U.S. patent number RE48,240 [Application Number 16/127,016] was granted by the patent office on 2020-10-06 for methods for testing milk.
This patent grant is currently assigned to Prolacta Bioscience, Inc.. The grantee listed for this patent is Prolacta Bioscience, Inc.. Invention is credited to Martin L. Lee, Elena M. Medo, David J. Rechtman.
United States Patent |
RE48,240 |
Medo , et al. |
October 6, 2020 |
Methods for testing milk
Abstract
The disclosure is related generally to methods for testing
mammary fluid (including milk) to establish or confirm the identity
of the donor of the mammary fluid. Such methods are useful in the
milk-bank business to improve safety.
Inventors: |
Medo; Elena M. (Lake Oswego,
OR), Lee; Martin L. (Studio City, CA), Rechtman; David
J. (Hermosa Beach, CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Prolacta Bioscience, Inc. |
City of Industry |
CA |
US |
|
|
Assignee: |
Prolacta Bioscience, Inc.
(Monrovia, CA)
|
Family
ID: |
37889534 |
Appl.
No.: |
16/127,016 |
Filed: |
September 10, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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13079932 |
Oct 2, 2012 |
8278046 |
|
|
|
12052253 |
May 17, 2011 |
7943315 |
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PCT/US2006/036827 |
Sep 20, 2006 |
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11947580 |
Oct 1, 2013 |
8545920 |
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60719317 |
Sep 20, 2005 |
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60731428 |
Oct 28, 2005 |
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Reissue of: |
13592678 |
Aug 23, 2012 |
8628921 |
Jan 14, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q
1/6809 (20130101); C12Q 1/6809 (20130101) |
Current International
Class: |
C12Q
1/68 (20180101); A23G 9/00 (20060101); A23L
3/015 (20060101); A01K 31/00 (20060101); C12Q
1/6809 (20180101); A23J 3/34 (20060101); A01N
61/00 (20060101) |
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International Searching Authority," 8 pages, from International
Patent Application No. PCT/US06/36827, United States Patent and
Trademark Office, Alexandria, Virginia, USA (mailed Sep. 5, 2007).
cited by applicant.
|
Primary Examiner: Campell; Bruce R
Attorney, Agent or Firm: Cooley LLP
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a .Iadd.reissue of U.S. application Ser. No.
13/529,678, filed Aug. 23, 2012, now U.S. Pat. No. 8,628,921, which
is a .Iaddend.continuation of U.S. application Ser. No. 13/079,932,
filed Apr. 5, 2011, now U.S. Pat. No. 8,278,046, which is a
continuation of U.S. application Ser. No. 12/052,253, filed Mar.
20, 2008, now U.S. Pat. No. 7,943,315, which is a
continuation-in-part and claims priority to the international
application PCT/US2006/036827 with an international filing date of
Sep. 20, 2006, which, in turn, claims priority under 35 U.S.C.
.[..sctn.119.]. .Iadd..sctn. 119 .Iaddend.from Provisional
Application Ser. Nos. 60/719,317, filed Sep. 20, 2005, and
60/731,428, filed Oct. 28, 2005.Iadd.; U.S. application Ser. No.
13/592,678 is also a continuation-in-part of U.S. application Ser.
No. 11/947,580, filed Nov. 29, 2007.Iaddend.. The disclosures of
all of each of which are incorporated herein by reference in their
entireties.
Claims
What is claimed is:
1. A method for determining whether a donated .[.mammary fluid.].
.Iadd.human breast milk .Iaddend.was obtained from a specific
subject.Iadd., wherein the specific subject is a qualified
donor.Iaddend., the method comprising: (a) testing a donated
biological sample from the specific subject to obtain at least one
reference identity marker profile for at least one .Iadd.identity
.Iaddend.marker.Iadd., wherein the specific subject has been
identified as a qualified donor by testing the specific subject for
absence of chronic illness.Iaddend.; (b) testing a sample of the
donated .[.mammary fluid.]. .Iadd.human breast milk .Iaddend.to
obtain at least one identity marker profile for the at least one
marker in step (a); (c) comparing the identity marker profiles
.Iadd.obtained in step (a) and (b).Iaddend., wherein.Iadd., if one
or more identity markers in the biological sample match one or more
identity markers in the donated human breast milk, such .Iaddend.a
match .[.between the identity marker profiles.]. indicates that the
.[.mammary fluid.]. .Iadd.donated human breast milk .Iaddend.was
obtained from the .[.specific subject.]. .Iadd.qualified
door.Iaddend.; and (d) processing the donated .[.mammary fluid.].
.Iadd.human breast milk from the qualified door .Iaddend.whose
identity marker profile has been matched with a reference identity
marker profile, wherein the processed donated .[.mammary fluid.].
.Iadd.human breast milk .Iaddend.comprises .Iadd.a caloric content
of 20 Cal/ounce; .Iaddend.a human protein constituent of 11-20
mg/mL; a human fat constituent of 35-55 mg/mL; and a human
carbohydrate constituent of 70-120 mg/mL.
.[.2. The method of claim 1, wherein the mammary fluid is human
breast milk..].
3. The method of claim 1 wherein the processing comprises: (a)
filtering the milk; (b) heat-treating the milk; (c) separating the
milk into cream and skim; (d) adding a portion of the cream to the
skim; and (e) pasteurizing.
.[.4. The method of claim 1, wherein the composition further
comprises one or more constituents selected from the group
consisting of: calcium, chloride, copper, iron, magnesium,
manganese, phosphorus, potassium, sodium, and zinc..].
5. The method of claim 1, wherein processing comprises separating
the milk into a cream portion and a skim portion, processing the
cream portion, and pasteurizing the cream portion.
6. The method of claim 1, further comprising nucleic acid typing,
wherein the nucleic acid typing comprises a method selected from
the group consisting of: STR analysis, HLA analysis, multiple gene
analysis, and a combination thereof.
7. The method of claim 1, wherein the donated .[.mammary fluid.].
.Iadd.breast milk .Iaddend.is frozen.
8. The method of claim 1, wherein the .[.mammary fluid.].
.Iadd.breast milk .Iaddend.sample comprises a mixture of one or
more .[.mammary fluid.]. .Iadd.breast milk .Iaddend.samples.
9. The method of claim 1, wherein the donated biological sample is
selected from a group consisting of: milk, saliva, buccal cell,
hair root, and blood.
10. The method of claim 1, wherein steps (a) through (c) are
carried out at a human breast milk donation center or at a milk
processing facility.
11. The method of claim 1, wherein steps (a) and (b) are carried
out at different facilities.
12. The method of claim 11, wherein step (a) is carried out at a
human breast milk donation facility and step (b) is carried out at
a milk processing facility.
13. A method for processing a donated human breast milk obtained
from a specific.Iadd., qualified donor .Iaddend.subject comprising:
(a) .Iadd.screening a donated biological sample from the subject
for the absence of chronic illness; (b) .Iaddend.testing a donated
biological sample from the specific subject to obtain at least one
reference identity marker profile for at least one marker; (.[.b.].
.Iadd.c.Iaddend.) testing a sample of the donated human breast milk
to obtain at least one identity marker profile for the at least one
marker in step (.[.a.]. .Iadd.b.Iaddend.); (.[.c.].
.Iadd.d.Iaddend.) comparing the identity marker profiles of steps
(.[.a.]. .Iadd.b.Iaddend.) and (.[.b.]. .Iadd.c.Iaddend.),
wherein.Iadd., if one or more identity markers in the biological
sample of step (b) match one or more identity markers in the
donated human breast milk of step (c), such .Iaddend.a match
.[.between the identity marker profiles.]. indicates that the
donated human breast milk was obtained from the specific subject;
(.[.d.]. .Iadd.e.Iaddend.) .Iadd.selecting for .Iaddend.processing
the donated human breast milk .Iadd.from the subject screened for
the absence of chronic illness and .Iaddend.whose identity marker
profile has been matched with a reference identity marker
profile.Iadd., wherein the selected subject is a qualified donor;
and (f) processing the donated human breast milk.Iaddend., wherein
the processing comprises: (i) filtering the donated human breast
milk; (ii) heat treating the donated human breast milk; (iii)
separating the donated human breast milk into cream and skim; (iv)
adding a portion of the cream to the skim to form a human milk
composition; and (v) pasteurizing the human milk composition to
produce a processed human breast milk composition; and wherein the
processed donated human breast milk comprises .Iadd.a caloric
content of 20 Cal/ounce; .Iaddend.a human protein constituent of
11-20 mg/mL; a human fat constituent of 35-55 mg/mL; and a human
carbohydrate constituent of 70-120 mg/mL.
14. A processed human milk composition suitable for administration
to an infant made by the process of claim 13.
.[.15. The method of claim 13, wherein the method further comprises
adding to the processed human breast milk one or more constituents
selected from the group consisting of: calcium, chloride, copper,
iron, magnesium, manganese, phosphorus, potassium, sodium, and
zinc..].
16. The method of claim 13, wherein the testing of the donated
human breast milk of step (.[.b.]. .Iadd.c.Iaddend.) and the
testing of the donated biological sample of step (.[.a.].
.Iadd.b.Iaddend.) comprises nucleic acid typing selected from the
group consisting of: STR analysis, HLA analysis, multiple gene
analysis, and a combination thereof.
17. The method of claim 13, wherein the donated biological sample
.Iadd.of step (a) and/or the donated biological sample of step (b)
.Iaddend.is selected from a group consisting of: milk, saliva,
buccal cell, hair root, and blood.
18. The method of claim 13, wherein steps (.[.a.].
.Iadd.b.Iaddend.) through (.[.c.]. .Iadd.e.Iaddend.) are carried
out at a human breast milk donation center or at a milk processing
facility.
19. The method of claim 13, wherein steps (.[.a.].
.Iadd.b.Iaddend.) and (.[.b.]. .Iadd.c.Iaddend.) are carried out at
different facilities.
20. The method of claim 19, wherein step (.[.a.]. .Iadd.b.Iaddend.)
is carried out at a human breast milk donation facility and step
(.[.b.]. .Iadd.c.Iaddend.) is carried out at a milk processing
facility.
.Iadd.21. The method of claim 1, wherein the human protein
constituent comprises about 11-13 mg/mL. .Iaddend.
.Iadd.22. The method of claim 1, wherein the human carbohydrate
constituent comprises about 80-105 mg/mL. .Iaddend.
.Iadd.23. The method of claim 1, wherein the processed donated
breast milk of step (d) does not comprise added non-human derived
nutritional components. .Iaddend.
.Iadd.24. The method of claim 1, wherein the method comprises
pooling the donated breast milk of step (c) whose identity marker
profile has been matched with the reference identity marker
profile, with other donated breast milk samples that have been
matched with the reference identity marker profile to obtain a pool
of identity matched breast milk. .Iaddend.
.Iadd.25. The method of claim 24, wherein the pool of identity
matched breast milk is in a volume of at least about 75 liters/lot
to about 2,000 liters/lot. .Iaddend.
.Iadd.26. The method of claim 1, wherein the processed breast milk
of step (d) has an osmolality of less than about 400 mOsm/kg
H.sub.2O. .Iaddend.
.Iadd.27. The method of claim 1, wherein the processing of step (d)
comprises filtering the donated breast milk by ultrafiltration.
.Iaddend.
.Iadd.28. The method of claim 27, wherein the ultrafiltration
filters out water, and wherein the processing step further
comprises washing the filters used during the ultrafiltration with
the water obtained by the ultrafiltration. .Iaddend.
.Iadd.29. The method of claim 26, wherein the processing of step
(d) further comprises reducing the bioburden of the human donor
milk. .Iaddend.
.Iadd.30. The method of claim 1, wherein the processing of step (d)
comprises concentrating the nutrients in the donated breast milk.
.Iaddend.
.Iadd.31. The method of claim 30, wherein the processing of step
(d) further comprises reducing the bioburden of the human donor
milk. .Iaddend.
.Iadd.32. The method of claim 13, wherein the processed donated
human breast milk comprises no added non-human derived nutritional
components. .Iaddend.
.Iadd.33. The method of claim 13, wherein the pool of identity
matched human donor milk is in a volume of at least about 75
liters/lot to about 2,000 liters/lot. .Iaddend.
.Iadd.34. The method of claim 13, wherein the processed milk has on
osmolality of less than about 400 mOsm/kg H.sub.2O. .Iaddend.
