U.S. patent number 4,748,129 [Application Number 06/645,458] was granted by the patent office on 1988-05-31 for assay method employing fluorescent cell incorporative dye.
This patent grant is currently assigned to Snytex (U.S.A.) Inc.. Invention is credited to Chiu C. Chang, Vartan Ghazarossian, Edwin F. Ullman.
United States Patent |
4,748,129 |
Chang , et al. |
May 31, 1988 |
Assay method employing fluorescent cell incorporative dye
Abstract
A method is provided for determining the presence in a sample of
a member of a specific binding pair ("sbp member") consisting of
ligand and its homologous receptor. The sample is combined in an
aqueous medium with (1) a complementary sbp member wherein at least
the sbp member or the complementary sbp member is bound to the
surface of a cell and (2) a fuorescent agent capable of being
incorporated into the cell. The presence of the sbp member is
indicated by a change in fluorescence of the unseparated cell
suspension as a result of agglutination of the cells. The present
invention has particular application to blood typing, for example,
for the determination of the presence of blood group antigens A, B,
AB, O, and D (Rh.sub.o) and antibodies to such antigens.
Inventors: |
Chang; Chiu C. (Sunnyvale,
CA), Ghazarossian; Vartan (Palo Alto, CA), Ullman; Edwin
F. (Atherton, CA) |
Assignee: |
Snytex (U.S.A.) Inc. (Palo
Alto, CA)
|
Family
ID: |
24589110 |
Appl.
No.: |
06/645,458 |
Filed: |
August 28, 1984 |
Current U.S.
Class: |
435/7.25;
436/536; 436/800; 436/805; 436/829; 436/520; 436/537; 436/807 |
Current CPC
Class: |
G01N
33/533 (20130101); G01N 33/80 (20130101); G01N
33/542 (20130101); G01N 33/555 (20130101); Y10S
436/80 (20130101); Y10S 436/807 (20130101); Y10S
436/805 (20130101); Y10S 436/829 (20130101) |
Current International
Class: |
G01N
33/536 (20060101); G01N 33/555 (20060101); G01N
33/542 (20060101); G01N 33/554 (20060101); G01N
33/533 (20060101); G01N 33/80 (20060101); G01N
033/554 (); G01N 033/555 () |
Field of
Search: |
;424/3,11
;436/519,520,536,537,800,805,807,829 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
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|
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|
|
|
A20106685 |
|
Oct 1983 |
|
EP |
|
2041517 |
|
Sep 1980 |
|
GB |
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Other References
Smith, D. S., FEBS Letters, vol. 77, No. 1, (1977), pp. 25-27.
.
Briggs, et al., Science, vol. 212, (1981), pp. 1266-1267. .
Briggs, et al., Proc. Natl. Acad. Sci. U.S.A., vol. 77, No. 8
(1980), pp. 4904-4908. .
Kinsland, et al.; J. Biochem. Biophys. Meth., vol. 9, (1984), pp.
81-83. .
Sprenger, et al., Angew. Chem. Internat. Edit, vol. 7, 530-535
(1968), Angew. Chem. 80, 541 (1968) German version. .
Sprenger, et al., Angew. Chem. Internat. Edit. vol. 5, 894 (1966),
Angew. Chem., 78, 938 (1966) German version. .
Maahs et al., Angew. Chem. Internat. Edit., vol. 5, 888-893 (1966),
Angew. Chem. 78, 927 (1966) German version. .
Sprenger, et al., Angew. Chem. Internat. Edit., vol. 6, 553-554
(1967), Angew. Chem., 79, 581 (1967). .
Angewandte Chemie International Edition, vol. 6, No. 1, Jan. 1967,
Weinheim, pp. 553-554. .
Angewandte Chemie International Edition, vol. 5, No. 1, Jan. 1966,
Weinheim, pp. 894-895..
|
Primary Examiner: Marantz; Sidney
Assistant Examiner: Wieder; Stephen C.
Attorney, Agent or Firm: Leitereg; Theodore J.
Claims
What is claimed is:
1. A method for determining in a sample the presence of a member of
a specific binding pair ("sbp member") consisting of ligand and its
homologous receptor, which method comprises-
(a) combining in an aqueous medium (1) a sample, (2) a
complementary sbp member wherein at least one of said sbp member or
said complementary sbp member is bound to the surface of a cell and
(3) a fluorescent dye capable of being incorporated into said cell
and
(b) without separating said cells from said aqueous medium
determining a change in fluorescence as a result of agglutination
of said cells, said change being an indication of the presence of
said sbp member.
2. The method of claim 1 wherein said sbp member is a cell surface
member.
3. The method of claim 1 wherein said complementary sbp member is a
receptor for a surface antigen.
4. The method of claim 1 wherein said cell is a red blood cell.
5. The method of claim 1 wherein said change in fluorescence is
determined by a non-flow cytometry technique.
6. The method of claim 1 wherein said change in fluorescence is
determined by a fiber optic cytometer.
