Secretome Profile-Facilitated In Vitro Fertilization
Secretome profiling improves the pregnancy success rate of in vitro fertilization processes, while reducing the risk of multiple births.
This application claims benefit of priority to U.S. Provisional Patent Application No. 61/321,448 filed Apr. 6, 2010, which is incorporated by reference to the same extent as though fully replicated herein.
SEQUENCE LISTINGThis application includes a Sequence Listing, as set forth in an ASCII-compliant text file named “CCRMProtein_ST25.txt”, created on Apr. 6, 2011, and containing 2263 kilobytes, which is incorporated by reference to the same extent as though fully replicated herein.
BACKGROUND1. Field of the Invention
The invention relates to the field of in vitro fertilization (IVF), which is a process by which mammalian egg cells are fertilized by sperm outside the womb. More particularly, evaluation of a secretome profile is used to enhance the pregnancy success rate when the fertilized egg is implanted into a patient's uterus.
2. Description of the Related Art
IVF infertility treatment offers infertile couples a chance to have a biologically related child. IVF may overcome female infertility due to problems of the fallopian tube or endometriosis. IVF also overcomes male infertility due to problems with sperm quality or quantity. The IVF process involves hormonally controlling the ovulatory process, removing eggs (termed ova) from the woman's ovaries and permitting the sperm to fertilize the eggs in a fluid medium. The fertilized egg, termed an embryo, is subsequently transferred to the patient's uterus with the intent of establishing a successful pregnancy. Due to expensive procedural costs, IVF is only attempted after the failure of less expensive fertility treatments.
IVF treatment begins with administration of hormonal medications to stimulate ovarian follicle production, such as gonadotropins hormones. The prevention of spontaneous ovulation involves using other hormones, such as GnRH antagonists or GnRH agonists that block the natural surge of luteinizing hormone. With adequate follicular maturation, administration of human chorionic gonadotropin hormone causes ovulation approximately 42 hours after the administration. However, the egg retrieval procedure takes place just prior to ovulation, in order to recover the eggs from the ovary. The egg retrieval proceeds using a transvaginal technique involving an ultrasound-guided needle that pierces the vaginal wall to reach the ovaries. After recovery of the follicles through the needle, the follicular fluid is provided to the IVF laboratory to identify eggs. Typically, the procedure retrieves between 10 and 30 eggs. The retrieval procedure takes approximately 20 minutes and is usually done under conscious sedation or general anesthesia.
For IVF, the fertilization of the egg (termed insemination) proceeds in the laboratory where the identified eggs and semen are usually incubated together in a culture media. The confirmation of fertilization proceeds by monitoring the eggs for cell division. For instance, a fertilized egg may show two pronuclei. In certain situations, such as low sperm count or motility, a single sperm may be injected directly into the egg using a method called intracytoplasmic sperm injection (ICSI). In another option known as gamete intrafallopian transfer, eggs are removed from the woman and placed in one of the fallopian tubes, along with the man's sperm. In this example, fertilization occurs within the women's body, a process termed in vivo fertilization.
Selected embryos are transferred to the patient's uterus through a thin, plastic catheter, which goes through the vagina and cervix. Typically, transfer of 6-8 cell stage embryos to the uterus occurs three days after embryo retrieval. Alternatively, embryos can be placed into an extended culture system with a transfer done at the blastocyst stage at approximately five days post-retrieval. Blastocyst stage transfers often result in higher pregnancy rates. Additionally, embryonic cryopreservation, or the storage of embryos in a frozen state, is feasible until uterine transfer. For example, the first term pregnancy derived from a frozen human embryo was reported in 1984.
Despite progressively improving IVF pregnancy rates, the majority of transferred human embryos result in implantation failure. For example, Canadian clinics reported an average pregnancy rate of 35% for one cycle, but a live birth rate of only 27% in 2006. Moreover, implantation success rates may decrease with the increasing maternal age, if donor eggs are not used. Various factors are associated with implantation failure, including embryo chromosome aneuploidies related to advanced maternal age and maternal factors such as endometrium response failure to hormone regulation.