.Iadd.35. The method of claim 13, wherein the processing step
comprises filtering the human donor milk. .Iaddend.
.Iadd.36. The method of claim 13, wherein the processing step
comprises filtering the human donor milk by ultrafiltration.
.Iaddend.
.Iadd.37. The method of claim 36, wherein the ultrafiltration
filters out water, and wherein the processing step further
comprises washing the filters used during the ultrafiltration with
the water obtained by the ultrafiltration. .Iaddend.
.Iadd.38. The method of claim 13, wherein the processing step
comprises concentrating the nutrients in the human donor milk.
.Iaddend.
.Iadd.39. The method of claim 38, wherein the processing step
further comprises reducing the bioburden of the human donor milk.
.Iaddend.
.Iadd.40. The method of claim 35, wherein the processing step
further comprises reducing the bioburden of the human donor milk.
.Iaddend.
.Iadd.41. The method of claim 13, wherein the human protein
constituent comprises about 11-13 mg/mL. .Iaddend.
.Iadd.42. The method of claim 13, wherein the human carbohydrate
constituent comprises about 80-105 mg/mL. .Iaddend.
.Iadd.43. A method for making concentrated, bioburden reduced,
identity-matched and health screened human donor milk from a
qualified human milk donor, wherein the human donor milk is safe
and provides standardized nutrition, the method comprising: (a)
screening a subject for drug use and/or chronic illness by testing
a biological sample from the subject; (b) selecting a qualified
donor, wherein the subject is a qualified donor if the biological
sample of step (a) did not test positive for drug use or chronic
illness; (c) testing a donated biological sample from the qualified
donor to obtain at least one DNA marker profile for at least one
DNA marker; (d) testing a sample of donated human breast milk from
the qualified donor to obtain at least one DNA marker profile for
the at least one DNA marker in step (c); (e) comparing the DNA
marker profiles obtained in (c) and (d), wherein, if DNA markers in
the biological sample of step (c) match DNA markers in the donated
human breast milk of step (d), such match indicates that the
donated human breast milk was obtained from the specific, health
screened, qualified donor, thereby obtaining identity matched,
health screened donor milk from a qualified human milk donor; (d)
pooling the identity matched, health screened donor milk obtained
in step (d) with other identity matched, health screened donor milk
from other qualified donors to obtain a pool of identity matched,
health screened human donor milk from qualified donors, comprising
at least about 75 liters, wherein the pool of identity matched,
health screened donor milk is not nutritionally sufficient for
preterm infants; (e) concentrating the pool of non-nutritionally
sufficient, identity matched, health screened human donor milk from
qualified donors to comprise a human protein constituent of 11-13
mg/mL; a human fat constituent of 35-55 mg/mL; a human carbohydrate
constituent of 70-120 mg/mL; a caloric content of 20 Cal/ounce; and
no added non-human derived vitamins or minerals to obtain a pool of
concentrated identity matched donor milk from qualified donors with
an osmolality of less than about 400 mOsm/Kg H.sub.2O; and (f)
treating the pool of concentrated, identity-matched, health
screened donor milk obtained in (e) to reduce the bioburden;
thereby obtaining concentrated, bioburden reduced,
identity-matched, health screened donor milk from qualified donors.
.Iaddend.
.Iadd.44. The method of claim 43, wherein the nucleic acid typing
comprises STR analysis, HLA analysis, multiple gene analysis, or a
combination thereof. .Iaddend.
.Iadd.45. The method of claim 43, wherein the donated biological
sample of step (a) and/or (c) is milk, saliva, buccal cell, hair
root, or blood. .Iaddend.
.Iadd.46. A processed human milk composition suitable for
administration to an infant made by the process of claim 43.
.Iaddend.
.Iadd.47. The processed human milk composition of claim 46, wherein
the infant is a preterm infant. .Iaddend.
.Iadd.48. The method of claim 43, wherein the concentrating step
comprises filtering the human donor milk. .Iaddend.
.Iadd.49. The method of claim 48, wherein the filtering step
comprises ultrafiltering the human donor milk. .Iaddend.
.Iadd.50. The method of claim 49, wherein the ultrafiltration
filters out water, and wherein the processing step further
comprises washing the filters used during the ultrafiltration with
the water obtained by the ultrafiltration. .Iaddend.
.Iadd.51. The method of claim 1, wherein the one or more identity
markers comprise DNA. .Iaddend.
.Iadd.52. The method of claim 51, wherein the one or more identity
markers comprise multiple DNA markers. .Iaddend.
.Iadd.53. The method of claim 1, wherein the specific subject has
been identified as a qualified donor by further screening for drug
use. .Iaddend.
.Iadd.54. The method of claim 1, wherein the specific subject has
been identified as a qualified donor by interview and/or by
biological sample testing for viral contamination. .Iaddend.
Description
TECHNICAL FIELD
The methods featured herein are related generally to methods of
testing mammary fluid (including milk) to establish or confirm the
identity of the donor of the mammary fluid.
BACKGROUND
Unlike blood donors, who give their donation under the direct
supervision of the blood bank personnel, human breast milk donors
tend to pump their milk for donation at home or other locations
convenient to them and then often store the breast milk in their
freezers until they have accumulated enough to bring to the
donation center. Thus, in the absence of direct supervision of the
donations, questions may arise as to the provenance of the donated
breast milk.
In order to establish that the breast milk provided by a donor is,
in fact, exclusively from that human female donor, some form of
testing to establish donor identity should occur.
Many different methods of DNA typing are known for identifying or
typing specimens from humans. Such methods include short tandem
repeats ("STR"), microsatellite repeats or simple sequence repeats
("SSR") analysis of human DNA; analysis of multiple human genes and
analysis of human lymphocyte antigen (HLA) genes and loci by
polymerase chain reaction (PCR) analysis, restriction length
polymorphism analysis and other methods.
It is known that humans possess antigens which are specific to that
individual. For example, the human leukocyte antigens (HLA) have
been used in the past for typing tissue for transplantation.
Such typing methods, among others, can be used to test for donor
identity in the methods featured herein.
SUMMARY
The methods and systems featured herein relate to diagnosing or
screening mammary fluid from any number of mammalian organisms. In
one aspect, the invention provides methods and systems for
diagnosing or screening human milk samples to confirm that the milk
is from a defined source.
The methods include obtaining a donated reference sample from a
potential mammary fluid donor, e.g., a human breast milk donor. The
sample can be analyzed at or around the time of obtaining the
sample for one or more markers that can identify the potential
donor. Alternatively, or in addition, the sample can be stored and
analyzed for identifying markers at a later time. When the
potential mammary fluid donor expresses the mammary fluid and
donates the fluid (e.g., by bringing or sending the fluid to the
donation center), the mammary fluid can be analyzed for the same
marker or markers as the donor's reference sample. The match
between the markers (and lack of any additional unmatched markers)
would indicate that the donated milk comes from the same individual
as the one who donated the reference sample. Lack of a match (or
presence of additional unmatched markers) would indicate that the
donated mammary fluid either comes from a non-tested donor or has
been contaminated with fluid from a non-tested donor.
The testing of the reference sample and of the donated mammary
fluid can be carried out at the donation facility and/or milk
processing facility. The results of the reference sample tests can
be stored and compared against any future donations by the same
donor.
Testing donors to confirm their identity improves safety of donated
milk. It ensures the provenance of the donated milk, which as
discussed above, is most often donated without supervision by the
donor center. Testing donor identity by the methods featured herein
allows for multiple donations by the same donor, whose identity can
be confirmed at the time of each donation. The donor can live at
any distance from the donation and/or processing facilities, as she
can send her milk at long distances, and her identity can be
confirmed based on reference samples or reference tests stored at
the donation and/or processing facility.
The mammary fluid tested by the methods featured herein can be
processed for further use. The donation facility and milk
processing facility can be the same or different facility. The
donated milk can be processed, e.g., to obtain human milk
fortifiers, standardized human milk formulations; human lipid
products, and/or compositions for total parenteral nutrition.
In one aspect, a method of determining whether a donated mammary
fluid was obtained from a specific subject is featured. The method
includes: (a) testing a donated biological sample from the specific
subject to obtain at least one reference identity marker profile
for at least one marker; (b) testing a sample of the donated
mammary fluid to obtain at least one identity marker profile for
the at least one marker in step (a); and (c) comparing the identity
marker profiles, wherein a match between the identity marker
profiles indicates that the mammary fluid was obtained from the
specific subject.
Embodiments can include one or more of the following features.
The method can further include: (d) processing the donated mammary
fluid whose identity marker profile has been matched with a
reference identity marker profile. The mammary fluid is human
breast milk. Processing can include generating a pasteurized milk
composition for administration to a human infant. The processing
can include: filtering the milk; heat-treating the milk; separating
the milk into cream and skim; adding a portion of the cream to the
skim; and pasteurizing.
The processed and pasteurized milk composition can include: a human
protein constituent of about 35-85 mg/mL; a human fat constituent
of about 60-110 mg/mL; and a human carbohydrate constituent of
about 60-140 mg/mL, and optionally, one or more constituents
selected from the group consisting of: calcium, chloride, copper,
iron, magnesium, manganese, phosphorus, potassium, sodium, and
zinc.
The processed and pasteurized milk composition can include: a human
protein constituent of about 11-20 mg/mL; a human fat constituent
of about 35-55 mg/mL; and a human carbohydrate constituent of about
70-120 mg/mL, and, optionally, one or more components selected from
the group consisting of: calcium, chloride, copper, iron,
magnesium, manganese, phosphorus, potassium, sodium, and zinc.
The processing can include separating the milk into a cream portion
and a skim portion, processing the cream portion, and pasteurizing
the cream portion.
The testing of the mammary fluid sample and the testing of the
biological sample can include a nucleic acid typing, e.g., STR
analysis, HLA analysis, multiple gene analysis, and a combination
thereof.
The donated mammary fluid can be frozen, and the method can include
obtaining the mammary fluid sample by drilling a core through the
frozen fluid. Alternatively, or in addition, the method can include
obtaining the mammary fluid sample by scraping the surface of the
frozen mammary fluid.
The method can further include isolating the mammary fluid prior to
step (b).
The mammary fluid sample can include a mixture of one or more
mammary fluid samples. The testing of the mammary fluid sample and
the testing of the biological sample can include antibody testing
to obtain a self-antigen profile. The sample of the donated mammary
fluid can include a selected solid fraction of the fluid. The
identity profiles can include peptide markers. The donated
biological sample can be, e.g., milk, saliva, buccal cell, hair
root, and blood.
A lack of a match between the identity marker profiles indicates
contamination of the mammary fluid by another mammal.
Steps (a) through (c) can be carried out at a human breast milk
donation center or at a milk processing facility.
In another aspect the disclosure is related to a method for
determining whether breast milk was obtained from a specific human
comprising testing a sample of the breast milk to obtain an
identity marker profile and testing a biological sample from the
human to obtain a reference identity marker profile and comparing
the identity marker profiles.
The disclosure provides a method for determining whether breast
milk was obtained from a desired source or specific human
comprising nucleic acid typing of a sample of the breast milk to
obtain a DNA type profile and nucleic acid typing of a biological
sample from the human to obtain a reference DNA type profile and
comparing the DNA type profiles.
In one embodiment, the method of nucleic acid typing of the
biological sample from the human is selected from STR analysis, HLA
analysis or multiple gene analysis. It is further contemplated that
nucleic acid typing method used for the breast milk sample is the
same as that used for the biological sample. It is contemplated
that the loci/alleles used for the DNA type profile will be the
same for both the reference DNA type profile and the breast milk
sample DNA type profile.
In one embodiment the breast milk will be frozen. It is further
contemplated that the method for obtaining the breast milk sample
from the frozen breast milk will be by drilling a core through the
frozen breast milk. Alternatively, it is contemplated that the
breast milk sample may be obtained by scraping the surface of the
frozen breast milk.
In another embodiment the breast milk will be liquid. It is
contemplated that the method for obtaining the breast milk sample
will be by isolating the breast milk sample by pipette or other
means.
In another embodiment the breast milk samples may be combined or
mixed prior to nucleic acid typing.
The disclosure also provides a method for determining whether
breast milk was obtained from a defined source (e.g., a specific
human) comprising testing of a sample of the breast milk to obtain
a self-antigen profile and testing a biological sample from the
human to obtain a reference self-antigen profile and comparing the
self-antigen profiles.
