtained as described in example 8, is shown
graphically. A The binding activity to K562 cells are measured in
three different dilutions for each clone. B and C. The binding
activity to lymphocytes and a blank ELISA plate is measured
similarity for each clone.
[0255] FIG. 7
Specificity of K562 Binding.
[0256] The data shown in FIG. 6 is used to evaluate the specificity
of the clones.
[0257] The rate of specificity is measured by the following
equations:
R1=K562-blan/lymph-blank
R2=(K562-Blank)-(Lympho-Blank)/Lympho-Blank
R1 is shown in FIGS. 7A and R2 in FIG. 78
EXAMPLES
Example 1
Preparation of Peripheral Blood from Pregnant Women
[0258] The maternal blood sample (usually 3 ml) is divided into
aliquots of 1 ml. Each aliquot is diluted 1:14 with 0.15 M NaCl,
and the cells are allowed to sediment over night at 4.degree. C.
After sedimentation, the upper 12 ml of the supernatants are
carefully removed and pooled.
[0259] The sedimented cells (total volume 2 ml/tube) are divided
into aliquots of 0.5 ml.
Pre-Fixation without Erythrocyte Lysis
[0260] Cells of the supernatant is pre-fixed in a paraformaldehyde
solution in PBS, with a final concentration of 0.5% of PFA for 10
minutes.
Recovering and Mounting on Slides
[0261] The cells are recovered by centrifugation for 10 minutes at
500 g. After centrifugation, the supernatant is discarded and the
cell pellet is re-suspended in 0.15 M NaCl (usually 20 .mu.l) and
smeared onto poly-L-lysine coated slides (usually 2 slides).
Pre-fixation with NH.sub.4Cl Mediated Erythrocyte Lysis
[0262] Each aliquot of the sedimented cells is diluted with 10 ml
of 29.degree. C. hot reagent mixture consisting of 1 part 10.sup.-3
M acetazolamide, 9 parts 0.15 M NaCl, and 90 parts 0.1844 M
NH.sub.4Cl. After incubation for 2 minutes at 29.degree. C., 200
.mu.l 30 mM NH.sub.4 HCO.sub.3 is added and the cell suspension is
incubated at 29.degree. C. until erythrocytes are lysed (usually
10-15 minutes).
[0263] The cells are then pre-fixed for 10 minutes by adding PFA to
an end concentration of 0.43%, that is 1.3 ml 4% PFA in PBS if the
sample are process directly from erythrocyte lysis.
Recovering and Mounting on Slides
[0264] The cells are recovered by centrifugation for 10 minutes at
500 g.
[0265] The cell pellet is resuspended in 0.15 M NaCl (usually 100
.mu.l isotonic salt solution per ml whole blood) and smeared onto
poly-L-lysine coated slides (usually 10 .mu.l cell suspension per
slide). After air-drying over night, the slides are sealed
individually in airtight plastic backs and stored at -20.degree. C.
until hybridization.
Example 2
Identification of Male Fetal Cells by Reverse Color Fish and
Automated Scanning
[0266] Slides are recovered from the freezer and the airtight
plastic back is removed. Before hybridization, slides are fixed and
permeabilised 10 minutes in -20.degree. C. cold methanol, 10 minute
in -20.degree. C. cold acetone and rinsed in phosphate buffered
saline (PBS). Slides are then fixed again for 10 minutes in 2% PBS
buffered paraformaldehyde (PFA) and rinsed for 5 minutes in PBS
before they are dehydrated 3 minutes each in 60%, 80% and 99.9%
ethanol and air-dried.
[0267] Chromosome-specific repeat probes, DXZ1 labeled with
spectrum green and DYZ1 labeled with spectrum orange (Vysis) are
used for the first hybridization. Hybridization mixture containing
both probes is prepared by mixing 1 part of the X-probe, 1 part of
the Y-probe, 1 part of distilled water, and 7 parts of
hybridization buffer (Vysis). For whole slide FISH, 28 .mu.l of the
hybridization mixture is added and covered by a 22.times.50 mm
cover slips. Cover slips are sealed with rubber cement and the
DNA's are denatured on a hot plate at 83.5.degree. C. for 7 minutes
and hybridized overnight in a humidified atmosphere at 42.degree.
