HCG ELISA Kit Medical Device Licence No.: 16416

Page HCG ELISA Kit Medical Device Licence No.: 16416 Enzyme immunoassay kit for the quantitative determination of Human Chorionic Gonadotropin (hcg) concentration in serum. Catalog Number: SL100294 96 tests For in vitro diagnostic use only. 396 N Summit Ave., Suite 2 Gaithersburg, MD 20877 U.S.A. Fax: (301) 330-6381 Email: info@signagenlabs.com Web Site: www.signagenlabs.com TABLE OF CONTENTS Rev. (06/04) HCG-D

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...Specificity 9...Correlation Study 10...Expected Normal Values 10 QUALITY CONTROL 11 REFERENCES 11 Rev. (06/04) HCG-D 1

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 concentration of hcg (human chorionic gonadotropin) in small volumes of serum (0.1 ml per assay). INTRODUCTION Human chorionic gonadotropin (hcg) is a glycoprotein produced by placenta trophoblastic cells whose primary function is maintaining the corpus luteum. The hcg has been detected in the urine and serum of pregnant women, six to fifteen days after conception. Its concentration rises throughout pregnancy, reaching peak levels of approximately 100 000 to 200 000 U/L at the end of the first trimester. The hcg has also been reported in conditions other than normal pregnancy including threatened abortion, ectopic pregnancy, multiple myeloma, malanoma, trophoblastic tumors and carcinomas of the stomach, liver, pancreas and breast. The hcg consists of an alpha polypeptide subunit and a beta polypeptide subunit. The alpha subunits for the four human glycoprotein hormones (LH, FSH, TSH, hcg) are nearly identical. The beta subunit, which also carries some amino acid sequences common among these hormones, is distinct enough to produce the biological and immunological specificity for each hormone. Although the beta subunit for these hormones is distinct, the common amino acid sequences can cause cross-reactivity and performance problems with some polyclonal hcg antisera. The use of purified antibodies in the SignaGen coated well ELISA hcg assay eliminates this interference by providing a homogeneous antibody with high affinity to hcg and virtually no cross-reactivity with LH, FSH or TSH. PRINCIPLES OF THE ASSAY The SignaGen coated well method for the quantitative measurement of hcg 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 peroxydase (HRP). The sample to be assayed (the antigen) is incubated with the antibody coated well. During the incubation, the hcg molecule is bind to the solid phase. After washing the excess of hormone add the enzyme substrate to the well. This step is for the formation of a sandwiches binding between well, antibody and the conjugate. Rev. (06/04) HCG-D 2

Standards of known hcg concentrations are run concurrently with the samples being assayed and a standard curve is plotted. The unknown hcg concentration in each sample is calculated from this curve. LIMITATIONS OF THE PROCEDURE 1. Reliable and reproducible results will be obtained when the assay procedure is carried out with a strict adherence to the exact procedure described within this package insert and good laboratory practice. 2. hcg determination is for diagnostic purposes. The hcg concentration should be used only as an adjunct to other data (ex.: results of other tests, clinical impressions, 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 intrinsic design of hcg ELISA assay, a dose hook effect is not seen until a concentration or even a decrease of 1,000,000 U/L. Samples greater than 200 U/L cannot accurately be quantitated by extrapolation. These samples should be diluted with 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 CGEL-1) 96 wells Pre-coated wells with anti-hcg immobilized into the well. 2. CONJUGATE (Part CGEL-2) 11 ml Anti-hCG conjugate with HRP, in a stabilizer solution, and thimerosal 0.1%. 3. ASSAY BUFFER (Part CGEL-3) 3 ml PBS Buffer with sodium azide as a preservative. 4. STANDARD 200 U/L (Part CGEL-4) 1 ml Prepared with human hcg in animal serum with 0.09% of sodium azide as preservative. Standards were calibrated against the WHO 3 rd IRP standard 75/537. 5. STANDARD 50 U/L (Part CGEL-5) 1 ml Prepared with human hcg in animal serum with 0.09% of sodium azide as preservative. Standards were calibrated against the WHO 3 rd IRP standard 75/537. 6. STANDARD 25 U/L (Part CGEL-6) 1 ml Prepared with human hcg in animal serum with 0.09% of sodium azide as preservative. Standards were calibrated against the WHO 3 rd IRP standard 75/537. Rev. (06/04) HCG-D 3