To overcome low implantation success rate, multiple embryos are commonly transferred during a single IVF procedure. The process for selecting embryos for transfer often involves grading methods developed in individual laboratories to judge oocyte and embryo quality. An arbitrary embryo score, involving the number and quality of embryos, may reveal the probability of pregnancy success post-transfer. For example, an embryologist may grade embryos using morphological qualities including the number of cells, clearness of cytoplasm, evenness of growth and degree of fragmentation. However, embryo selection based on morphological qualities is not precise. Often, several embryos selected for these general qualities are implanted to improve the chance of pregnancy. The number of embryos transferred depends upon the number available, the age of the woman and other health and diagnostic factors.
The transfer of multiple embryos, however, often results in multiple pregnancies, a major complication of IVF. In general, multiple pregnancies, specifically, more than twins, hold maternal and fetal risks. For example, multiple births are associated with increased risk of pregnancy loss, neonatal morbidity, obstetrical complications, and prematurity with potential for long term damage. Some countries implemented strict limits on the number of transferred embryos to reduce the risk of high-order multiples (e.g., triplets or more). However, these limitations are not universally followed or accepted.
SUMMARYIn one embodiment, a system for enhancing the pregnancy success rate of in vitro fertilization includes a means for determining the secretome profile of an embryo to identify proteins, polypeptides, oligopeptides or protein fragments implicated in implantation success and a means for recommending whether to implant the embryo, such as a blastocyst, on the basis of the secretome profile.
The CDC data from 2006 shows that use of donor eggs in a 40 year old woman will result in a live birth 54% of the time. This compares to 20.6% using the woman's own eggs. Thus, an opportunity exists for the instrumentalities of this disclosure to facilitate success rates approximating the use of donor eggs when using one's own eggs, and may even permit higher success rates due to better selection of viable embryos.
In an embodiment, a system is provided for enhancing the pregnancy success rate of in vitro fertilization. The system includes an electronic system to configured to gather secretome data as a secretome profile of an embryo by quantitating proteins implicated in implantation success. A model is provided for use in recommending whether to implant the embryo on the basis of this secretome profile. The secretome date may be, for example, provided by use of mass spectroscopy or ELISA measurements.
According to one aspect of the system, the secretome profile may be provided by using proteins as embryo secretions in culture media that may be linked to changed odds of implantation success, for example, as may be found in one or more sequences found in SEQ ID Nos. 1-404. The sequences of SEQ ID Nos. 261-404 are preferred for this use. Particularly preferred are the sequences of SEQ ID Nos. 310, 311, 313, 317, 318, 319, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 345, 346, 347, 348, 349, 350, 368, 371, 374, 383, 391, 397, 398, 399, 402, 403, and 404. The sequences of SEQ ID Nos. 310, 311, 313, 318, 319, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 345, 346, 347, 348, 349, 350, 368, 371, 374, 391, 397, 398, 399, 402, 403, and 404 are associated with increased odds of implantation success, whereas those of SEQ ID Nos. 317 and 383 are associated with failure or aneuploidy.
In an embodiment, a method of in vitro fertilization entails determining the secretome profile of a candidate embryo by use of these sequences to ascertain proteins implicated in implantation success. This provides data that may be submitted to a model that associates one or more of these proteins with changes odds of implantation success or failure. A recommendation for implantation of the embryo may then be provided based upon the modeling outcome. The embryo may be conditionally implanted on the basis of the recommendation.
In an embodiment, there is an improved ELISA assay kit with a plurality of microwells for the quantitation of protein content in a sample. The microwells are constructed and arranged to quantitate for a plurality of proteins selected from these sequences.
The following definitions are provided to facilitate understanding of certain terms used herein and are not meant to limit the scope of the present disclosure.
The term “secretome panel” refers to a collection of individual proteins, polypeptides, oligopeptides or protein fragments that are differentially expressed and secreted by an embryo. The proteins, polypeptides, oligopeptides or protein fragments of the secretome panel are selected based on predictions of developmental competence and implantation potential of an embryo.
The term “spent media” refers to media surrounding an embryo that accumulates proteins secreted from the embryo.
The term “secretome profiling” refers to qualitative and quantitative analysis of a secretome panel collected from spent media.
There will now be shown and described a system for enhancing the pregnancy success rate of in vitro fertilization involving non-invasive secretome profiling of an embryo. Secretome profiling may be performed on embryos at differing embryonic developmental stages, such as day one through day six embryos.
