WO2009014987A2 - Identification and quantification of biomarkers for evaluating the risk of preterm birth - Google Patents
Identification and quantification of biomarkers for evaluating the risk of preterm birth Download PDFInfo
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- WO2009014987A2 WO2009014987A2 PCT/US2008/070320 US2008070320W WO2009014987A2 WO 2009014987 A2 WO2009014987 A2 WO 2009014987A2 US 2008070320 W US2008070320 W US 2008070320W WO 2009014987 A2 WO2009014987 A2 WO 2009014987A2
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/81—Protease inhibitors
- G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- G01N2333/811—Serine protease (E.C. 3.4.21) inhibitors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/36—Gynecology or obstetrics
- G01N2800/368—Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour
Definitions
- Preterm delivery affects more than 10% of all pregnant mothers. It is also one of the leading causes of illness and death associated with newborns. Compared with babies born at term, infants born prematurely experience a 40-fold increase in neonatal death, and may be at significantly increased risk for major medical complications such as cerebral palsy, chronic respiratory illness, blindness and deafness. Furthermore, long-term neurologic and developmental problems have been identified in as many as 70% of children with birth weight less than 1.5 lbs. It has been estimated that these complications are associated with billions of dollars of direct costs and unrealized potential each year just in the United States.
- Described herein are methods for evaluating the risk of preterm birth in pregnant subjects.
- the methods involve detecting and quantifying one or more biomarkers associated with preterm birth in a biological sample from the subject.
- biomarkers useful in predicting preterm birth are also described in detail.
- preterm birth includes the delivery of a baby prior to full gestation. For example, delivery of the baby less than 37 weeks of gestation is considered a preterm birth.
- preterm birth is synonymous with preterm delivery and premature delivery.
- biomarker may be used to refer to a naturally- occurring biological molecule present in pregnant women at varying concentrations useful in predicting the risk of preterm birth.
- the biomarker can be a peptide present in higher or lower amounts in a subject at risk of preterm birth relative to the amount of the same biomarker in a subject who did not experience preterm birth.
- the biomarker can include other molecules besides peptides including small molecules such as but not limited to biological amines and steroids.
- peptide may be used to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another.
- the peptide is not limited by length, and thus “peptide” can include polypeptides and proteins.
- isolated refers to material that has been removed from its original environment, if the material is naturally occurring. For example, a naturally-occurring peptide present in a living animal is not isolated, but the same peptide, which is separated from some or all of the coexisting materials in the natural system, is isolated. Such isolated peptide could be part of a composition and still be isolated in that the composition is not part of its natural environment.
- An “isolated” peptide also includes material that is synthesized or produced by recombinant DNA technology.
- the term “specifically immunoreactive” refers to a measurable and reproducible specific immunoreaction such as binding between a peptide and an antibody that is determinative of the presence of the peptide in a biological sample or in a heterogeneous population of peptides and other biologies.
- the term “specifically immunoreactive” may include specific recognition of structural shapes and surface features. Thus, under designated conditions, an antibody specifically immunoreactive to a particular peptide does not bind in a significant amount to other peptides present in the sample.
- a variety of immunoassay formats can be used to determine antibodies specifically immunoreactive to a particular peptide.
- solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a peptide. See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, which is incorporated herein by reference, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
- the term “antibody” refers to an immunoglobulin specifically immunoreactive to a given antigen.
- the term “antibody” is intended to include whole antibodies of any isotype (IgG, IgA, IgM, IgE, etc), and fragments thereof.
- An “antibody” as sued herein also includes an antibody preparation.
- Antibodies may be labeled with detectable labels using a variety of techniques as is known in the art. The label can be a radioisotope, fluorescent compound, chemiluminescent compound, enzyme, or enzyme co-factor, or any other labels known in the art.
- the antibody that binds to the peptide of interest may not be labeled, but may instead be detected by binding of a labeled secondary antibody that specifically binds to the primary antibody.
- the term “detect” refers to the quantitative measurement of undetectable, low, normal, or high serum concentrations of one or more biomarkers such as, for example, peptides and other biological molecules.
- the terms “quantify” and “quantification” may be used interchangeably, and refer to a process of determining the quantity or abundance of a substance in a sample (e.g., a biomarker), whether relative or absolute.
- the term "about” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “a little above” or “a little below” the endpoint without affecting the desired result.
- Described herein are methods for identifying pregnant subjects that are at risk for preterm birth.
- Particular biomarkers have been identified that may be utilized to identify pregnant subjects during early to mid-pregnancy that may be at risk for preterm birth. Such markers may allow the diagnostic distinction between preterm birth and other conditions that exhibit similar symptoms. Early identification of subjects at greater risk for preterm birth would be of considerable value, as such subjects could be more closely monitored.
- Biomarkers indicative of preterm birth may be quantifiable in the subject.
- biomarkers may be tested at from about 20 weeks to about 34 weeks gestation.
- biomarkers may be tested at from about 24 weeks to about 32 weeks gestation. It should be noted that these ranges should not be seen as limiting, as such testing may be performed at any point during pregnancy. Rather these ranges are provided to demonstrate periods of the gestational cycle where such testing is most likely to occur in a majority of subjects.
- Useful biomarkers in identifying subjects at risk for preterm birth include various peptides and other biological molecules.
- biomarkers associated with preterm birth complications may include biological molecules and peptides found to be statistically different (p ⁇ 0.01) from control subjects (i.e., pregnant women that did not experience preterm birth complications), and a p (probability) value ⁇ 0.02 served as the cutoff.
- nonlimiting examples of peptides associated with preterm birth may include peptides having amino acid sequences of QLGLPGPPDVPDHAAYHPF (SEQ ID NO 1), NVHSAGAAGSRMNFRPGVLSSRQLGLPGPPDVPDHAAYHPF (SEQ ID NO 2), NVHSAGAAGSRM (O) NFRPGVLSSRQLGLPGPPD VPDHAA YHPF (SEQ ID NO 3), where M (O) represents an oxidized methionine, and NVHSGSTFFKYYLQGAKIPKPEASFSPR (SEQ ID NO 4).
