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E1-198 Testosterone

E1-198 Testosterone
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    9428 Eton Ave, Unit O Chatsworth, CA 91311 Tell :( 818)717-1840 Fax :( 818)717-1842 Total Testosterone Catalog No. E1-198 INTENDED USE The Immunospec TESTOSTERONE Quantitative is a solid phase enzyme linked immunosorbent assay (ELISA). This test provides quantitative measurement of testosterone in the human serum. (For professional use only)   SUMMARY AND EXPLANATION OF TEST Testosterone is formed in the Leydig Cell, then capillaries and veins carry it to the periphery or it traverses testicular myoid cells and enters the seminiferous tubules where it its involved in spermatogenesis. In the seminiferous tubules testosterone stimulates primary spermatocytes to form secondary spermatocytes and finally young spermatocytes. Testicular secretion accounts for 95 percent of the circulating testosterone present in men. In the female, the ovary and adrenal secrete small amounts of testosterone; however, the majority of testosterone in the blood derives from metabolism of androstenedione 1 . The measurement of serum testosterone in man and boy is related to the investigation of testicular dysfunction and is used to monitor the treatment of patients with congenital adrenal hyperplasia 2 . In woman, serum testosterone is useful evaluating hirsutism, alopecia and menstrual disorder. The plasma concentration of testosterone also is part of the evaluation of newborns or young infants with ambiguous genitia, and isolated micropenis 3 . The traditional assay of plasma testosterone involves extraction of steroids, chromatographic purification and RIA 4 . Immunospec TESTOSTERONE Quantitative provides an enzyme immunoassay system. This system is used to establish an ELISA method for detection of total testosterone in serum. PRINCIPLE OF THE ASSAY The Immunospec TESTOSTERONE Quantitative test is based on the principle of competitive solid phase enzyme immunoassay. The patient sample competes with enzyme-labeled-testosterone for a fixed and limited number of antibody sites on the microtiter wells. In the assay procedure, the testosterone standard or patient serum is incubated with the testosterone antibody and the testosterone-horseradish peroxidase conjugate in the anti-rabbit IgG coated well. In this solid-phase system, the antibody bound testosterone will remain on the well while unbound testosterone will be removed by washing. A color is developed when the substrate, TMB, is mixed with the antibody bound testosterone-horseradish peroxidase enzyme conjugate. After a short incubation, the enzyme reaction is stopped and the intensity of the color measured with microreader at 450 nm. When high levels of patient testosterone are present, less enzyme conjugate is bound; then less color development is observed. WARNING AND PRECAUTION 1.   The Immunospec TESTOSTERONE Quantitative is designed for in vitro use only. 2.   The components in this kit are intended for use as an integral unit. The components of different lots should not be mixed. It is advised not to exchange strips of different plates even if of the same lot. The kits may have been shipped or stored under different conditions, and the characteristics of the plates may result slightly different.    STORAGE AND STABILITY Store the kits at 2-8 o C in the refrigerator. Keep microwells in dry bag with desiccants. The reagents are stable until expiration of the kit. The calibrators and conjugate are stable for 8 weeks after first opening. Chromogen substrate solution should be colorless; if the solution turns blue, it must be replaced. Do not expose these reagents to strong light during storage or usage. MATERIALS PROVIDED 1.   Microwell strips (96 wells): anti-rabbit IgG coated wells. 2.   Enzyme Conjugate (6 mL)(red): Testosterone conjugated to horseradish Peroxidase. 3.   Testosterone Antibody Solution (6 mL) (blue). 4.   Reference Standard Set (0.5 ml each vial): Prepared 0, 0.5, 1, 2.5, 10 and 20 ng/mL in the human serum base. 5.   Washing Buffer Concentrate (50 mL) (20X): Working Washing Buffer: Prepared by adding the Concentrate 50 mL into 950 ml of distilled water. 