C.
[0268] The next day hybridized slides are washed for 2 minutes in
0.4.times.SSC/0.3% Tween 20 at 73.degree. C. and for 1 minute in
2.times.SSC/0.1% Tween 20 at room temperature. The slides are then
mounted in Vectashield with 0.64 g/ml DAPI as counter stain.
[0269] Cells containing a red signal located in a DAPI stained
nucleus is identified by automatic scanning at 20.times.
magnification of slides using scan function 5 in the MDS
slide-scanning system developed by Applied imaging. After scanning,
cells identified by the scanner are inspected visually by automatic
relocation using the multi-filter acquisition setup. Cells that
have one red signal (a putative Y-signal) but two green X-signals
are discarded, while cells that have one red signal and one green
X-signal or a split green X-signal are classified as candidate
fetal male cells.
[0270] The true fetal origin of the candidate fetal cells is
analyzed by re-hybridization of candidate cells with the same
probes in reverse colors. First, the cover slips are removed and
the slides are washed in 4.times.SSC/0.1% Tween 20 for 10 minutes,
rinsed in 2.times.SSC, dehydrated through 60%, 80%, and 99.9%
ethanol and air-dried.
[0271] Hybridization mixture containing 1 part of the
chromosome-specific repeat probes, DXZ1 labeled with spectrum red,
1 part of the chromosome-specific repeat probe DYZ1 labeled with
spectrum green, 1 part distilled water, and 7 parts hybridization
buffer (Vysis) is used for re-hybridization.
[0272] For whole slide FISH, 28 .mu.l of the hybridization mixture
is added and covered by a 22.times.50 mm cover slips. For selective
FISH, 2.5 .mu.l hybridization mixture is applied at positions on
the slides where candidate fetal cells had been found and the
mixture is spread by covering with a 10 mm circular cover slip.
Cover slips are then sealed with rubber cement, and DNA's is
denatured on a hot plate at 83.5.degree. C. for 7 minutes and
hybridized overnight in a humidified atmosphere at 42.degree.
C.
[0273] After re-hybridization slides are washed for 2 minutes in
0.4.times.SSC/0.3% Tween 20 at 73.degree. C. and for 1 minute in
2.times.SSC/0.1% Tween 20 at room temperature. The slides are then
mounted in Vectashield containing 0.6 .mu.g/ml DAPI as
counterstain,
[0274] Re-hybridized slides are placed in the scanning microscope
and re-hybridized candidate fetal cells are relocated using the
multifilter acquisition setup and FISH signals are analyzed
visually. Re-hybridized candidate fetal cells where the red signal
remains red and the green signal has switched to red are classified
as false fetal cells, whereas re-hybridized candidate fetal cells
where the red signal has switched to green and the green signal to
red are classified as true fetal cells.
Y Chromosome Control Procedure
[0275] Blood samples are drawn before an invasive procedure. Only
blood samples from women pregnant with a male fetus is analyzed for
the presence of male (XY) fetal cells using reverse color FISH with
sex chromosome specific X and Y probe mixtures. The gender of the
fetus is established by interphase fluorescent in situ
hybridization (FISH) on part of the CVS sample/amniocytes or by
routine ultrasound scanning.
Probes
[0276] The X-Y specific probes are obtained from Vysis inc. The
DYZ1 probe recognises Satellite III DNA on Yq12, while DXZ1
recognise Alpha Satellite DNA Xp11.1-q11.
Example 3
Generation of Foetal Cell Specific Antibody Fragments
[0277] Foetal cell specific antibody fragments against cell markers
are generated by use of phage display.
[0278] Based on the identification of foetal cells by FISH, the
localization of the foetal cell on the slides is known. The
identified foetal cell is subjected to biopanning whereby single
chain fragment variable (scFv) antibody fragments are
generated.
[0279] The selection of antibody fragments may be preformed as
follows. [0280] 1. The slide is blocked for 1 hour in 2% MPBS
(skimmed milk powder in 1.times.PBS) at room temperature in order
to reduce the amount of unspecific binding. This is done by
subjecting the slide into MPBS in a holder for slides. [0281] 2.
