A portion of the work set forth herein was supported by grants NIHGMS-48829 and NSF MCB-9406656 and by the Medical University of South Carolina.

1. INTRODUCTION

The present invention relates to methods for determining predisposition towards disease, diagnosing diseases or disorders, monitoring the ablation of cancers and monitoring relaxin-related therapeutic drug programs. The present invention is based, in part, on the observation that elevated relaxin levels correlate with the occurrence, prognosis, and clinical course of various human malignancies, as well as pregnancy. The present invention further relates to assays and kits for the determination of relaxin levels in body fluids or tissue samples, and uses thereof.

2. BACKGROUND OF THE INVENTION

The peptide hormone relaxin (RLX) is a member of the insulin-related family of hormones which includes insulin (Blundell and Humbel, 1980, Nature 287:781- 787) , insulin-like-growth factor-I and -II (Blundell and Humbel, 1980, Nature 287:781-787) , bombyxin (Jhoti et al . , 1987, FEBS Lett . 219:419-425) . and molluscan insulin-related peptide (Smit et al . , 1989, Nature 331:535-538) .

Generally, each of the peptides of the insulin- related family share primary and structural homology. For example, with exception of the molluscan insulin- related peptide, these peptides share the structural feature of three disulfide bonds at corresponding locations. Ryle, et al . , 1955, Biochem . J. 60:541-

556; Schwabe et al . , 1977, Science 197:914-917; Iwai et al . , 1989, J. Biochem . (Tokyo) 106:949-951: Nagasawa et al . , 1987, Peptide Chemistry 123-126. Likewise, with exception of the single-chain insulin- like growth factors, the hormones of this family consist of two polypeptide chains linked by two interchain disulfide bonds. Notwithstanding their similar structure, the peptides comprising the insulin-related family are functionally distinct. Cooper et al. , 1989, Prog. Growth Factor Res . .1:99- 105.

Relaxin, comprising approximately 6000 daltons, has been purified from a variety of species including porcine, murine, equine, shark, tiger, rat, dogfish and human. In the human, two genes encoding relaxin have been identified, designated as (HI) and (H2) . Hudson et al. , 1983, Nature 301:628-631; Hudson et al . , 1984, EMBO J . 2:2333-2339; U.S. Patents Nos. 4,758,516 and 4,871,670. Only one of the genes, the gene encoding relaxin (H2) , has been found to be transcribed in the ovary. It is still unclear whether the (HI) gene is expressed at another tissue site, or whether it represents a non-transcribed pseudo-gene. Methods for making relaxin are described in U.S. Patents No. 4,835,251 and in co-pending U.S. Serial Nos. 07/908,766 (PCT US90/02085) and 08/080,354 (PCT US94/0699) .

Although most abundantly found in the female during pregnancy in the corpora lutea, relaxin has also been detected in the non-pregnant female as well as in the male. For example, relaxin has also been isolated from or identified in the placenta (Stewart and Papkoff, 1986, Endocrinology 119:1093-1099) and seminal fluid (Sarosi et al . , 1983, Endocrinology 112:1860-1861; Weiss, 1989, Biol . Re prod. 40:197-

PATENT

The effects of RLX (H2) on the uterus, cervix and pubic symphysis (i.e., the mediation of connective tissues to obtain the required changes in organ structure during pregnancy and parturition) has been documented. See e .g. , Hisaw, 1926, Proceedings of the Society for Experimental Biology and Medicine 23:661- 663; McLennan et al . , 1981, Lancet i:220-223; Bryant-Greenwood, 1982, Endocrine Reviews 3_:62-90. In vitro, relaxin also has been shown to have a stimulatory effect on sperm motility (for reviews, see , Weiss, 1989, Biol . Reprod . 40:197-200) .