7. The method of claim 1 wherein said fluorescent dye is a membrane
soluble dye.
8. The method of claim 1 wherein said fluorescent dye is a squarate
dye.
9. The method of claim 1 wherein said fluorescent dye exhibits a
fluorescence when incorporated into said cell at least three times
the fluorescence of the unincorporated fluorescent dye.
10. The method of claim 1 wherein said fluorescent dye is
incorporated into said cells prior to combining with said aqueous
medium.
11. A method for determining in a sample of whole blood the
presence of a member of a specific binding pair ("sbp member"),
which method comprises
(a) combining in an aqueous medium (1) a sample, (2) a
complementary sbp member wherein at least one of said sbp member or
said complementary sbp member is bound to the surface of a cell and
(3) a fluorescent dye capable of being incorporated into said cells
and
(b) without separating said cells from said aqueous medium,
determining a change in fluorescence as an indication of the
presence of said sbp member.
12. The method of claim 11 wherein said change in fluorescence is
determined by a non-flow cytometric technique.
13. The method of claim 11 wherein said change in fluorescence is
determined by a fiber optic cytometer.
14. The method of claim 11 wherein said fluorescent dye is a
membrane soluble dye.
15. The method of claim 11 wherein said fluorescent dye exhibits a
fluorescence when incorporated into said cells at least three times
the fluorescence of the unincorporated fluorescent dye.
16. The method of claim 11 wherein said fluorescent dye is a
squarate dye.
17. The method of claim 11 wherein said cells are red blood
cells.
18. The method of claim 11 wherein said sbp member is a surface
antigen.
19. The method of claim 18 wherein said surface antigen is selected
from the antigens indicative of blood types selected from the group
consisting of type A and type B.
20. The method of claim 18 wherein said surface antigen is the D
blood group antigen.
21. The method of claim 18 wherein said surface antigen is an
immunoglobulin.
22. The method of claim 11 wherein said change in fluorescence is a
result of agglutination of said cells.
23. The method of claim 11 wherein said determination of Step b is
compared to a sample having a known amount of a red blood cell
antigen.
24. The method of claim 11 wherein said fluorescent dye has a molar
extinction coefficient of greater than 10,000 M.sup.-1 CM.sup.-1 at
633 nm.
25. The method of claim 11 wherein said fluorescent dye exhibits at
least three times the fluorescence in a lipid environment relative
to an aqueous environment.
26. The method of claim 11 wherein said fluorescent dye is
incorporated into said cells prior to combining with said aqueous
medium.
27. The method of claim 11 wherein said fluorescent dye is a
squarate dye of the formula selected from the group consisting of
##STR3## wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are
independently selected from the group consisting of a alkyl of from
2 to 16 carbon atoms and aralkyl of from 2 to 16 carbon atoms and
aralkyl of from 2 to 16 atoms wherein the groups may be the same or
different and may be substituted with a substituent selected from
the group consisting of OH, CONHR.sup.1, SR.sup.1, OR.sup.1 wherein
R.sup.1 is lower alkyl of from 1 to 6 carbon atoms, F, Cl, Br, I,
NO.sub.2, oxocarbonyl and CN, R.sub.5, and R.sub.6 are
independently selected from the group consisting of hydrogen,
methoxy, and hydroxyl, or ##STR4## wherein R.sub.1, R.sub.2, and
R.sub.3 are independently selected from the group consisting of
alkyl of from 2 to 16 carbon atoms and aralkyl of from 2 to 16
carbon atoms wherein the groups may be the same or different and
may be substituted with a substituent selected from the group
consisting of OH, CONHR.sup.1, SR.sup.1, OR.sup.1 wherein R.sup.1
is lower alkyl of from 1 to 6 carbon atoms, F, Cl, Br, I, NO.sub.2,
oxocarbonyl, and CN.
28. A method for determining in a sample of whole blood the
presence of a member of a specific binding pair ("sbp member"),
which method comprises
(a) combining in an aqueous medium (1) a sample, (2) a
complementary sbp member wherein at least one of said sbp member or
said complementary sbp member is bound to the surface of a cell and
(3) a fluorescent dye capable of being incorporated into said cells
and
(b) without separating said cells from said aqueous medium,
determining a change in fluorescence by a non-flow cytometric
technique as a result of agglutination of said cells, said change
being an indication of the presence of said sbp member.
29. The method of claim 28 wherein said fluorescent dye is
incorporated into said cells prior to combining with said aqueous
medium.
30. The method of claim 28 wherein said fluorescent dye is a
squarate dye.
31. In a method for typing red blood cells comprising treating red
blood cells under conditions to achieve an agglutination of said
cells and determining the extent of agglutination as an indication
of the presence or absence of a particular red blood cell type, the
improvement which comprises combining said cells with a fluorescent
dye capable of being incorporated into said cells prior to said
determining.
32. The method of claim 31 wherein said fluorescent dye is a
squarate dye.
33. The method of claim 31 wherein said fluorescent dye has an
absorption wavelength of greater than 600 nanometers.