- the proteomic techniques used to identify biomarkers as disclosed in
- a method for testing a pregnant subject for potential preterm birth may include detecting the difference in concentration or amount of one or more biomarkers associated with preterm birth present in a biological sample compared to a control (i.e., the relative concentration or amount of the biomarker(s) in a pregnant woman that does not experience preterm birth).
- proteomic systems and methods can be used to identify and quantify the biomarkers.
- Such a method may include fractionating each of a plurality of biological samples to form a plurality of elutions, obtaining a plurality of mass spectra from each of the plurality of elutions at a plurality of elution times, and finding a molecular ion peak of interest that appears to be quantitatively different between biological samples.
- the method may additionally include identifying a mass spectrum reference peak corresponding to an endogenous reference molecule that is substantially consistent between biological samples, the endogenous reference molecule having an elution time and a mass to charge ratio that are substantially similar to the peak of interest, and compensating for non-biological variation for each biological sample across the plurality of elutions by normalizing the peak of interest to a mass spectrum peak of the endogenous reference molecule.
- the method may further include conducting collision-induced fragmentation studies that use each of a plurality of collision energies one run at a time and summing resulting pluralities of fragment ion mass spectra without averaging to form a single cumulative daughter fragment mass spectrum, and use the daughter fragment mass spectrum to establish amino acid sequence data which is then used in identifying a peptide corresponding to a peak of interest in the single aligned mass spectrum.
- a biological sample containing the biomarker(s) of interest can be fractionated to form a plurality of elutions, obtaining a plurality of mass spectra from each of the plurality of elutions at a plurality of elution times, and identifying a mass spectrum alignment peak corresponding to an endogenous alignment molecule that elutes in each of the plurality of elutions.
- the method may further include aligning the pluralities of mass spectra from each elution by aligning the mass spectrum alignment peak from each of the plurality of elutions, summing the pluralities of aligned mass spectra to form a single aligned mass spectrum, and identifying a peptide corresponding to a peak of interest in the single aligned mass spectrum.
- aligning the pluralities of mass spectra may further include visually aligning the pluralities of mass spectra.
- fractionating each of the plurality of biological molecules present in a plurality of biological samples may be accomplished by numerous methods, for example by capillary liquid chromatography (cLC). Specific methods and parameters for detecting and quantifying the biomarkers described herein are provided in the Examples.
- a peak is chosen as a reference if it can be shown to be quantitatively similar between comparison groups, elutes from the column in the same elution window as the candidate biomarker, is similar in its mass to charge ratio to that of the candidate biomarker, and is sufficiently abundant that every specimen will have a quantity that is more than 3 times the level of noise.
- the reference peaks described here are for quantitative correction of peak height or area that is related to specimen processing, chromatographic loading, ionization efficiency or instrumental sensitivity fluctuations but not due to biologic differences in peak quantity.
- This reference is termed an internal quantitative control.
- external controls can be used to facilitate the quantification of the biomarker.
- a compound in a known amount can be added to the biological sample so that a ratio of biomarker to control can be calculated. The ratio can then be compared to ratios from control samples in order to assess the risk of preterm birth.
- biomarkers SEQ ID NOS 1-4
- SEQ ID NO 1 SEQ ID NO 1
- the reference i.e., internal control
- the neutral parent mass was 2017.07 mass units
- the chromatographic elution time was 15.5 min.
- the elution time is provided as a fraction of its elution time relative to the internal time control (0.9968, i.e.
- the second internal quantitative control served as a reference for the two biomarkers SEQ ID NO 2 (m/z 857) and SEQ ID NO 3 (m/z 860).
- the m/z of the reference molecule was 842.39 in its +5 charge state with a neutral parent mass of 4206.07 mass units.
- the chromatographic elution time was approximately 15.8 min. However, given elution time variability its elution time is more appropriately described in relation to the elution times of the internal time control and the biomarker SEQ ID NO 2 (m/z 857).
- the internal quantitative control eluted a factor of 0.0159 times the elution time its own elution time after the elution of the internal time control (or a ratio of 1.0161 of the time control marker).
- the internal quantitative marker came off by a factor of 0.0539 times its own elution time after the biomarker (or a factor of 1.0700 of the elution time of the biomarker).
- the reference used for the biomarker SEQ ID NO 4 (m/z 795) had an m/z of 595.3 for its +1 charge state for the monoisotopic peak.
- the neutral parent mass was 594.32 mass units, and the chromatographic elution time was 18.8 min.
- the elution time is provided as a fraction of its elution time relative to the two internal time controls, the one that precedes it (1435.2) and the one that follows it (2009.95), i.e. it elutes 0.607 of the way through that interval specified by the two boundary time alignment markers.
- elution time retention time
- retention time can also be expressed as a function of internal time controls. This is determined by the relative position of the peak of interest between the time maker that precedes the biomarker and the time marker that follows the peak of interest. This determination is deemed an R f value.
- R f values are calculated as follows:
- R f (elution time of biomarker - elution time of preceding time marker)/(elution time of following time marker - elution time of preceding time marker).
- the second biomarker (SEQ ID NO 2), which is a peptide, has a mass ion peak (m/z) at 857, a mean mass of 4279.25 Daltons, a mean elution time of 17.20 + 2.04 minutes, and a R f value of 0.781 ⁇ 0.086.
- SEQ ID NO 2 is also referred to herein as "biomarker 2.”
- the third biomarker (SEQ ID NO 3), which is a peptide, has a mass ion peak (m/z) at 860, a mean mass of 4295.25 Daltons, a mean elution time of 16.13 + 1.97 minutes, and a R f value of 0.695 ⁇ 0.134.
- SEQ ID NO 3 is also referred to herein as "biomarker 3.”
- the fourth biomarker (SEQ ID NO 4), which is a peptide, has a mass ion peak (m/z) at 795, a mean mass of 3968.96 Daltons, a mean elution time of 15.52 + 0.15 minutes, and a R f value of 0.0252 ⁇ 0.021.
- SEQ ID NO 4 is also referred to herein as "biomarker 4.”
- the method includes detecting at least one biomarker described herein associated with a preterm birth in a biological sample from the subject, where the at least one biomarker has an amino acid sequence that is identical with or homologous to a sequence, a sequence represented by SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, or SEQ ID NO 4 and quantifying an abundance of the at least one biomarker in the biological sample.