The disclosure also provides an article of manufacture or kit
comprising a container, a label on the container and a reagent for
detecting or measuring identity markers, wherein the label on the
container indicates that the reagent can be used to determine the
identity marker profile of breast milk.In one embodiment, the
reagent comprises PCR materials (a set of primers, DNA polymerase
and 4 nucleoside triphosphates) that hybridize with the gene or
loci thereof. The kit may further comprise additional components,
such as reagents, for detecting or measuring the detectable entity
or providing a control. Other reagents used for hybridization,
prehybridization, DNA extraction, visualization and the like may
also be included, if desired. In another embodiment, the regent is
an antibody for detecting self-antigens.
Unless defined otherwise, technical and scientific terms used
herein have the same meaning as commonly understood by one of skill
in the art to which this invention belongs.
One skilled in the art will recognize many methods and materials
similar or equivalent to those described herein, which could be
used in the practice of this invention. Indeed, the invention is no
way limited to the methods and materials described herein. For
purposes of the methods featured herein, the following terms are
defined.
"Mammary fluid" includes breast milk and/or colostrum expressed
from lactating female subjects. Whole mammary fluid, selected
liquid or solid fractions of the mammary fluid, whole cells or
cellular constituents, proteins, glycoproteins, peptides,
nucleotides (including DNA and RNA polynucleotides) and other like
biochemical and molecular constituents of the mammary fluid can be
used in the present methods. The mammary fluid may be obtained from
any number of species of female subjects including, but not limited
to, humans, bovines, goats and the like.
"Identity marker" includes a marker that can be used to identify an
individual subject from other subjects in a population. Such
markers are present in the cells found in mammary fluid. Such
markers could include, but are not limited to, genes, alleles,
loci, antigens polypeptides or peptides.
An "identity marker profile" comprises a profile of a number of
identity markers. The profile identifies the individual human or
subject from other humans with a sufficient degree of certainty. It
is contemplated that the identity marker profile identifies at
least one human from 100,000 humans, or 1 human from 1 million
humans or 1 human from 5 million humans.
"Nucleic acid typing" refers to a method of determining the DNA
type profile of a biological or milk sample. Such methods include,
but are not limited to: STR analysis, HLA analysis or multiple gene
analysis of genes/alleles/loci present in a polynucleotide sample
of the biological or milk sample.
"DNA type profile" refers to a profile of a human's or subject's
genomic DNA, which is sufficient to distinguish the individual
human or subject from other humans with a sufficient degree of
certainty. It is contemplated that the DNA profile identifies at
least one human from 100,000 humans, or 1 human from 1 million
humans or 1 human from 5 million humans. Generally the methods
featured herein involve identifying alleles of at least 5
loci/genes or at least 10 loci/genes or at least 13 loci/gene.
An "allele" comprises one of the different nucleic acid sequences
of a gene at a particular locus on a chromosome. One or more
genetic differences can constitute an allele. Examples of HLA
allele sequences are set out in Mason and Parham (1998) Tissue
Antigens 51:417-66, which list HLA-A, HLA-B, and HLA-C alleles and
Marsh et al (1992); and Hum. Immunol. 35:1, which list MLA Class II
alleles for DRA, DRB, DQA1, DQB1, DPA1, and DPB1.
A "locus" comprises a discrete location on a chromosome. The loci
may be part of a gene or part of repeat sequence. Exemplary human
leukocyte antigens (HLAs) loci are the class I MHC genes designated
HLA-A, HLA-B and HLA-C; nonclassical class I genes including HLA-E,
HLA-F, HLA-G, HLA-H, HLA-J and HLA-X, MIC; and class II genes such
as HLA-DP, HLA-DQ and HLA-DR. Exemplary STR loci are: CSF1PO,
D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, DI8S51, D21S11,
DYS19, F13A1, FES/FPS, FGA, HPRTB, THO1, TPDX, DYS388, DYS391,
DYS392, DYS393, D2S1391, D18S535, D2S.1338, D19S433, D6S477,
D1S518, D14S306, D22S684, F13B, CD4, D12S391, D10S220 and D7S523
(see, e.g., U.S. Pat. No. 6,090,558).
A method of HLA analysis or human leukocyte antigen analysis is a
method that permits the determination or assignment of one or more
genetically distinct human leukocyte antigen (HLA) genetic
polymorphisms by any number of methods known in the art. Some
methods contemplated are described below.
A method of STR analysis is a method that permits the determination
or assignment of one or more genetically distinct STR genetic
polymorphisms by any number of methods known in the art. Some
methods contemplated are described herein.
A method of multiple gene analysis is a method that permits the
determination or assignment of one or more genetically distinct
genetic polymorphisms of human genes by any number of methods known
in the art. Such genes may or may not include the HLA genes. Some
methods contemplated are described herein.
A number of amplification techniques are known in the art.
Amplifying refers to a reaction wherein a template nucleic acid, or
portions thereof, is duplicated at least once. Such amplification
techniques include arithmetic, logarithmic, or exponential
amplification. The amplification of a nucleic acid can take place
using any nucleic acid amplification system, both isothermal and
thermal gradient based including, but not limited to, polymerase
chain reaction (PCR), reverse-transcription-polymerase chain
reaction (RT-PCR), ligase chain reaction (LCR), self-sustained
sequence reaction (3SR), and transcription mediated amplifications
(TMA). Typical nucleic acid amplification mixtures (e.g., PCR
reaction mixture) include a nucleic acid template that is to be
amplified, a nucleic acid polymerase, nucleic acid primer
sequence(s), nucleotide triphosphates, and a buffer containing all
of the ion species required for the amplification reaction.
Amplification products obtained from an amplification reaction
typically comprise a single stranded or double stranded DNA or RNA
or any other nucleic acid products of isothermal and thermal
gradient amplification reactions that include PCR, LCR, and the
like.
A "template nucleic acid" refers to a nucleic acid polymer that is
sought to be copied or amplified. The template nucleic acid(s) can
be isolated or purified from a cell, tissue, and the like. The
template nucleic acid can comprise genomic DNA, eDNA, RNA, or the
like.
Primers are used in some amplification techniques. A primer
comprises an oligonucleotide used in an amplification reaction
(e.g., PCR) to amplify a target nucleic acid. The primer is
typically single stranded. The primer may be from about 5 to 30
nucleic acids in length, more commonly from about 10 to 25 nucleic
acids in length.
An STR locus-specific primer is an oligonucleotide that hybridizes
to a nucleic acid target variant that defines or partially defines
that particular STR locus.
An HLA allele-specific primer is an oligonucleotide that hybridizes
to a nucleic acid target variant that defines or partially defines
that particular HLA allele.
HLA locus-specific primer is an oligonucleotide that permits the
amplification of an HLA locus or that can hybridize specifically to
an HLA locus.
An allele-specific primer is an oligonucleotide that hybridizes to
a target nucleic acid variant that defines or partially defines
that particular gene allele.
A locus-specific primer is an oligonucleotide that permits the
amplification of a gene locus or that can hybridize specifically to
a gene locus.
A forward primer and a reverse primer constitute a pair of primers
that can bind to a template nucleic acid and under proper
amplification conditions produce an amplification product. If the
forward primer is binding to the sense strand then the reverse
primer is binding to antisense strand. Alternatively, if the
forward primer is binding to the antisense strand then the reverse
primer is binding to sense strand. In essence, the forward or
reverse primer can bind to either strand so long as the other
reverse or forward primer binds to the opposite strand.
Any number of detectable labels can be used to detect a target
nucleic acid by use of amplification or other techniques. A
detectable label refers to a moiety that is attached through
covalent or non-covalent techniques to an oligonucleotide or other
detection agent. Examples of detectable labels include a
radioactive moiety, a fluorescent moiety, a chemiluminescent
moiety, a chromogenic moiety and the like. Fluorescent moieties
comprise chemical entities that accepts radiant energy of one
wavelength and emits radiant energy at a second wavelength.
Various hybridization techniques can be used in the methods
described herein. Hybridizing or hybridization refers to the
binding or duplexing of a molecule to a substantially complementary
polynucleotide or fragment through bonding via base pairing.
Hybridization typically involves the formation of hydrogen bonds
between nucleotides in one nucleic acid and a complementary second
nucleic acid. Methods of hybridization can include highly
stringent, moderately stringent, or low stringency conditions.
The term "stringent conditions" refers to conditions under which a
capture oligonucleotide, oligonucleotide or amplification product
will hybridize to its target nucleic acid. "Stringent hybridization
conditions" or "highly stringent conditions" are sequence dependent
and will be different with different environmental parameters
(e.g., salt concentrations, and presence of organics). Generally,
stringent conditions are selected to be about 5.degree. C. to
20.degree. C. lower than the thermal melting point (T.sub.m) for a
specific nucleic acid at a defined ionic strength and pH. Stringent
conditions are about 5.degree. C. to 10.degree. C. lower than the
thermal melting point for a specific nucleic acid bound to a
complementary nucleic acid. The T.sub.m is the temperature (under
defined ionic strength and pH) at which 50% of a nucleic acid
hybridizes to a matched probe. Longer oligonucleotides hybridize at
higher temperatures. Typically, stringent conditions will be those
in which the salt concentration comprises about 0.01 to 1.0 M Na
(or other salts) at pH 7.0 to 8.3 and the temperature is at least
about 300 C for short probes (e.g., 10 to 50 nucleotides).
Stringent conditions may also be achieved with the addition of
destabilizing agents such as formamide. An extensive guide to the
hybridization and washing of nucleic acids is found in Tijssen
(1993) Laboratory Techniques in Biochemistry and Molecular
Biology--Hybridization with Nucleic Acid Probes parts I and II,
Elsevier, N.Y.; Choo (ed) (1994) Methods Molecular Biology Volume
33, In Situ Hybridization Protocols, Humana Press Inc., New Jersey;
Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed.
1989); Current Protocols in Molecular Biology, Ausubel et al.,
eds., (1994).
Hybridization conditions for a particular probe, primer and target
are readily determinable by one of ordinary skill in the art, and
generally is an empirical calculation dependent upon probe length,
washing temperature, and salt concentration. In general, longer
probes require higher temperatures for proper annealing, while
shorter probes need lower temperatures. Hybridization generally
depends on the ability of single stranded nucleic acids to anneal
with a complementary strands present in an environment below their
inciting temperature. The higher the degree of desired homology
between a probe and hybridizable target, the higher the relative
temperature which can be used. As a result, it follows that higher
relative temperatures would tend to make the reaction conditions
more stringent, while lower temperatures less so.
Hybridization wash conditions are ordinarily determined empirically
for hybridization of each probe or set of primers to a
corresponding target nucleic acid. The target nucleic acid and
probes/primers are first hybridized (typically under stringent
hybridization conditions) and then washed with buffers containing
successively lower concentrations of salts, or higher
concentrations of detergents, or at increasing temperatures until
the signal to noise ratio for specific to non-specific
hybridization is high enough to facilitate detection of specific
hybridization. Stringent temperature conditions will usually
include temperatures in excess of about 30.degree. C. more usually
in excess of about 37.degree. C., and occasionally in excess of
about 45.degree. C. Stringent salt conditions will ordinarily be
less than about 1000 mM, usually less than about 500 mM, more
usually less than about 400 mM, typically less than about 300 mM,
typically less than about 200 mM, and more typically less than
about 150 mM. However, the combination of parameters is more
important than the measure of any single parameter. See, e.g.,
Wetmur et al., J. Mol Biol 31:349-70 (1966), and Wetmur, Critical
Reviews Biochemistry and Molecular Biology 26 (34):227-59
(1991).
In one embodiment, highly stringent conditions comprise
hybridization in 50% formamide, 6.times.SSC (0.75 M NaCl, 0.075 M
sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium
pyrophosphate, 5.times. Denhardt's solution, sonicated salmon sperm
DNA (100/.mu.g/ml), 0.5% SDS, and 10% dextran sulfate at 420 C,
with washes at 42.degree. C. in 2.times.SSC (sodium chloride/sodium
citrate) and 0.1% SDS, followed by a high-stringency wash
comprising of 0.2.times.SSC containing 0.1% SDS at 42.degree.
C.
