Human FSH ELISA Kit Medical Device Licence No.: 16418

Transcription

1 Human FSH ELISA Kit Medical Device Licence No.: Enzyme immunoassay kit for the quantitative determination of Human Follicular Stimulating Hormone (FSH) concentration in serum. Catalog Number: SL tests For in vitro diagnostic use only. 396 N Summit Ave., Suite 2 Gaithersburg, MD U.S.A. Fax: (301) info@signagenlabs.com Web Site: Rev. (06/04) FSH-D

2 TABLE OF CONTENTS Page INTENDED USE 2 INTRODUCTION 2 PRINCIPLE OF THE ASSAY 2 LIMITATIONS OF THE PROCEDURE 3 REAGENTS PROVIDED 3 MATERIALS REQUIRED BUT NOT SUPPLIED 4 PRECAUTIONS 5 SAMPLE PREPARATION 5 ASSAY PROCEDURE 5 CALCULATION OF RESULTS 7 TYPICAL DATA 7...Example 8 PERFORMANCE CHARACTERICS 8...Sensitivity 8...Precision 8...Accuracy 9...Linearity 9...Specificity 10...Correlation Study 10...Expected Normal Values 11 QUALITY CONTROL 11 REFERENCES 11 Rev. (06/04) FSH-D 1

3 INTENDED USE Enzyme immunoassay (EIA) permits the routine quantitative determination of many protein hormones in body fluids and provides an accurate, sensitive, reproducible, rapid and specific assay. This enzyme immunoassay method makes it possible to measure very low concentrations of hfsh (human chorionic gonadotropin) in small volumes of serum (0.1 ml per assay). INTRODUCTION Follicle-stimulating hormone (hfsh) is gonadotrophin secreted by the anterior pituitary gland. The biological action of hfsh is to stimulate the growth and maturation of ovarian follicles, to provoke in association with luteinizing hormone (hlh) the production of estrogens and to stimulate growth of semineferous tubules and spermatogenesis. The secretion of hfsh is under the control of gonadotrophin releasing hormone (GnRH) secreted by the hypothalamus. hfsh is a glycoprotein (M.W daltons) composed of two polypeptide chains (alpha and beta). The amino-acid sequence of the alpha subunit is almost identical to those of hlh, htsh and hcg and it is the beta subunit, which confers its immunological and biological specificity. In clinical terms, the hfsh enzyme immunoassay method is especially valuable for experimental investigation of various diseases: pituitary tumors, Klinefelter s syndrome, and gonad dysgenesis in women and delayed puberty. PRINCIPLES OF THE ASSAY The ANOGEN coated well method for the quantitative measurement of hfsh in serum is a two-site immunoenzymatic assay, commonly referred to as a «sandwich» assay. The system utilizes a solid phase coupled antibody (antibody-coated well) and a second antibody conjugate with peroxidase (HRP). The sample to be assayed (the antigen) is incubated with the antibody coated well. During the incubation, the FSH molecule is bound to the solid phase. After washing away the excess of hormone, add the enzyme substrate to the well. This step is for the formation of a «sandwich» binding between well, antibody and the conjugate. Standards of known hfsh concentrations are run concurrently with the samples being assayed and a standard curve is plotted. The unknown hfsh concentration in each sample is calculated from this curve. Rev. (06/04) FSH-D 2