- the abundance of the biomarker is measured following processing and separation as a function of a reference molecule also present in the biological sample that serves as an internal control.
- the term "abundance" as used herein represents the number of ions of a particular mass measured by the mass spectrometer in a given mass spectrum or the sum of the number of ions of a specific mass observed in several mass spectra representing the full elution interval. Normalization of biomarker abundance to this internal control reduces non-biological variation and improves the ability to utilize biomarkers in risk prediction. Stated another way, by choosing a molecule for a reference that is present in a biological sample in an abundance that is relatively constant from one subject to another, variability in the processing of biological samples can be corrected for, particularly when comparing runs conducted on different days that may be spread out over long periods of time. As such, the relative abundance of a biomarker may vary depending on the particular biomarker involved. A particular cutoff value may therefore be established for each biomarker/reference ratio such that ratios of the biomarker peak abundance to the reference peak abundance above or below a certain value may be predictive of a substantially increased risk of preterm birth.
- Testing for potential preterm birth may also be accomplished by comparing the abundance of one or more biomarkers in a biological sample from a subject with a known abundance of those same biomarkers that is indicative of a normal birth.
- preterm birth may occur if a subject has a measured abundance of SEQ ID NO 1 that is less than about 50% of the abundance of the control at least 22 weeks gestation.
- preterm birth may occur if a subject has a measured abundance of SEQ ID NO 1 that is less than about 30% of the abundance of the control at least 22 weeks gestation.
- preterm birth may occur if a subject has a measured abundance of SEQ ID NO 1 that is less than about 10% of the abundance of the control at least 22 weeks gestation
- preterm birth may occur if a subject has a measured abundance of SEQ ID NO 2 that is less than about 50% of the abundance of the control at least 22 weeks gestation. In another aspect, preterm birth may occur if a subject has a measured abundance of SEQ ID NO 2 that is less than about 30% of the abundance of the control at least 22 weeks gestation. In yet another aspect, preterm birth may occur if a subject has a measured abundance of SEQ ID NO 2 that is less than about 10% of the abundance of the control at least 22 weeks gestation.
- preterm birth may occur if a subject has a measured abundance of SEQ ID NO 3 that is less than about 55% of the abundance of the control at least 22 weeks gestation.
- preterm birth may be suggested if a subject has a measured abundance of SEQ ID NO 3 that is less than about 35% of the abundance of the control at least 22 weeks gestation.
- preterm birth may occur if a subject has a measured abundance of SEQ ID NO 3 that is less than about 15% of the abundance of the control at least 22 weeks gestation.
- preterm birth may occur if a subject has a measured abundance of SEQ ID NO 4 that is less than about 50% of the abundance of the control at least 22 weeks gestation. In another aspect, preterm birth may be suggested if a subject has a measured abundance of SEQ ID NO 4 that is less than about 30% of the abundance of the control at least 22 weeks gestation. In yet another aspect, preterm birth may occur if a subject has a measured abundance of SEQ ID NO 4 that is less than about 10% of the abundance of the control at least 22 weeks gestation.
- biomarker of interest Any type of biological sample that may contain a biomarker of interest may be screened, including such non-limiting examples as serum, plasma, blood, urine, cerebrospinal fluid, amniotic fluid, synovial fluid, cervical vaginal fluid, lavage fluid, tissue, and combinations thereof.
- biomarkers 1-4 are present in most pregnant women, many pregnant women that go on to experience preterm birth had lower blood serum concentrations of one or more of these biological molecules during pregnancy as compared to women that had normal births. For example, biomarkers 1-4 either alone or collectively were less abundant in PTB cases than in the controls. Thus, a comparison of the abundance of one or more of these biomarkers in a biological sample from a subject against a known control concentration from subjects that did not experience preterm birth, or against a known biomarker concentration from the subject being tested, may be predictive of preterm birth. Those subjects having a higher or lower abundance of one or more of these biomarkers may have an increased risk of preterm birth, and can thus be identified early enough to allow appropriate treatment. The abundance of a particular biomarker in predicting preterm birth is described in detail below.
- log ratios were taken. For example, the log ratio of log
- 676.7/673.36 (biomarker I/reference peak) yielded a mean control of 0.579+0.101 and a mean PTB of -0.015 ⁇ 0.090.
- the log ratio of log 856.8/842.8 (biomarker 2/ reference peak) yielded a mean control (subjects who did not experience preterm birth) of 0.231+0.102 and a mean PTB (subjects at risk for preterm birth) of -0.149 ⁇ 0.095 (Table 4 in Examples). Referring to Table 4 in the Examples, the log ratios of the other biomarkers were calculated.
- the log ratio of log 860.0/842.8 (biomarker 3/ reference peak) yielded a mean control of 0.201+0.096 and a mean PTB of -0.204 ⁇ 0.088.
- the log ratio of log 794.8/595.3 (biomarker Al reference peak) yielded a mean control of 0.582+0.637 and a mean PTB of 0.274+0.656.
- a subject at risk for preterm birth would most likely exhibit an decrease in biomarker 1, a decrease in biomarker 2, a decrease in biomarker 3, and a decrease in biomarker 4 either individually or collectively.
- it may be predictive of a substantially increased risk of preterm birth if the ratio of the abundance of SEQ ID NO 1 (m/z 677) to the abundance of a reference molecule at m/z 673 is measured to be less than about 1.0 at least 22 weeks gestation. In another aspect, it may be predictive of a substantially increased risk of preterm birth if the ratio of the abundance of SEQ ID NO 1 (m/z 677) to the abundance of a reference molecule at m/z 673 is measured to be less than about 0.8 at least 22 weeks gestation.
- it may be predictive of a substantially increased risk of preterm birth if the ratio of the abundance of SEQ ID NO 1 (m/z 677) to the abundance of a reference molecule at m/z 673 is measured to be less than about 0.6 at least 22 weeks gestation.
- it may be predictive of a substantially increased risk of preterm birth if the ratio of the abundance of SEQ ID NO 2 (m/z 857) to the abundance of a reference molecule at m/z 843 is measured to be less than about 0.44 at least 22 weeks gestation.