Non-limiting examples of proteins comprising the secretome panel include proteins involved in amino acid metabolism, lipid metabolism, carbohydrate metabolism, signal transduction, apoptosis, transcription and combinations thereof. In one example, an embryo secretes a protein that is involved in amino acid metabolism, thereby indicating potential for successful embryo implantation. In another example, an embryo secretes a protein that is involved in apoptosis, thereby indicating a reduced potential for successful blastocyst implantation. Nonlimiting examples of the number of proteins comprising the secretome panel includes ≧250 proteins, ≧100 proteins, ≧50 proteins, ≧20 proteins or ≧10 proteins.
Secretome profiling involves assessment of a secretome panel. Secretome profiling is a noninvasive method for predicting developmental competence and viability of an embryo. Comparison of secretome profiles between developing embryos and degenerating embryos, both at the same developmental stage, reveals significant differences in protein expression. Moreover, secretome profiling provides a molecular perspective of the functioning biochemical pathways present during embryo development. A noninvasive secretome profiling assay correlates embryonic secretome to embryonic viability, thereby facilitating single embryo transfer during in vitro fertilization.
In one embodiment, secretome profiling occurs by assessing a secretome panel via Enzyme-Linked Immunosorbent Assay (ELISA). In another embodiment, secretome profiling proceeds by assessing a secretome panel via mass spectrometry.
Non-limiting examples of solid-state substrates used in ELISA include microwell plates, such as 96-well plates, 384-well plates and 8-well strips, microarray slides and nitrocellulose membranes. In one embodiment, glass microarray surfaces contain chemical functional groups such as epoxy, amine or aldehyde. In one embodiment, microwell plate material comprises polystyrene.
The term “capture antibody” refers to an antibody secured, either covalently or non-covalently, to a solid-state substrate. The capture antibody recognizes and binds to a specific antigen such as a protein, a polypeptide, an oligopeptide, a protein fragment, a carbohydrate or a small molecule. In one example, a solid-state substrate is functionalized with capture antibodies by passive adsorption or by specific binding. For example, specific binding of capture antibodies may occur using biotinylated capture antibodies and streptavidin-coated solid-state substrates. In another example, solid-state substrates are functionalized with antigens via passive adsorption.
Non-limiting examples of ELISA types include direct, indirect, competitive, and sandwich. In one example, proteins from the secretome panel are detected on a solid-state substrate using either a primary labeled antibody or a secondary labeled antibody. In another example, proteins from the secretome panels are detecting using a sandwich ELISA wherein a protein from the secretome panel is bound between two primary antibodies, namely the capture antibody and the detection antibody. Non-limiting examples of antibody labels include enzymes and fluororphores. In one embodiment, an enzyme that is conjugated to a detection antibody binds to a substrate producing either a chromogenic, fluorescent or chemiluminescent signal that is proportional to the quantity of protein from the secretome panel.
It will be appreciated that perceptive use of the instrumentalities described herein may result in a better selection of healthy embryos, such as blastocysts, for implantation. Thus, fewer blastocysts need to be implemented, such that there is lower risk of multiple pregnancies while achieving a higher overall pregnancy success rate.
The following descriptions will show and describe, by way of non-limiting examples, a process for improving pregnancy success rates with lower incidence of multiple births. The following examples describe secretome profiling of spent media from human blastocysts to provide implantation recommendation. It is to be understood that these examples are provided by way of illustration and should not be unduly construed to limit the scope of what is disclosed herein.
Example 1 Secretome Profiling of Human Embryos Using Mass SpectrometryThe following nonlimiting example teaches by way of illustration, not by limitation, secretome profiling of a human embryo using mass spectrometry (MS). Human cleavage-stage embryos were cultured in 10 μL drops of G1 supplemented with 2.5 mg/mL recombinant albumin under oil at 37° C., 6% CO2, 5% O2 for 24 hours. The embryos were washed twice in G2 culture media and further cultured in 10 μL drops of G2 supplemented with 2.5 mg/mL recombinant albumin under oil at 37° C., 6% CO2, 5% O2 for 48 hours with a fresh drop of G2 media added after 24 hours. Spent media samples of blastocysts were transferred into 0.65 mL Eppendorf tubes. Control groups comprised media cultured and collected under the same conditions but without embryos.