7. STANDARD 5 U/L (Part CGEL-7) 1 ml Prepared with human hcg in animal serum with 0.09% of sodium azide as preservative. Standards were calibrated against the WHO 3 rd IRP standard 75/537. 8. STANDARD - 0 U/L (Part CGEL-8) 25 ml Prepared with human hcg in animal serum with 0.09% of sodium azide as preservative. Standards were calibrated against the WHO 3 rd IRP standard 75/537. 9. SUBSTRATE (Part CGEL-9) 11 ml Buffered solution with TMB. 12. WASH BUFFER (Part CGEL-10) 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. 13. STOP SOLUTION (Part CGEL-11) 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 or 405 nm 5. Multichannel pipette with a repeater 6. Deionized or distilled water 7. Absorbent paper Rev. (06/04) HCG-D 4

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 hcg 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, freezing is recommended. Patient samples for which the expected values are higher than 200 U/L must be diluted with the animal serum (started with 1/10 dilution). ASSAY PROCEDURE DO NOT INTERCHANGE REAGENTS BETWEEN KITS BEARING DIFFERENT LOT NUMBERS. ALL REAGENTS AND PATIENT SAMPLES SHOULD BE BROUGHT TO 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 in each well. 2. Pipette 100 µl of standard, control or patient sample into the corresponding wells. Rev. (06/04) HCG-D 5

3. Incubate for 30 minutes on the plate shaker (100 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 re-inverting, blot the plate against absorbing paper. Wash each well three times with 300 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-hcg antibody conjugate with HRP in each well. 6. Incubate for 30 minutes on the plate 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 to each wells (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 a stopping solution and shake the microplate for 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 Solution A 1,A 2 0 U/L B 1,B 2 5 U/L C 1,C 2 25 U/L D 1,D 2 50 U/L E 1,E 2 200 U/L F 1,F 2 Serum G 1,G 2 Serum etc etc 1 3 1 3 2 25 µl 100 µl INCUBATE DECANT & 100 µl INCUBATE DECANT & 100 µl INCUBATE 100 µl READ AT 414 1. Incubate 30 minutes on the plate shaker (100 ± 10 rpm) at room temperature (22 ± 2 C) 2. Incubate 15 minutes on the plate shaker (100 ± 10 rpm) at room temperature (22 ± 2 C) 3. Wash 3 times with a multichannel pipette (refer to the washing procedure) Rev. (06/04) HCG-D 6

CALCULATION OF RESULTS DO NOT ATTEMPT TO SUBSTITUTE ANY PART OF THIS SAMPLE DATA FOR YOUR OWN. Examine data for acceptance consistency with quality control guidelines. Aberrant values may be rejected. Refer to the sample data and calculations, Table II and graphic. The absorbance value of the standard zero should not be exceeding 0.120 however, it is an indication of careless washing and the assay must be repeated. For each standard, control and unknown sample, the optical density value is averaged (if there is a duplicate). Subtract the means of the absorbance values of the zero standard from 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 -- -- 5 U/L 0.054 -- 25 U/L 0.447 -- 50 U/L 0.931 -- 200 U/L 2.800 -- Serum 0.107 7.7 Serum 0.532 29.3 etc Rev. (06/04) HCG-D 7

EXAMPLE OF hcg STANDARD CURVE (plotted from data on Table II) O.D. hcg Concentration (U/L) PERFORMANCE CHARACTERISTICS 1. SENSITIVITY: Sensitivity is defined as the minimum concentration of hcg, which can be statistically distinguished from standard 0. This value is 2.5 U/L. 2. PRECISION & REPRODUCIBILITY: a) Intra-assay variation: The precision of the assays was verified by assaying 8 replicates of two different sera. The results were: Parameters Samples 1 2 Number of determinations (N) 8 8 Mean (U/L) 9.1 89.0 Standard deviation (U/L) 0.34 1.74 Coefficient of variation (%) 3.7 1.9 Rev. (06/04) HCG-D 8