The terms "complement," "complementarity" or "complementary," as
used herein, are used to describe single-stranded polynucleotides
related by the rules of antiparallel base-pairing. For example, the
sequence 5'-CTAGT-3' is completely complementary to the sequence
5'-ACTAG-3'. Complementarity may be "partial," where the base
pairing is less than 100%, or complementarity may be "complete" or
"total," implying perfect 100% antiparallel complementation between
two polynucleotides. By convention in the art, single-stranded
nucleic acid molecules are written with their 5' ends to the left,
and their 3' ends to the right.
The term "complementary base pair" refers to a pair of bases
(nucleotides) each in a separate nucleic acid in which each base of
the pair is hydrogen bonded to the other. A "classical"
(Watson-Crick) base pair contains one purine and one pyrimidine;
adenine pairs specifically with thymine (A-T), guanine with
cytosine (G-C), uracil with adenine (U-A). The two bases in a
classical base pair are said to be complementary to each other.
"Substantially complementary" between a probe or primer nucleic
acid and a target nucleic acid embraces minor mismatches that can
be accommodated by reducing the stringency of the hybridization
media to achieve the desired degree of hybridization and
identification of hybridized target polynucleotides.
A "capture oligonucleotide" useful for identification of a target
nucleic acid refers to a nucleic acid or fragment that can
hybridize to a polynucleotide, oligonucleotide, amplification
product, or the like, and has the ability to be immobilized to a
solid phase. A capture oligonucleotide typically hybridizes to at
least a portion of an amplification product containing
complementary sequences under stringent conditions.
An "HLA locus-specific capture oligonucleotide" is a capture
oligonucleotide that is complementary to and hybridizes to a
conserved region of an HLA locus. For example, the capture
oligonucleotide can be specific for the HLA-A locus and will
hybridize to one or more conserved regions or subsequences of the
HLA-A locus.
Similarly, an "STR locus-specific capture oligonucleotide" is a
capture oligonucleotide that is complementary to and hybridizes to
a conserved region of an STR locus. A locus-specific capture
oligonucleotide is a capture oligonucleotide that is complementary
to and hybridizes to a conserved region of a genetic locus.
A capture oligonucleotide is typically immobilized on a solid phase
directly or indirectly. Such immobilization may be through covalent
and/or non-covalent bonds.
The term "amplicon", is used herein to mean a population of DNA
molecules that has been produced by amplification, e.g., by
PCR.
The term "subject" refers to a lactating mammalian subject. The
subject may be a human, bovine, goat and the like. For example, the
screening as to the origination of milk products from non-human
mammals may be important for the tracing of products to a
particular bovine, for example, for FDA or other purposes.
"Mutation" as used herein sometimes refers to a functional
polymorphism that occurs in the population, and is strongly
correlated to a gene. "Mutation" is also used herein to refer to a
specific site and type of functional polymorphism, without
reference to the degree of risk that particular mutation poses to
an individual for a particular disease.
A "self-antigen" is an antigen which identifies the individual
subject from other subjects in the population. Exemplary
self-antigens include the major histocompatibility antigens (MHC)
antigens or the blood type antigens (ABO).
A "self antigen profile" means a profile of a human's or subject's
self-antigens which is sufficient to distinguish the individual
subject from other subjects with a sufficient degree of
certainty.
The term "antibody" is used in its broadest sense and covers
polyclonal antibodies, monoclonal antibodies, single chain
antibodies and antibody fragments.
.Iadd.This disclosure features human milk compositions, e.g.,
pasteurized human milk compositions, and methods of making and
using such compositions. The compositions include human milk
fortifiers (e.g., PROLACTPLUS.TM. Human Milk Fortifiers, e.g.,
PROLACT+4.TM., PROLACT+6.TM., PROLACT+8.TM., and/or
PROLACT+10.TM.), which are produced from human milk and contain
various concentrations of nutritional components, e.g., protein,
fat, carbohydrates, vitamins, and/or minerals. These fortifiers can
be added to the milk of a nursing mother to provide an optimal
nutritional content of the milk for, e.g., a preterm infant.
Depending on the content of mother's own milk, various
concentrations of the fortifiers can be added to mother's milk. For
example, the protein concentration of the mother's milk can be
increased with the use of the fortifier. As mentioned above, the
fortifiers of the present disclosure are generated from human milk
and, therefore, provide infants with human-derived nutrients.
.Iaddend.
.Iadd.The disclosure also features standardized human milk
formulations (exemplified by PROLACT20.TM., NEO20.TM., and/or
PROLACT24), which are produced from human milk. Methods of making
and using such compositions are also described herein. These
standardized human milk formulations can be used to feed, e.g.,
preterm infants, without mixing them with other fortifiers or milk.
They provide a nutritional human-derived formulation and can
substitute for mother's milk. Human milk formulations can contain
various caloric contents, for example, PROLACT24.TM. (a full-feed
whole milk product) can contain about 24 Cal/oz or about 81 Cal/100
mL. .Iaddend.
.Iadd.The methods featured herein are used to process large volumes
of donor milk, e.g., about 75-2,000 liters/lot of starting
material. .Iaddend.
.Iadd.In one aspect, the disclosure features a pasteurized human
milk composition that includes: a human protein constituent of
about 35-85 mg/mL; a human fat constituent of about 60-110 mg/mL;
and a human carbohydrate constituent of about 60-140 mg/mL. The
carbohydrate constituent can include lactose. The composition can
further include IgA and/or one or more constituents selected from
the group consisting of: calcium, chloride, copper, iron,
magnesium, manganese, phosphorus, potassium, sodium, and zinc. In
one embodiment, the composition can be mixed with raw human milk to
provide a nutritional composition, wherein the raw human milk
comprises about 80%, about 70%, about 60%, or about 50% of the
nutritional composition. .Iaddend.
.Iadd.Embodiments can include one or more of the following
features. .Iaddend.
.Iadd.In one embodiment, the composition can include the protein
constituent of about 55-65 mg/mL; the fat constituent of about
85-95 mg/mL; and the carbohydrate constituent of about 70-120
mg/mL. The carbohydrate constituent can include lactose. The
composition can further include IgA and/or one or more constituents
selected from the group consisting of: calcium (e.g., at a
concentration of about 4.0-5.5 mg/mL or at 2.00-2.90 mg/mL);
chloride (e.g., at a concentration of about 0.35-0.95 mg/mL or at
about 0.175-0.475 mg/mL); copper (e.g., at a concentration of about
0.0005-0.0021 mg/mL or at about 0.00025-0.001 mg/mL); iron (e.g.,
at a concentration of about 0.001-0.007 mg/mL or at about
0.0005-0.0025 mg/mL); magnesium (e.g., at a concentration of about
0.180-0.292 mg/mL or at about 0.090-0.170 mg/mL); manganese (e.g.,
at a concentration of about 0.010-0.092 mcg/mL or at about
0.005-0.046 mcg/mL;); phosphorus (e.g., at a concentration of about
2.00-3.05 mg/mL or at about 1.00-2.90 mg/mL, e.g., at about
1.00-1.50 mg/mL); potassium (e.g., at a concentration of about
1.90-2.18 mg/mL or at about 0.95-1.41 mg/mL); sodium (e.g., at a
concentration of about 0.75-0.96 mg/mL or at about 0.375-0.608
mg/mL); and zinc (e.g., at a concentration of about 0.0200-0.0369
mg/mL or at about 0.010-0.0198 mg/mL). In one embodiment, the
composition can be mixed with raw human milk to provide a
nutritional composition, wherein the raw human milk comprises about
80%, about 70%, about 60%, or about 50% of the nutritional
composition. .Iaddend.
.Iadd.In another aspect, the disclosure features a pasteurized
human milk composition that includes: a human protein constituent
of about 11-20 mg/mL, e.g., about 11-13 mg/mL; a human fat
constituent of about 35-55 mg/mL; and a human carbohydrate
constituent of about 70-120 mg/mL, e.g., about 80-105 mg/mL. The
carbohydrate constituent can include lactose. The caloric content
of the composition can be about 0.64 to about 1.10 Cal/mL.
.Iaddend.
.Iadd.Embodiments can include one or more of the following
features. .Iaddend.
.Iadd.In one embodiment, the pasteurized human milk composition can
further include one or more of the following components: calcium
(e.g., at a concentration of about 0.40-1.50 mg/mL); chloride
(e.g., at a concentration of about 0.30-0.80 mg/mL); copper (e.g.,
at a concentration of about 0.0005-0.0021 mg/mL); iron (e.g., at a
concentration of about 0.001-0.005 mg/mL); magnesium (e.g., at a
concentration of about 0.03-0.13 mg/mL); manganese (e.g., at a
concentration of about 0.01-0.092 mcg/mL); phosphorus (e.g., at a
concentration of about 0.15-0.631 mg/mL); potassium (e.g., at a
concentration of about 0.60-1.20 mg/mL); sodium (e.g., at a
concentration of about 0.20-0.60 mg/mL); and/or zinc (e.g., at a
concentration of about 0.0025-0.0120 mg/mL). .Iaddend.
.Iadd.The disclosure also features method of making various human
milk compositions. .Iaddend.
.Iadd.In one aspect, the disclosure features a method for obtaining
a pasteurized human milk composition. The method includes: (a)
genetically screening human milk for one or more viruses; (b)
filtering the milk; (c) heat-treating the milk, e.g., at about
63.degree. C. or greater for about 30 minutes; (d) separating the
milk into cream and skim; (e) adding a portion of the cream to the
skim; and (f) pasteurizing. .Iaddend.
.Iadd.Embodiments include one or more of the following features.
.Iaddend.
.Iadd.In one embodiment, the method can further include filtering
the skim through filters after step (d), e.g., to filter the water
out of the skim. After filtering the skim after step (d), the
filters used in the filtering can be washed to obtain a post wash
solution. The post wash solution can be added to the skim.
.Iaddend.
.Iadd.The genetic screening in step (a) can be polymerase chain
reaction and/or can include screening for one or more viruses,
e.g., HIV-1, HBV, and/or HCV. The milk can be filtered through an
about 200 micron screen in step (b). The method can further include
running cream, e.g., about 30-50% of cream, through a separator
following step (d). The composition of post wash and skim can
include about 7.0-7.2% of protein. .Iaddend.
.Iadd.The method can further include carrying out mineral analysis
of the portion of the composition obtained after step (e). The
method can also include adding to the composition obtained after
step (e) one or more minerals selected from the group consisting
of: calcium, chloride, copper, iron, magnesium, manganese,
phosphorus, potassium, sodium, and zinc. Adding of the one or more
minerals can include heating the composition. .Iaddend.
.Iadd.The method can also include cooling the composition after
step (f), carrying out biological testing of a portion of the
composition after step (f), and/or carrying out nutritional testing
of a portion of the composition after step (f). .Iaddend.
.Iadd.The human milk of step (a) can be pooled human milk. The
methods featured herein can be carried out with large volumes of
the starting material, e.g., human milk, e.g., pooled human milk.
The volumes can be in the range of about 75-2,000 liters/lot of
starting material. .Iaddend.
.Iadd.The composition obtained after step (f) can include about
8.5-9.5% fat, about 6.3-7.0% protein, and about 8.0-10.5% lactose.
.Iaddend.
.Iadd.In another aspect, the disclosure features a method for
obtaining a pasteurized human milk composition. The method
includes: (a) genetically screening human milk for one or more
viruses; (b) filtering the milk; (c) adding cream; and (d)
pasteurizing. .Iaddend.
.Iadd.Embodiments can include one or more of the following
features. .Iaddend.
.Iadd.In one embodiment, the genetic screening in step (a) can be
polymerase chain reaction. The genetic screening can include
screening for one or more viruses, e.g., HIV-1, HBV, and/or HCV.
.Iaddend.
.Iadd.The milk can be filtered through an about 200 micron screen
in step (b). The method can further include ultra filtering the
whole milk after step (b) through filters. The filters used during
ultra filtering can be post washed. .Iaddend.
.Iadd.The composition can be cooled after step (d). Biological
and/or nutritional testing of the composition can be carried out
after step (d). .Iaddend.
.Iadd.Human milk of step (a) can be pooled human milk. The methods
featured herein can be carried out with large volumes of the
starting material, e.g., human milk, e.g., pooled human milk. The
volumes can be in the range of about 75-2,000 liters/lot of
starting material. .Iaddend.
.Iadd.The method can also include adding to the composition
obtained after step (c) one or more minerals selected from the
group consisting of: calcium, chloride, copper, iron, magnesium,
manganese, phosphorus, potassium, sodium, and zinc. In one
embodiment, the composition obtained after step (d) can include
about 11-20 mg/mL protein, about 35-55 mg/mL fat, and about 70-120
mg/mL carbohydrates. .Iaddend.