- it may be predictive of a substantially increased risk of preterm birth if the ratio of the abundance of SEQ ID NO 3 (m/z 860) to the abundance of a reference molecule at m/z 843 is measured to be less than about 0.6 at least 22 weeks gestation. In another aspect, it may be predictive of a substantially increased risk of preterm birth if the ratio of the abundance of SEQ ID NO 3 (m/z 860) to the abundance of a reference molecule at m/z 843 is measured to be less than about 0.4 at least 22 weeks gestation.
- it may be predictive of a substantially increased risk of preterm birth if the ratio of the abundance of SEQ ID NO 3 (m/z 860) to the abundance of a reference molecule at m/z 843 is measured to be less than about 0.2 at least 22 weeks gestation.
- it may be predictive of a substantially increased risk of preterm birth if the ratio of the abundance of SEQ ID 4 (m/z 795) to the abundance of reference molecule at m/z 595 is measure to be less than about 0.6 at least 22 weeks gestation. In another aspect, it may be predictive of a substantially increased risk of preterm birth if the ratio of the abundance of SEQ ID NO 4 (m/z 795) to the abundance of a reference molecule at m/z 595 is measured to be less than about 0.4 at least 22 weeks gestation.
- it may be predictive of a substantially increased risk of preterm birth if the ratio of the abundance of SEQ ID NO 4 (m/z 795) to the abundance of a reference molecule at m/z 595 is measured to be less than about 0.2 at least 22 weeks gestation.
- the log ratios calculated above may be used to statistically predict the risk of pregnant women at risk of experiencing preterm birth.
- One common measure of the predictive power of a biomarker is its sensitivity and specificity.
- "Sensitivity” as used herein is a statistical term defined as the true positive rate (e.g., the percentage of pregnant women who later experience preterm birth that are correctly identified by the biomarker).
- the term "specificity” as used herein is defined as the true negative rate (e.g., the percentage of pregnant women with uncomplicated pregnancies correctly identified).
- the range of values for the specific biomarker are considered from lowest to highest and at each point the percent of subjects correctly identified as positive and at that same point the percent of controls incorrectly identified as positive.
- the range of values for the specific biomarker may be calculated by taking the actual quantitative value from the lowest to highest for a specific data set. This is termed a receiver operator curve (ROC).
- ROC receiver operator curve
- the false positive rate can be limited to 20%, which is commonly considered the maximum value tolerated for a clinical test.
- the false positive rate i.e., the percentage of women with uncomplicated pregnancies identified by the biomarker at risk for experiencing preterm birth
- the threshold at a false positive rate is calculated from the true negative rate subtracted from 100%.
- the threshold at a false positive rate of 20% or less which is equivalent to a specificity of 80% or higher, determines the threshold used to determine whether someone is at risk or is not at risk.
- a threshold for each of the four log ratios was determined for the identification of subjects at risk for preterm birth.
- the threshold for each was calculated such that there would be a specificity (a true negative rate) of 80% or more, which is the same as a false positive rate of no more than 20%.
- the four ratios independently provided sensitivity (true positive) and specificity (true negative) rates (Table 5).
- the ratio of biomarker I/reference peak provided the greatest sensitivity (65%) and specificity (85%) with respect to predicting preterm birth.
- biomarkers identified herein are powerful tools in predicting the risk of preterm birth.
- the biomarkers described herein can be predictive of preterm birth.
- the predictive value of a test for preterm birth may be improved by screening for and quantifying multiple biomarkers.
- a biological sample from a subject may be screened for at least two biomarkers having amino acid sequences that are identical with or homologous to sequences represented by SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, or SEQ ID NO 4.
- a biological sample from a subject may be screened for at least three biomarkers having sequences that are identical with or homologous to sequences represented by SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, and SEQ ID NO 4.
- the predictive value may also vary depending on the type of test or assay being utilized, some of which are discussed in more detail herein.
- biomarkers By assessing the presence and amount of multiple biomarkers (i.e., two or more), it is possible to produce fingerprints useful in predicting preterm birth. For example, the determination and quantification of at least two biomarkers can increase the predictive value of the methods described herein. Although fewer women who develop PTB may be included, it is more indicative of the risk of experiencing preterm birth.
- Any type of biological sample that may contain a peptide of interest may be screened, including such non-limiting examples as serum, plasma, blood, urine, cerebrospinal fluid, amniotic fluid, synovial fluid, cervical vaginal fluid, lavage fluid, tissue, and combinations thereof. In one aspect, however, it may be convenient to screen for peptides in a serum sample obtained from a subject. In another aspect, it may be convenient to screen for peptides in a blood sample obtained from the subject.
- an isolated peptide may have an amino acid sequence that is identical with or homologous to a sequence represented by SEQ ID NO 1 SEQ ID NO 2, SEQ ID NO 3, or SEQ ID NO 4.
- Peptide synthesis is well known in the art, and it is understood that one of ordinary skill in the art would be capable of using a variety of techniques to synthesize the peptides disclosed herein once in possession of the peptide sequences.
- Such techniques may include, without limitation, liquid- phase syntheses and solid-phase synthesis methods, as well as various methods of chemical ligation, such as prior thiol capture, native chemical ligation, expressed protein ligation, acyl initiated capture, and Staudinger ligation methods to name a few. Additionally, peptides may also be synthesized using recombinant DNA technologies.
- the proteomics techniques described above can be used to identify and quantify the biomarkers; however, other methods capable of detecting and/or quantifying the biomarker in a biological sample according can be used herein.
- One potential type of peptide assay includes immunoassays. Numerous immunoassay protocols are known that utilize antibodies to screen a biological sample for specific peptides, including homogenous, and nonhomogenous, as well as competitive and noncompetitive methods. For example, such techniques may include the use of solid supports, immunoprecipitation, etc. Generally, however, immunoassays for the detection of peptides often involve using labeled antibodies. Such labels may include any type of material known, including fluorescent labels, chemiluminescent labels, radioactive labels, enzyme labels, etc.
- antibodies that are specifically immunoreactive to the biomarkers described herein can be used.
- an antibody that is immunologically specific to a peptide having an amino acid sequence that consists of SEQ ID NO 1 is provided.
- an antibody that is immunologically specific to a peptide having an amino acid sequence that consists of SEQ ID NO 2 is provided.