Several assays have been developed to measure RLX activity. For example, bioassays have been used for the measurement of active relaxin during pregnancy and non-pregnancy. Steinetz et al . , 1960, Endocrinology j7:102-115; Sarosi et al . , 1983, American Journal of Obstetrics and Gynecology 145:402-405. These bioassays, however, generally lack sensitivity, specificity and precision (Sherwood, 1979, Methods of Hormone Radioimmunoassay edn 2.:875-886), thus necessitating the development of sensitive and specific immunoassays. Sherwood et al . , 1975, Endocrinology 107:691-696; O'Bryne and Steinetz, 1976, Proceedings of the Society for Experimental Biology and Medicine 152:272-276: Loumaye et al . , 1978, Gynecologic and Obsteric Investigation .9:262- 267; Sherwood and Crnekovic, 1979, Endocrinology 104:893-897 ; Sherwood et al . , 1980, Endocrinology 107:691-698; Eddie et al . , 1986, Lancet 1:1344-1346; Lucas, et al . , 1989, Journal of Endocrinology 120:449- 457. To this end, several antibodies have been prepared to relaxin. Schwabe et al . , 1978, Recent Progress in Hormone Research 3_4:123-211; Bryant- Greenwood, 1982, Endocrine Reviews 3.:62-90. Additionally, a synthetic analogue of hRLX with an accessible tyrosine has been generated, allowing

the direct iodination for the use in a sensitive radioim unoassay (RIA) to quantify serum hRLX concentrations during pregnancy. Eddie et al . , 1986, .Lancet 1:1344-1346. Moreover, Bodsch and Struck have developed an enzyme immunoassay using porcine RLX. This assay has not been characterized or extensively applied. Bodsch and Struck, 1980, Fresenius Zeitschrift fur Analytiεche Chemie 301:133-134. Finally, Lucas et al . have developed a double- antibody enzyme-linked immunosorbent assay (ELISA) for the detection of hRLX in serum and plasma which is as sensitive and specific as the RIA described above. Lucas et al . , 1989, Journal of Endocrinology 120:449- 457. Lucas et al . report use of this ELISA for the determination of whether the hRLX (H2) analogue crossed the placenta during its infusion into a pregnant rhesus monkey, and to measure hRLX concentrations in various plasma and serum samples from men, non-pregnant and pregnant women, as well as in corpora luteal extracts from pregnant women.

Despite the breadth of information available regarding relaxin assays, the art does not discuss or suggest the use of such assays for the diagnosis of human malignancies or the long term monitoring of pregnancy, which are the subject of the present invention.

Similarly, in comparison to the knowledge available regarding RLX's structure and biological function as it relates to pregnancy, only minimal data is available regarding the peptide's full range of activity or the control mechanisms for the synthesis and/or secretion of relaxin. Specifically, what is known includes the report of Goldsmith et al . providing that relaxin synthesis may be stimulated in vitro with a mixture of hCG, progesterone and human

placental lactogen. Goldsmith et al . , 1982, Annuals of New York Academy of Sciences 380:60-74. It has also been reported that secretion of relaxin is stimulated in women treated with hCG. Quaglarello et al . , 1980, J. Clin . Endocrinol and Metab . 51:74-77.

3. SUMMARY OF THE INVENTION

The present invention is directed to methods for the diagnosis of human malignancies and to determine and monitor the clinical course thereof. In one embodiment of the present invention, such malignancies include ovarian cancers, prostate cancers, testicular cancers, and breast cancer. In yet other embodiments of the present invention, the malignancies are grouped on the basis of whether such malignancies share a common relaxin producing phenotype and may be detected by the expression of relaxin.

More specifically, the present invention is directed to: (1) the determination of elevated relaxin levels in the body fluids and/or tissue samples of patients to determine that patient's predisposition towards malignancies, (2) diagnosing such malignancies, (3) monitoring the advent of metastases following ablation of the malignancy, (4) monitoring therapeutic drug programs comprising the use of relaxin and (5) imaging and thereby identifying tumor and/or metastatic sites.

The basis of the present invention is the inventors' unexpected discovery that certain malignant tissues including ovarian, cervix, prostate, testicular, breast and other cancers having a common relaxin producing phenotype (e . g . colon- and lung cancers) secrete relaxin.