Micro-drops of spent media were depleted of human serum albumin (HSA) using Cibracron Blue Activated SwellGel Discs (Themo Fisher Scientific, Rockford, Ill.). The proteins in the spent media were separated by 1D gel electrophoresis (Invitrogen, Carlsbad, Calif.), followed by Coomassie staining. Twenty five individual bands were cut out from each sample lane and a standard in-gel digestion protocol was used based on previously used methods ((1.) Rosenfeld, J., Capdevielle, J., Guillemot, J. C. & Ferrara, P. In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal. Biochem. 203, 173-179 (1992), (2.) Hellman, U., Wernstedt, C., Gonez, J. & Heldin, C. H. Improvement of an “In-Gel” digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing. Anal. Biochem. 224, 451-455 (1995)). Iodoacteamide (IAM) was used for cysteine alkylation. The samples were analyzed on a LTQ-FT Ultra Hybrid Mass Spectrometer (Thermo/Finnigan; Waltham, Mass.) with a method based on a previously described protocol from Hansen et al., (Hansen KC, Kiemele L, Maller O, O'Brien J, Shankar A, Formetti J, Schedin P. An in-solution ultrasonication-assisted digestion method for improved extracellular matrix proteome coverage. Mol Cell Proteomics. 8(7):1648-57 (2009)).
Spent media samples were analyzed on a LTQ-FT Ultra Hybrid Mass Spectrometer (Thermo/Finnigan; Waltham, Mass.). Peptide desalting and separation was achieved using a dual capillary/nano pump HPLC system (Agilent 1200, Palo Alto, Calif.). On this system 8 μL of spent media sample was loaded onto a trapping column (ZORBAX 300SB-C18, dimensions 5×0.3 mm 5 μm) and washed with 5% acetonitrile (ACN), 0.1% formic acid (FA) at a flow rate of 15 μL/min for 5 minutes. At this time, the trapping column was put online with the nano-pump at a flow rate of 350 nL/min. An 85 minute gradient, from 8% ACN to 40% ACN, was used to separate the peptides. The column was made from an in-house pulled 360/100 nm (outer/inner diameter) fused silica capillary packed with Jupiter C18 resin (Penomenex; Torrance, Calif.). The column was kept at a constant 40° C. using an in-house built column heater. Data acquisition was performed using the instrument supplied Xcalibur (version 2.0.6) software. The LC runs were monitored in positive ion mode by sequentially recording survey MS scans (m/z 400-2000), in the ICR cell, while three MS2 were obtained in the ion trap via CID for the most intense ions. After two acquisitions of a given ion within 45 seconds, the ion was excluded for 150 seconds.
For data analysis, The Raw Distiller program (UCSF) was used to create a de-isotoped centroided peak lists from the raw spectra into the mascot format using the default settings. The peak lists were searched against the SwissProt Human database (51.6, Homo sapiens 15720 sequences) using Mascot™ server (Version 2.2, Matrix Science, Boston, Mass.). The search parameters are the same as those followed in Hansen et al., (Hansen K C, Kiemele L, Maller O, O'Brien J, Shankar A, Formetti J, Schedin P. An in-solution ultrasonication-assisted digestion method for improved extracellular matrix proteome coverage. Mol Cell Proteomic. 8(7): 1648-57 (2009)). The Mascot results were loaded into Scaffold (v 2.06) and the runs were compared.
In one embodiment, MS analysis of spent media reveals a secretome panel of 261 individual proteins (Table 1). In one example, secretome profiling using the secretome panel of 261 proteins (Table 1), as analyzed by MS, identifies the potential for developmental competence and implantation success of a human embryo.
In one embodiment, analysis of spent media via MS reveals a secretome panel of 37 individual proteins (Table 2). Secretome profiling via MS of 37 individual proteins (Table 2) from spent media of human embryos correlates with embryonic viability and euploidy. The term “euploidy” refers to having a chromosome number that is an exact multiple of the haploid number for a human embryo, namely 23 pairs of chromosomes.
In Tables 1 and 2 below, the entries for “Entry Name” and “Accession Number” refer to identifiers for published sequence data that is stored in bioinformatic databases including the Uniprot Knowledgebase, Swiss-Prot and TrEMBL. The information is made freely available to the world and is coordinated by the Swiss Institute of Bioinformatics, which is centrally administered in Lausanne, Switzerland with offices in Bern, Geneva and Zurich. The sequence information represented by these identifiers together with the representative publications is hereby incorporated by reference to the same extent as though fully replicated herein. For those proteins having isoforms, the sequences for Table 1 include consensus sequences for the primary isoforms, while sequences for Table 2 include also all other isoforms available at the time of filing.