4 LIMITATIONS OF THE PROCEDURE 1. Reliable and reproducible results will be obtained when the assay procedure is carried out with strict adherence to the procedure described within this package insert and good laboratory practice. 2. hfsh determination is for diagnostic purposes. The hfsh concentration should be used only as an adjunct to other data (ex: results of other tests, clinical impression, etc.) available to the physician who can take into consideration the history of the patient. Each laboratory should compile its own normal ranges, if possible. This kit is suitable for use with serum of human origin only. 3. Due to the intrinsic design of ELISA assay, the dose hook effect is not seen until a concentration of U/L. Meanwhile, samples greater than 100 U/L cannot accurately be quantitated by extrapolation. These samples should be diluted with the 0 U/L standard and re-assayed to give accurate concentrations. 4. A maximal total pipetting time of 10 minutes per run is suggested. REAGENTS PROVIDED All reagents provided are stored at 2-8 C. Refer to expiration date on the label. 96 tests 1. MICROTITER PLATE (Part FSEL-1) 96 wells Pre-coated wells with mouse anti-hfsh immobilized in the well. 2. CONJUGATE (Part FSEL-2) 11 ml Anti-hFSH conjugate with HRP in a stabilizer solution, thimerosal 0.1%. 3. ASSAY BUFFER (Part FSEL-3) 3 ml PBS Buffer with sodium azide as a preservative. 4. STANDARD 100 U/L (Part FSEL-4) 1 ml Prepared with human FSH in animal serum with 0.09% sodium azide as preservative. These standards were calibrated against the WHO 2 nd IRP standard with 78/ STANDARD 50 U/L (Part FSEL-5) 1 ml Prepared with human FSH in animal serum with 0.09% sodium azide as preservative. These standards were calibrated against the WHO 2 nd IRP standard with 78/ STANDARD 20 U/L (Part FSEL-6) 1 ml Prepared with human FSH in animal serum with 0.09% sodium azide as preservative. These standards were calibrated against the WHO 2 nd IRP standard with 78/549. Rev. (06/04) FSH-D 3

5 7. STANDARD 5 U/L (Part FSEL-7) 1 ml Prepared with human FSH in animal serum with 0.09% sodium azide as preservative. These standards were calibrated against the WHO 2 nd IRP standard with 78/ STANDARD 2 U/L (Part FSEL-8) 1 ml Prepared with human FSH in animal serum with 0.09% sodium azide as preservative. These standards were calibrated against the WHO 2 nd IRP standard with 78/ STANDARD - 0 U/L (Part FSEL-9) 2 ml Prepared with human FSH in animal serum with 0.09% sodium azide as preservative. These standards were calibrated against the WHO 2 nd IRP standard with 78/ SUBSTRATE (Part FSEL-10) 11 ml Buffered solution with TMB. 11. WASH BUFFER (Part FSEL-11) 100 ml Concentrated solution of saline phosphate buffer with thimerosal as a preservative. Dilute each bottle to one (1) liter with deionized or distilled water. 12. STOP SOLUTION (Part FSEL-12) 25 ml 0.5 M Sulfuric Acid (H 2 SO 4 ). CAUTION: Caustic Material! WARNING: Sodium azide may react with lead and copper to form explosive azides. Flush with copious quantities of water. MATERIALS REQUIRED BUT NOT SUPPLIED 1. Precision pipettes (100 µl) with disposable tips 2. 8 channels pipette (100 µl) with disposable tips 3. Plate shaker set at 100 ± 10 rpm 4. Microplate reader with filter at 414 nm or 405 nm 5. Multichannel pipette with a repeater 6. Deionized or distilled water 7. Absorbent paper Rev. (06/04) FSH-D 4

6 PRECAUTIONS 1. All materials in this kit may be used only for in vitro clinical or laboratory tests not involving internal or external administration of the material to human or animals. 2. The Standards and Enzyme Conjugate contain products derived from human blood. Handle the materials as though they were capable of transmitting infectious diseases, since no known test method can offer absolute assurance that such products will not transmit infectious agents even tested non-reactive. 3. Reagents are matched in each kit and therefore reagents from different lot numbers should not be mixed. Do not use after the expiration date. 4. Optimal results will be obtained by strict adherence to this protocol. Respect laboratory quality control rules. 4. The kit containing sodium azide and thimerosal as preservatives. These are toxic and therefore all reagents should be handled carefully to avoid ingestion or skin contact. 5. The stopping solution contains sulfuric acid. This solution should be handle with caution, avoiding contact with skin. 6. Prior to assay, warm all reagents to ambient temperature by allowing them to stand at room temperature. Gently mix all reagents. SAMPLE PREPARATION Serum must be used in this hfsh procedure. No additives or preservatives are necessary to maintain the integrity of the specimen. Store at 2-8 C and assay within one week after collection. If the assay cannot be performed within one week, storage at 20 C is recommended. ASSAY PROCEDURE DO NOT INTERCHANGE REAGENTS BETWEEN KITS BEARING DIFFERENT LOT NUMBERS. ALL REAGENTS AND PATIENT SAMPLES SHOULD BROUGHT TO ROOM TEMPERATURE (22 ± 2 C) BEFORE ASSAYING ALL REAGENTS AND PATIENT SAMPLES SHOULD BE MIXED BY SWIRLING OR GENTLY VORTEXING. DO NOT INDUCE FOAMING. Refer to the assay procedure, Table I. 1. Pipette 25 µl of Assay Buffer in each well. 2. Pipette 100 µl of standard, control or patient sample into the corresponding wells. 3. Incubate for 30 minutes on the plate shaker (100 ± 10 rpm) at room temperature (22 ± 2 C). 4. Wash manually, precautions must be taken to avoid cross-contamination between wells. Decant the well contents by inverting the plate over a container and without reinverting, blot the plate against absorbing paper. Wash each well three times with 300 Rev. (06/04) FSH-D 5