The present invention is further directed to assays and kits for the determination of relaxin levels in the plasma, serum, urine, and/or tissue of

patients for the diagnosis of human malignancies, including ovarian cancers, prostate cancers, testicular cancers, breast cancers, colon cancers, and lung cancers. More specifically, the assays and kits of the present invention are used for the determination of predisposition or presence of such malignancies, and/or to determine and monitor the ablation of cancer by known treatment specimens.

In another embodiment, the present invention is directed to monitoring the course of human pregnancy by monitoring relaxin levels during the pregnancy term. In yet another embodiment of the present invention, the assays and kits of the present invention are comprised of labelled antibodies for use in imaging.

. DEFINITIONS

As used in the present specification, the following words and phrases are generally intended to have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.

The term "relaxin" means human relaxin, including full length relaxin or a portion of the relaxin molecule that retains biological activity [as described in U.S. Patent No. 5,023,321, preferably recombinant human relaxin (H2)] and other active agents with relaxin-like activity, including relaxin- like factor [as described in co-pending application entitled "Relaxin-Like Factor And Methods And Uses

Thereof," filed concurrently herewith] and agents that competitively displace bound relaxin from a receptor. Relaxin can be made by any method known to those skilled in the art, preferably as described in U.S. Patents No. 4,835,251 and in co-pending U.S. Serial

Nos. 07/908,766 (PCT US90/02085) „and 08/080,354 (PCT US94/0699) .

5. DETAILED DESCRIPTION OF THE INVENTION

5 Relaxin has been considered primarily as a hormone of reproduction. More specifically, it has been widely reported that relaxin modulates the restructuring of connective tissues in target organs to obtain the required changes during pregnancy and

10 parturition.

5.1. Identification Of Correspondence Between Malignancies And Relaxin Levels

The subject invention is based on the inventors' unexpected discovery that a variety of malignant

15 tissues, including ovarian, cervix, prostate, testicular, breast and other cancers including subsets of colon and lung cancers, secrete relaxin, either fortuitously or for undefined physiological purposes.

More specifically, a broad survey of blood

20 samples derived from patients having different types of malignancies has revealed that a certain subset of such malignancies secretes relaxin. One group of such malignancies (i.e. ovarian, cervix, prostate, testicular and breast cancer) is believed to have a

25 common relaxin producing phenotype. Evidence also supports a connection between relaxin production and/or secretion and other malignancies, including a subset of colon and oat fat cell lung tumors.

As has been determined by the present inventors, ₃₀ the secretion level for relaxin appears to directly correlate with the aggressive nature of the cancer and the progression of the disease. Thus, assessment of the relaxin levels secreted by such tumors is a valuable tool for diagnosis and prognosis of the _ _c relevant malignancies.

In a preferred embodiment, the present invention is directed to the determination of relaxin secretion related to the predisposition or presence of cancers which share a common relaxin producing phenotype (e.g. ovarian, prostate, testicular and breast cancer) . The advantages of the present invention are particularly poignant in the case of the group of cancers related to ovarian cancer as the ability of detect and provide early diagnosis of such cancer is particularly beneficial. Ovarian cancer is typically only successfully treated when identified at a very early stage. Notwithstanding early diagnosis, ovarian cancer is further problematic in that assessing the amount of residual ovarian cancer and monitoring its response to treatment is extremely difficult. For example, while many germ cell tumors can be successfully subclassified and the course of the disease monitored by utilizing the markers α- fetoprotein (Sell et al . , 1976, International J. Cancer 18 _. :574-80) , and /3-human chorionic gonadotropin (Perlin et al . , 1975, Cancer 37:215-225) , such specific and accurate markers are not available for the stromal and epithelial varieties or ovarian cancer. Thus, there is a substantial need for methods of early diagnosis, and also for reliable, non-invasive methods of monitoring treatment to support investigation of new therapies.