34. The method of claim 31 wherein said fluorescent dye is a
squarate dye of the formula selected from the group consisting of
##STR5## wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are
independently selected from the group consisting of a alkyl of from
2 to 16 carbon atoms and aralkyl of from 2 to 16 carbon atoms and
aralkyl of from 2 to 16 atoms wherein the groups may be the same or
different and may be substituted with a substituent selected from
the group consisting of OH, CONHR.sup.1, SR.sup.1, OR.sup.1,
wherein R.sup.1 is lower alkyl, F, Cl, Br, I, NO.sub.2,
oxocarbonyl, and CN, R.sub.5, and R.sub.6 are independently
selected from the group consisting of hydrogen, methoxy, and
hydroxyl, or ##STR6## wherein R.sub.1, R.sub.2, and R.sub.3 are
independently selected from the group consisting of alkyl of from 2
to 16 carbon atoms and aralkyl of from 2 to 16 carbon atoms wherein
the groups may be the same or different and may be substituted with
a substituent selected from the group consisting of OH,
CONHR.sup.1, SR.sup.1, OR.sup.1, wherein R.sup.1 is lower alkyl of
from 1 to 6 carbon atoms, F, Cl, Br, I, NO.sub.2, oxocarbonyl, and
CN.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
There is a continuing need for improved assay methods for the
detection of an analyte in a sample. The analyte generally is a
member of a specific binding pair consisting of ligand and its
homologous receptor. Exemplary of sbp members are antigens and
antibodies.
The mammalian red blood cells carry numerous antigens some of which
must be accurately identified in both patient and donor for medical
procedures such as transfusions. Accurate determination of blood
groups, A, B, AB, O and D (Rh.sub.o) is critically important. Also
antibodies present in the blood to such antigens can be of
diagnostic interest.
Conventionally, agglutination techniques are used on a microscope
slide or in a tube. Improved rapid accurate screening of blood is
desirable in view of the large numbers of samples which must be
tested.
2. Description of the Prior Art
Identification of red blood cell antigens by agglutination
techniques is standard, e.g., C. Hudson and F. C. Hay, Practical
Immunology, Second Edition, Blackwell Scientific Publications,
Oxford (1980), p. 139. U.S. Pat. No. 3,862,303 is exemplary of
immunologial detection and identification of serological factors
using carrier particles such as latex beads. Smith, FEBS Letters
77,25 (1977) describes a fluorescent immunoassay.
U.S. patent application Ser. No. 434,761, filed Oct. 15, 1982, now
U.S. Pat. No. 4,550,017 concerns Fluorescence Screening for Blood
Typing.
The use of laser beams and slits to differentiate particles based
on their relative size by the correlation of fluorescence
fluctuations in a relatively large sample volume is described by
Briggs et al, Science, 212: 1266-1267, 1981, and by Nicoli et al.,
Proc. Natl. Acad. Sci., USA, 77: 4904-4908, 1980.
Various squarate dyes are discussed by Sprenger, et al., Angew.
Chem., 80, 541 (1968) [Angew. Chem. Internatl. Edit, Vol. 7:
530-535, 1968]; Sprenger, et al., Angew. Chem., 79; 581, 1967
[Angew. Chem. Internatl. Edit, Vol. 6: 553-554, 1967]; Sprenger, et
al., Angew. Chem. internat. Edit, 5: 894, 1966; and Maahs, et al.,
ibid., 5: 888, 1966.
The employment of a merocyanine dye for the detection of malignant
leukocyte cells is described in U.S. Pat. No. 4,424,201.
U.S. Pat. No. 3,853,987 discloses an immunological reagent and
radioimmunoassay. In a preferred method for detecting clumping of
his reagents, the patentee dilutes a suspension of the reaction
mixture and passes it through a flow cell in a
spectrophotofluorometer.
SUMMARY OF THE INVENTION
A method is provided for determining the presence in a sample of a
member of a specific binding pair ("spb member") consisting of
ligand and its homologous receptor. The sample is combined in an
aqueous medium with (1) a complementary sbp member wherein at least
the spb member or the complementary sbp member is bound to the
surface of a cell and (2) a fluorescent agent capable of being
incorporated into the cell. The presence of the sbp member is
indicated by a change in fluorescence of the unseparated cell
suspension as a result of agglutination of the cells.
The present invention has particular application to blood typing,
for example, for the determination of the presence of blood group
antigens A, B, AB, O, and D (Rh.sub.o) and antibodies to such
antigens, as well as antibodies to antigens M, N, S, s, Lewis,
Lutheran, Kell, Duffy, Kidd, etc.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
The subject invention provides a novel method for determining the
presence of an analyte, usually a sbp member, in a sample without a
separation step. The method employs a complementary sbp member
where at least one of either the sbp member or the complementary
sbp member is bound to the surface of a cell. Also employed in the
method is a fluorescent cell incorporative agent. Preferably, when
the sbp member in the sample is not bound to the surface of a cell,
the fluorescent agent is combined with the sample by first
incorporating the agnet into cells bound to a complementary sbp
member and then combining the combination with the sample. When the
sbp member on the sample is bound to the surface of a cell, the
fluorescent agent is generally added to the sample prior to
combining the sample with the complementary sbp member. Thus, the
term "combining with the sample" is meant to include combining
together two or more of the reagents mentioned above prior to
combining the remaining reagents. The term "reagents" includes the
sample, the complementary sbp member, and the fluorescent cell
incorporative agent, and may further include any additional agents
required for the successful operation of the subject method.