.Iadd.In another aspect, the disclosure features a kit that
includes the pasteurized human milk compositions featured herein
(e.g., a fortifier) and a graduated container (e.g., a bottle, a
syringe, and a can) for mixing the featured compositions with raw
human milk. .Iaddend.
.Iadd.In yet another aspect, the disclosure features a method of
obtaining a nutritional milk composition. The method includes
adding the pasteurized human milk compositions featured herein
(e.g., fortifiers) to raw human milk, thereby increasing the
nutritional concentration of the raw human milk. The caloric
composition of the raw human milk can be increased by about 2-10
Cal/oz. .Iaddend.
.Iadd.In another aspect, the disclosure features a method of
providing supplemental nutrients to a premature human infant, the
method comprising adding the compositions (fortifiers) featured
herein to raw human milk to obtain a mixture and administering the
mixture to the premature infant. .Iaddend.
.Iadd.The terms "premature", "preterm" and "low-birth-weight (LBW)"
infants are used interchangeably and refer to infants born less
than 37 weeks gestational age and/or with birth weights less than
2500 gm. .Iaddend.
.Iadd.By "whole milk" is meant milk from which no fat has been
removed. .Iaddend.
.Iadd.By "bioburden" is meant microbiological contaminants and
pathogens (generally living) that can be present in milk, e.g.,
viruses, bacteria, mold, fungus and the like. .Iaddend.
.Iadd.The compositions of the present disclosure are generated from
human donor milk, e.g., pooled milk, which undergoes rigorous
genetic screening, processing (e.g., to concentrate nutrients in
the fortifier compositions, and/or to reduce bioburden), and
pasteurization. The milk can be supplemented with various minerals
and/or vitamins. Thus, the disclosure also features methods of
obtaining and processing milk from human donors. .Iaddend.
The details of one or more embodiments of the methods featured
herein are set forth in the description below. Other features,
objects, and advantages of the methods will be apparent from the
description and the claims.
All patents, patent applications, and references cited herein are
incorporated in their entireties by reference.
DETAILED DESCRIPTION
In one aspect, the methods featured herein are used to determine
milk origination. For example, in order to ensure that human breast
milk received from a specific human actually comes from that human,
methods of identity testing are needed on samples of milk received
from each human. Testing donors to confirm their identity improves
safety of donated milk. It ensures the provenance of the donated
milk, which as discussed above, is most often donated without
supervision of personnel of the organization that will be receiving
the milk, e.g., a milk bank center. Testing donor identity by the
methods featured herein allows for multiple donations by the same
donor, whose identity can be confirmed at the time of each
donation. The donor can live at any distance from the donation
and/or processing facilities, as she can send her milk at long
distances, and her identity can be confirmed based on reference
samples or reference test results stored at the donation and/or
processing facility.
As part of the qualification process for donating milk, each
potential milk donor will be identified by biological methods
(e.g., biological fingerprinting, as described herein). The
identifying characteristics of the individual (i.e., at least one
identity marker) will also be present in the donor's milk. Such
characteristics will be used to match the donated milk with a
specific donor.
Obtaining a Reference Biological Sample
The methods featured herein include, inter alia, obtaining at least
one donated reference sample from a potential mammary fluid donor,
e.g., a human breast milk donor. Such sample may be obtained by
methods known in the art such as, but not limited to, a cheek swab
sample of cells, or a drawn blood sample, milk, saliva, hair roots,
or other convenient tissue. Samples of reference donor nucleic
acids (e.g., genomic DNA) can be isolated from any convenient
biological sample including, but not limited to, milk, saliva,
buccal cells, hair roots, blood, and any other suitable cell or
tissue sample with intact interphase nuclei or metaphase cells. The
sample is labeled with a unique reference number. The sample can be
analyzed at or around the time of obtaining the sample for one or
more markers that can identify the potential donor. Results of the
analysis can be stored, e.g., on a computer-readable medium.
Alternatively, or in addition, the sample can be stored and
analyzed for identifying markers at a later time.
It is contemplated that the biological reference sample may be DNA
typed by methods known in the art such as STR analysis of STR loci,
HLA analysis of HLA loci or multiple gene analysis of individual
genes/alleles (further discussed below). The DNA-type profile of
the reference sample is recorded and stored, e.g., on a
computer-readable medium.
It is further contemplated that the biological reference sample may
be tested for self-antigens using antibodies known in the art or
other methods to determine a self-antigen profile. The antigen (or
another peptide) profile can be recorded and stored, e.g., on a
computer-readable medium.
Testing a Sample of Donated Mammary Fluid
A subject desiring to donate mammary fluid will express the mammary
fluid (breast milk) using standard procedures. The mammary fluid is
typically collected in containers useful for shipping and storage.
The mammary fluid can be frozen prior to donation. The mammary
fluid may be frozen at the donation facility or processing facility
for later analysis and use or analyzed without freezing. One or
more of the containers with donated fluid can be used for obtaining
a test sample. The test sample is taken for identification of one
or more identity markers.
Methods of obtaining a sample of expressed frozen fluid include a
stainless steel boring tool used to drill a core the entire length
of the container. Alternatively, a sample may be scraped from the
surface of the frozen mammary fluid. The container may contain a
separate portion for collection of a sample of the expressed
mammary fluid, and this portion may be removed as the sample for
testing. Where the mammary fluid is in liquid form it is
contemplated that the method for obtaining the test sample will be
by pipette or other means.
A sample of the donated the mammary fluid is analyzed for the same
marker or markers as the donor's reference sample. The marker
profiles of the reference biological sample and of the donated
mammary fluid are compared. The match between the markers (and lack
of any additional unmatched markers) would indicate that the
donated milk comes from the same individual as the one who donated
the reference sample. Lack of a match (or presence of additional
unmatched markers) would indicate that the donated mammary fluid
either comes from a non-tested donor or has been contaminated with
fluid from a non-tested donor.
The donated mammary fluid sample and the donated reference
biological sample can be tested for more than one marker. For
example, each sample can be tested for multiple DNA markers and/or
peptide markers. Both samples, however, need to be tested for at
least some of the same markers in order to compare the markers from
each sample.
Thus, the reference sample and the donated mammary fluid sample may
be tested for the presence of differing identity marker profiles.
If there are no identity marker profiles other than the identity
marker profile from the expected subject, it generally indicates
that there was no fluid (e.g., milk) from other humans or animals
contaminating the donated mammary fluid. If there are signals other
than the expected signal for that subject, the results are
indicative of contamination. Such contamination will result in the
mammary fluid (e.g., milk) failing the testing.
The testing of the reference sample and of the donated mammary
fluid can be carried out at the donation facility and/or milk
processing facility. The results of the reference sample tests can
be stored and compared against any future donations by the same
donor.
It is contemplated that samples from a number of milk containers
from the same subject may be pooled for identity marker testing. It
is contemplated that at least 2 samples, at least 5 samples or at
least 8 samples may be pooled for testing.
It is contemplated that the test sample of the donated mammary
fluid may be tested by nucleic acid typing using methods known in
the art, such as, STR analysis of STR loci, HLA analysis of HLA
loci or multiple gene analysis of individual genes/alleles to
obtain the DNA-type of the milk sample. The donated mammary fluid
can also be tested for peptide profiles, e.g., antigen profile.
The DNA-type or another biological profile (i.e., identity profile
(s)) of the donated mammary fluid test sample (s) will be compared
to the reference DNA-type or another biological profile for the
putative donor. A match or identity of the DNA-type or biological
profile will indicate that the mammary fluid was obtained from a
same (i.e., a specified subject).
Use of the Donated and Tested Mammary Fluid
The mammary fluid tested by the methods featured herein can be
processed for further use. The donation facility and milk
processing facility can be the same or different facility. The
donated milk whose provenance has been confirmed can be processed,
e.g., to obtain human milk fortifiers, standardized human milk
formulations, and/or human lipid compositions. As discussed above,
testing the mammary fluid to confirm the identity of the donor
ensures safety of the mammary fluid and any products derived from
such fluid.
Processing of human milk to obtain human milk fortifiers (e.g.,
PROLACTPLUS.TM. Human Milk Fortifiers, e.g., PROLACT+4.TM.,
PROLACT+6.TM., PROLACT+8.TM., and/or PROLACT+10.TM., which are
produced from human milk and contain various concentrations of
nutritional components) and the compositions of the fortifiers are
described in U.S. patent application Ser. No. 11/947,580, filed on
Nov. 29, 2007, the contents of which are incorporated herein in
their entirety. These fortifiers can be added to the milk of a
nursing mother to provide an optimal nutritional content of the
milk for, e.g., a preterm infant. Depending on the content of
mother's own milk, various concentrations of the fortifiers can be
added to mother's milk.
Methods of obtaining standardized human milk formulations
(exemplified by PROLACT20.TM., NEO20.TM., and/or PROLACT24) and
formulations themselves are also discussed in U.S. patent
application Ser. No. 11/947,580, filed on Nov. 29, 2007, the
contents of which are incorporated herein in their entirety. These
standardized human milk formulations can be used to feed, e.g.,
preterm infants, without mixing them with other fortifiers or
mills. They provide a nutritional human-derived formulation and can
substitute for mother's milk.
Compositions that include lipids from human milk, methods of
obtaining such compositions, and methods of using such compositions
to provide nutrition to patients are described in PCT Application
PCT/US07/86973 filed on Dec. 10, 2007, the contents of which are
incorporated herein in their entirety.
Methods of obtaining other nutritional compositions from human milk
that can be used with the methods featured herein are discussed in
U.S. patent application Ser. No. 11/012,611, filed on Dec. 14,
2004, and published as U.S. 2005/0100634 on May 12, 2005, the
contents of which are incorporated herein in their entirety.
Processing of milk that has been tested with the methods featured
herein can be carried out with large volumes of donor milk, e.g.,
about 75 liters/lot to about 2,000 liters/lot of starting
material.
The methods featured herein can also be integrated with methods of
facilitating collection and distribution of human milk over a
computer network, e.g., as described in U.S. patent application
Ser. No. 11/526,127, filed on Sep. 22, 2006, and published as U.S.
2007/0098863 on May 3, 2007; and in U.S. patent application Ser.
No. 11/679,546, filed on Feb. 27, 2007, and published as U.S.
2007/0203802 on Aug. 30, 2007. The contents of both applications
are incorporated herein in their entireties.
.Iadd.As discussed above, donor milk is screened to ensure the
identity of the donors and reduce the possibility of contamination.
Donor milk is pooled and further screened (step 1), e.g.,
genetically screened (e.g., by PCR). The screening can identify,
e.g., viruses, e.g., HIV-1, HBV, and/or HCV. Milk that tests
positive is discarded. After the screening, the composition
undergoes filtering (step 2). The milk is filtered through about a
200 micron screen and then ultrafiltered. During ultrafiltration,
water is filtered out of the milk (and is referred to as permeate)
and the filters are postwashed using the permeate. Post wash
solution is added to the milk to recover any lost protein and
increase the concentration of the protein to, e.g., about 1.2% to
about 1.5%. Cream from another lot (e.g., excess cream from a
previous fortifier lot) is added in step 3 to increase the caloric
content. At this stage of the process, the composition generally
contains: about 3.5% to 5.5% of fat; about 1.1% to 1.3% of protein;
and about 8% to 10.5% of carbohydrates, e.g., lactose.
.Iaddend.
.Iadd.At this stage, the composition can be frozen and thawed out
for further processing later. .Iaddend.
.Iadd.Optionally, if the human milk formulation is to be fortified
with minerals, a mineral analysis of the composition is carried out
after step 3. Once the mineral content is known, a desired amount
of minerals can be added to achieve target values. .Iaddend.
.Iadd.In step 4, the composition is pasteurized. Pasteurization
methods are known in the art. For example, the product can be
pasteurized in a tank that is jacketed. Hot glycol can be use to
heat up the tank. The product temperature can be about 63.degree.
C. or greater and the air temperature above the product about
66.degree. C. or greater. The product is pasteurized for a minimum
of about 30 minutes. Other pasteurizing techniques are known in the
art. .Iaddend.
