QUANTITATIVE DETERMINATION NEW PRODUCT OF HUMAN CYSTEINE-RICH SECRETORY PROTEIN 1 (CRISP-1)

Human Cysteine-Rich Secretory Protein 1 ELISA

High sensitivity (0.028 ng/ml) Excellent analytical characteristics Validated for human seminal plasma samples Preliminary population data

REPRODUCTION HUMAN CYSTEINE-RICH SECRETORY PROTEIN 1 ELISA

Introduction

Cysteine-Rich Secretory Protein 1 (CRISP-1, AEG-like protein, sperm-egg fusion process [3]. An additional role for CRISP-1 ARP, Acidic epididymal glycoprotein homolog, AEGL1), 28,5 is to inhibit the uptake of ions, such as Ca2+, required for kDa epididymal protein, is a member of the cysteine-rich capacitation [4]. secretory proteins (CRISPs) family. This family of proteins is defined by the presence of 16 conserved cysteines that fold In the rat, CRISP-1 comprises two forms referred to as Proteins into two domains: a CAP domain, which has been implicated D and E. One difference between the D and E forms is an in cell-cell adhesion, and a CRISP domain, which has been additional single O-linked N-acetyl galactosamine on an shown to regulate several classes of ion channels across amino residue in protein E that is not present on D form. The multiple species [1]. studies demonstrate that the more abundant D form interacts with spermatozoa transiently, consistent with a capacitation- CRISPs show a strong expression bias to the mammalian suppressing function, and the E form binds tightly and could male reproductive tract and the venom of poisonous reptiles. act in the female reproductive tract [5]. Within mammals, CRISPs are also expressed in lower levels within non-reproductive tissues including secretory glands, Findings indicate vasectomy has an effect on CRISP-1 skeletal muscle, the spleen and the thymus [1]. In the male expression, because after vasectomy, the flux and composition reproductive tract CRISPs have been implicated in many of the epididymal fluid are modified. CRISP-1 was detected aspects of male germ cell biology spanning haploid germ cell in the seminal plasma of normal and vasovasostomized men, development, epididymal maturation, capacitation, motility but not in vasectomized individuals. The soluble concentration and the actual processes of fertilization [1]. of CRISP-1 was significantly higher in the seminal plasma of vasovasostomized men when compared with normal men [6]. Human CRISP-1 is produced throughout the epididymis, whereupon it is incorporated into epididymosomes and Moreover, another study has detected different levels of presumably transferred onto the sperm surface. CRISP-1 was CRISP-1 in two types of azoospermia. The seminal plasma also detected as a soluble form in the seminal plasma [2]. samples from normospermic and nonobstructive azoospermic donors are CRISP-1 positive, whereas CRISP-1 is absent or The molecular mechanism of action of the mammalian CRISP present at low levels in samples from patients with obstructive proteins remains uknown, but certain non-mammalian CRISP azoospermia [7]. proteins can block ion channels [2]. CRISP-1 gene knockout mouse sperm showed reduced ability to fertilize ZP-intact and ZP-free eggs, suggesting the importance of CRISP-1 in HUMAN CYSTEINE-RICH SECRETORY PROTEIN 1 ELISA QUANTITATIVE DETERMINATION OF HUMAN CYSTEINE-RICH SECRETORY PROTEIN 1 (CRISP-1)

BioVendor Human Cysteine-Rich Secretory Protein 1 ELISA (RD191260200R)