In carrying out one embodiment of the present method, the sample,
the complementary sbp member, and the fluorescent cell
incorporative agent are combined in an aqueous assay medium and a
change in fluorescence of the mixture as a result of agglutination
of the cells is then determined. The presence of the sbp member in
the sample is indicated by this change in fluorescence.
The present method is adaptable to a wide variety of assay
determinations for a wide variety of sbp member analytes. It is of
special interest where at least one of the sbp member and
complementary sbp member are a normal component of the cell
surface. Cell surface sbp members include naturally occurring
membrane components such as antigens, cell wall antigens,
particularly bacterial cell walls, cell surface receptors including
receptors for activating, growth, and inhibition factors,
antibodies, HLA antigens, Fc receptors, hormone receptors, ion
channels, glycolipids, lipoproteins, complement components, viral
antigens, membrane bound enzymes, peptidoglycan, fungal antigens,
idiotypic antigens and the like. Cell types of interest include
leukocytes, bacteria, fungal cells, erythrocytes, reticulocytes,
lymphocytes including monocytes, macrophage, B cells, T cells,
eosinophils, etc. A particular adaptation of the present method is
in the area of blood typing. Blood group antigens, as well as
antibodies thereto, may be ascertained using the method described
above.
The subject invention provides a novel method and is particularly
useful for typing red blood cells or identifying red blood cell
antigens and the antibodies thereto by using the red blood cells as
a carrier of incorporated fluorescence where the cells agglutinate
during the assay method. A change in fluorescence as a result the
agglutination is determined and is an indication of the presence of
a particular red blood cell antigen or antibody thereto. Substances
which bind to red blood cell antigens, normally antibodies or
lectins, are required to cause agglutination of the cells. In one
embodiment of the present invention for determining blood group
antigens, whole blood is combined with a fluorescent cell
incorporative agent and a receptor for the antigen of interest in
an aqueous medium, e.g., an appropriate buffer. If the antigen of
interest is present on the surface of the red blood cells in the
blood agglutination will occur and a change in fluorescence will be
observed as an indication of the presence of the antigen of
interest.
The receptor which is employed binds preferentially to the blood
group surface antigens of interest. Thus, there will be a
fluorometrically measurable change when a given antigen is present
as compared to when that antigen is absent in a given red blood
cell sample. For example, in the A, B, O system, if anti-A antibody
were used, agglutination would occur and there would be a change in
fluorescence if the analyte contained the A antigen of type A or
type AB blood over that where the analyte contained blood types B
or O.
In addition to antibodies, certain lectins are known to bind in
varying degrees to red blood cell surface antigens, and are
convenient receptors for use in the present assays.
The subject method can also be used for determining the presence of
antibodies to a red blood cell antigen. In this approach, red blood
cells having the surface antigen homologous to the antibody in
question are employed in the assay. The antigen bearing cells and a
fluorescent agent are preferably combined first and then combined
with the sample in an aqueous medium. If the antibody in question
is present in the sample, a change in fluorescence will occur as
the result of agglutination. In this situation, a change in
fluorescence would indicate the presence of the antibodies in
question.
The present method is also important in cross-match determinations.
In such a determination, blood from a donor and blood from a
potential recipient are mixed and the mixture is treated in
accordance with the method of the invention. Preferably, the cells
from the donor will be combined with the fluorescent agent prior to
combining with the patient's sample. Generally, a positive signal
indicates that the match is incompatible.
The present method is simple and can be performed in a reasonably
short period of time. For the most part, the reagents can be added
simultaneously. Background interferences from free fluorescent
agent and from endogenous materials in a sample are minimal. The
change in fluorescence may be determined over a continuous
background. A particular advantage of the present method is that no
separation or washing step is necessary. The assay medium or
mixture may be observed directly for a change in fluorescence as a
result of agglutination of the cells.
Before proceeding further a number of terms will be defined.
"Analyte"--the compound or composition to be measured, which is a
sbp member and may be a ligand, which is mono- or polyvalent, that
is, having one or a plurality of determinant sites, haptenic and
antigenic, a single compound or plurality of compounds which share
at least one common epitopic or determinant site; or a
receptor.
"Sbp member"--a member of a specific binding pair, consisting of
two different molecules, where one of the molecules has an area on
the surface or in a cavity which specifically binds to a particular
spatial and polar organization of the other molecule. The two
members of a specific binding pair are referred to as ligand and
receptor (antiligand) and are also referred to as homologous.