- an antibody that is immunologically specific to a peptide having an amino acid sequence that consists of SEQ ID NO 3 is provided.
- an antibody that is immunologically specific to a peptide having an amino acid sequence that consists of SEQ ID NO 4 is provided.
- the antibodies may be polyclonal, monoclonal, or recombinant, and they may be produced by any method known in the art. Antibody fragments are also considered to be within the scope of the present invention.
- the antibodies according to aspects of the present invention can be from any animal origin including birds and mammals. In one aspect, for example, antibodies may be derived from human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, chicken, etc.
- polyclonal antibodies may be utilized to detect and quantify one or more biomarkers described herein in a biological sample to evaluate the risk of preterm birth. Polyclonal antibodies can be produced by various procedures that are well known to those of ordinary skill in the art.
- polyclonal antibodies may be produced in an in vivo host animal such as a rabbit, a rat, a mouse, a sheep, a goat, etc.
- the host animal is immunized with either free or carrier-coupled peptides, for example, by intraperitoneal and/or intradermal injection.
- Injection material is typically an emulsion containing about 100 ⁇ g of peptide or carrier protein.
- various adjuvants may be used to increase the immunological response.
- adjuvants may include, without limitation, Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, poly anions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as bacille Calmette- Guerin (BCG) and corynebacterium parvum. These and other adjuvants are well known in the art.
- BCG Bacille Calmette- Guerin
- Several booster injections may be required, in some cases at intervals of about two weeks, to provide a useful titer of antibody which can be detected.
- the titer of antibodies in serum from an immunized animal can be increased by selection of antibodies, e.g., by adsorption of the peptide onto a solid support and elution of the selected antibodies according to methods well known in the art.
- monoclonal antibodies may be utilized to detect and quantify one or more biomarkers in a biological sample to evaluate the risk of preterm birth.
- a monoclonal antibody refers to an antibody that recognizes only one species of antigen. These antibodies are generated by daughter cells of a single antibody-producing hybridoma.
- a monoclonal antibody typically displays a single binding affinity for any epitope with which it immunoreacts.
- a monoclonal antibody may contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different epitope, e.g., a bispecific monoclonal antibody.
- Monoclonal antibodies may be obtained by a variety of methods known to those skilled in the art.
- the antibodies useful herein can be monospecific or multispecific (e.g., bispecific, trispecific, or of greater multispecificity).
- Multispecific antibodies can be specific for different epitopes of a peptide, or they can be specific for both a peptide of interest, and a heterologous epitope, such as a heterologous peptide or solid support material.
- antibodies can also be prepared from any region of the biomarkers described herein.
- monoclonal antibodies can be prepared using well- established methods.
- monoclonal antibodies are prepared using hybridoma technology.
- an immunizing agent e.g., a peptide according to aspects of the present invention
- lymphocytes may be immunized in vitro.
- the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell.
- Immortalized cell lines are often transformed mammalian cells, particularly myeloma cells of rodent, rabbit, bovine and human origin. Often rat or mouse myeloma cell lines are employed.
- the hybridoma cells may be cultured in a suitable culture medium that may contain one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
- a suitable culture medium may contain one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
- the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT)
- HGPRT or HPRT the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium") to inhibit growth of HGPRT-deficient cells.
- the culture medium in which the hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies.
- the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art.
- RIA radioimmunoassay
- ELISA enzyme-linked immunoabsorbent assay
- the monoclonal antibodies may be isolated or purified from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, affinity chromatography, etc.
- Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567, which is hereby incorporated by reference. Other methods for generating antibodies known to those of skill in the art are considered to be within the scope of the present invention. Accordingly, in one aspect a method for testing a pregnant subject for a potential preterm birth is provided.
- Such a method may include obtaining a biological sample from the subject, contacting the biological sample with an at least one antibody under conditions that allow formation of antibody-antigen complexes, the at least one antibody being immunologically specific to at least one peptide having an amino acid sequence that is identical or homologous to one of the biomarkers described herein, and assaying for formation of the antibody-antigen complexes to detect and quantify the at least one biomarker in the biological sample.
- the presence and amount of the biomarker of interest in the biological sample would provide an indication of the risk of preterm birth.
- immunoassays are known that are capable of detecting and/or quantifying a peptide in a biological sample.
- the immunoassay may be a competitive assay.
- a labeled peptide having the sequence of the peptide being tested for is contacted with an antibody specific for at least a portion of the peptide sequence to allow the formation of an antibody- antigen (or peptide) complex.
- a biological sample is added to the peptide/antibody mixture to allow any peptide of interest present in the biological sample to compete with the labeled peptide, resulting in a decrease in the strength of the label.
- Competitive assays may include one-step or two-step protocols, which are well known in the art.
- the immunoassay may be a noncompetitive, or sandwich assay. Such assays generally provide higher levels of assay sensitivity and specificity.
- Noncompetitive assay formats may also utilize one- or two-step protocols.
- an assay includes antibodies immobilized on a physical support, where the immobilized antibodies are immunologically specific to the peptide (i.e., biomarker) of interest.
- the biological sample is added to the support along with labeled antibody that is also immunologically specific to the peptide of interest.
- Peptide present in the biological sample will bind to the immobilized antibody along the support.
- Labeled antibody also binds to the peptide of interest, and thus is also immobilized to the physical substrate via the peptide and the immobilized antibody.
- the label on the labeled antibody can then be detected to quantify the amount of biomarker in the biological sample and compared to a control (i.e., a pregnant subject that does not experience preterm birth).
- a control i.e., a pregnant subject that does not experience preterm birth.
- non-immobilized labeled antibody can be washed away prior to detection of the label.
- the strength of the label is proportional to the amount to amount of biomarker present in the biological sample.
- Such a substrate can include any suitable substrate for immobilization of a detection material, such as an antibody or an antibody anchor.
- a suitable substrate may include any solid support, such as any solid organic, biopolymer, or inorganic support material that is capable of forming bonds
- organic solid support materials may include, without limitation, polymers such as polystyrene, nylon, phenol-formaldehyde resins, acrylic copolymers such as polyacrylamide, etc.
- biopolymer support materials may include, without limitation, cellulose, polydextrans, agarose, collagen, chitin, etc.