.Iadd.After cooling to about 2 to 8.degree. C., the product is
filled into containers of desired volumes and various samples of
the human milk formulation are taken for nutritional and bioburden
analysis. The nutritional analysis ensures proper content of the
composition. A label generated for each container reflects the
nutritional analysis. The bioburden analysis tests for presence of
contaminants, e.g., total aerobic count, B. cereus, E. coli,
Coliform, Pseudomonas, Salmonella, Staphylococcus, yeast, and/or
mold. The product is packaged and shipped once the analysis is
complete and desired results are achieved. .Iaddend.
.Iadd.The standardized human milk formulations featured herein can
be used in lieu of mother's own milk to feed the infants, e.g.,
premature infants. They include various nutritional components for
infant growth and development. .Iaddend.
.Iadd.In one embodiment, the standardized human milk formulation
can include: a human protein constituent of about 11-20 mg/mL; a
human fat constituent of about 35-55 mg/mL; and a human
carbohydrate constituent of about 70-120 mg/mL. In a particular
embodiment, the formulation can contain: a human protein
constituent of about 11-13 mg/mL; a human fat constituent of about
35-55 mg/mL; and a human carbohydrate constituent of about 80-105
mg/mL. The total caloric content of the formulations can be, e.g.,
from about 0.68 Cal/mL to about 0.96 Cal/mL. .Iaddend.
.Iadd.The milk formulation can be supplemented with vitamins and/or
minerals. In one embodiment, the composition can include: calcium
concentration of about 0.40-1.50 mg/mL; chloride concentration of
about 0.30-0.80 mg/mL; copper concentration of about 0.0005-0.0021
mg/mL; iron concentration of about 0.001-0.005 mg/mL; magnesium
concentration of about 0.03-0.13 mg/mL; manganese concentration of
about 0.01-0.092 mg/mL; phosphorus concentration of about
0.15-0.631 mg/mL (e.g., about 0.15-0.60 mg/mL); potassium
concentration of about 0.60-1.20 mg/mL; sodium concentration of
about 0.20-0.60 mg/mL; and zinc concentration of about
0.0025-0.0120 mg/mL. .Iaddend.
.Iadd.The human milk formulations can contain various caloric
content, e.g., 67 Kcal/dL (20 Calorie per ounce), and 81 Kcal/dL
(24 Calorie per ounce). An exemplary human milk formulation (e.g.,
PROLACT24.TM.) can include the following constituents: human milk,
calcium glycerophosphate, potassium citrate, calcium gluconate,
calcium carbonate, magnesium phosphate, sodium chloride, sodium
citrate, zinc sulfate, cupric sulfate, and manganese sulfate. This
exemplary composition can have the following characteristics per
100 ml: about 81 Cal; about 4.4 g of total fat; about 20.3 mg of
sodium; about 60.3 mg of potassium; about 8 g total carbohydrates;
about 5-9 g of sugars; about 2.3 g of protein; about 180-250 IU of
Vitamin A; less than about 1.0 mg of Vitamin C; about 40.0-150.0 mg
of calcium; about 100-150 mcg of iron; about 15-50 mg of
phosphorus; about 3-10 mg of magnesium; about 25-75.0 mg of
chloride; about 1.2 mcg of zinc; about 140-190 mcg of copper; less
than about 60.2 mcg of manganese; and Osmolarity of about 322
mOsm/Kg H.sub.2O. Milk formulations with other constituents and
constituents of different concentrations are encompassed by this
disclosure. .Iaddend.
.Iadd.The osmolality of human milk fortifiers and standardized milk
formulations featured herein can affect adsorption, absorption, and
digestion of the compositions. High osmolality, e.g., above about
400 mOsm/Kg H.sub.2O, has been associated with increased rates of
necrotizing enterocolitis (NEC), a gastrointestinal disease that
affects neonates (see, e.g., Srinivasan et al., Arch. Dis. Child
Fetal Neonatal Ed. 89:514-17, 2004). The osmolality of the human
milk composition and fortifier (once mixed with raw milk) of the
disclosure is typically less than about 400 mOsm/Kg H.sub.2O.
Typically the osmolality is from about 310 mOsm/Kg of water to
about 380 mOsm/Kg of water. The osmolality can be adjusted by
methods known in the art. .Iaddend.
Nucleic Acid Identity Marker Profiles
As discussed above, samples of reference donor nucleic acids (e.g.,
genomic DNA) are isolated from any convenient biological sample
including, but not limited to, milk, saliva, buccal cells, hair
roots, blood, and any other suitable cell or tissue sample with
intact interphase nuclei or metaphase cells.
Methods for isolation of nucleic acids (e.g., genomic DNA) from
these various sources are described in, for example, Kirby, DNA
Fingerprinting, An Introduction, W. H. Freeman & Co. New York
(1992). Nucleic acids (e.g., genomic DNA) can also be isolated from
cultured primary or secondary cell cultures or from transformed
cell lines derived from any of the aforementioned tissue
samples.
Samples of RNA can also be used. RNA can be isolated as described
in Sambrook et al., supra, RNA can be total cellular RNA, mRNA,
poly A+ RNA, or any combination thereof. For best results, the RNA
is purified, but can also be unpurified cytoplasmic RNA. RNA can be
reverse transcribed to form DNA which is then used as the
amplification template, such that PCR indirectly amplifies a
specific population of RNA transcripts. See, e.g., Sambrook et al.,
supra, and Berg et al., Hum. Genet. 85:655-658 (1990).
Short tandem repeat (STR) DNA markers, also referred to as
microsatellites or simple sequence repeats (SSRs) or DNA tandem
nucleotide repeat ("DTNR"), comprise tandem repeated DNA sequences
with a core repeat of 2-6 base pairs (bp). STR markers are readily
amplified during PCR by using primers that bind in conserved
regions of the genome flanking the repeat region.
Commonly sized repeats include dinucleotides, trinucleotides,
tetranucleotides and larger. The number of repeats occurring at a
particular genetic locus varies from a few to hundreds depending on
the locus and the individual. The sequence and base composition of
repeats can vary significantly, including a lack of consistency
within a particular nucleotide repeat locus. Thousands of STR loci
have been identified in the human genome and have been predicted to
occur as frequently as once every 15 kb. Population studies have
been undertaken on dozens of these STR markers as well as extensive
validation studies in forensic laboratories. Specific primer
sequences located in the regions flanking the DNA tandem repeat
region have been used to amplify alleles from STR loci via the
polymerase chain reaction ("PCR"). The PCR products include the
polymorphic repeat regions, which vary in length depending on the
number of repeats or partial repeats, and the flanking regions,
which are typically of constant length and sequence between
samples.
The number of repeats present for a particular individual at a
particular locus is described as the allele value for the locus.
Because most chromosomes are present in pairs, PCR amplifications
of a single locus commonly yields two different sized PCR products
representing two different repeat numbers or allele values. The
range of possible repeat numbers for a given locus, determined
through experimental sampling of the population, is defined as the
allele range, and may vary for each locus, e.g., 7 to 15 alleles.
The allele PCR product size range (allele size range) for a given
locus is defined by the placement of the two PCR primers relative
to the repeat region and the allele range. The sequences in regions
flanking each locus must be fairly conserved in order for the
primers to anneal effectively and initiate PCR amplification. For
purposes of genetic analysis di-, tri-, and tetranucleotide repeats
in the range of 5 to 50 are typically utilized in screens. Forensic
laboratories use tetranucleotide loci (i.e., 4 bp in the repeat)
due to the lower amount of "stutter" produced during PCR (Stutter
products are additional peaks that can complicate the
interpretation of DNA mixtures by appearing in front of regular
allele peaks). The number of repeats can vary from 3 or 4 repeats
to more than 50 repeats with extremely polymorphic markers. The
number of repeats and hence the size of the PCR product, may vary
among samples in a population making STR markers useful in identity
testing of genetic mapping studies.
There are 13 core STR loci identified in the United States CODIS
database. These STR loci are THO1, TPDX, CSF1PO, VWA, FGA, D3S1358,
D5S818, D7S820, D13S317, D16S539, D8S1179, D18S51 and D21S11. The
sex-typing marker amelogenin, is also included in the STR
multiplexes that cover the 13 core STR loci. The 13 CODIS STR loci
are covered by the Profiler Plus.TM. and COfiler.TM. kits from
Applied Biosystems (ABI) (Foster City, Calif.). It is contemplated
that the following STR loci may be used in this invention: CSF1PO,
D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11,
DYS19, F13A1, FESfFPS, FGA, HPRTB, THO1, TPDX, DYS388, DYS391,
DYS392, DYS393, D2S1391, D18S535, D2S1338, D19S433, D6S477, D1S518,
D14S306, D22S684, F13B, CD4, D12S391, D10S220 and D7S523 (the
sequence of each loci is incorporated herein by reference). With
the exception of D3 S1358, sequences for the STR loci of this
invention are accessible to the general public through GenBank (see
U.S. Pat. No. 6,090,558, incorporated herein by reference). Other
STR loci have been developed by commercial manufacturers and
studied extensively by forensic scientists. These include all of
the GenePrint.TM. tretranucleotide STR systems from Promega
Corporation (Madison Wis.).
Many different primers have been designed for various STR loci and
reported in the literature. These primers anneal to DNA segments
outside the DNA tandem repeat region to produce PCR products
containing the tandem repeat region. These primers were designed
with polyacrylamide gel electrophoretic separation in mind as a
method of detection/measurement, because DNA separations have
traditionally been performed by slab gel or capillary
electrophoresis. STR multiplex analysis is usually performed with
PCR amplification and detection of multiple markers. STR
multiplexing is most commonly performed using spectrally
distinguishable fluorescent tags and/or non-overlapping PCR product
sizes. Multiplex STR amplification in one or two PCR reactions with
fluorescently labeled primers and measurement with gel or capillary
electrophoresis separation and laser induced fluorescence detection
is a standard method. The STR alleles from these multiplexed PCR
products typically range in size from 100-800 bp with commercially
available lots.
Gel-based systems are capable of multiplexing the analysis of 2 or
more STR loci using two approaches. The first approach is to size
partition the different PCR product loci. Size partitioning
involves designing the PCR primers used to amplify different loci
so that the allele PCR product size range for each locus covers a
different and separable part of the gel size spectrum. As an
example, the PCR primers for Locus A might be designed so that the
allele size range is from 250 to 300 nucleotides, while the primers
for Locus B are designed to produce an allele size range from 340
to 410 nucleotides.
The second approach to multiplexing 2 or more STR loci on gel-based
systems is the use of spectroscopic partitioning. Current state of
the art for gel-based systems involves the use of fluorescent dyes
as specific spectroscopic markers for different PCR amplified loci.
Different chromophores that emit light at different color
wavelengths provide a method for differential detection of two
different PCR products even if they are exactly the same size, thus
2 or more loci can produce PCR products with allele size ranges
that overlap. For example, Locus A with a green fluorescent tag
produces an allele size range from 250 to 300 nucleotides, while
Locus B with a red fluorescent tag produces an allele size range of
270 to 330 nucleotides. A scanning, laser-excited fluorescence
detection device monitors the wavelength of emissions and assigns
different PCR product sizes, and their corresponding allele values,
to their specific loci based on their fluorescent color.
It is contemplated that a mass spectrometry approach to STR typing
and analysis, examining smaller nucleic acid oligomers may be used
because the sensitivity of detection and mass resolution are
superior with smaller oligomers. Application of STR analysis to
time of flight-mass spectrometry (TOF-MS) requires the development
of primer sets that produce small PCR products 50 to 160
nucleotides in length, typically about 50 to 100 nucleotides in
length. Amplified nucleic acids may also be used to generate single
stranded products that are in the desired size range for TOF-MS
analysis by extending a primer in the presence of a chain
termination reagent. A typical class of chain termination reagent
commonly used by those of skill in the art is the dideoxynucleotide
triphosphates. Again, application of STR analysis to TOF-MS
requires that the primer be extended to generate products of 50 to
160 nucleotides in size, and typically about 50 to 100 nucleotides
in length (see U.S. Pat. No. 6,090,558 incorporated by
reference).
A biotinylated cleavable oligonucleotide is used as a primer in
each assay and is incorporated through standard nucleic acid
amplification (i.e., PCR) methodologies into the final product
which is measured in the mass spectrometer. This process is
described in, for example, U.S. Pat. No. 5,700,642 and U.S. Pat.