"Ligand"--any organic compound for which a receptor naturally
exists or can be prepared;
"Receptor" (antiligand)--any macromolecular compound or composition
capable of recognizing (having an enhanced binding affinity to) a
particular spatial and polar organization of a molecule, i.e.,
epitopic or determinant site. Illustrative receptors include
naturally occurring receptors, e.g., thyroxine binding globulin,
antibodies, enzymes, Fab fragments, lectins, and the like. The term
antibody is employed in this case as illustrative of, and to more
generally denote, receptor.
"Complementary sbp member"--the homologous member of a specific
binding pair where the sbp member is an analyte.
"Cell"--any one of the minute protoplasmic masses which make up
organized tissue, comprising a mass of protoplasm surrounded by a
membrane including nucleated and unnucleated cells and
organelles.
"Fluorescent cell incorporative agent"--a compound of molecular
weight less than 2000 capable of being incorporated into a cell and
thereby causing the cell to be fluorescent, for example, a cell
membrane soluble dye, a DNA intercalating dye, a vital dye or the
like.
The fluorescent cell incorporative agent is preferably more
fluorescent after incorporation into a cell. It may be capable of
incorporation into the cell by virtue of being soluble in the cell
membrane or of being transportable across the cell membrane and
undergoing a chemical reaction that inhibits transport out of the
cell. Fluorescent agents with high protein or carbohydrate affinity
may also be useful in the present invention. Where the cells
possess deoxyribonucleic acid (DNA), one may also employ
fluorescent agents having an affinity for DNA. The fluorescent cell
incorporative agent may be a hydrophobic dye that is subsequently
rendered water soluble by chelation with a water compatible
chelator.
The fluorescent cell incorporative agent should preferably have an
absorption maximum greater than 450 nm, more preferably greater
than 540 nm, to provide maximum avoidance of biological
interference. For the most part, the absorption wavelength maximum
should be 320 to 1000 nm, preferably 600 to 800 nm.
The molar extinction coefficient for the fluorescent cell
incorporative agent at the wavelength of the exciting light should
be as high as practical and should be greater than 1,000,
preferably greater than 10,000, per mole per centimeter.
Fluorescent cell incorporative agents are chosen to have a high
quantum yield, normally greater than 0.05, preferably greater than
0.3 when incorporated in cells. The choice of the excitation
wavelength depends on minimizing background fluoresence from the
sample, maximizing fluorescence of the stained cells, and
maximizing the intensity and reliability of the light source and
filters. Particularly advantageous wavelengths are 488 nm, 515 nm
and 633 nm because of the availability of these wave lengths from
Argon, Argon, and Helium/Neon (He/Ne) lasers, respectively. In
general, longer wavelengths minimize background. A He/Ne laser is
particularly desirable and dyes with a high quantum yield and a
high molar coefficient of extinction at 633 nm are therefore
preferred.
In addition, it is desirable that the fluorescent cell
incorporative agent have a large Stokes shift, preferably greater
than 15 nm, more preferably greater than 30 nm. That is, it is
preferred that there be a substantial spread or difference in
wavelengths for the such fluorescent agent between its absorption
maximum and emission maximum.
The fluorescent agent should remain substantially incorporated in
the cell during the time of the assay, particularly where cells
containing such fluorescent agent are to be mixed with a sample
containing other cells. Furthermore, it is preferable that the
fluorescent cell incorporative agent exhibit much stronger
fluorescence when incorporated into a cell than when in an aqueous
environment. By much stronger fluorescence is meant that the
product of the extinction coefficient and the quantum yield at a
given excitation wavelength be much greater when the fluorescent
agent is incorporated in the cell than when the fluorescent agent
is not incorporated in the cell. Although an increase in
fluorescence is not required, it is usually preferable that the
fluorescence of the cell incorporated agent be at least three,
preferably at least ten, times that of the unincorporated
fluorescent agent.
A further characteristic of the fluorescent cell incorporative
agent is that it not interfere with binding of the sbp members,
e.g., binding of the antigen and antibody. The fluorescent cell
incorporative agent should preferably also exhibit a high affinity
for the cell.
The number of fluorescent cell incorporative agent molecules per
cell should be sufficient to conduct a meaningful assay, generally
being about 10.sup.2 to 10.sup.7 of such molecules per cell,
preferably 10.sup.3 to 10.sup.6 of such molecules per cell.
As mentioned above, a preferred class of fluorescent cell
incorporative agents comprises fluorescent dyes that are soluble in
the cell membrane, which means that the dyes are hydrophobic and
will usually be amphiphilic to provide for sufficient water
solubility to permit the agent to be incorporated in the cells in a
reasonable time. A preferred group of membrane soluble dyes
includes certain squarate dyes that have an absorption maximum
greater than 600 nm and an appropriate molar extinction coefficient
and Stoke's shift.