The slide is washed one time in 1.times.PBS [0282] 3. Biopanning:
the scFv phage library (McCafferty et al. 1990) is incubated with
the slide in 2% MPBS for one hour, with gentle shaking. A suitable
library may have a semi-synthetic V.sub.H+V.sub.L repertoire and
two single framework repertoires. An input of 10.sup.12-10.sup.13
phages is used, to cover the diversity of the repertoire. [0283] 4.
The slide is washed 2 times with 1.times.PBS Tween (1.times.PBS
added 0.05% Tween 20) followed by 3 washes with 1.times.PBS, for 3
minutes each with gently shaking. [0284] 5. Capture of the cell may
be performed by either by Laser capture microdissection (LCM) (Lu
et al., 2004) or by micro-dissection. [0285] 6. Bound phages are
eluted from the retrieved cell. Trypsin is added to the cell, (100
.mu.l, 1 mg/ml) for 10 min. [0286] 7. Eluted phages are incubated
with E. coli. TG-1 (supE hsdD5 (lac-proAB)thi F'[traD36 pro AB'
lacI.sup.q lacZ]) for 45 min at 37.degree. C., to allow infection
of the cells through the F-pillus. [0287] 8. The infected culture
is plated on TYE agar plates containing 1% glucose and 100 .mu.g/ml
ampicillin. The plate is incubated at 30.degree. C. over night,
allowing colonies to form. [0288] 9. All colonies are picked and
grown monoclonal in 96 well microtitre plates. Each well containing
cells producing one antibody fragment displayed on phage. Each will
be grown in large scale for testing of specificity.
Example 4
Characterisation of Foetal Cell Specific Binding Members
[0289] Following isolation of binding members the specificity of
the binding members for foetal cell specific is evaluated.
[0290] The testing of antibody fragment specificity may be
performed by a series of steps as describe here below.
[0291] 1. Binding members recognizing adult blood cells are
excluded. This is performed by testing the phage displayed antibody
fragments in Enzyme-linked immunosorbent assays (ELISA) against
adult blood cells from a non-pregnant person. ELISA plates are
coated by adding 10.000-100.000 blood cells per well and plates are
allowed to dry. According to the invention this is the most
suitable method of coating the ELISA plates with blood cells,
although other methods may be applied. Phages binding these
non-foetal blood cells are following excluded.
[0292] 2. The remaining binding members are tested in ELISA against
different cell cultures such as embryonic, foetal and stem cell
cultures. If any cell lines are similar or related to the
identified foetal cell, this may preferably be used for the
ELISA,
[0293] 3. The selected binding members are subcloned in suitable
vectors, such as the pKBJ3 vector (Jensen et al., 2002) which are
used for phages, for large scale expression of the binding members.
These binding members such as scFv antibodies are used for staining
of foetal blood samples to analyse the specificity and localization
of the antigen, Once the specificity of the antibody is confirmed,
the next step the identification of the cell marker/antigen is
performed.
Example 5
Identification of Cell Marker/Antigen
[0294] Immunoprecipitation and/or 2D PAGE followed by Mass
Spectroscopy for identification of foetal cell antigens recognized
by the identified binding member. Immunoprecipitation is preformed
by biotinylating the antibodies and incubating these with
streptavidin coated DYNA beads, followed by washing away the
unbound antibodies. The beads are then incubated with foetal cells
(or cell culture cells if these are recognised by the antibody). A
detergent is added and the beads retrieved binding the antigen to
the antibody. This sample is analysed by SDS-PAGE, the band(s) cut
out and analysed mass spectroscopy whereby the antigen is
identified. When the antibody is capable of recognising the antigen
in denatured condition, a 2D gel analysis may be performed. Cells
are run on the gel and the proteins transferred to a nitrocellulose
membrane. A western blot is performed with the antibody, revealing
a "spot" that is cut out and analysed by mass spectroscopy.