5.2. Uses Of Assays And Methods

The methods and assays of the present invention for the measurement of relaxin in body fluids such as blood or blood fractions, urine and ascites fluid, or tissue samples may be used for a variety of clinical purposes.

First, determination of relaxin secretion can be employed to determine the presence of several human malignancies which can be characterized by the secretion of relaxin. For example, in the preferred embodiment of the present invention, the methods and assays of the present invention may be used for the diagnosis of ovarian, breast, prostate, testicular and other types of cancer having common relaxin producing phenotype, and further a certain subset of colon and lung cancers.

Second, determination of relaxin levels may be useful for the determination of predisposition for such types of cancer. For example, to the extent that elevated relaxin levels in the body fluid of the patient can readily be detected in general standard check-ups, they will indicate predisposition for malignancies which are related to the secretion of relaxin. Patients identified to be predisposed can then be monitored on a regular basis for changes in relaxin levels in their body fluids, facilitating an early diagnosis of a malignancy. Early start of suitable therapeutic treatment will significantly increase the chances to successfully cure the disease. The methods and assays of the present invention can also be used to determine and monitor the ablation of cancer by known treatment regimens. More specifically, such methods and assays can be used as follow-up therapy to detect metastases following, for example, surgical removal of malignant tumors. Additionally, the assays and methods of the present invention can be conducted using labelled, and preferably radiolabelled, antibodies for use in imaging to, for example, identify and/or locate tumors and metastases. The methods and assays of the present invention can be used outside the context of cancer. For

example, the present invention can be used to monitor pregnancy by measuring relaxin levels throughout the pregnancy term. It has been observed that relaxin levels during normal healthy pregnancy steadily increase or remain constant throughout the pregnancy term. In contrast, in unhealthy pregnancies, the relaxin levels will decrease or fluctuate. The present invention, as it pertains to monitoring pregnancy, relies on this observation. As further example, the methods and assays of the present invention can also be used to monitor therapeutic drug programs comprising relaxin administration.

5.3. Applicable In Vitro Assays Several assays have been developed for the determination of- relaxin levels in body fluids, such as plasma, serum, and urine, and/or tissue samples. In addition to the assays described hereinbelow, a detailed description of such assays can be found in the co-pending U.S. Patent applications entitled "Relaxin-Like Factor, Methods and Uses Thereof" and "Relaxin Analogs, Methods And Uses Thereof," both of which are filed concurrently herewith. In a preferred embodiment of the present invention, relaxin levels are determined using antibody based immunoassays. Generally, immunoassays, including radioimmunoassayε and ELISA, known in the art, may be employed for the determination of relaxin levels. For example, the ELISA may be conducted according to known protocols which are described in, for example, Voller, et al. , 1988, "Enzyme-Linked Immunosorbent Assay," In: Manual Of Clinical Immunology, 2d Ed. , edited by Rose and Friedman, pp. 359-371, Am. Soc. of Microbiology, Washington, D.C. Said protocols may be modified to specifically detect

for the presence of relaxin. See e .g . , Lucas et al . , supra .

Preferred protocols are set forth in Section 6 of the instant application. 5.4. Assay Test Samples

A variety of different biological samples may be employed for the measurement of relaxin secreted by malignant tissues, such as ovarian, breast, prostate, testicular and other cancers having common relaxin producing phenotype.

Blood, a convenient biological sample and readily to obtain, is suitable for diagnostic assays for the determination of elevated relaxin levels related to the occurrence of malignancies. Relaxin can be measured in either whole blood, plasma or serum. As such, blood or blood fractions are suitable diagnostic samples for the diagnosis of many types of cancer which correlate with the secretion of relaxin, such as ovarian, prostate, breast, cervix, testicular, and other types and subsets of cancer. Since readily to obtain, blood or blood fractions are preferred for determination of predisposition or presence of such malignancies, as well as follow-up therapy to determine the onset of metastases following ablation of the cancer.