Exemplary of such dyes by way of illustration and not limitation
are those of the formula: ##STR1## wherein R.sub.1, R.sub.2,
R.sub.3 and R.sub.4, are each independently selected from the group
consisting of alkyl of from 2 to 16, preferably lower alkyl of from
2 to 6, carbon atoms and aralkyl of from 2 to 16, preferably 2 to
6, carbon atoms wherein the groups may be the same or different and
may be substituted with OH, CONHR.sup.1, SR.sup.1, OR.sup.1 wherein
R.sup.1 is lower alkyl of from 1 to 6 carbon atoms, F, Cl, Br, I,
NO.sub.2, oxocarbonyl, CN, etc. and R.sub.5 and R.sub.6 are
independently selected from the group consisting of hydrogen,
methoxy, and hydroxyl. Some of these dyes are disclosed by
Sprenger, et al., Angew. Chem. internat. Edit., 5: 894, 1966.
Squarate dyes of the following formula are also exemplary of
particular fluorescent cell incorporation agents in accordance with
the present invention: ##STR2## wherein R.sub.1, R.sub.2, and
R.sub.3 have been defined above. Some of these dyes are disclosed
by Sprenger, et al. in Angew. Chem. internat. Edit., 6: 553-554,
1967.
Other squarate dyes having the appropriate characteristics
identified above for the fluorescent cell incorporative agent will
be suggested to those skilled in the art.
The water solubility of the dyes can be enhanced by complexing the
dye with a water solubility enhancing compound such as
.beta.-cyclodextrin and the like according to the teaching of, for
example, Kinsland et al., J. Biochem. Biophys. Methods (1984) 9:
81-83.
Another class of fluorescent cell incorporative agents that may be
used in the present invention are the "vital dyes". These dyes are
derivatives of fluorescent dyes, which are membrane soluble and
usually weakly fluorescent but are capable of becoming membrane
insoluble and highly fluorescent after transport across the cell
membrane. Once these derivatives are inside the cell, enzymes, for
example, within the cell act upon the fluorescent dye derivative to
prevent it from being transported out of the cell as readily as it
was transported in. Exemplary of such dye derivatives are esters,
such as acetates and the like, of fluorescent compounds such as the
xanthene dyes, which include the fluoresceins derived from
3,6-dihydroxy-9-phenylxanthhydrol and rosamines and rhodamines,
derived from 3,6-diamino-9-phenylxanthene. The rhodamines and
fluoresceins have a 9-O-carboxyphenyl group, and are derivatives of
9-O-carboxyphenylxanthene. These compounds are commercially
available with or without substituents on the phenyl group.
Other examples of fluorescent compounds are the naphthylamines,
having an amino group in the alpha or beta position, usually alpha
position. Included among the naphthylamino compounds are
1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene
sulfonate and 2-p-toluidinyl-6-naphthalene sulfonate. Other
fluorescent compounds of interest include coumarins, e.g.,
umbelliferone.
Another class of fluorescent cell incorporative dyes that may be
employed in the present invention are those possessing an affinity
for DNA or RNA. As mentioned above, such dyes may be used in assays
in which cells possessing DNA are involved. Exemplary of such dyes
are DNA intercalating agents, which are generally well known
compounds and are, for the most part, commercially available.
Representative of such agents are acriflavine, acriflavine
hydrochloride, and like acridine derivatives, and ethidium halides
such as ethidium bromide.
Another class of fluorescent cell incorporative agents are those
dyes or stains having a high affinity for proteins or
carbohydrates. Such agents include, by way of example and not
limitation, pyrene and naphthalene sulfonates, stilbene
dimaleimides, salicylate maleimides, pyrene and coumarin
isothiocyanates, dansyl compounds such as dansyl azide and so
forth, anthracene carboxaldehyde carbohydrazones, common bacterial
stains and the like. Examples of the above agents are found in the
March 1981 catalog of Molecular Probes, Inc., Junction City, Oreg.,
and in Kodak Biological Stains and Related Products, publication
number JJ-281-51.
The invention will next be described in detail using a blood sample
as exemplary of the assay sample and blood group antigens, or
antibodies thereto, as exemplary of sbp members that may be
determined in accordance with the present method. This description
is by way of illustration only and is not meant to limit the scope
of the present invention.
In carrying out an assay for a blood group antigen in accordance
with the present invention, a blood sample optionally in a buffered
aqueous medium comprising greater than 5%, preferably greater than
20%, more preferably greater than 50%, blood by volume is employed.
The pH of the buffered aqueous medium is usually about 5 to 9,
preferably about 6 to 8. The sample is mixed with appropriate
amounts of a fluorescent cell incorporative agent and a
complementary sbp member, which amounts generally should be
sufficient to result in a meaningful assay. The amount of the
fluorescent cell incorporative agent depends upon the nature of the
cells and the nature of the agent. Usually, about 0.1 to 100 .mu.g,
preferably about 1 to 10 .mu.g, of fluorescent cell incorporative
agent are employed per ml of blood. The amount of complementary sbp
member employed is determined empirically and is usually between
0.01 and 1000 times the amount of sbp member, preferably 0.1 to 100
times the amount of sbp member.