Example 6
Identification of Foetal Cells by the Presence of Cell Type
Specific Proteins
[0295] Antibody staining using isolated antibodies specifically
recognizing foetal cells. Slides are fixed 10 minutes in
-20.degree. C. cold methanol, 10 minutes in -20.degree. C. cold
acetone and rinsed in PBS. They are then fixed 10 minutes in 2% PBS
buffered paraformaldehyde, rinsed in PBS and dehydrated in
increasing concentrations of ethanol. After air drying, slides are
pre-incubated for 10 minutes at RT with 100 .mu.l of blocking
buffer consisting of 4.times.SSC containing 1% BSA and 0.5%
blocking reagent (Boehringer Manheim). Slides are then incubated
for 30 minutes at RT with 100 .mu.l antibody solution diluted in
blocking buffer according to manufactures instructions. After
antibody incubation, slides are washed three times for 5 minutes in
4.times.SSC. To detect antibody bound to foetal cells, the slides
are incubated with 100 .mu.l biotinylated goat anti-mouse antibody
(Dako, Glostrup, Denmark, diluted 1:25 in blocking buffer) for 30
minutes at RT, washed two times for 5 minutes in 4.times.SSC, and
incubated with 100 .mu.l of streptavidin conjugated to FITC or Cy3
(DAKO, Glostrup, Denmark, diluted 1:100 in blocking buffer) for 30
minutes at RT. After washing two times for 5 minutes each in
4.times.SSC and one time for 5 minutes in 2.times.SSC, slides were
air dried and mounted with antifade Vectashield containing
4,6-diamidino-2-phenylindole (DAPI) to counterstain the nuclei.
Example 7
Chromosome Analysis of Foetal Cells from Maternal Blood
[0296] Analysis of numerical chromosome 13, 18, 21, X and Y
abnormalities in foetal cells from maternal blood is done by FISH
using the AneuVision DNA Probe Kit from Vysis Inc. Before
hybridization, the slides are fixed 10 minutes in -20.degree. C.
cold methanol, 10 minutes in -20.degree. C. cold acetone and rinsed
in PBS. They are then fixed 10 minutes in 2% PBS buffered
paraformaldehyde, rinsed in PBS and dehydrated in increasing
concentrations of ethanol and air-dried. 10 d of the probe mixture
is added and covered by a 22.times.22 mm coverslip. The coverslip
is sealed with rubber cement and the DNA's are denatured on a hot
plate for 1 minute at 75.degree. C. and hybridized over night in a
humidified atmosphere at 37-42.degree. C. The next day hybridized
slides are washed at 72.degree. C. for 2 minutes in
0.4.times.SSC/0.3% Tween 20 followed by 1 minute in
2.times.SSC/0.1% Tween at room temperature. The slides are mounted
in Vectashield with 0.6 .mu.g/ml ml DAPI as counterstain.
Example 8
Use of Phage-Library for Generation of Antibody Fragments Specific
for H562 Cells
[0297] Slide with double hybridized male blood with a spike of K562
cells on top is used as.
Incubation procedure [0298] Slides are submerged in PBS and the
coverslide is removed. [0299] Slides are blocked for 1-2 hours in
2% MPBS (2% Marvel skimmed milk powder in PBS) [0300] Wash slides
two times with PBS [0301] Incubate with phage library for 1 hour;
approximately 10.sup.13 phage added in 2% MPBS. [0302] Slides are
washed 3 times with PBS and stored in a 10% glycerol solution to
avoid drying out of the slide.
UV Irradiation
[0302] [0303] Slide is placed in slideholder on microscope, and an
area of cells comprising target antigenic material is selected and
complete drying of the slide during the procedure is avoided.
[0304] Shadow-stick, puck, or other UV shielding protection is
placed over selected area. [0305] Slide is irradiated with UV-C
(254 nm) light for 5 min; distance approximately 2 cm. [0306] The
protected area is marked, so that the location is known for the
elution of phages.
Phage Elution
[0306] [0307] 50 .mu.l of 1 mg/ml trypsin in buffer (100 mM
Tris-HCl, 1 mM CaCl.sub.2) is added to the area on the slide with
the protected cell(s) for 10 min. [0308] Alternatively the phages
may be eluted at a low pH Buffer for elution: 50 mM Glycine, pH
2.2; neutralize min 1 h with Tris-HCL. [0309] The liquid is
transferred to an eppendorf tube. Area washed with 200 .mu.l of PBS
that is added to the same tube
Phage Infection
[0310] 800 .mu.l of TG-1 culture in log-phase is added to half the
eluted phage (the other half is retained for infection the
following day). Incubated at 37.degree. C. for 30-45 min. [0311]
Cells are spun down, 800 .mu.l of the supernatant is removed. The
remaining is resuspended and plated on TYE plates containing
ampicillin. [0312] Plates are incubated at 3000 over night and
analysed next day.