7 µl of washing solution. At the last wash, decant completely the washing solution by tapping the plate against absorbing paper until no trace of water is visible on the paper. 5. Pipette 100 µl of anti-hfsh antibody conjugate with HRP in each well. 6. Incubate for 30 minutes on the place shaker (100 ± 10 rpm) at room temperature (22 ± 2 C). 7. Decant and wash (refer to step no. 3). 8. Pipette 100 µl of the enzyme substrate solution into each well (TMB Cat. No. : ES- 5001). 9. Incubate for 15 minutes on the plate shaker (100 ± 10 rpm) at room temperature (22 ± 2 C). Protect from direct light source. 10. Add 100 µl of the stopping solution and shake the microplate to homogenize. 11. Measure the absorbance at 414 nm or 405 nm using a microplate reader. NOTE: READ THE ABSORBANCES IMMEDIATELY AFTER COMPLETING THE ASSAY. TABLE I Wells Identification Assay Buffer Assay volume Conjugate Substrate Stop. Sol. A 1,A 2 0 U/L B 1,B 2 2 U/L C 1,C 2 5 U/L D 1,D 2 20 U/L E 1,E 2 50 U/L F 1,F U/L G 1,G 2 Serum H 1,H 2 Serum etc etc 25 µl 100 µl INCUBATE DECANT & WASH 100 µl INCUBATE DECANT & 100 µl INCUBATE 100 µl READ AT Incubate 30 minutes on the plate shaker (100 ± 10 rpm) at room temperature (22 ± 2 C) 2. Wash 3 times with multichannel pipette (refer to the washing procedure) 3. Incubate 15 minutes on the plate shaker (100 ± 10 rpm) at room temperature (22 ± 2 C) Rev. (06/04) FSH-D 6

8 CALCULATION OF RESULTS DO NOT ATTEMPT TO SUBSTITUTE ANY PART OF THESE SAMPLE DATA FOR YOUR OWN. Examine data for acceptance consistent with quality control guidelines. Aberrant values should be rejected. Refer to the sample data and calculations, Table II and graphic. The absorbance value of the standard zero should not be exceeding however, it is an indication of careless washing and the assay must be repeated. For each standard, control and unknown sample, the optical density values are averaged (if there are duplicate). Subtract the means of the absorbance values of the zero standard from mean absorbance values of other standards, controls and samples. On millimeter paper using the ordinate for the optical density and the abscissa for the standard concentrations (U/L), a smooth standard curve is plotted. The values of the control and of unknown samples are read directly from the standard curve. TYPICAL DATA EXAMPLE Results of a typical standard run are shown below: TABLE II WELLS OPTICAL DENSITY at 414 nm CONCENTRATION (U/L) 0 U/L U/L U/L U/L U/L U/L Serum Serum Serum etc Rev. (06/04) FSH-D 7