b) Inter-assay variation: Reproducibility of the protocol was established by assaying two different sera in replicates in successive runs. The results were: Parameters Samples 1 2 Number of determinations (N) 16 16 Mean (U/L) 8.7 92.6 Standard deviation (U/L) 0.59 7.5 Coefficient of variation (%) 6.8 8 3. ACCURACY: or recovery study: known amounts of hcg were added to a human serum sample determine recovery performance of the assay. The data obtained are indicated below: Samples hcg added (U/L) Expected value (U/L) Observed value (U/L) % of recovery 1 ( 5.1 U/L) 11.7 16.8 17.9 107 25.9 31.0 30.0 97 2 (11.9 U/L) 25.9 37.8 40.3 107 52.6 64.5 69.2 107 3 (78.4 U/L) 11.7 90.2 84.2 94 25.9 104.3 112.4 108 4. SPECIFICITY: cross-reactivity between the anti-hcg used in this kit with various polypeptide hormones are as follows hcg hlh hgh htsh hfsh hprl 100% * 0.15% * <0.001% * <0.001% * <0.001% * 0.04% * * by weight It was assessed by calculating the ratio of results obtained in a presence of 1 000 µg/l of hlh, hgh, htsh, hfsh and hprl. These cross-reactions data indicate that for all practical purpose the presence of these hcg analogs in serum does not interfere with measurement of serum hcg when this antiserum is used. Rev. (06/04) HCG-D 9

5. CORRELATION STUDY: Clinical samples were analyzed by the SignaGen hcg- ELISA kit in parallel with a similar method. The results of this study are as follows: N = 26 Intercept = -1.9 Slope = 1.08 Correlation coefficient = 0.96 REGRESSION LINE FROM CORRELATION STUDY Similar method (U/L) Rev. (06/04) HCG-D 10 hcg-elisa (U/L) 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. RANGE OF EXPECTED hcg CONCENTRATIONS Approximate gestational age Approximate hcg range U/L Recommended dilutions 0 2 weeks 0 300 -- 2 3 weeks 100 1 000 1 :10 3 4 weeks 500 6 000 1 :10, 1 :100 1 st trimester 10 000 200 000 1 :100, 1 :1 000 2 nd trimester 3 000 50 000 1 :100, 1 :1 000 3 rd trimester 1 000 50 000 1 :100, 1 :1 000

SERUM CONCENTRATION of hfsh WOMEN - excluding mid-cycle peak 0.2 10 U/L - mid-cycle peak 6-15 U/L - post-menopausal up to 100 U/L MEN - normal 0.2 8 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. Krieg, A.F. Pregrancy Tests and Evaluation of Placental Function. Clinical Diagnosis and Management by Laboratory Methods, 16th ed. Henry, J.B. editor, W.B. Saunders Co. Philadelphia. app. 680, 1979. 2. Brody, S., and Carlstrom, G., Immunoassay of Human Chorionic Gonadotropin in Normal and Pathologic Pregnancy. J. Clin. Endocrinol. Metab., 22: 564, 1962. 3. Braunstein, G.D., Rasor, J., Adleer, D., et al: Serum Human Chorionic Gonadotropin Levels Throughout Normal Pregnancy. Am. J. Obstet. Gynecol., 126: 678, 1976. 4. Skinner, M.S., Seckinger, D., Evaluation of Beta-Subunit Chorionic Gonadotropin as an Aid in Diagnosis of Trophoblastic Disease. Ann. Clin. Lab. Sci., 9 (4): 347-52, 1979. 5. Braunstein, G.D., Vaitukaitis, J.L., Carbone, P.P., and Ross, G.T., Ectopic Production of Human Chorionic Gonadotropin by Neoplasms. Ann. Intern. Med., 78: 39-45, 1973. 6. Bélanger, A., Côté, J., Lavoie, M., et al. The Production of High Affinity Monoclonal Antibodies to Human Chorionic Gonadotropin and their application to Immunoradiometric Assay., J. of Immunoassay, 7 (1-2): 37-55, 1986. Rev. (06/04) HCG-D 11