Selection of Antigen Specific Phages
[0313] Phages from the selected area of the slides are eluted
according to the scheme here below.
TABLE-US-00002 Slide number Elution First Half Second Half Total 1
Trypsine 4 4 8 2 Trypsine 3 1 4 3 Trypsine 5 17 22 4 Acid 1 2 3 5
Acid 0 3 3 6 Acid 0 1 1 7 Trypsine 12 353 365 8 Trypsine 1 2 3 9
Acid 0 0 0 10 Acid 0 10 10 -stick Trypsine 0 7 7 Total Amount 26
400 426
[0314] Following 384 antibody fragments (4 plates) are tested
monoclonally in ELISA against K562 (fixed) and cells from
lymphoprep (fixed) are selected.
[0315] The procedure is repeated several times and phage derived
antibody fragments are test in ELISA. Below are the results from a
series of selections. ELISA results are shown in the tables here
below and graphically in FIGS. 6 and 7.
Serie 1
TABLE-US-00003 [0316] S1.1B1 S1.1C7 S1.1D8 S2.1A2 S2.1A6 S2.1A8
S2.2G6 S2.4A10 S2.4B3 S2.4D1 K562 1:10 0.06 1.041 0.057 0.125 0.336
1.97 0.074 0.051 0.215 0.145 1:10 0.061 0.778 0.068 0.146 0.369
2.007 0.083 0.063 0.243 0.187 1:50 0.036 0.175 0.047 0.06 0.081
1.459 0.054 0.056 0.108 0.075 1:50 0.037 0.144 0.044 0.055 0.089
1.443 0.052 0.049 0.102 0.075 1:200 0.032 0.061 0.036 0.053 0.05
0.838 0.044 0.038 0.049 0.043 1:200 0.035 0.074 0.043 0.047 0.061
0.85 0.048 0.049 0.061 0.058 0 0.04 0.05 0.048 0.054 0.055 0.058
0.058 0.06 0.063 0.062 0 0.038 0.046 0.047 0.051 0.05 0.054 0.053
0.057 0.057 0.054 Lympho 1:10 0.131 0.484 0.119 0.191 0.314 0.857
0.135 0.102 0.493 0.476 1:10 0.149 0.58 0.135 0.203 0.318 0.92
0.161 0.1 0.51 0.526 1:50 0.067 0.149 0.064 0.084 0.082 0.254 0.064
0.062 0.203 0.212 1:50 0.06 0.158 0.063 0.07 0.088 0.274 0.069
0.061 0.215 0.182 1:200 0.04 0.064 0.039 0.048 0.052 0.08 0.05
0.043 0.069 0.094 1:200 0.041 0.068 0.05 0.056 0.061 0.096 0.062
0.053 0.093 0.098 0 0.038 0.055 0.049 0.059 0.059 0.062 0.057 0.065
0.074 0.069 0 0.038 0.053 0.05 0.059 0.058 0.062 0.062 0.066 0.067
0.067 Blank 1:10 0.03 0.062 0.039 0.048 0.056 0.078 0.048 0.041
0.055 0.046 1:10 0.032 0.056 0.041 0.052 0.06 0.079 0.047 0.047
0.055 0.047 1:50 0.035 0.049 0.046 0.051 0.051 0.055 0.057 0.055
0.057 0.054 1:50 0.034 0.045 0.041 0.046 0.047 0.053 0.049 0.049
0.051 0.048 1:200 0.031 0.039 0.038 0.039 0.04 0.039 0.042 0.041
0.045 0.041 1:200 0.034 0.047 0.044 0.049 0.047 0.051 0.05 0.053
0.055 0.051 0 0.033 0.047 0.043 0.052 0.05 0.055 0.054 0.058 0.06