- inorganic support materials may include, without limitations, glass beads (porous and nonporous), stainless steel, metal oxides including porous ceramics such as Zr ⁇ 2 , Ti ⁇ 2 , AI 2 O 3 , and NiO, sand, etc.
- Such specific assay methods may include protocols such as radioimmunoassays (RIA), enzyme immunoassays (EIA), enzyme linked immunosorbent assays (ELISA), fluorescence immunoassays (FIA), fluorescence polarization immunoassays (FPIA), nephelometric inhibition immunoassays (NIA), microparticle enzyme immunoassays (MEIA), chemiluminescent magnetic immunoassays (CMIA), etc.
- RIA radioimmunoassays
- EIA enzyme immunoassays
- ELISA enzyme linked immunosorbent assays
- FPIA fluorescence immunoassays
- FPIA fluorescence polarization immunoassays
- NIA nephelometric inhibition immunoassays
- MEIA microparticle enzyme immunoassays
- CLIA chemiluminescent magnetic immunoassays
- Various detectable labels may be coupled to the antibodies according to aspects
- Appropriate labels may include, without limitation, radionuclides (e.g., n 1, 131 1, 3 S, 3 H, 32 P, etc.), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, luciferase, beta-glactosidase, etc.), fluorescent moieties or proteins (e.g., fluorescein, rhodamine, phycoerythrin, GFP, BFP, etc.), or luminescent moieties (e.g., Qdot® nanoparticles supplied by the QUANTUM DOT CORP., Palo Alto, Calif.).
- radionuclides e.g., n 1, 131 1, 3 S, 3 H, 32 P, etc.
- enzymes e.g., alkaline phosphatase, horseradish peroxidase, luciferase, beta-glactosidase, etc.
- MS mass spectrometry
- kits for testing a biological sample from a pregnant subject to evaluate the risk of preterm birth Such kits may be employed by hospitals, clinics, reference laboratories, doctor's offices, etc. to help make medical decisions and, if necessary, provide available therapies or interventions. Additionally, such kits may also allow the diagnosis, prognosis, or risk assessment of other medical conditions associated with preterm birth.
- kits for testing a pregnant subject for potential preterm birth may include at least one monoclonal antibody capable of selectively binding to at least one biomarker having an amino acid sequence SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, and SEQ ID NO 4, and an indicator functionally associated with the antibody to assay formation of an antibody-antigen complex between the at least one monoclonal antibody and the at least one biomarker that can be used to quantify the concentration of the at least one biomarker.
- the kit may further include any reagents necessary or beneficial for the particular testing assay being utilized.
- the kit may contain any means of detecting and quantifying biomarkers in the biological sample, and the contents of the kit may necessarily vary depending on the type of detection assay being used.
- the kit can include antibodies for binding peptides of interest, or fragments thereof, solid substrates, additional antibodies for detection of antibody-antigen complexes, etc. As has been suggested, antibodies or antibody fragments may be present in free form or immobilized to a substrate such as a plastic dish, a test tube, a test rod, beads, etc.
- the kit can also include suitable reagents for the detection of and/or for the labeling of positive or negative controls, wash solutions, dilution buffers and the like, as well as instructions.
- reaction conditions e.g., component concentrations, desired solvents, solvent mixtures, temperatures, pressures and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions.
- Acetonitrile Precipitation Two volumes of HPLC grade acetonitrile (400 ⁇ L) were added to 200 ⁇ L of serum, vortexed vigorously for 5 sec and allowed to stand at room temperature for 30 min. Samples from (Serum collection) were then centrifuged for 10 min at 12,000 rpm in and IEC Micromax RF centrifuge (Thermo Fisher Scientific, Waltham, MA) at room temperature. An aliquot of supernatant was then transferred to a microcentrifuge tube containing 300 ⁇ L HPLC grade water. The sample was vortexed briefly to mix the solution, which was then lyophilized to -200 ⁇ L in a Labconco CentriVap Concentrator (Labconco Corporation, Kansas City, MO).
- the volume of water added prior to lyophilization aids in the complete removal of acetonitrile from the solution. This is necessary because acetonitrile is incompatible with the assay used to determine protein concentration.
- Supernatant protein concentration were determined using a Bio-Rad microtiter plate protein assay performed according to manufacturer's instructions. An aliquot containing 4 ⁇ g of protein was transferred to a new microcentrifuge tube and lyophilized to near dryness. Samples were brought up to 20 ⁇ L with HPLC water and then acidified using 20 ⁇ L 88% formic acid.
- Acetonitrile treated (post precipitation) serum samples (40 ⁇ L) were loaded into 250 ⁇ L conical polypropylene vials closed with polypropylene snap caps having septa (Dionex Corporation, Sunnyvale, CA), and placed into a FAMOS ® autosampler 48 well plate kept at 4 0 C.
- the FAMOS ® autosampler injected 5 ⁇ L of each serum sample onto a liquid chromatography guard column using HPLC water acidified with 0.1% formic acid at a flow rate of 40 ⁇ L/min. Salts and other impurities were washed off of the guard column with the acidified water.
- the FAMOS ® autosampler draws up three times the volume of what is loaded onto the column, it was necessary to inject the samples by hand when sample volume was limited. This was accomplished by injecting 10 ⁇ L volume sample onto a blank loop upstream of the guard column and programming the FAMOS ® autosampler to inject a 10 ⁇ L sample of HPLC water in place of the sample. The serum sample was loaded onto the guard column an desalted as if it had been loaded from the conical vials.
- Capillary liquid chromatography was performed to fractionate the sample.
- Capillary LC uses a 1 mm (16.2 ⁇ L) microbore guard column (Upchurch Scientific, Oak Harbor, WA) and a 15 cm x 250 ⁇ m i.d. capillary column assembled in-house. The guard column was dry-packed and the capillary column was slurry packed using POROS Rl reversed-phase media (Applied Biosystems, Framingham, MA). Column equilibration and chromatographic separation were performed using an aqueous phase (98% HPLC grade H 2 O, 2% acetonitrile, 01.% formic acid) and an organic phase (2% HPLC H 2 O, 98% acetonitrile, 0.1% formic acid).
- MS calibrations were performed using an external control daily prior to running samples. If needed, settings were adjusted to optimize signal to noise ratio and to maximize sensitivity.
