Human T4-HRP ELISA Kit Medical Device Licence No.: 21177

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1 Human T4-HRP ELISA Kit Medical Device Licence No.: Enzyme immunoassay kit for the quantitative determination of T4 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) Web Site: Rev. (06/04) T4-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 4 SAMPLE PREPARATION 5 PREPARATION OF REAGENTS 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 9...Expected Normal Values 10 QUALITY CONTROL 11 REFERENCES 11 Rev. (06/04) T4-D 1

3 INTENDED USE The T4-EIA kit from ANOGEN, catalog number KTSP contains sufficient material to assay at least 96 tests. INTRODUCTION 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 T4 (thyroxine) in small volumes of serum (0.01 ml per assay). PRINCIPLES OF THE ASSAY Thyroxine (T4) and triiodothyronine (T3) are secreted from the thyroid gland and regulated by a sensitive feedback system involving the hypothalamus and pituitary gland (1-3). The hypothalamus releases the thyrotropin releasing hormone (TRH), which stimulates the pituitary to release the thyroid stimulating hormone (TSH). This causes the thyroid to release T3 and T4 and these in turn regulate the release of TRH and TSH via a feedback control mechanism (4). Thyroxine levels are generally found to be high in the serum of untreated patients with hyperthyroidism (4). Therefore, T4 may act as an indicator of the thyroidal state. Circulating T4 is almost exclusively bound by TBG. In order to quantitate total thyroxine in serum, the T4 must first be released from the native serum binding protein. This protein must also be inhibited from further participation in the assay. The ANOGEN Coated well immunoenzymatic assay for the quantitative measurement of serum T4 utilizes a solid phase coupled antibody and a conjugated T4. The sample to be assayed is incubated with the solid phase coupled antibody and conjugated T4. The conjugated T4 competes with T4 from the sample for available binding sites on the antibody. After the incubation period, the wells are decanted. Both conjugated and unconjugated T4 bound to the antibody during the incubation remain on the solid phase. The substrate and the stopping solution are added to provide a color. The wells are counted in a microplate reader. Standards of known T4 concentrations are run concurrently with the samples being assayed and a standard curve is plotted. The unknown T4 concentration in each sample is calculated from this curve. Rev. (06/04) T4-D 2

4 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. T4 determination is for diagnostic purposes. The T4 concentration should be used only as an adjunct to other date (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. A maximal total pipetting time of ten (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 T4EL-13) 96 wells Pre-coated wells with anti-t4 immobilized into the well. 2. CONJUGATE (Part T4EL-14) 2 ml Concentrated T4-HRP conjugate in a stabilizer solution. 3. ASSAY BUFFER (Part T4EL-15) 40 ml HEPES buffer ph 7.2, bovine serum albumin, ANS, sodium salicylate and preservative. 4. STANDARD 30 µg% (Part T4EL-16) 0.5 ml Prepared with human T4 in human serum. 5. STANDARD 12 µg% (Part T4EL-17) 0.5 ml Prepared with human T4 in human serum. 6. STANDARD 4 µg% (Part T4EL-18) 0.5 ml Prepared with human T4 in human serum. 7. STANDARD 1 µg% (Part T4EL-19) 0.5 ml Prepared with human T4 in human serum. 8. STANDARD 0 µg% (Part T4EL-20) 1 ml Prepared with human T4 in human serum. Rev. (06/04) T4-D 3

5 9. SUBSTRATE (Part T4EL-21) 11 ml Buffered solution with TMB. 12. WASH BUFFER (Part T4EL-22) 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 T4EL-23) 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 (10 µl) with disposable tips or 2. a SMI pipette 3. 8 channels pipette (50, 100 and 150 µl) with disposable tips 4. Microplate reader with filter at 414 nm or 405 nm 5. 8 channels repeater pipette 6. Deionized or distilled water 7. Absorbent paper 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. Respect laboratory quality control rules. 3. Reagents are matched in each kit and therefore reagents from different lot numbers should not be mixed. 4. This should not be used after the expiration date. 5. Optimal results will be obtained by strict adherence to this protocol. 6. 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. 6. The stopping solution contains sulfuric acid. This solution should be handle with caution, avoiding contact with skin. 7. Prior to assay, warm all reagents to ambient temperature by allowing them to stand at room temperature. Gently mix all reagents. Rev. (06/04) T4-D 4

6 SAMPLE PREPARATION Serum must be used in this T4 procedure. No additive or preservative 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. PREPARATION OF REAGENTS 1. Washing solution: Dilute each bottle (100 ml) to 1 liter of deionized or distilled water. 2. T4-HRP Conjugate: Just before use, dilute concentrated T4-HRP conjugate with T4 Assay Buffer in the proportion of 1 volume of T4-HRP in 10 volumes of T4 Assay Buffer. (Dilution 1/11). The diluted T4-HRP conjugate is not stable, prepare just enough for the required number of tests. Suggested volume of diluted T4-HRP per number of wells: No. of wells No. of strip Volume (ml) Concentrated T4-HRP Volume (ml) T4 Assay Buffer Discard unused portion of diluted T4-HRP conjugate after completing the addition of the reagent to the wells. 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 a) Pipette 10 µl of standard, control or patient sample into the corresponding wells. b) Pipette 150 µl of diluted T4-HRP conjugate in each well. For the dilution procedure refer to section VIII, Reagent preparation. Rev. (06/04) T4-D 5