0.058 0 0.034 0.043 0.042 0.045 0.042 0.049 0.046 0.05 0.049 0.05
K562 1:10 0.0605 0.9095 0.0625 0.1355 0.3525 1.9885 0.0785 0.057
0.229 0.166 1:50 0.0365 0.1595 0.0455 0.0575 0.085 1.451 0.053
0.0525 0.105 0.075 1:200 0.0335 0.0675 0.0395 0.05 0.0555 0.844
0.046 0.044 0.055 0.051 0 0.039 0.048 0.0475 0.0525 0.0525 0.056
0.0555 0.0585 0.06 0.058 Lympho 1:10 0.14 0.532 0.127 0.197 0.316
0.8885 0.148 0.101 0.5015 0.501 1:50 0.0635 0.1535 0.0635 0.077
0.085 0.264 0.0665 0.0615 0.209 0.197 1:200 0.0405 0.066 0.0445
0.052 0.0565 0.088 0.056 0.048 0.081 0.096 0 0.038 0.054 0.0495
0.059 0.0585 0.062 0.0645 0.0655 0.0705 0.068 Blank 1:10 0.0335
0.0525 0.0435 0.0515 0.0555 0.067 0.052 0.051 0.056 0.051 1:50
0.0325 0.042 0.0395 0.0425 0.0435 0.046 0.0455 0.045 0.048 0.045
1:200 0.0335 0.047 0.0435 0.0505 0.0485 0.053 0.052 0.0555 0.0575
0.055 0 0.034 0.043 0.042 0.045 0.042 0.049 0.046 0.05 0.049 0.05
K562-Blank/Lympho-Blank 1:10 0.2535 1.7873 0.2275 0.5773 1.1401
2.339 0.276 0.12 0.3883 0.256 1:50 0.129 1.0538 0.25 0.4348 1 6.445
0.3571 0.45455 0.354 0.2 1:200 0 1.0789 -4 -0.333 0.875 22.6 -1.5
1.53333 -0.106 -0.096 0 1.25 0.4545 0.7333 0.5357 0.6364 0.5385
0.5135 0.54839 0.5116 0.444
(K562-Blank)-(Lympho-Blank)/Lympho-Blank 1:10 -0.746 0.7873 -0.772
-0.423 0.1401 1.339 -0.724 -0.88 -0.612 -0.744 1:50 -0.871 0.0538
-0.75 -0.565 0 5.445 -0.643 -0.5455 -0.646 -0.8 1:200 -1 0.0789 -5
-1.333 -0.125 21.6 -2.5 0.53333 -1.106 -1.096 0 0.25 -0.545 -0.267
-0.464 -0.364 -0.462 -0.486 -0.4516 -0.488 -0.556
Serie 2
[0317] The data obtained in Serie 2 is shown in the tables here
below and in FIGS. 8 and 9.
TABLE-US-00004 S3.1A4 S3.1A6 S3.1A9 S3.1B2 S3.1F6 S3.1G4 S3.2E9
S3.2G2 S3.3E3 S3.4G2 K562 1:10 1.555 0.403 2.127 0.628 0.186 0.249
0.122 0.124 0.474 0.167 1:10 1.73 0.436 2.352 0.689 0.257 0.346
0.184 0.165 0.532 0.248 1:50 0.73 0.134 1.551 0.072 0.114 0.138
0.081 0.058 0.185 0.118 1:50 0.673 0.13 1.548 0.201 0.102 0.133
0.085 0.073 0.158 0.09 1:200 0.211 0.044 0.545 0.054 0.027 0.054
0.037 0.02 0.055 0.042 1:200 0.215 0.039 0.512 0.061 0.036 0.056
0.042 0.022 0.055 0.045 0 0.01 0.019 0.013 0.022 0.019 0.02 0.021
0.022 0.022 0.023 0 0.011 0.01 0.01 0.012 0.01 0.02 0.017 0.018
0.015 0.016 Lympho 1:10 0.734 0.175 0.202 0.11 0.267 0.361 0.208
0.179 0.357 0.27 1:10 0.808 0.184 0.241 0.123 0.175 0.288 0.17
0.147 0.302 0.327 1:50 0.271 0.045 0.062 0.041 0.074 0.075 0.061