No. 6,090,558 (see also Butler et al., International Journal of
Legal Medicine 112(1) 45-59 (1998)). The STR assay involves a PCR
amplification step where one of the primers is replaced by a
cleavable biotinylated primer. The biotinylated PCR product is then
captured on streptavidin-coated magnetic beads for post-PCR sample
cleanup and salt removal, followed by mass spectrometry
analysis.
Single nucleotide polymorphisms (SNPs) represent another form of
DNA variation that is useful for human identity testing. SNPs are
the most frequent form of DNA sequence variation in the genomes of
organisms and are becoming increasingly popular genetic markers for
genome mapping studies and medical diagnostics. SNPs are typically
bi-allelic with two possible nucleotides (nt) or alleles at a
particular site in the genome. Because SNPs are less polymorphic
(i.e., have fewer alleles) than the currently used STR markers,
more SNP markers are required to obtain the same level of
discrimination between samples. Approximately 30-50 unlinked SNPs
may be required to obtain the matching probabilities of 1 in 100
billion as seen with the 13 CODIS STRs.
A SNP assay typically involves a three-step process: (1) PCR
amplification (2) phosphatase removal of nucleotides, and (3)
primer extension using a biotinylated cleavable primer with
dideoxynucleotides for single-base addition of the nucleotide (s)
complementary to the one(s) at the SNP site (Li et al.,
Electrophoresis 20(6): 1258-1265 (1999)).
Simultaneous analysis of multiple SNP markers (i.e. multiplexing)
is possible by simply putting the cleavage sites at different
positions in the various primers so that they do not overlap on a
mass scale.
The most common means for amplification is polymerase chain
reaction (PCR), as described in U.S. Pat. Nos. 4,683,195,
4,683,202, 4,965,188 each of which is hereby incorporated by
reference in its entirety. If PCR is used to amplify the target
regions in blood cells, heparinized whole blood should be drawn in
a sealed vacuum tube kept separated from other samples and handled
with clean gloves. For best results, blood should be processed
immediately after collection; if this is impossible, it should be
kept in a sealed container at about 4.degree. C. until use. Cells
in other physiological fluids may also be assayed. When using any
of these fluids, the cells in the fluid should be separated from
the fluid component by centrifugation.
To amplify a target nucleic acid sequence in a sample by PCR, the
sequence must be accessible to the components of the amplification
system. One method of isolating target DNA is crude extraction
which is useful for relatively large samples. Briefly, mononuclear
cells from samples of blood, buccal cells, or the like are
isolated. The pellets are stored frozen at -200C until used (U.S.
Patent Publication No. 20040253594 which is incorporated by
reference).
The pellets may be resuspended in lysis solution from the
PUREGENE.RTM. DNA isolation kit (Cat#D-5000, GENTRA, Minneapolis,
Minn.) containing 100/ug/ml of proteinase K. After incubating at
55.degree. C. overnight, DNA extraction is performed according to
manufacturers recommendations. The DNA samples are resuspended in
aqueous solution and stored at -200C.
When the sample contains a large number of cells, extraction may be
accomplished by methods as described in Higuchi, "Simple and Rapid
Preparation of Samples for PCR", in PCR Technology, p. 31-43
Ehrlich, H. A. (ed.), Stockton Press, New York.
A relatively easy procedure for extracting DNA for PCR is a salting
out procedure adapted from the method described by Miller et al.,
Nucleic Acids Res. 16:1215 (1988), which is incorporated herein by
reference. Nucleated cells are resuspended in 3 ml of lysis buffer
(10 mM Tris-HCl, 400 mM NaCl, 2 mM Na2 EDTA, pH 8.2). Fifty .mu.l
of a 20 mg/ml solution of proteinase K and 200 ul of a 20% SDS
solution are added to the cells and then incubated at 37.degree. C.
overnight. Following adequate digestion, one ml of a 6M NaCl
solution is added to the sample and vigorously mixed. The resulting
solution is centrifuged for 15 minutes at 3000 rpm. The pellet
contains the precipitated cellular proteins, while the supernatant
contains the DNA. The supernatant is removed to a 15 ml tube that
contains 4 ml of isopropanol. The contents of the tube are mixed
gently until the water and the alcohol phases have mixed and a
white DNA precipitate has formed. The DNA precipitate is placed in
distilled water and dissolved. (U.S. Patent Publication No.
20040253594 which is incorporated by reference).
Kits for the extraction of high-molecular weight DNA for PCR
include PUREGENE.RTM. DNA Isolation kit (D-5000) GENTRA, a Genomic
Isolation Kit A. S.A. P..RTM. (Boehringer Mannheim, Indianapolis,
Ind.), Genomic DNA Isolation System (GIBCO BRL, Gaithdrsburg, Md.),
ELU-QUIK.RTM. DNA Purification Kit (Schleicher & Schnell,
Keene, N.H.), DNA Extraction Kit (Stratagene, LaJolla, Calif.),
TURBOGEN.RTM. Isolation Kit (Invitrogen, San Diego, Calif.), and
the like. Use of these kits according to the manufacturer's
instructions is generally acceptable for purification of DNA prior
to practicing the methods of the invention (U.S. Patent Publication
No. 20040253594 which is incorporated by reference).
The concentration and purity of the extracted DNA can be determined
by spectrophotometric analysis of the absorbance of a diluted
aliquot at 260 nm and 280 nm.
After extraction of the DNA, PCR amplification may proceed. The
first step of each cycle of the PCR involves the separation of the
nucleic acid duplex formed by the primer extension. Once the
strands are separated, the next step in PCR involves hybridizing
the separated strands with primers that flank the target sequence.
The primers are then extended to form complementary copies of the
target strands. For successful PCR amplification, the primers are
designed so that the position at which each primer hybridizes along
a duplex sequence is such that an extension product synthesized
from one primer, when separated from the template (complement),
serves as a template for the extension of the other primer. The
cycle of denaturation, hybridization, and extension is repeated as
many times as necessary to obtain the desired amount of amplified
nucleic acid (U.S. Patent Publication No. 20040253594 which is
incorporated by reference).
In one embodiment of PCR amplification, strand separation is
achieved by heating the reaction to a sufficiently high temperature
for a sufficient time to cause the denaturation of the duplex but
not to cause an irreversible denaturation of the polymerase (see
U.S. Pat. No. 4,965,188, incorporated herein by reference). Typical
heat denaturation involves temperatures ranging from about
80.degree. C. to about 105.degree. C. for times ranging from
seconds to minutes. Strand separation, however, can be accomplished
by any suitable denaturing method including physical, chemical, or
enzymatic means. Strand separation may be induced by a helicase,
for example, or an enzyme capable of exhibiting helicase activity.
For example, the enzyme RecA has helicase activity in the presence
of ATP. The reaction conditions suitable for strand separation by
helicases are known in the" art (see Kuhn et al., 1979,
CSH-Quantitative Biology, 43:63-67; and Radding, 1982, Ann. Rev,
Genetics 16:405-437, incorporated by reference).
Template-dependent extension of primers in PCR is catalyzed by a
polymerizing agent in the presence of adequate amounts of four
deoxyribonucleotide triphosphates (typically dATP, dGTP, dCTP, and
dTTP) in a reaction medium comprised of the appropriate salts,
metal cations, and pH buffering systems. Suitable polymerizing
agents are enzymes known to catalyze template-dependent DNA
synthesis. In some cases, the target regions may encode at least a
portion of a protein expressed by the cell. In this instance, mRNA
may be used for amplification of the target region. Alternatively,
PCR can be used to generate a cDNA library from RNA for further
amplification, the initial template for primer extension is RNA.
Polymerizing agents suitable for synthesizing a complementary
copy-DNA (cDNA) sequence from the RNA template are reverse
transcriptase (RT), such as avian myeloblastosis virus RT, Moloney
murine leukemia virus RT, or Thermus thermophilus (Tth) DNA
polymerase, a thermostable DNA polymerase with reverse
transcriptase activity marketed by Perkin Elmer Cetus, Inc.
Typically, the genomic RNA template is heat degraded during the
first denaturation step after the initial reverse transcription
step leaving only DNA template. Suitable polymerases for use with a
DNA template include, for example, E. coli DNA polymerase I or its
Klenow fragment, T4 DNA polymerase, Tth polymerase, and Taq
polymerase, a heat-stable DNA polymerase isolated from Thermus
aquaticus and commercially available from Perkin Elmer Cetus, Inc.
The latter enzyme is widely used in the amplification and
sequencing of nucleic acids. The reaction conditions for using Taq
polymerase are known in the art (U.S. Patent Publication No.
20040253594 which is incorporated by reference).
Allele-specific PCR differentiates between target regions differing
in the presence or absence of a variation or polymorphism. PCR
amplification primers are chosen which bind only to certain alleles
of the target sequence. This method is described by Gibbs, Nucleic
Acid Res. 17:2437-2448 (1989) (U.S. Patent Publication No.
20040253594 which is incorporated by reference).
Further diagnostic screening methods employ the allele-specific
oligonucleotide (ASO) screening methods, as described by Saiki et
al., Nature 324:163-166 (1986). Oligonucleotides with one or more
base pair mismatches are generated for any particular allele. ASO
screening methods detect mismatches between variant target genomic
or PCR amplified DNA and non-mutant oligonucleotides, showing
decreased binding of the oligonucleotide relative to a mutant
oligonucleotide. Oligonucleotide probes can be designed that under
low stringency will bind to both polymorphic forms of the allele,
but which at high stringency, bind to the allele to which they
correspond. Alternatively, stringency conditions can be devised in
which an essentially binary response is obtained, i.e., an ASO
corresponding to a variant form of the target gene will hybridize
to that allele, and not to the wild-type allele (U.S. Patent
Publication No. 20040253594 which is incorporated by
reference).
Target regions of a subject's DNA can be compared with the mammary
fluid sample by ligase-mediated allele detection. Ligase may also
be used to detect point mutations in the ligation amplification
reaction described in Wu and Wallace., Genomics 4:560-569 (1989).
The ligation amplification reaction (LAR) utilizes amplification of
specific DNA sequence using sequential rounds of template dependent
ligation as described in Barany, Proc. Nat. Acad. Sci. 88:189-193
(1990) and U.S. Patent Publication No. 20040253594 which are
incorporated by reference.
Amplification products generated using the polymerase chain
reaction can be analyzed by the use of denaturing gradient gel
electrophoresis. Different alleles can be identified based on the
different sequence-dependent melting properties and electrophoretic
migration of DNA in solution. DNA molecules melt in segments,
termed melting domains, under conditions of increased temperature
or denaturation. Each melting domain melts cooperatively at a
distinct, base-specific melting temperature (TM). Melting domains
are at least 20 base pairs in length, and may be up to several
hundred base pairs in length.
Differentiation between alleles based on sequence specific melting
domain differences can be assessed using polyacrylamide gel
electrophoresis, as described in Myers et al., Chapter 7 of Erlich,
ed., PCR Technology, W.H. Freeman and Co., New York (1989)
incorporated by reference.
Generally, a target region to be analyzed by denaturing gradient
gel electrophoresis is amplified using PCR primers flanking the
target region. The amplified PCR product is applied to a
polyacrylamide gel with a linear denaturing gradient as described
in Myers et al., Meth. Enzymol. 155:501-527 (1986), and Myers et
al., in Genomic Analysis, A Practical Approach, K. Davies Ed. IRL
Press Limited, Oxford, pp. 95-139 (1988). The electrophoresis
system is maintained at a temperature slightly below the Tm of the
melting domains of the target sequences.
In an alternative method of denaturing gradient gel
electrophoresis, the target sequences may be initially attached to
a stretch of GC nucleotides, termed a GC clamp, as described by
Myers in Chapter 7 of Erlich, PCT Technology Stockton Press, It is
contemplated that at least 80% of the nucleotides in the GC clamp
are either guanine or cytosine. The GC clamp may be at least 30
bases long. This method is particularly suited to target sequences
with high Tm's.
Generally, the target region is amplified by the polymerase chain
reaction as described above. One of the oligonucleotide PCR primers
carries at its 5' end, the GC clamp region, at least 30 bases of
the GC rich sequence, which is incorporated into the 5' end of the
target region during amplification. The resulting amplified target
region is run on an electrophoresis gel under denaturing gradient
conditions as described above. Nucleic acid fragments differing by
a single base change will migrate through the gel to different
positions, which may be visualized by ethidium bromide
staining.