- the cLC system was coupled directly to a mass spectrometer. Effluent from the capillary column was directed into a QSTAR Pulsar I quadrupole orthogonal time-of-flight mass spectrometer through an IonSpray source (Applied Biosystems). Data was collected for m/z 500 to 2500 beginning at 5 min and ending at 55 min. The delay in start time was programmed because, with a flow rate of 5 ⁇ L/min, it takes over 5 min for sample to get from the guard column to the mass spectrometer, and thus no useful data can be obtained before 5 min. Data collection, processing and preliminary formatting are accomplished using the Analyst QS ® software package with BioAnalyst add-ons (Applied Biosystems).
- Mass spectra were obtained every 1 sec throughout the entire cLC elution period for each specimen from 5 minutes to 55 minutes.
- the elution profile of the cLC fractionated protein depleted serum of each subject reported as the total ion chromatogram, was inspected to insure that it was consistent with previously run human sera.
- Specimens having an overall abundance less than 50% of normal or greater than 200% normal or lacking the characteristic series of three broad ion intense regions were rerun or omitted if there was inadequate specimen to redo the analysis.
- Analyst® the software program supporting the Q-Star (q-TOF) mass spectrometer, allows for compilation of 16 individual liquid chromatographic runs and the comparison of mass spectra within those runs at similar elution times. Ten two-minute windows were established as described above over the 20 minute period of useful elution to allow data file size to remain manageable. The two minute windows were aligned as is also described above. Of the 10 two minute elution intervals, the first to be analyzed was the second two-minute window, chosen because there were typically more peptide species present.
- Peptides were identified by the characteristic appearance of their multiply charged states, which appear as a well defined cluster of peaks having a Gaussian shape with the individual peaks being separated by less than 1 mass/charge unit rather than a single peak or peaks separated by 1 mass/charge unit.
- Groups of 8 subjects experiencing PTB and 8 subjects from controls (no PTB) were color coded and overlaid. The data was then visually inspected and molecular species that seemed to be dominated by one color were recorded. The software used was limited to visualizing only 16 samples. For a sampling size larger than 16, multiple comparisons of data sets were made. For a compound to be considered further, the same apparent difference between the two groups was needed to be observed in at least two thirds of the data sets.
- Molecules that appeared to be different between the two study groups were then individually inspected. These candidate species were all peptides. Prior to extracting quantitative data, the mass spectrum was examined to insure that the peptide peak had the same m/z and also represented the same charge state to further insure that the same peptide was being considered. Additionally, a second nearby peak, which did not demonstrate differences in abundance between the two groups, was selected as a reference. This peak was used to normalize the candidate peak of interest and correct for variability in specimen processing, specimen loading and ionization efficiencies .
- the molecular species are then 'extracted' by the Analyst® software to determine the peak maxima of the individual molecular species in each individual run. This feature did not limit inspection of a specific m/z to a two minute elution window and consequently the peak used to align cLC elution time may be used additionally to insure the location in the elution profile was the same and hence insure that the same molecular species was selected each time.
- the peak height for each molecular species was considered a reasonable estimate of its abundance.
- the abundance of each candidate compound was tabulated and the calculated value of each candidate species was ratioed to the nearby reference species. Because a single species was being considered, univariate statistical analysis was employed in evaluating possible differences in this peptide's abundance between the two groups.
- biomarker candidates were identified visually in an initial process where multiple mass spectra were overlaid with PTB cases and controls each assigned a color. Those peaks that appear to be predominantly one color were studied further. The individual spectra were then submitted to peak height determination by the computer equipped with Analyst ® software (Applied Biosystems) which is the operating system for the QqTOF mass spectrometer (Applied Biosystems). The quantity of the biomarkers was then tabulated. In addition, a second peak that occurred in the same time window, which was not quantitatively different between cases and controls, was also selected. This represented a endogenous control to allow for reduction of non-biologic variability. This was accomplished by dividing the quantity of the candidate peak by the quantity of the endogenous control. The magnitude of the ratio for each specimen was recorded and statistical differences were sought using a Student's t test comparing PTB cases and controls.
- the R f values were more reliable than the actual elution times. Elution times may vary with new columns or with the altered performance of an existing column with fouling, but the R f was not altered by these changes.
- the R f values of the five biomarkers are provided in Table 3.
- a threshold for each of the four log ratios in Table 4 was determined in order to identify subjects at risk of developing PTB. The threshold for each was calculated such that there would be a specificity (a true negative rate) of 80% or more. As stated, this is the same as a false positive rate of no more than 20%. Using these mathematically determined thresholds the four ratios independently provided the following sensitivity (true positive) and specificity (true negative) rates as summarized in Table 5.
- Sensitivity is a statistical term defined as the true positive rate or specifically in this case the percentage of pregnant women who later develop PTB that are correctly identified by the biomarker.
- the specificity is defined as the true negative rate or in this case the percentage of pregnant women with uncomplicated pregnancies correctly identified.
- a numeric threshold must be established.
- numeric value typically the range of values for the biomarker are considered from lowest to highest and at each point the percent of subjects correctly identified as positive and at that same point the percent of controls incorrectly identified as positive. This is termed a receiver operator curve (ROC).
- ROC receiver operator curve
- the false positive rate is limited to 20%. This is commonly considered the maximum tolerated for a clinical test.
- the false positive rate (the percentage of women with uncomplicated pregnancies, the control group, identified by the biomarker as at risk for later PTB) is calculated from the true negative rate being subtracted from 100%. Whatever the threshold is at a false positive rate of 20% or less (which is equivalent to a specificity of 80% or higher) determines the threshold used to determine whether someone is at risk or is not at risk.
- a threshold for each of the four ratios was determined that allowed for the identification of subjects at risk of later PTB. The threshold for each was calculated such that there would be a specificity (a true negative rate) of 80% or more. As stated this is the same as a false positive rate no more than 20%. Using these mathematically determined thresholds the four ratios independently provided the following sensitivity (true positive) and specificity (true negative) rates as summarized in Table 5. Combinations of peaks did not significantly improve on the ability of the peak at 677 to predict later PTB .