Where the fluorescent cell incorporative agent has low water
solubility, the blood sample may first be mixed with the
fluorescent agent in a suitable organic polar solvent to facilitate
incorporation of such agent into the cell. The organic solvent will
generally have from 1 to 6 carbon atoms and from 1 to 3 heteroatoms
selected from the group consisting of oxygen, nitrogen, and sulfur.
Exemplary of such solvents are dimethylformamide,
dimethylsulfoxide, hexamethylphosphoramide, and the like. Next, the
mixture is combined with the complementary sbp member.
The sample, fluorescent cell incorporative agent, and complementary
sbp member are combined and incubated under conditions that will
provide for agglutination of the cells when the sbp member of
interest is present. Incubation times may vary widely depending on
the surface density of the sbp member, the concentration of the
cells and the complementary sbp member and the reaction conditions
including the addition of agglutination enhancers such as
polybrene, dextran or dextran derivatives, low ionic strength
medium, serum albumin, polyethyleneglycol, and the like. Desirable
incubation times are about 10 to 600 sec, preferably about 10 to
200 sec, at mild temperatures usually about 10.degree. to
37.degree. C.
In reverse blood typing for the determination in a whole blood
sample of antibodies to a particular blood group antigen, the
sample is combined in an aqueous buffered medium with red blood
cells of the particular type, A or B, of interest. The fluorescent
cell incorporative agent is preferably incorporated into such cells
prior to combining with the sample, in a manner and amounts similar
to that for incorporation of such agent into the red blood sample
as described above. The medium is then held for a period and at a
temperature as mentioned above.
Following the above holding period, the medium is examined to
determine any change in fluorescence as a result of agglutination
of the cells.
To this end one may use a non-flow cytometric technique in which a
small diameter beam of light produced by means of slits or
preferably a laser is used to differentiate particles based on
their relative size. This technique employs fluorescent pulse
height analysis or correlation of fluorescence fluctuations:
Briggs, et al., "Homogeneous Fluorescent Immunoassay," Science,
212, 1266-1267 (1981) and Nicoli, et al., "Fluorescence Immunoassay
Based on Long Time Correlations of Number Fluctuations," Proc.
Natl. Acad. Asci. USA, 77(8), 4904-4908 (1980).
A preferred method for determining a change of fluorescence in
accordance with the present invention involves the use of the fiber
optic cytometer described in U.S. patent application Ser. No.
397,285 filed July 12, 1982, the disclosure of which is
incorporated herein in its entirety. In the application, method and
apparatus are provided for determining the presence of particles in
a dispersion in relation to the detection of the presence or amount
of the material of interest. An optical fiber is used to define a
relatively small volume from which fluorescent light can be
received and counted. The volume is related to the volume in which
there is likely to be only a single particle which results in a
predetermined fluctuation. By employing a variety of techniques,
which allow for changes in fluorescence fluctuations in relation to
the presence of an analyte in a sample, the amount of analyte
present may be determined. The fluctuations are observed over a
period of time in a static mode or by sampling a plurality of
volumes in the sample. By comparing the observed results with
results obtained with assay solutions having a known amount of
analyte, the amount of analyte can be quantitatively
determined.
As a control, a known amount of blood group antigen or antibody in
question is incorporated into an appropriate medium and treated as
described above for the sample containing the unknown analyte. The
change in fluorescence for the control is compared with the change
in fluorescence for the unknown sample as an indicator of the
presence of the blood group antigen or antibody thereto in
question.
EXAMPLES
The invention is further demonstrated by the following illustrative
examples.
EXAMPLE 1
Preparation of
2-(p-Diethylamino-m-hydroxyphenyl)-4-(4-diethylimmonio-2-hydroxy-2,5-cyclo
hexadienylidene)-3-oxo-1-cyclobutenolate (DEAS)
DEAS was prepared as follows: Squaric acid (741 mg, 65 mmole) was
mixed with stirring, with 2.16 g, 13 mmole
3-N,N-diethylamino-phenol in 90 ml of n-butanol:toluene (2:1). The
mixture was refluxed overnight with azeotropic removal of water.
Progress of the reaction was followed by thin layer chromatography
(tlc) using methanol:toluene (1:9). Next, the reaction mixture was
distilled to remove about 40 ml of toluene, and then the reaction
mixture was cooled to room temperature. Crystalline product was
separated by filtration and dried at room temperature to give 2.5 g
of product. UV (DMF) .lambda. max 650 nm, .epsilon.=240,000,
fluorescence (DMF) 650/666 nm.
EXAMPLE 2
Assay for the Determination of the D (Rho) Blood Group Antigen
A saturated solution of DEAS in dimethylformamide (DMF) was
prepared and then diluted 1:10 (by volume) with DMF. Fifty .mu.l of
the diluted DEAS solution was mixed dropwise with 1 ml of an O
(Rho) positive whole blood sample under continuous vortexing. Ten
.mu.l of this mixture was mixed with 10 .mu.l of antibody
(commercially available typing reagent) specific for the D
(Rh.sub.o) blood group antigen. The mixture was held for one minute
at ambient temperature and then diluted with 1.5 ml of phosphate
buffer containing serum albumin and sucrose.
