El i aad go Enzyme Immunoassay for quantitative ‘cantration in human eerum and plasma, (for Jinvltrodiagnostic use only) ‘SUMMARY: Thyroxine hormone is secreted in the thyroic important comaonent of important hyperthyrol isease, in those with myxedema, the Mleveliedecreased _ AEST PRINCIPLE: Competitive E1A (Quanta ) In a competive EIA, enzyme competes ized antibody coated on the micro wel Unbound antigen fraction is then washed away. The enzyme actvity in the antivody bound ion which is inversely proportional to the weasured by ing calibrators fof known antigen values, dose response cune ‘may be generated. from which the antigen ‘coneentrationafan unknown can be obtained KIT CONTENTS: 41, TéReaction Coated Microplate (96 well) ne 96-weil microplate coated with sheep ant {thyroxine serum and packaged in an aluminum Daguithacessicant Storeat23°C, 2. Té-Enzyme Conjugate (1.Smilvial)-[2) ‘One vial of thyroxine-horseradsh peroxidase (HRP) conjugate ina bovine burst ‘alr A preservative has been added. Store at 26 Btoreat 28°C. of serum reference fr tyro tions of 0 (4A) 2.0 (4B). 5:0 *5.0(dE]and25.0[@F gi. Store 28°C, Apreservativehas baen added. 5. Substrate A (7mivial)-[5] ‘One botle containing tetramethylbenzidine (TMia)inbuter. Store at23°C. 6. Substrate 8 (7mivial)-[6) ‘One bats containing hydrogen peroxide (+.0,) Inbufler. Storost2 °C. 7. Wash Solution Concentrate (2% ‘One val containing a surfactant in buflored saline. preservative has been added, Store at 230°C. 8. Stop Solution (@mitvial) [8] (One botie containing a strong acid (1N HCI. Storeat 230°C. 9, Productinser. PRECAUTIONS: 4. Al Reagents are for in vo diagnostic uso only 2. Please handle all reagants and controls provided in the kit 25 potenlaly infectous although they are non reactive for HIV 1 and 2, HBsAg and HCV ay FDA appraveditests, 3. The stop solution providedinthe ktisastrong acid (1N HC). Please wear gloves and face ‘mask avoid contact with the skin, 4. Please use a disposable container or acid vwashed tubes for preparing the substrate vith INHOlor INH 2, Micro plate washer or a squeeze bottle (optiona, ‘Quality control material Timer. Micro plate cover for incubation steps. ‘Storage container for storage of wash bur. 7. Vacuum aspirator (optional) for wash steps. 8. Deionized water. tes capable of delivering 25 1004 10. Dispensers) for repetitive dl ‘and 0.3 mi. volumes witha pri than 1.5% (optional. 11, Containers for mixing reagents. ‘SAMPLE COLLECTION AND PREPARATION : ‘Serum or plasma may be used forthe test, Serum jasma should be prepared trom a whole blood specimen obtained by approved aseptic technique. If testing cannot be done wi hour after sample collection, refigerate ‘maximumof 48 hours) the specimen mec PREPARATION OF REAGENTS AND STORAGE: 1. Working Conjugate Solution lugate solution ty ‘The prepared reagents should be slored at2. 8°C and must be used preparation 2. Wash Solution Preservedat21-25°Cfor60 days When stored at 2.8°C the wash solution concentrate may get crystalized, use that cnly after thawing properly by keeping at 37°C forsome time. 3. Working Substrate Solution Determine the amount ofreagentneededand Brepare by mixing equal portions of Substrate and Substrate Bin a sutable container. For example, add Iml ofA and tml of B per two {2)eight well strips (A sight excess of solution Ismade, Discarc the unused portion). Note: Do not use the working substrate if Itlooks blue. ASSAY PROTOCOL: Before starting the assay, allow al the reagents, serum references & controls 10 reach room temperature, 1. Format the micro plate wells for each callorator and patient specimen to be assayed. Replace the unused 2. Add 25 ul of the calibrator and the patient specimen to the assigned wes enzyme conjugate reagentto the bottom 3. Shake the microplate gently for 20-30 ‘seconds & cover 6. Incubate for 60 min. at controlled room temperature (21-25°C), 7. Aspirate the contents ofthe wells & f complotely (approximately 300 pl) with diluted- washing solution. Repeat the aspiration and washing procedure two mere times. After the ast wash, blotthe micro plat on absorbent issue fo remove excess quid is. Aways add reagent inthe same mize reaction time aiference 9. Incubate at controled room temperature (21 25°C} forfteen minutes, 10, Add 80 lof Stop Solution to each well and Wor 15-20 seaonds, 11, Read the absorbance in each well at 450 mm {using a reference nave length of 620-630 ‘Amo minimize well imperfections) in a micro plate reader. (QUALITY PARAMETERS: Every control with known concent hypothyroid, euthyroid and hyperthyroid range ‘ust be included in every run. Each laboratory must establish lls own acceplable assay performance limits. Run to. run reproducibly Must be observed in a batch. If there is any ‘deviation from tne established data, t could be ‘due to degradation in the kit components. or change inthe experimental cond The absorbance ofthe calibrator 4A (0.0) should be> 1.3 or an assay tobe valid CALCULATION OF RESULTS: A & samples reference B. Plot @ point to absorbance of against concentration of each calibrator on thexaxle 3 absorbance value for each sample responding concentration obiaines, GUIDELINES: A. Performanceoftheassay 1. Same sequence of reagent addition should be maintained throughout assay deitcan be avoided 2. Donottouchthebottom ofthe wells 3. Improperwashing may leactofaultyresuts. 4. Lipemic, hemolyzed_ and contaminated specimen should not be used, 8B. Interpretation of results 1. The concentration obtained ofthe calibrators valuesinygid 2 Total serum thyroxine concentration obiainedis reguated by many factorsike theTBG. Thus total thyroxine concentration ne Ie nat sufiient to assess clinical Slandard deviation, Intra Assay Reproducibility: (Valve in ygial) 3 sets pooled samples (24 in each) were studied ‘and gave a coeficient of 3 sets of pooled samples were studied for inter assay reproducblly and gave a coeficient of ‘variation of 8.3% for low value samples and <5% for normal andhigh samples, Accuracy: ‘Stacan"™ Té results wore compared with 2 coated ‘The correlation coefficient and least square regression equation were completed for Sasa” Téin comparison wit the reference method ‘A very high degree of correlation was found between Btaeen'T4 and ine reference method ‘SENSITIVITY AND SPECIFICITY: Sensitivity 80 pg. Equivalent to a sample containig a concentration of.4 us, Specificity The extent of cross reactivity of Etseea™ 4 with & interfering substances was! {L-Thyroxine 1.00 - Thyroxine -0.98 4- Trlodathyronine- 1.5% increase in 0.0. 1 Triodothyronine - 3% increase in O.D. at 100 Indio beas follows, roxine” Clinical Endocrinol, 33,865 2. Storing, L., Diagnosis and Treatment of Thyroid Disease, Cleveland CRC Press, P. 3, Charkes ND, "The many causes of subclinical hyperthyroidism. "Thyroid 1996;6:301-306, Manutaetred by urbe