Urine is another readily available biological sample for the .determination of elevated relaxin levels related to the occurrence of malignancies. As true for blood derived diagnostic samples, urine appears to be suitable for diagnosis of the whole variety of cancers which relate to the secretion of relaxin.

In a method for determining the predisposition towards or presence of ovarian malignancies, ascites fluids of the patient are used as a sample. Ascites fluids provide the most concentrated levels of relaxin

and therein appear to be the mosl abundant and the easiest to measure. Ascites fluid can readily be obtained by methods known by the skilled artisan.

Additionally, tumor derived tissue samples can be obtained for the determination of relaxin production. The preferred use for the determination of relaxin production by a certain tissues will be to locate the malignancy responsible for the elevation of relaxin levels in the body fluids of the patient. In a preferred method, the tumor derived tissue sample is obtained by small needle biopsy.

6. EXAMPLES

6.1. Assessment of Relaxin Secretion by Human Malignancies

In order to assess the correlation of the presence of various human malignancies with the secretion of relaxin, blood samples from random patients, collected from the Deaconness Hospital

Oncology Serum Bank were subjected to assays for the determination of the relaxin levels. The double coded samples were considered to represent a reasonable sampling of the bank's contents. No discrimination between men and women has been made for tumors of the non-reproductive organs. The results are set forth in

Table I.

Table I -.

Relaxin Concentrations in Sera of Patients with

Various Malignancies

Tumor Number of Number of Relaxin Concentration Samples Positives (ng/ml

Ovary (Epithelioid) 7 1 2.5

Renal 10 0 -

Cervix 7 2 0.625; 0.513

Choriocarcinoma 3 0 -

Lung - oat fat cell 10 1 0.723

Lung - other 10 0 -

Sarcoma 7 0 -

Lymphoma 10 0 VV-

Pancreatic 10 0 -

Prostate 8 1 0.400

Breast 11 0 -

Testicular 2 0 -

Hepatoma 2 0 -

Colon 10 1 0.24; (0.34*)

Bladder 9 0 -

Total 116 6 -

Pregnancy 713 713 0.1 to 0.7 (22)

* Repeated determination

As evidenced by this data, several tumor types, i.e. ovarian carcinoma, cervix carcinoma, a certain subset of lung carcinoma, prostate carcinoma, and colon carcinoma, secrete relaxin in concentrations essentially equivalent to those found in pregnancy. Ovarian tumors in fact produce higher levels of relaxin than found in pregnancy. Further data suggest that a certain subset of testicular as well as breast cancers which have a common relaxin producing phenotype with prostate and ovarian cancer are

indicated by elevated levels of relaxin in body fluids.

A detailed analysis of a patient with ovarian carcinoma revealed that the pathological diagnosis was

"metatastic poorly differentiated adenocarcinoma suggestive of ovarian malignancy." Comparison with the other tumors confirmed that this was the most primitive tumor in the group. Thus, particularly high levels of relaxin appear to indicate poor prognosis of the disease.

Several samples of serum from prior admissions of this ovarian cancer patient were also examined for their relaxin levels. The results of these assays are tabulated in Table II.

Table II

Serial Serum Relaxin Levels in a Patient with Ovarian Carcinoma

Date Relaxin Level (ng/rn])

08/27/79 0.81

10/31/79 0.83

04/29/80 1.25

08/05/8 12.5

As intervening therapy, the patient received Adiamycin, Cytoan and cis-platinum throughout the course of the disease. Prior to enrollment in the study, the patient additionally received L- phenylalanine mustard. Upon entry into the study, the tumor mass was very large and non-operable.

The data set forth in Table II clearly show correlation of the disease progression, i.e. increase of the tumor mass, and the relaxin levels in the patient's serum. Moreover, in correlation with the clinical impression that the therapy was not effective, the relaxin level constantly increased with progression of the disease.