The medium was analyzed for a change in fluorescence as a result of
agglutination of cells by means of the limited volume method and
apparatus for particle counting disclosed in U.S. Ser. No. 397,285,
filed July 12, 1982, now U.S. Pat. No. 4,564,598.
The single fiber end of a "Y"-shaped fiber optics coupler obtained
from Kaptron, Inc., Palo Alto, Calif. (Splitter-Monitor, Model
FOMS-850-P), was submerged in the medium. The fiber had a diameter
of 50 microns and produced an excitation cone with a half angle of
12.degree. and an effective sampling volume of 1.times.10.sup.-7
ml. Excitation light from a He-Ne laser (632.9 nm) was fed into one
of the two branch fibers. The portion of the fluorescence emitted
from the cells which entered the submerged fiber end was split at
the fiber juncture to transmit equal halves back along the two
branch fibers. The portion traveling through the second branch
fiber was then read on a high-gain EMI photo-multiplier after
filtering out interference within gate times of one millisecond at
the rate of one every 0.1 second for periods of time ranging from
50 to 500 seconds. The average number of fluorescent pulses per
gate time was then determined by computer.
Two types of control runs were made to establish a standard
emission level.
(a) Samples that were types as D (Rh.sub.o) negative by
conventional type were assayed in the same way.
(b) A commercially available "Rh control" reagent which includes
all the ingredients of a D (Rh.sub.o) typing reagent except for the
antibody was used in the above assay in place of the antibody
reagent.
The results from samples from five positive and five negative
individuals are summarized in Table 1.
TABLE 1 ______________________________________ Type Signal*
______________________________________ D (Rh.sub.o) positive 84 108
72 46 74 D (Rh.sub.o) negative 18 23 22 17 20 Control 21 19 17
______________________________________ *Signal was obtained by
fluctuation analysis as described in the specification, Signals
greater than 40 were regarded as positive.
EXAMPLE 3
Assay for the Determination of the Antibody Specific for the A
Blood Group Antigen
Whole type A blood was centrifuged at 2800 rpm and the supernatant
and buffy coat of the white cells were removed by aspiration. The
packed cells were washed free of plasma using isotonic buffered
saline and suspended at 50% hematocrit in buffer containing 10%
bovine serum albumin.
A saturated solution of DEAS in DMF was prepared and diluted 1:10
(by volume) with DMF. Fifty .mu.l of the diluted DEAS solution was
mixed dropwise under continuous vortexing with 1 ml of the above
type A cell suspension.
Ten .mu.l of the above suspension was mixed with 20 .mu.l of a
whole blood sample. The mixture was held for one minute at ambient
temperature diluted with 3 ml of buffer containing serum albumin
and dextran, and analyzed as described above in Example 2 using the
limited volume method and apparatus for particle counting. The
results are summarized in Table 2.
TABLE 2 ______________________________________ Group* Signal**
______________________________________ A 18 23 25 13 18 B 112 110
91 107 56 AB 35 25 19 22 18 O 106 134 81 116 80 Control*** 15 18 16
______________________________________ *Samples from five separate
individuals having the listed blood groups were tested. **Signal
was obtained by fluctuation analysis as described in specification,
Signals greater than 40 were regarded as positive. ***Control
signals were obtained by reacting the A cells with blood from
individuals who were known to be of AB type.
EXAMPLE 4
The assay of Example 2 was repeated for blood group antigens A, B,
and O using antibody specific for the A(.alpha.A) and B(.alpha.B)
blood group antigens and antibodies obtained from type O
individuals (.alpha.A,B), respectively. DEAS was complexed with
.beta.-cyclodextrin following the teaching of Kinsland, supra. The
results are summarized in Table 3.
TABLE 3 ______________________________________ Blood Type Reagent
Signal* ______________________________________ A .alpha.A 98
.alpha.B 13 .alpha.A, B 252 Control - no reagent 11 B .alpha.A 10
.alpha.B 65 .alpha.A, B 272 Control - no reagent 16 O .alpha.A 18
.alpha.B 20 .alpha.A, B 34 Control - no reagent 13
______________________________________ *Signal was obtained by
fluctuation analysis as described in the specification. Signals
greater than 40 were regarded as positive.
The above data demonstrate that the method of the invention has
utility for assaying for a wide variety of analytes and has
particular utility in blood typing. The method is simple and rapid.
Generally, the method may be performed in a single step. The
effects, on the sensitivity of the assay, of background
interference from other components of a sample are minimized. The
result of the assay may be obtained without a separation or washing
step.
Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity and
understanding, it will be obvious that certain changes or
modifications may be practiced within the scope of the appended
claims.
* * * * *