6.   Solution A (11 mL): Buffer solution containing hydrogen peroxide. 7.   Solution B (11 mL): Tetramethylbenzidine solution. 8.   Stop Solution: 2 N HCl.  9.   Well holder for securing individual wells. MATERIALS REQUIRED BUT NOT PROVIDED 1.   Microwell reader. 2.   Pipetor with tip for 10 uL, 50 uL and 100 uL. SPECIMEN COLLECTION AND HANDLING Collect blood by venipuncture, allow clotting, and separating the serum by centrifugation at room temperature. If sera cannot be assayed immediately, they can be stored at 2 - 8 o C or frozen for few days. Do not use hyperlipemic, hemolyzed, contaminated or heat inactivated sample as they may cause erroneous results. PREPARATION FOR ASSAY 1.   Before beginning the test, bring all specimens and reagents to room Temperature. 2.   Have all reagents and samples ready before the start of the assay. 3.   Once the test is begun it must be performed without any interruption to get the most reliable and consistent results. 4.   Use new disposable tips for each specimen. ASSAY PROCEDURE 1.   Secure the desired number of coated wells in the holder. 2.   Dispense 10 uL of standards, controls or serum samples into appropriate wells. Save one well for the blank (do not add any standards or enzyme conjugate). 3.   Dispense 50 uL of antibody solution into each well except the blank well. 4.   Dispense 50 uL of enzyme conjugate into each well except the blank well. 5.   Incubate for 60 minutes at room temperature. 6.   Remove incubation mixture and rinse the wells with working washing buffer 5 times. 7.   Dispense 100 uL of Solution A and 100 uL Solution B into each well including the blank well. 8.   Incubate for 30 minutes at room temperature. 9.   Stop reaction by adding 50 uL of 2 N HCl  to each well and read O.D. 9.   Record absorbencies. PROCEDURAL NOTE 1.   Azid and thimerosal at concentration higher than 0.01% interfere in this test; therefore, the assay of control sera or samples containing the above compound may give high results. 2.   It is very important to wash the microwell thoroughly and remove the last droplets of water to achieve the best results. 3.   Absorbance is a function of the time and temperature of incubations. It is recommended to have all reagents and samples caps remove, all needed wells secured in holder and assigned. It will ensure the equal elapsed time for each pipetting without interruption. 4.   For the same reason the size of the assay run should be limited. It is suggested running no more than 20  patient samples with a set of reference standards in duplicate. CALCULATION OF RESULTS 1.   Calculate the index A/Ao x 100 for standards, controls, and patient samples. A is the absorbance of each standard, control or patient sample and Ao is the average absorbance of the replicates of 0 ng/ml testosterone standard. 2.   Plot the concentration (X) of each reference standard against its index (Y) A/Ao x 100 on the logit-log paper. Draw a point to point line through the mean of the duplicate point. 3.   Obtain the value of patient testosterone by standard curve. The following data is for demonstration purpose only and must not be used in place of data for each assay. Calibratr or Controls (ng/ml) Absorbncy (450 nm) A/Ao x 100 Calculated Results (ng/ml) 0 2.218 2.380 0.5 1.913 1.878 82 1.0 1.457 1.489 64 2.5 0.974 0.934 41 10.0 0.437 0.464 20 20.0 0.292 0.239 12 Control 1 1.857 2.013 84 0.44 Control 2 0.478 0.492 21 8.2 10%100%0.512.51020Testosterone (ng/mL)    A   /   A  o   X   1   0   0   %  LIMITATION Extrapolation of testosterone values beyond the standard curve may yield variable results samples containing >30 ng/mL testosterone can be diluted with the 0 calibrator and retest. Calibrators and controls from other manufacturers may contain serum preservatives such as sodium azide, which is  incompatible with Immunospec ELISA reagents should not be used. Whenever laboratory data conflict with clinical finding or impressions, clinical judgment should be exercised and additional evaluation undertaken. EXPECTED VALUE 1.   It is recommended that each laboratory should determine its own normal and abnormal range. 