6.2. Assays for the Determination of Relaxin Levels in Biological Samples

6.2.1. Radioi munoassay

The radioimmunoassay for the determination of relaxin levels in biological samples is performed with a rabbit anti-relaxin antibody and a monotyrosyl relaxin tracer. The second antibody is a goat commercial anti-rabbit immunoglobulin D antibody, coupled to a Whatman cellulose powder with aid cyanogen bromide. Samples and standards are preincubated with the first antibody for three hours.

The ¹²⁵ I-labeled tyrosyl relaxin tracer is added and incubation continued for an additional two hours. The second antibody is added and the incubation continued for another hour with vigorous shaking. Samples are centrifuged and the amount of radioactivity in the pellets is measured in a gamma-counter. The amount of relaxin in unknown samples and standards is inversely proportional to the amount of radioactivity in the pellet. Relaxin in unknown samples can then be quantitated by comparison against a standard curve generated with known concentrations of relaxin.

Monotyrosyl relaxin is prepared by reacting N- formyltyrosine-N-hydroxysuccinimide (1.5 equivalents) with one equivalent of porcine relaxin in 50% dioxane,

0.2 M N-ethyl-morpholine at pH 8.5. The reaction runs for four hours at room temperature. The relaxin is desalted on a Sephadex G-25 column in 1 M acetic acid.

The tyrosine content is measured by amino acid analysis.

Although the antibody described herein is a highly specific antibody, it has to be emphasized that what is being measured is "immuno-reactive relaxin." In fact, human relaxin(s) may differ and, in fact, immuno-reactivity may differ from biological activity. Furthermore, "relaxin" secreted by malignant tissues

may differ from that secreted by,the normal corpus luteum. However, there is no reasonable alternative to this method because of its sensitivity (200 pg/ml) . Antibodies prepared utilizing appropriate antigens will help answer these questions. 6.2.2. ELISA The following protocol was used to measure the presence of relaxin in a sample.

Materials And Methods . The following equipment and reagents were used: A. Equipment

* 96-well microtiter plate (Costar Plate No. 9018 or equivalent)

* Mini-orbital shaker (Bellco Glass, Inc. No 7744-50099 or equivalent)

* Microliter pipets (Gilson or equivalent)

* Microliter multichannel pipetter (Titertek or equivalent)

* Plate sealing tape (Dynatech No. 1-220-30-1 or equivalent)

* Plate washer (Dynawasher II or equivalent)

* Plate reader (Bio-tek EL309 or equivalent) * Computer with 4-parameter date reduction software (optional) B. Reagents

* Phosphate Buffered Saline (PBS) (refer to SOP)

* Buffer Diluent (PBS/0.5% BSA/0.05% Polysorbate 20/0.01% Thimerosal) * Serum Diluent (pooled normal human male serum or equivalent prepared according to the following steps: a. Spin at 625 x g for 20 minutes if necessary and filter through a 0.45 mm filter before use. New pools must be compared to previously used pools.

b. Add 0.01% Thimerosal to pool, said product being stored at -10°C or below and expiring five years from the date of preparation. c. Store thawed serum at 2-8°C. d. Expiration of thawed serum is 1 month from the thaw date.

* Plasma diluent (pooled normal human male plasma or equivalent) prepared according to the following steps: a. Spin at 625 x g for 20 minutes before use. New pools must be compared to previously used pools. b. Add 0.01% Thimerosal to pool. c. Store at -10°C or below (Expiration is 2 years form the date of preparation. d. Store thawed plasma at 2-8°C. Expiration of thawed plasma is 1 month from the thaw date.

* Wash Buffer (PBS/0.05% Polysorbate 20)

* 20X PBS/1.0% Polysorbate 20 which is stored at 2-8°C and wherein expiration is 1 month from the date of preparation.

* Coating Buffer (0.05 M Sodium Carbonate Buffer, pH 9.6 ± 0.1) which is stored at 2-8°C (wherein expiration is two weeks from the date of preparation) .

* Substrate Diluent (Sodium Phosphate/Citrate buffer), pH 5.0 ± 0.1 (19.2 g/1 citric acid in 0.2 M Sodium phosphate, dibasic (28.4 g Na ₂ HP0 ₄ /l) ) which is stored at 2-8°C and wherein expiration is one month from the date of preparation.