The following nonlimiting example teaches by way of illustration, not by limitation, the fabrication and employment of a customized immunoassay test kit for secretome profiling of a human embryo. Immunoassay test kit fabrication occurs by modifying the well surfaces of a 96-well microtiter plate. Each separate well of the microtiter plate is incubated with an unlabelled capture antibody that recognizes one specific protein from the secretome panel. Custom capture antibodies are purchased from Rockland Immunochemicals, Inc. After incubation for 12 hours at 4° C., capture antibodies passively adsorb to the well surface. Subsequently, all wells of the 96-well plate are washed three times with a buffer comprising 1× phosphate buffer saline and 0.1% Tween-20 (PBST), blocked with 1% bovine serum albumin (BSA) for 1 hour at ambient temperature and washed three times with PBST.
Human cleavage-stage embryos are cultured in 10 μL drops of G1 supplemented with 2.5 mg/mL recombinant albumin under oil at 37° C., 6% CO2, 5% O2 for 24 hours. The embryos are washed twice in G2 culture media and further cultured in 10 μL, drops of G2 supplemented with 2.5 mg/mL recombinant albumin under oil at 37° C., 6% CO2, 5% O2 for 48 hours with a fresh drop of G2 media added after 24 hours. Spent media samples of blastocysts are transferred into 0.65 mL Eppendorf tubes. Control groups comprise media cultured and collected under the same conditions but without embryos.
Secretome profiling proceeds using spent media culture in a sandwich ELISA format. The spent media culture is diluted into phosphate buffer saline (PBS) buffer and further distributed to separate wells of said customized microtiter plate. After incubation for one hour at room temperature, the samples are aspirated and all wells are washed four times with PBST buffer. Primary antibodies, each recognizing a distinct protein from the secretome panel, are distributed into wells that correspond to appropriate capture antibodies and permitted to incubate for one hour at room temperature. The wells are washed four times with PBST and incubated with a secondary antibody conjugated with an enzyme. The term “secondary antibody” refers to an antibody that binds to primary antibodies and may be conjugated with detection probes such as enzymes or fluorophores. In one embodiment, the secondary antibody contains the horseradish peroxidase (HRP) enzyme that converts the chromogenic substrate, 3,3′,5,5′-tetramethylbenzidene (TMB), into a blue product quantified at 655 nm on a spectrophotometer. In another embodiment, the HRP enzymatic reaction is stopped with a solution containing sulfuric acid and quantified at 450 nm.
In one embodiment, secretome profiling of a secretome panel of 261 individual proteins (Table 1), each selected for significant developmental competence and implantation potential, are monitored using said ELISA immunoassay test kit. In another embodiment, secretome profiling of a secretome panel comprising 37 proteins (Table 2) are monitored using said ELISA immunoassay test kit. Secretome profiling of the 37 individual proteins (Table 2) from spent media of human embryos correlates with embryonic viability and euploidy.
Changes may be made in the above methods and systems without departing from the scope hereof. It should be noted that the matter contained in the above description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. The following claims are intended to cover all generic and specific features described herein, as well as all statements of the scope of the present method and system and reasonable variations thereof, which, as a matter of language, might be said to fall therebetween.
Claims
1. A system for enhancing the pregnancy success rate of in vitro fertilization, comprising:
- means for determining the secretome profile of an embryo to identify proteins implicated in implantation success; and
- means for recommending whether to implant the embryo on the basis of the secretome profile.
2. The system of claim 1, further comprising data representative of a secretome profile of an embryo acquired by ELISA.
3. The system of claim 2, further comprising the secretome profile of an embryo acquired using a secretome panel selected from the group consisting of SEQ ID Nos. 1-403.
4. The system of claim 3, further comprising the secretome profile of an embryo acquired using a secretome panel selected as one or more members of the group consisting of SEQ ID Nos. 1-404.
5. The system of claim 3, further comprising the secretome profile of an embryo acquired using a secretome panel selected as one or more members of the group consisting of SEQ ID Nos. 261-404.
6. The system of claim 3, further comprising the secretome profile of an embryo acquired using a secretome panel selected as one or more members of the group consisting of SEQ ID Nos. 310, 311, 313, 317, 318, 319, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 345, 346, 347, 348, 349, 350, 368, 371, 374, 383, 391, 397, 398, 399, 402, 403, and 404.