2.   Serum samples from 100 Normal women were assayed and 90% of the values were found to be less than 2.2 ng/mL. The values from 100 normal men sera were from 3 to 20 ng/mL. 3.   Danazol metaboltes may compatitively displace testosterone fromplasma protein; therefore, the value for testosterone in danazol- treated patients should be appropriately corrected before interpretation 5 . 4.   Men with low testosterone concentration are usually hypogonadal. However, because variations in the testosterone transport protein, sex hormone-binding globulin directly influence the total testosterone concentration confirmation of a low testosterone with other alternated methods are recommended 6 . QUALITY CONTROL Each laboratory should utilize controls at several levels to monitor assay performance. The controls should be treated as unknown values obtained should be in agreement with the assigned values of the controls. Controls can be obtained from commercially available sources but should not contain sodium azide as preservative. PERFORFORMANCE CHARACTERISTICS ACCURACY Mixing an aliquot of pooled serum and testosterone standards performed recovery studies. The testosterone values were measured and percentage of recovery was determined. Initial Value (ng/mL) Concentration Spiked (ng/mL) Expected Values (ng/mL) Observed Values (ng/mL) Recovry (%) 0.4 0.4 0.4 0.4 100% 0.4 5.0 2.75 2.9 105% 0.4 14.0 7.20 7.15 99% 10 0.4 5.20 5.1 98% 10 5.0 7.50 8.0 107% 10 14.0 12.0 12.0 100% PRECISION Intra-assay: Ten samples each from three pooled sera were assay in a single run while three pooled sera were assayd in duplicate 10 times in 5 days for Inter-assay. Intra-Assay Pool A Pool B Pool C N 10 10 10 Mean (ng/mL) 0.80 7.4 12 S.D. (ng/mL) 0.09 0.65 0.9 C.V. (%) 11% 8.8% 7.5% Inter-Assay Pool A Pool B Pool C N 10 10 10 Mean (ng/mL) 0.6 6.5 14 S.D. (ng/mL) 0.08 0.54 1.1 C.V. (%) 13% 8.3% 8%  SPECIFICITY The following compounds were tested for cross reactivity of the assay. Compounds Percent Cross-Reactivity Testosterone 100% Androstenedione 0% Androsterone 0% Dihydrotestosterone 5% Dihydroepiandrosterone 0% Estriol 0% Estridiol 0% Estrone 0% Progesterone 0% FSH 0% LH 0% SENSITIVITY The sensitivity obtained from this study was less than 0.1 ng/mL. The minimal detectable concentration of testosterone is estimated to be 0.125 ng/mL. The minimal detectable concentration is defined as that concentration of testosterone which corresponds to absorbance value that is two standard deviations less than the mean absorbance value of twenty replicate determinations of the zero diluent. REFERENCES 1.   DM Styne and MM Grumbach. Puberty in the Male and Female: Its physiology and disorders. In Reproductive Endocrinology: Pathophysiology and Clinical Management, SSC Yen, RB Jaffe (eds).Philadelphia, Saunders, 1978, pp 189-240. 2.   M Sanchez-Carbayo, M Mauri, R Alfayate, C Miralles, and F Soria. Elecsys Testosterone Assay Evaluated. Clin Chem 44(8): 1744 -1746, 1998. 3.   JS Fuqua, ES Sher, CJ Migeon, and D Berkovitz. Assay of Plasma Testosterone During the First Six Months of Life: Importance of Chromatographic Purification of Steroids. 41(8): 1146 –1149. 4.   R L fitzgerald and DA Herold. Serum Total Testosterone: Immunoassay Compared with Negative Chemical Ionization Gas Chromatography-mass Spectrometry. Clin Chem 42(5): 749-755, 1996. 5.   RV Haning Jr, IH Carlson, J Cortes, WE Nolten and S Meier. Danazol And Its Principal Metabolites Interfere with Binding of Testosterone, Cortisol, and Thyroxin by Plasma Proteins, Clin Chem 28(4): 696, 1996. 6.   SJ Winters, DE Kelley and B Goodpaster. The Analog Free Testosterone Assay: Are the Results in Men Clinically Useful? Clin Chem 44(21): 2178-2182, 1998. S:\ImmunoSpec\Immuno doc\Immunospec Inserts\Immunospec ELISA E1-198 Testosterone European Authorized Representative: CEpartner4U , Esdoornlaan 13, 3951DB Maarn . The Netherlands. Tel.: +31 (0)6.516.536.26  Manufacturer:   IMMUNOSPEC CORPORATION 9428 Eton Ave,Unit O Chatsworth,CA,91311,USA (818) 717-1840  PI   E1-198 Revision 4 Revision date: 08/2005  
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