* Substrate Stock [lOx o-phenylenediamine (OPD) ]

* OPD (Sigma P1526 or equivalent) 4.0 g

* Substrate Diluent qs to 1.0 1 (stored in aliquots at -10°C or below wherein expiration is 1 month from the date of preparation) .

* 4 mM Hydrogen peroxide,(H ₂ 0 ₂ ) in PBS

* 30% v/v H ₂ 0 ₂ (e.g. Kodak 1024), 400 μl

* PBS qs to 1.0 1 (stored at 2-8°C wherein expiration is 1 month from the date of preparation. * Substrate Solution: Any of the following preparations may be used. Substrate Solution (OPD/Substrate Diluent) without H ₂ 0 ₂ added can be stored at ambient temperature for up to one hour. Substrate Solution must be at ambient temperature before adding to plate.

(A) Substrate Solution from Substrate Stock per late

Substrate Stock (lOx OPD) 1.0 ml

Substrate Diluent 9.0 ml 3% v/v H ₂ 0 ₂ 40.0 μl

(B) Fresh Substrate Solution per plate OPD (Sigma P1526 or equivalent) 5.0 mg Substrate Diluent 12.5 ml

3% v/v H ₂ 0 ₂ 50.0 μl

(C) Substrate Solution using OPD tablet per plate OPD, 5 mg tablet (Sigma P6912 or 1 tablet equivalent) 4 mM H ₂ 0 ₂ in PBS 12.5 ml

Expiration is 20 minutes from the time of preparation.

* Antibody to Relaxin (anti-Relaxin) (stored in aliquots at -60°C or below wherein expiration is 5 years from the date of preparation; After thawing, store at 2-8°C wherein expiration of thawed aliquots is 2 months from date of thaw) .

* Current approved Relaxin Reference Standard (stored in aliquots at -60°C or below wherein expiration is as assigned by the analytical Standard Committee) . * Standard Stock: Dilute Reference Standard to 10 μg/ml with Buffer Diluent. Store in aliquots at -60°C or below. Expiration is 2 years from the date of preparation. After thawing store at 2-8°C and expiration is 1 month from the date of thaw. * Antibody to Relaxin conjugated to horseradish peroxidase per SOP (anti-Relaxin- HRP) (wherein aliquots are stored at -60°C or below and expiration is 3 years from the date of preparation) . After thawing, store at 2-8°C. Expiration of thawed aliquots is 2 weeks from the date of thaw. Stock I: Dilute anti-Relaxin-HRP approximately 1/10 with Buffer Diluent. Store in aliquots at -60°C or below. Expiration is 2 years from the date of preparation.

After thawing, store at 2-8°C. Expiration of thawed aliquots is 1 week from the date of thaw.

* Non-immune Goat Immunoglobulin (IgG) (Cappel Lot #6006-0080 or equivalent) reconstituted to approximately 0.5 mg/ml according to product instructions which is stored at -10°C or below. Expiration is two years from the date of preparation. After thawing, store at 2-8°C. Expiration of thawed aliquots is 1 month from the date of thaw.

* 4.5 N Sulfuric Acid which is stored at ambient temperature. Expiration is one year from the date of preparation.

Standard, Control and Sample Preparation . * Standard dilution schedule

NOTE: Comparable dilutions to equivalent concentration may be made. Assuming concentration of Standard Stock I to be 10 μg/ml, dilutions will be as follows:

(A) Standard A: 100,000 pg/mL. Add 40 μl standard Stock I to 3.96 ml Buffer Diluent. Mix well.

Store at 2-8°C.

Expiration is 12 hours after time of preparation.