9 EXAMPLE OF hfsh STANDARD CURVE (plotted from data on Table II) O.D. x 10 3 PERFORMANCE CHARACTERISTICS FSH CONCENTRATION (U/L) 1. SENSITIVITY: Sensitivity is defined as the minimum concentration of hfsh, which can be statistically distinguished from standard 0. This value is 1.5 U/L. 2. PRECISION & REPRODUCIBILITY: a) Intra-assay variation: The precision of the assays was verified by assaying eight (8) replicates of three (3) different sera. The results were: Parameters Samples Number of determinations (N) Mean (U/L) Standard deviation (U/L) Coefficient of variation (%) Rev. (06/04) FSH-D 8

10 b) Inter-assay variation: Reproducibility of the protocol was established by assaying three different sera in replicates in successive runs. The results were: Parameters Samples Number of determinations (N) Mean (U/L) Standard deviation (U/L) Coefficient of variation (%) ACCURACY: or recovery. Known amounts of hfsh were added to a human serum sample to determine recovery performance of the assay. The data obtained were: Samples hfsh Add (U/L) Expected value (U/L) Observed value (U/L) % of recovery (3.57 U/L) (9.33 U/L) (32.12 U/L) LINEARITY: or dilution study; two (2) serum samples were diluted and run in the ANOGEN hfsh-elisa kit. The results are as follows: Samples Dilution factor Theoretical value (U/L) Experimental value (U/L) 1 1/ / / / / / / / / / Rev. (06/04) FSH-D 9

11 5. SPECIFICITY: Cross-reactivity between the anti-hfsh used in this kit with various polypeptide hormones is as follows: hfsh hlh hgh htsh hcg hprl 100%* 0%* 0% * 0%* 0%* 0%* * by weight It was assessed by calculating the ratio of results obtained in a presence of 2000 µg/l of hlh, hgh, htsh, hprl and hcg. These cross-reactivity data indicate that for all practical purpose the presence of these hfsh analogs in serum does not interfere with the measurement of serum hfsh when this antiserum is used. 6. CORRELATION STUDY: Clinical samples were analyzed by the ANOGEN hfsh- ELISA kit in parallel with a similar method. The results of this study are as follows: N = 45 Intercept = Slope = 0.95 Correlation coefficient = 0.99 REGRESSION LINE FROM CORRELATION STUDY Similar method (U/L) hfsh ELISA (U/L) Rev. (06/04) FSH-D 10

12 6. EXPECTED NORMAL VALUES : It is recommended that, as with any assay, expected values for given populations be determined by each laboratory over a suitable period of time and that a statistically significant number of assays be collected before definitive clinical significance is attached to the results of the assay. SERUM CONCENTRATION of hfsh WOMEN - excluding mid-cycle peak U/L - mid-cycle peak 6-15 U/L - post-menopausal up to 100 U/L MEN - normal U/L QUALITY CONTROL Good laboratory practice requires that quality control specimens be run with each calibration curve to check the assay performance. Commercial controls are suitable. Any material used should be assayed repeatedly to establish mean values and acceptable ranges to assure proper performance. REFERENCES 1. Hojo, H. et R.S. Ryan, Monoclonal Antibodies against Human Follicle-stimulating Hormone. Endocrinology, 117/6, 2428, (1985). 2. Reichert Jr. L.E. et D.N. Ward, On the Isolation and Characterization of the Alpha and Beta Subunits of Human Pituitary Follicle-stimulating Hormone. 3. Pierce, J.G. et T.F. Parsons, Glycoprotein Hormones: Structure and Functions. Annual Rev. Bioche,., 50, 465, (1981). 4. Cargille, C.M., Ross. G.T. et T. Yoshimi, Daily Variations in plasma Follicle-stimulating Hormone, Luteinizing Hormone and Progesterone in the Normal Menstrual Cycle. J. Clin. Endocrinol. Metab., 29, 12, (1969). 5. Gostin, J.P. (1990) "A Decade of development in Immunoassay Methodology" CL.N. Chem 3618, Nakane, P.K. et A. Kawaoi (1971) "Peroxidase-Labeled antibody a new Method of conjugation". Journal of Histochemistry and Cytochemistry, Vol. 22, No. 12, O'Sullivan, M.J. Bridges, J.W. et V. Marks (1979) "Enzyme Immunoassay a review" Annals of Clinical Biochemistry 16, Rev. (06/04) FSH-D 11