Temperature gradient gel electrophoresis (TGGE) is based on the
same underlying principles as denaturing gradient gel
electrophoresis, except the denaturing gradient is produced by
differences in temperature instead of differences in the
concentration of a chemical denaturant. Standard TGGE utilizes an
electrophoresis apparatus with a temperature gradient running along
the electrophoresis path. As samples migrate through a gel with a
uniform concentration of a chemical denaturant, they encounter
increasing temperatures. An alternative method of TGGE, temporal
temperature gradient gel electrophoresis (TTGE or tTGGE) uses a
steadily increasing temperature of the entire electrophoresis gel
to achieve the same result. As the samples migrate through the gel
the temperature of the entire gel increases, leading the samples to
encounter increasing temperature as they migrate through the gel.
Preparation of samples, including PCR amplification with
incorporation of a GC clamp, and visualization of products are the
same as for denaturing gradient gel electrophoresis (see, e.g.,
U.S. Patent Application No. 20040253594).
The human leukocyte antigen complex (also known as the major
histocompatibility complex) spans approximately 3.5 million base
pairs on the short arm of chromosome 6. It is divisible into 3
separate regions which contain the class I, the class II and the
class III genes. In humans, the class I HLA complex is about 2000
kb long and contains about 20 genes. Within the class I region
exist genes encoding the well characterized class I MHC molecules
designated HLA-A, HLA-B and HLA-C. In addition, there are
nonclassical class I genes that include HLA-E, HLA-F, HLA-G, HLA-H,
HLA-J and JLA-X as well as a new family known as MIC. The class II
region contains three genes known as the HLA-DP, HLA-DQ and HLA-DR
loci. These genes encode the chains of the classical class II MHC
molecules designated HLA-DR, DP and DQ. In humans, nonclassical
genes designated DM, DN and DO have also been identified within
class II. The class III region contains a heterogeneous collection
of more than 36 genes. Several complete components are encoded by
three genes including the TNFs (see, e.g., U.S. Pat. No. 6,670,124
incorporated by reference).
Any given copy of human chromosome 6 can contain many different
alternative versions of each of the preceding genes and thus can
yield proteins with distinctly different sequences. The loci
constituting the MHC are highly polymorphic, that is, many forms of
the gene or alleles exist at each locus. Several hundred different
allelic variants of class I and class II MHC molecules have been
identified in humans. However, any one individual only expresses up
to 6 different class I molecules and up to 12 different class II
molecules.
The foregoing regions play a major role in determining whether
transplanted tissue will be accepted as self (histo-compatible) or
rejected as foreign (histoincompatible). For instance, within the
class H region, three loci, i.e., HLA-DR, DQ and DP are known to
express functional products. Pairs of A and B genes within these
three loci encode heterodimeric protein products which are
multi-allelic and allorcactive. In addition, combinations of
epitopes on DR and/or DQ molecules are recognized by alloreactive T
cells. This reactivity has been used to define "Dw" types by
cellular assays based upon the mixed lymphocyte reaction (MLR). It
is contemplated that matching of the HLA type of the reference
biological sample with the mammary fluid sample may be used to
determine whether the mammary fluid sample originated from the
donor.
One nucleic acid typing method for the identification of these
alleles has been restriction fragment length polymorphism (RFLP)
analysis discussed herein (see, also, U.S. Pat. No. 6,670,124).
In addition to restriction fragment length polymorphism (RFLP),
another approach is the hybridization of PCR amplified products
with sequence-specific oligonucleotide probes (PCR-SSO) to
distinguish between HLA alleles (see, Tiercy et al., (1990) Blood
Review 4:9-15). This method requires a PCR product of the HLA locus
of interest be produced and then dotted onto nitrocellulose
membranes or strips. Then each membrane is hybridized with a
sequence specific probe, washed, and then analyzed by exposure to
x-ray film or by colorimetric assay depending on the method of
detection. Similarly to the PCR-SSP methodology, probes are made to
the allelic polymorphic area responsible for the different HLA
alleles. Each sample must be hybridized and probed at least 100-200
different times for a complete Class I and II typing. Hybridization
and detection methods for PCR-SSO typing include the use of
nonradioactive labeled probes, microplate formats, and the like
(see, e.g., Saiki et al. (1989) Proc. Natl. Acad. Sci., U.S.A. 86:
6230-6234; Erlich et al. (1991) Eur. J. Immunogenet. 18(1-2):
33-55; Kawasaki et al. (1993) Methods Enzymol. 218:369-381), and
automated large scale HLA class II typing (see, e.g., U.S. Pat. No.
6,670,124).
Another typing method comprises sequence specific primer
amplification (PCR-SSP) which may be used in the methods of the
invention (see, Olemp and Zetterquist (1992) Tissue Antigens 39:
225-235). In PCR-SSP, allelic sequence specific primers amplify
only the complementary template allele, allowing genetic
variability to be detected with a high degree of resolution. This
method allow determination of HLA type simply by whether or not
amplification products (collectively called an "amplicon") are
present or absent following PCR. In PCR-SSP, detection of the
amplification products is usually done by agarose gel
electrophoresis followed by ethidium bromide (EtBr) staining of the
gel (see, e.g., U.S. Pat. No. 6,670,124).
Another HLA typing method is SSCP--Single-Stranded Conformational
Polymorphism. Briefly, single stranded PCR products of the
different HLA loci are run on non-denaturing Polyacrylamide Gel
Electrophoresis (PAGE). The single strands will migrate to a unique
location based on their base pair composition. By comparison with
known standards, a typing can be deduced. It is the only method
that can determine true homozygosity, (see, e.g., U.S. Pat. No.
6,670,124) (Orita et al., Proc. Nat. Acad. Sci 86:2766-2770
(1989)).
The identification of a DNA sequence can be made without an
amplification step, based on polymorphisms including restriction
fragment length polymorphisms ("RFLP") in a subject. Hybridization
probes are generally oligonucleotides which bind through
complementary base pairing to all or part of a target nucleic acid.
Probes typically bind target sequences lacking complete
complementarity with the probe sequence depending on the stringency
of the hybridization conditions. The probes are typically labeled
directly or indirectly, such that by assaying for the presence or
absence of the probe, one can detect the presence or absence of the
target sequence. Direct labeling methods include radioisotope
labeling, such as with 32P or 35S. Indirect labeling methods
include fluorescent tags, biotin complexes which may be bound to
avidin or streptavidin, or peptide or protein tags. Visual
detection methods include photoluminescents, Texas red, rhodamine
and its derivatives, red leuco dye and
3,3',5,5'-tetra-methylbenzidine (TMB), fluorescein, and its
derivatives, dansyl, umbelliferone and the like or with horse
radish peroxidase, alkaline phosphatase and the like (see, e.g.,
U.S. Patent Publication No. 20040253594, U.S. Patent Publication
No. 20050123947, which are incorporated by reference).
One or more additional restriction enzymes and/or probes and/or
primers can be used. Additional enzymes, constructed probes, and
primers can be determined by routine experimentation by those of
ordinary skill in the art and are intended to be within the scope
of the invention.
Although the methods described herein may be in terms of the use of
a single restriction enzyme and a single set of primers, the
methods are not so limited. One or more additional restriction
enzymes and/or probes and/or primers can be used, if desired.
Additional enzymes, constructed probes and primers can be
determined through routine experimentation, combined with the
teachings provided and incorporated herein.
The reagents suitable for applying the methods of the invention may
be packaged into convenient kits. The kits provide the necessary
materials, packaged into suitable containers. Typically, the
reagent is a PCR set (a set of primers, DNA polymerase and 4
nucleoside triphosphates) that hybridize with the gene or loci
thereof. Typically, the PCR set is included in the kit. Typically,
the kit further comprises additional means, such as reagents, for
detecting or measuring the detectable entity or providing a
control. Other reagents used for hybridization, prehybridization,
DNA extraction, visualization etc. may also be included, if
desired.
Other Identity Markers Profiles
It is further contemplated that the mammary fluid sample may be
tested for self-antigens (or other peptides and polypeptides)
present in the mammary fluid to establish a self-antigen profile
(identity marker profile). The self-antigen profile of the mammary
fluid sample will be compared to the reference self-antigen profile
for the individual human. A match or identity of the self-antigen
profile will indicate that the mammary fluid was obtained from the
specific subject.
The various antigens that determine self are encoded by more than
40 different loci, such as the major histocompatibility complex
(MHC), also called the human leukocyte antigen (HLA) locus, and the
blood group antigens, such as ABO.
Methods are known in the art for screening humans for ABO blood
group type. The blood-group antigens are expressed on red blood
cells, epithelial cells and endothelial cells.
Testing for HLA type can be conducted by methods known in the art
such as serological and cellular typing.
It is contemplated that the antigens could be identified by a
microcytotoxicity test. In this test, white blood cells are
distributed in a microtiter plate and monoclonal antibodies
specific for class I and class II MHC antigens are added to
different wells. Thereafter, complement is added to the wells and
cytotoxicity is assessed by uptake or exclusion to various dyes
(e.g., trypan blue or eosin Y) by the cells. If the white blood
cells express the MHC antigen for a particular monoclonal antibody,
then the cells will be lysed on addition of complement and these
dead cells will take up the dye (see, Terasaki and McClelland,
(1964) Nature, 204:998 and U.S.
Pat. No. 6,670,124). HLA typing based on antibody-mediated
microcytotoxicity can thus indicate the presence or absence of
various MHC alleles (See Kuby Immunology 4th Ed., Freeman and
Company, pp 520-522).
The detection of antigens may be selected from, but is not limited
to, enzyme-linked immunosorbent assay, solid phase radiobinding
immunoassays where the antibodies may be directed against soluble
antigens or cell surface antigens, autoradiography, competitive
binding radioimmunoassay, immunoradiometric assay (IRMA) electron
microscopy, peroxidase antiperoxidase (PAP) labeling, fluorescent
microscopy, alkaline phosphatase labeling and peroxidase
labeling.
In the case where the detection method (s) use optical microscopy,
the cells from the biological sample or the mammary fluid sample
are mounted and fixed on a microscope slide. In this case, the step
of detecting the labelled antibody is detecting a resulting
colouration of the self-antigen with an optical microscope (see,
e.g., U.S. Pat. No. 6,376,201).
The following example provides an embodiment of the methods
described herein and should not be understood as restrictive.
Example 1
Testing of a Human Breast Milk Donor
A woman who wishes to donate her breast milk will provide a
biological reference sample prior to (or at the time of) her first
donation. The biological sample will include a convenient tissue
type, e.g., blood, cheek cell, hair etc. The sample will be donated
under supervision of another individual(s), e.g., bank milk
personnel. The sample will be labeled for later reference. The
reference sample will be tested for a specific marker profile,
e.g., nucleic acid and/or peptide profile. The sample will be
tested for one or more markers. Results of the tests will be
stored, e.g., on a computer-readable medium for future reference.
Any remaining sample will be stored. The woman can also be screened
(using the reference sample or another sample) for, e.g., drug use,
viruses, bacteria, parasites, and fungi etc., to determine her
health. The woman will be given a label corresponding to the
reference sample to keep with her and use with her donated
milk.
Alternatively, the sample will be stored without testing, and will
be tested at a later date, for example, together with the donated
breast milk.
The woman will express her milk for donation and either forward the
milk to the milk bank or processing facility or store the milk in
her refrigerator, e.g., the freezer, for donation with other
samples at a later date. The donated milk will be labeled with the
label given to the woman and matching the reference sample.
A sample of the donated milk that arrives at the milk bank or
processing facility will be tested for at least one of the same
markers as the reference sample. The marker profile of the
reference sample will be compared to the marker profile of the
donated milk sample. If the profiles will match, the identity of
the donor will be confirmed. If the profiles will not match, the
results will be an indication that the donated milk is contaminated
with another woman's milk or that it does not come from the woman
whose reference sample was taken.
The milk whose provenance (i.e., origin) will be confirmed by the
matched profiles will be further processed, e.g., pasteurized,
e.g., into human milk fortifiers, standardized human milk
compositions, and/or human lipid compositions. Such compositions
will be administered to human infants, e.g., premature infants,
whose mothers may not be able to provide them with adequate
nutrition.
The reference sample and/or results of the reference sample tests
will be stored for any future donation by the corresponding
mother.
A number of embodiments of the invention have been described.
Nevertheless, it will be understood that various modifications may
be made without departing from the spirit and scope of the
invention. Accordingly, other embodiments are within the scope of
the following claims.
* * * * *
References