- inter- alpha trypsin inhibitors a protein super family termed the inter- alpha trypsin inhibitors. More specifically, the peptides appear to be derived from two different proteins that are currently considered isoforms of inter-alpha trypsin inhibitor heavy chain 4, isoform 1 (ITIH4-1) and isoform 2 (ITIH4-2). The two isoforms have some sequence homology but also have sections of amino acids that are not found in the other. The two isoforms do not simply represent a truncation one of the other.
- ELISA Assay I The following ELISA assay can be utilized to detect and quantify a biomarker of interest in a biological sample.
- a first antibody immunologically specific to the peptide of interest is adsorbed onto the surface of a 96 well microtiter plate. 25 microliters of serum or standard of known, graded concentration of the peptide of interest is added to individual wells. The serum is incubated with the first antibody for 30 min. The first antibody coated on the well surface binds the antigen, immobilizing it. 200 micoliters of a second solution containing a second antibody that is also immunologically specific to the antigen is added to each well. The second antibody has been labeled with a marker such as horseradish peroxidase or a chemiluminescent precursor.
- a marker such as horseradish peroxidase or a chemiluminescent precursor.
- the wells are incubated for 30 minutes to allow binding of the second antibody to the antigen-first antibody complex to form an antibody-antigen-antibody 'sandwich' which is itself bound to the well surface.
- the well is then carefully and fully washed to remove any unbound second antibody.
- a solution containing a specific substrate to the second antibody label is added.
- horseradish peroxidase a color change occurs in the well corresponding to the amount of bound second antibody.
- the chemiluminescent marker the substrate is converted from a non- chemiluminescing molecular species into a chemiluminescent product that glows.
- the light emitted by the product is proportional to the amount of antigen present in the well and is measured by a 'plate reader,' a specialized spectrometer that measures the light emitted at a specific wavelength and records its intensity.
- the following ELISA assay can be utilized to detect and quantify a biomarker of interest in a biological sample.
- This assay is similar to the ELISA Assay I, with the exception that the second antibody is labeled with a biotin molecule.
- a solution containing streptavidin bound to horseradish peroxidase is added to the wells to allow reaction with the biotin molecule.
- an uncolored substrate is converted to a colored product.
- the intensity of the color measured as its absorbance of light of a particular wavelength, is proportional to the amount of antigen present in the well.
- the concentration of an unknown can be estimated by comparison of its absorbance to a plot of absorbance versus concentration of series of calibrating standards of known, graded concentrations of antigen.
Abstract
Description
Claims
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CA2694112A CA2694112A1 (en) | 2007-07-20 | 2008-07-17 | Identification and quantification of biomarkers for evaluating the risk of preterm birth |
US12/669,343 US10247736B2 (en) | 2007-07-20 | 2008-07-17 | Identification and quantification of biomarkers for evaluating the risk of preterm birth |
AU2008279355A AU2008279355A1 (en) | 2007-07-20 | 2008-07-17 | Identification and quantification of biomarkers for evaluating the risk of preterm birth |
EP08796232A EP2179288A4 (en) | 2007-07-20 | 2008-07-17 | Identification and quantification of biomarkers for evaluating the risk of preterm birth |
JP2010518302A JP5363481B2 (en) | 2007-07-20 | 2008-07-17 | Biomarker identification and quantification to assess the risk of preterm birth |
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WO2011022526A1 (en) * | 2009-08-20 | 2011-02-24 | University Of Utah Research Foundation | Identification and quantification of biomarkers for evaluating the risk of preterm birth |
US10247736B2 (en) | 2007-07-20 | 2019-04-02 | Brigham Young University | Identification and quantification of biomarkers for evaluating the risk of preterm birth |
US10392665B2 (en) | 2015-06-19 | 2019-08-27 | Sera Prognostics, Inc. | Biomarker pairs for predicting preterm birth |
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US11662351B2 (en) | 2017-08-18 | 2023-05-30 | Sera Prognostics, Inc. | Pregnancy clock proteins for predicting due date and time to birth |
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US10247736B2 (en) | 2007-07-20 | 2019-04-02 | Brigham Young University | Identification and quantification of biomarkers for evaluating the risk of preterm birth |
WO2011022526A1 (en) * | 2009-08-20 | 2011-02-24 | University Of Utah Research Foundation | Identification and quantification of biomarkers for evaluating the risk of preterm birth |
EP2467716A1 (en) * | 2009-08-20 | 2012-06-27 | University of Utah Research Foundation | Identification and quantification of biomarkers for evaluating the risk of preterm birth |
CN102822669A (en) * | 2009-08-20 | 2012-12-12 | 犹他州大学研究基金会 | Identification and quantification of biomarkers for evaluating the risk of preterm birth |
JP2013502584A (en) * | 2009-08-20 | 2013-01-24 | ユニバーシティ オブ ユタ リサーチ ファウンデーション | Identification and quantification of biomarkers for risk assessment of preterm birth |
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CN102822669B (en) * | 2009-08-20 | 2015-02-25 | 犹他州大学研究基金会 | Identification and quantification of biomarkers for evaluating the risk of preterm birth |
EP2972308B1 (en) * | 2013-03-15 | 2020-09-16 | Sera Prognostics, Inc. | Biomarkers and methods for predicting preterm birth |
US10392665B2 (en) | 2015-06-19 | 2019-08-27 | Sera Prognostics, Inc. | Biomarker pairs for predicting preterm birth |
US10961584B2 (en) | 2015-06-19 | 2021-03-30 | Sera Prognostics, Inc. | Biomarker pairs for predicting preterm birth |
US11662351B2 (en) | 2017-08-18 | 2023-05-30 | Sera Prognostics, Inc. | Pregnancy clock proteins for predicting due date and time to birth |
Also Published As
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US10247736B2 (en) | 2019-04-02 |
WO2009014987A3 (en) | 2009-03-05 |
KR20100058496A (en) | 2010-06-03 |
EP2179288A2 (en) | 2010-04-28 |
CA2694112A1 (en) | 2009-01-29 |
US20100297679A1 (en) | 2010-11-25 |
EP2179288A4 (en) | 2010-07-21 |
AU2008279355A1 (en) | 2009-01-29 |
JP2010534341A (en) | 2010-11-04 |
JP5363481B2 (en) | 2013-12-11 |
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