Intended use Test principle The RD191260200R Human Cysteine-Rich Secretory In the BioVendor Human Cysteine-Rich Secretory Protein 1 Protein 1 ELISA is a sandwich enzyme immunoassay for the ELISA, Standards and samples are incubated in microtitration quantitative measurement of human Cysteine-Rich Secretory wells pre-coated with polyclonal anti-human CRISP-1 Protein 1. antibody. After 60 minutes incubation followed by washing, The total assay time is less than 4 hours biotin labelled polyclonal anti-human CRISP-1 antibody is added and incubated with the captured CRISP-1 for 60 The kit measures CRISP-1 protein in human seminal plasma minutes. After another washing, streptavidin-HRP conjugate Assay format is 96 wells is added. After 30 minutes incubation and the last washing Standard is recombinant protein based step, the remaining conjugate is allowed to react with the Components of the kit are provided ready to use, substrate solution (TMB). The reaction is stopped by addition concentrated or lyophilized of acidic solution and absorbance of the resulting yellow product is measured. The absorbance is proportional to the concentration of CRISP-1. A standard curve is constructed by Clinical application plotting absorbance values against CRISP-1 concentrations of Standards and concentrations of unknown samples are Reproduction determined using this standard curve.

3.0 HUMAN CYSTEINE-RICH SECRETORY PROTEIN 1 ELISA CAT. NO.: RD191260200R 2.5 Assay format Sanwich ELISA, Biotin-labelled antibody, 96 wells/kit 2.0

Samples Seminal plasma 1.5 Standards 0.156 - 5 ng/ml 1.0 Limit of detection 0.028 ng/ml 0.5 Absorbance at 450 nm - 630 0.0 0.1 110 Concentration of Human CRISP-1 (ng/ml) QUANTITATIVE DETERMINATION OF HUMAN CYSTEINE-RICH SECRETORY PROTEIN 1 (CRISP-1)

Precision Intra-assay (Within-Run) (n=8) Inter-assay (Run-to-Run) (n=7) Sample Mean SD CV Sample Mean SD CV (µg/ml) (µg/ml) (%) (µg/ml) (µg/ml) (%) 1 3.17 0.15 4.78 1 10.16 0.57 5.56 2 11.57 0.4 3.43 2 18.72 0.88 4.71

Spiking recovery Linearity Serum samples were spiked with different amounts of human Serum samples were serially diluted with Dilution Buffer and CRISP-1 and assayed. assayed. Sample Observed Expected Recovery O/E Sample Dilution Observed Expected Recovery O/E (µg/ml) (µg/ml) (%) (µg/ml) (µg/ml) (%) 6.02 - - - 17.46 - - 10.80 10.70 100.9 2x 8.91 8.73 102.1 1 1 16.34 15.39 106.1 4x 4.37 4.37 100.0 24.95 24.77 100.7 8x 2.16 2.18 99.0 10.85 - - - 24.62 - - 15.56 15.53 100.1 2x 12.68 12.31 103.0 2 2 19.95 20.22 98.7 4x 6.21 6.15 100.9 30.47 29.6 102.9 8x 3.14 3.08 101.9

Summary of protocol • Reconstitute Master Standard and prepare set of Standards • Dilute samples (15000x) • Add 100 µl Standards and samples • Incubate at RT for 1 hour/300 rpm • Wash plate 3 times • Add 100 µl Biotin Labelled Antibody • Incubate at RT for 1 hour/300 rpm • Wash plate 3 times • Add 100 μl Streptavidin-HRP Conjugate • Incubate at RT for 30 min/300 rpm • Wash plate 3 times • Add 100 μl Substrate Solution • Incubate at RT for 10 min • Add 100 μl stop solution • Read absorbance and calculate results HUMAN CYSTEINE-RICH SECRETORY PROTEIN 1 ELISA

Cross-reactivity The antibodies used in this ELISA are specific for human CRISP-1 with no detectable crossreactivity to human CRISP-2 and CRISP-3 at 10 ng/ml.

Preliminary Population Data

The range of the seminal plasma samples from normospermic donors (N=19) has been determined using this Human Cysteine- Rich Secretory Protein 1 ELISA kit in our laboratory: Mean concentration of CRISP-1 from this normospermic donors was 18.6 ng/ml (SD =7.9). The data quoted in these instructions should be used for guidance only. It is recommended that each laboratory include its own panel of control samples in the assay. Each laboratory should establish its own normal and pathological reference ranges for human CRISP-1 levels with the assay.