7 2. Incubate for forty (40) minutes on the plate shaker (100 ± 10 rpm) at room temperature (22 ± 2 C). 3. 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 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. 4. Pipette 100 µl of the TMB enzyme substrate solution to each well. 5. Incubate for twenty (20) minutes on the plate shaker (100 ± 10 rpm) at room temperature (22 ± 2 C). Protect from direct light source. 6. Add 50 µl of a stopping solution and shake the microplate for homogenizing. 7. Measure the absorbance at 414 nm or 405 nm using a microplate reader. NOTE: READ THE ABSORBANCES IMMEDIATELY AFTER COMPLETING THE ASSAY. TABLE 1 Wells Identification Assay volume Diluted T4-HRP Conjugate Substrate Stop Solution A 1,A 2 0 µg% B 1,B 2 1 µg% C 1,C 2 4 µg% D 1,D 2 12 µg% E 1,E 2 30 µg% F 1,F 2 Serum G 1,G 2 Serum H 1,H 2 etc 10 µl 150 µl INCUBATE DECANT & WASH 100 µl INCUBATE 50 µl READ AT Incubate 40 minutes on the plate shaker (100 ± 10 rpm) at room temperature (22 ± 2 C) 2. Incubate 20 minutes on the plate shaker (100 ± 10 rpm) at room temperature (22 ± 2 C) 3. Wash 3 times (refer to the washing procedure) Rev. (06/04) T4-D 6

8 CALCULATION OF RESULTS DO NOT ATTEMPT TO SUBSTITUE 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. For each standard, control and unknown sample, the optical density value is averaged (if there are duplicates). On millimeter paper using the ordinate for the optical density (or the %B/B 0 ) and the abscissa for the standard concentrations (µg%), 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 (µg%) 0 µg% µg% µg% µg% µg% Serum Serum Serum etc Rev. (06/04) T4-D 7

9 FIGURE 1 EXAMPLE OF T4 STANDARD CURVE (plotted from data on Table II) %B/B 0 T4 CONCENTRATION (µg%) PERFORMANCE CHARACTERISTICS 1. SENSITIVITY: Sensitivity is defined as the minimum concentration of T4 that can be statistically distinguished from standard 0 µg%. This method will reliably detect T4 concentrations as low as 0.4 µg% 2. PRECISION & REPRODUCIBILITY: a) Intra-assay variation: The precision of the assays was verified by assaying 10 replicates of 3 different sera. The results were: Parameters Samples Number of determinations (N) Mean (µg%) Standard deviation (µg%) Coefficient of variation (%) Rev. (06/04) T4-D 8

10 b) Inter-assay variation: Reproducibility of the protocol was established by assaying 3 different sera in replicates in successive runs. The results were as follows: Parameters Samples Number of determinations (N) Mean (µg%) Standard deviation (µg%) Coefficient of variation (%) ACCURACY: or recovery study: Known amounts of T4 were added to a human serum sample determine recovery performance of the assay. The data obtained are indicated below: Samples Expected value (µg%) Observed value (µg%) % of recovery SPECIFICITY: Results are expressed as the ratio of T4 Concentration to the concentration of the cross-reactant that will displace 50% of T4. CROSS-REACTANT % CROSS-REACTIVITY L-Thyroxine (T4) 100% D-Thyroxine ,3,5 -Triiodo L-Thyronine (T3) ,3,5 - Triiodo L-Thyronine (Reverse T3) ,3 5 -Triiodo Thyropropionic Acid ,5-Diiodo L-Thyronine Iodo-L-Tyrosine <0.01 3,5-Diiodo-L-Tyrosine <0.01 Thyroglobulin 0.03 Diphenylhydantoin <0.01 Acetylsalicylic Acid < CORRELATION STUDY: Clinical samples were analyzed by the ANOGEN T4-ELISA kit in parallel with a similar method. The results of this study are as follows: N = 45 Intercept = 0.60 Slope = 1.11 Correlation coefficient = 0.96 Rev. (06/04) T4-D 9

11 REGRESSION LINE FROM CORRELATION STUDY T4-EIA (µg%) Similar method (µg%) 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 in a statistically significant number of assays before definitive clinical significance is attached to the results of the assay. However, the following may be used as a guide to preliminary interpretation. Hypothyroid: Euthyroid: Hyperthyroid: Less than 4.2 µg% 4.2 to 12 µg% Greater than 12 µg% Rev. (06/04) T4-D 10

12 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. Bowers, C.Y., Chally, C., Gual, C., et al. Biochem. Biophys. R., 39, 353 (1970). 2. Fleisher, N., Burgus, R., Vale, W., et al. J. Clin. Endocr., 31, 109 (1970). 3. Anderson, M.S., Bowers, C.Y., Kastin, A.J., et al. N.E.J. Med., 285, 1279 (1971). 4. Snyder, R.J. and R.D. Utiger, J. Clin. Inv., 51, 2077 (1972). 5. Mitsuma, T., Colucci, J. Shenkman, L., et al. Biochem. Biophy.R. 46, 2107 (1972). 6. Chopra, I.J.J. Clin. Endorcr., 35, 938 (1972). 7. Stein, R.B., and L. Price, J. Clin. Endocr. and Metab., 34, 225 (1972). Rev. (06/04) T4-D 11

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