0.055 0.096 0.101 1:50 0.274 0.043 0.059 0.04 0.073 0.105 0.056
0.048 0.128 0.037 1:200 0.068 0.019 0.021 0.017 0.024 0.038 0.025
0.022 0.03 0.028 1:200 0.076 0.021 0.023 0.017 0.025 0.037 0.026
0.024 0.033 0.03 0 0.014 0.017 0.018 0.018 0.019 0.025 0.02 0.021
0.023 0.022 0 0.01 0.016 0.014 0.023 0.019 0.024 0.022 0.026 0.025
0.027 Blank 1:10 0.037 0.02 0.022 0.021 0.022 0.019 0.023 0.019
0.031 0.022 1:10 0.039 0.016 0.017 0.015 0.018 0.025 0.019 0.022
0.032 0.019 1:50 0.014 0.015 0.014 0.015 0.017 0.017 0.017 0.018
0.02 0.016 1:50 0.012 0.015 0.011 0.011 0.011 0.012 0.013 0.011
0.014 0.011 1:200 0.009 0.01 0.011 0.011 0.012 0.01 0.01 0.009
0.011 0.01 1:200 0.01 0.011 0.01 0.011 0.011 0.011 0.015 0.011
0.012 0.011 0 0.011 0.013 0.013 0.015 0.016 0.016 0.016 0.016 0.017
0.017 0 0.011 0.013 0.013 0.016 0.016 0.017 0.016 0.017 0.017 0.017
K562 1:10 1.6425 0.4195 2.2395 0.6585 0.2215 0.2975 0.153 0.1445
0.503 0.2075 1:50 0.7015 0.132 1.5495 0.1365 0.108 0.1355 0.083
0.0655 0.1715 0.104 1:200 0.213 0.0415 0.5285 0.0575 0.0315 0.055
0.0395 0.021 0.055 0.0435 0 0.0105 0.0145 0.0115 0.017 0.0145 0.02
0.019 0.02 0.0185 0.0195 Lympho 1:10 0.771 0.1795 0.2215 0.1165
0.221 0.3245 0.189 0.163 0.3295 0.2985 1:50 0.2725 0.044 0.0605
0.0405 0.0735 0.09 0.0585 0.0515 0.112 0.214 1:200 0.072 0.02 0.022
0.017 0.0245 0.0375 0.0255 0.023 0.0315 0.099 0 0.012 0.0165 0.016
0.0205 0.019 0.0245 0.021 0.0235 0.024 0.0625 Blank 1:10 0.0265
0.0155 0.0155 0.015 0.0175 0.021 0.018 0.02 0.026 0.0175 1:50
0.0105 0.0125 0.011 0.011 0.0115 0.011 0.0115 0.01 0.0125 0.0135
1:200 0.0105 0.012 0.0115 0.013 0.0135 0.0135 0.0155 0.0135 0.0145
0.0105 0 0.011 0.013 0.013 0.016 0.016 0.017 0.016 0.017 0.017
0.0105 K562-Blank/Lympho-Blank 1:10 2.1706 2.4634 10.796 6.3399
1.0025 0.911 0.7895 0.8706 1.5717 0.6762 1:50 2.6374 3.7937 31.081
4.2542 1.5565 1.5759 1.5213 1.3373 1.598 0.4514 1:200 3.2927 3.6875
49.238 11.125 1.6364 1.7292 2.4 0.7895 2.3824 0.3729 0 -0.5 0.4286
-0.5 0.2222 -0.5 0.4 0.6 0.4615 0.2143 0.1731
(K562-Blank)-(Lympho-Blank)/Lympho-Blank 1:10 1.1706 1.4634 9.7961
5.3399 0.0025 -0.089 -0.211 -0.129 0.5717 -0.324 1:50 1.6374 2.7937
30.081 3.2542 0.5565 0.5759 0.5213 0.3373 0.598 -0.549 1:200 2.2927
2.6875 48.238 10.125 0.6364 0.7292 1.4 -0.211 1.3824 -0.627 0 -1.5
-0.571 -1.5 -0.778 -1.5 -0.6 -0.4 -0.538 -0.786 -0.827
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