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References

1. Koppers A.J., Reddy T., O’Bryan M.K.: The role of cysteine-rich secretory proteins in male fertility. Asian Journal of Andrology, 13, 111-117 (2011) 2. Kratzschmar J., Haendler B., Eberspaecher U., Roosterman D. Donner P., Schleuning W.D.: The human cysteine-rich secretory protein (CRISP) family Primary structure and tissue distribution of CRISP-1, CRISP-2 and CRISP-3. Eur. J. Biochem., 236, 827-836 (1996) 3. Roberts K.P., Johnston D.S., Nolan M.A., Wooters J.L., Waxmonsky N.C., Piehl L.B., Ensrud-Bowlin K.M., Hamilton D.W.: Structure and function of epididymal protein cysteine-rich secretory protein-1. Asian Journal of Andrology, DOI:10.1111/j., 1745-7262 (2007) 4. Batruch I., Leckde I., Kagedan D., Smith C.R., Brendan J.M.: Proteomic Analysis of Seminal Plasma from Normal Volunteers and Post-Vasectomy Patients Identifies over 2000 Proteins and Candidate Biomarkers of the Urogenital Systém. Journal of proteome research, 10, 941-95, (2011). 5. Roberts K.P., Ensrud K.M.,Wooters J.L., Nolan M.A., Johnston D.S., Hamilton D.W.: Epididymal secreted protein Crisp-1 and sperm function. Molecular and Cellular Endokrinology, 250, 122-127 (2006) 6. Légaré Ch., Boudreau L., Thimon V., Thabet M., Sullivan R.: Vasectomy Affects Cysteine-Rich Secretory Protein Expression Along the Human Epididymis and Its Association With Ejaculated Spermatozoa Following Vasectomy Surgical Reversal. Journal of Andrology, 31, 573-583 (2010) 7. Légaré Ch., Cloutier F., Makosso-Kallyth S., Laflamme N., Jarvi K., Tremblay R.R., Sullivan R.: Cysteine-rich secretory protein 1 in seminal plasma: potential biomarker for the distinction between obstructive and nonobstructive azoospermia. Fertility and Sterility, 100 (5), 1253-1260 (2013) 8. Gibbs G.M., Roelants K. and O’Bryan M.K.: The CAP Superfamily: Cysteine-Rich Secretory Proteins, Antigen 5, and Pathogenesis-Related 1 Proteins-Roles in Reproduction, Cancer, and Immune Defense. Endocrine Reviews, 29, 865-897 (2008) 9. Cohen J.D., Da Ros V.G., Busso D., Ellerman D.A, Maldera J.A., Goldweic N., Cuasnicú S.P.: Participation of epididymal cysteine-rich secretory proteins in sperm-egg fusion and thein potential use for male fertility regulation. Asian Journal of Andrology, DOI: 10.1111/j.1745-7262 (2007) 10. Lavers A.E., Swanlund D.J., Hunter B.A., Tran M.L., Prior J.L., Roberts K.P.: Acute Effect of Vasectomy on the Function of the Rat Epididymal Epithelium and Vas Referend. Journal of Andrology, 27, 826-836 (2006) 11. Roberts K.P., Ensrud K.M., Hamilton D.W.: A Comparative Analysis of Expression and Processing of the Rat Epididymal Fluid and Sperm-Bound Forms of Proteins D and E. Biology of reprodution, 67, 525-533 (2002) 12. Maldera J.A., Vasen G., Ernesto J.I., Weigel-Mun˜oz M., Cohen D.J., Cuasnicu P.S.: Evidence for the Involvement of Zinc in the Association of CRISP1 with Rat Sperm During Epididymal Maturation. Biology of Reproduction, 85, 503-510 (2011) 13. Luo J., Yang J., Cheby Y., Li W., Yin T.L., Xu W.M., Zou Y.J.: Immunogenicity Study of Plasmid DNA Encoding Mouse Cysteine-Rich Secretory Protein-1 (mCRISP1) as a Contraceptive Vaccine. America Journal of Reproductive Imunology, 68, 47-55 (2012)

Contact Information

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Date of issue March 2014 about BioVendor products.