7. The system of claim 3, further comprising the secretome profile of an embryo acquired using a secretome panel selected as one or more members of the group consisting of SEQ ID Nos. 310, 311, 313, 318, 319, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 345, 346, 347, 348, 349, 350, 368, 371, 374, 391, 397, 398, 399, 402, 403, and 404.
8. The system of claim 3, further comprising the secretome profile of an embryo acquired using a secretome panel selected as one or more members of the group consisting of SEQ ID Nos. 317 and 383.
9. The system of claim 1, further comprising the secretome profile of an embryo acquired using a secretome panel selected as one or more members of the group consisting of SEQ ID Nos. 1-404.
10. The system of claim 1, further comprising the secretome profile of an embryo acquired using a secretome panel selected as one or more members of the group consisting of SEQ ID Nos. 261-404.
11. The system of claim 1, further comprising the secretome profile of an embryo acquired using a secretome panel selected as one or more members of the group consisting of SEQ ID Nos. 310, 311, 313, 317, 318, 319, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 345, 346, 347, 348, 349, 350, 368, 371, 374, 383, 391, 397, 398, 399, 402, 403, and 404.
12. The system of claim 1, further comprising the secretome profile of an embryo acquired using a secretome panel selected as one or more members of the group consisting of SEQ ID Nos. 310, 311, 313, 318, 319, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 345, 346, 347, 348, 349, 350, 368, 371, 374, 391, 397, 398, 399, 402, 403, and 404.
13. The system of claim 1, further comprising the secretome profile of an embryo acquired using a secretome panel selected as one or more members of the group consisting of SEQ ID Nos. 317 and 383.
14. A method of in vitro fertilization, comprising:
- determining the secretome profile of a candidate embryo to ascertain proteins implicated in implantation success;
- modeling on basis of the secretome profile to assess candidate embryo viability for
- recommending whether to implant the candidate embryo on the basis of the viability assessment; and
- conditionally implanting the candidate embryo on the basis of the recommendation.
15. The method of claim 14, wherein the step of determining the secretome profile includes using a secretome panel selected as one or more members of the group consisting of SEQ ID Nos. 1-404.
16. The method of claim 14, wherein the step of determining the secretome profile includes using a secretome panel selected as one or more members of the group consisting of SEQ ID Nos. 261-404.
17. The method of claim 14, wherein the step of determining the secretome profile includes using a secretome panel selected as one or more members of the group consisting of SEQ ID Nos. 310, 311, 313, 317, 318, 319, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 345, 346, 347, 348, 349, 350, 368, 371, 374, 383, 391, 397, 398, 399, 402, 403, and 404.
18. The method of claim 14, wherein the step of determining the secretome profile includes using a secretome panel selected as one or more members of the group consisting of SEQ ID Nos. 310, 311, 313, 318, 319, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 345, 346, 347, 348, 349, 350, 368, 371, 374, 391, 397, 398, 399, 402, 403, and 404.
19. The method of claim 14, wherein the step of determining the secretome profile includes using a secretome panel selected as one or more members of the group consisting of SEQ ID Nos. 317 and 383.
20. In an ELISA assay kit comprising a plurality of microwells for the quantitation of protein content in a sample, the improvement comprising:
- the microwells being constructed and arranged to quantitate for a plurality of proteins selected from SEQ ID Nos. 1-404.
21. The kit claim 20, wherein the plurality of proteins are selected from the group consisting of SEQ ID Nos. 261-404.
22. The kit claim 20, wherein the plurality of proteins are selected from the group consisting of SEQ ID Nos. 310, 311, 313, 317, 318, 319, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 345, 346, 347, 348, 349, 350, 368, 371, 374, 383, 391, 397, 398, 399, 402, 403, and 404.
23. The kit claim 20, wherein the plurality of proteins are selected from the group consisting of SEQ ID Nos. 310, 311, 313, 318, 319, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 345, 346, 347, 348, 349, 350, 368, 371, 374, 391, 397, 398, 399, 402, 403, and 404.
24. The kit claim 20, wherein the plurality of proteins are selected from the group consisting of SEQ ID Nos. 317 and 383.
Type: Application
Filed: Apr 6, 2011
Publication Date: Oct 6, 2011
Inventors: William B. Schoolcraft (Englewood, CO), Mandy Katz-Jaffe (Denver, CO), Susanna McReynolds (Parker, CO)
Application Number: 13/081,463
International Classification: A61B 17/435 (20060101); C12M 1/34 (20060101);