(B) as set forth in Table III

TABLE III

Standard pp/mL Standard to Add Assay Diluent to Add

B 2500 12.5 μL A 0.488 mL

C 1250 0.35 mL B 0.35 mL

D 625 0.35 mL C 0.35 mL

E 313 0.35 mL D 0.35 mL

F 156 0.35 mL E 0.35 mL

G 78 0.35 mL F 0.35 mL

H 39 0.35 mL G 0.35 mL

I 20 0.35 mL H 0.35 mL

J 0 -- 0.35 mL

Use C through J for the standard curve. Store at 2-8°C.

Expiration is 4 hours from time of preparation.

* Control preparation: Dilute controls in appropriate Assay Diluent to fall within the low, middle and high areas of the standard curve.

20

Store in aliquots at -fjO°C or below.

Expiration is 1 year from the date of preparation. * Sample preparation: Make appropriate dilutions in appropriate Assay Diluent to fall within the range of the standard curve.

(A) Serum and plasma samples to be assayed at dilutions of less than 1/20 should be run in assays using either Serum or Plasma Diluent depending on the sample matrix.

(B) Serum and plasma samples with dilutions of greater than 1/20 may also be diluted in Buffer Diluent, but a minimum dilution of 1/20 is required for optimal recovery in an assay using Buffer Diluent.

Protocol. The following protocol was used to conduct the ELISA described herein.

1. Dilute anti-Relaxin antibody in Coating Buffer to yield the full range of the standard curve concentrations. Prepare fresh before use. Pipette 100 μl of diluted antibody into each well of microtiter plate. Incubate at 2-8°C for 12 hours to 24 hours for assays using Serum or Plasma Diluent; 12- 72 hours for assays using Buffer Diluent.

2. Remove solution from wells by inverting plate over waste reservoir. Wash each well with approximately 200 μl of Wash Buffer three times using platewasher, do not aspirate plates. Rotate plates 180 degrees and repeat wash cycle. Blot thoroughly.

3. Add approximately 200 μl of Buffer Diluent to each well. Incubate for 1-2 hours at ambient temperature with agitation.

4. Wash as in Step 2. with only one wash cycle.

5. Pipette goal IgG to a inal concentration of 0.02 mg/ml to all diluted standards, controls, and samples except those in a buffer matrix.

6. Pipette 100 μl per well of diluted standards (in duplicate) , controls (in duplicate) , and samples into appropriate wells. Incubate for 12-24 hours at 2-8°C.

7. Wash as in Step 2.

8. Dilute anti-Relaxin-HRP in Buffer Diluent to yield a significant OD range between the highest and lowest standards. Pipette 100 μl of diluted conjugate to each well; incubate at ambient temperature for 4 hours ± 10 min with agitation.

9. Wash as in Step 2. 10. Pipette 100 μl per well of substrate

Solution. Incubate for 15 to 20 minutes for assays using Serum or Plasma Diluents, or 10 to 15 minutes for assays using Buffer Diluents, in the dark at ambient temperature. 11. Pipette 100 μl per well of 4.5 N Sulfuric Acid.

12. Read ODs on plate reader using two filters, 490-492 nm for absorbance and 405 nm for reference.

Calculations . The following methodology was used to calculate the results of the assay:

* Sample concentration is determined by entering data into a 4-parameter logistic curve fitting program.

* If computerized data reduction is not available, plot ODs versus standard concentration on semi-log paper and read the concentration of controls and samples from the generated curve. Extrapolation should not be conducted beyond the standard curve.

The present invention is not to be limited in scope by the exemplified embodiments which are intended as illustrations of single aspects of the invention, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

All references cited within the body of the instant specification are hereby incorporated by reference in their entirety. In addition, the publications listed below are of interest in connection with various aspects of the invention and are incorporated herein as part of the disclosure:

1. Adham et al . , 1993, J . Biol . Chem . 268:26668-26672; 2. Adler et al . , 1973, Methods Enzymol . 27:675- 735;

3. Burkhardt et al . , 1994, Genomicε 20:13-19

4. Eigenbrot et al . , 1991, J. Mol . Biol .

221:15-21; 5. Ellman, 1959, Arch. Biochem . Biophys . 82:70- 77;

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