Free T3 (Triiodothyronine) MICRO-ELISA Test Kit

Intended use:The EIA FREE TRIIODOTHYRONINE (fT₃) test is a solid phase competitive enzyme immunoassay (EIA) Kit for the in vitro quantitative determination of free triiodothyronine (T₃) concentration in human serum.

Summary and explanation of the test:The principal tests used in the laboratory evaluation of thyroid function are Total Thyroxin (T₄), Total Triiodothyronine (T₃), T-Uptake (T-Up), a calculated Free Thyroxin Index (FTI) and Thyroid Stimulating Hormone (TSH). The results of these tests are interrelated and help the clinician in making a diagnosis. Clinical hypothyroidism results from underproduction of thyroid hormones by the thyroid gland, consequently an abnormally low circulating T₄ and T₃ concentration in blood. Clinical hyperthyroidism results from excessive production of thyroid hormones and resulting elevation of T₄ and T₃ concentrations.

96 wells T₃ ANTIBODY COATED WELLS Coated with anti T₃ (murine monoclonal); contained in a pack with silica gel desiccant. 1 bottle 12 ml Free T₃-ENZYME CONJUGATE T₃-labeled horseradish peroxidase in a buffered protein solution; contains a preservative. 6 vials 0.75 ml Free T₃ SERUM STANDARDS, 0.0, 1.8, 3.3, 5.7, 8.6 AND 18.0 pg/dl. Human serum with added T₃; contains a preservative. NOTE: Exact value of Standards are lot specific and are listed on the vial label. 1 bottle 50 ml WASH BUFFER CONCENTRATE (20X) Buffered detergent solution; contains a preservative. Dilute bottle to 1000 ml with deionized water. 1 bottle 12 ml SUBSTRATE CHROMOGEN Buffered hydrogen peroxide and 3,3',5,5' tetramethylbenzidine (TMB) solution. 1 bottle 12 ml STOP SOLUTION 3 N HCl.

Additional Materials required, not included in kit:
Disposable tip precision pipettes 0.050, and 0.1 ml
microtiter plate reader
Absorbent paper
Distilled or deionized water

Store all components at 2° 8°C when not in use. The wells, substrate/chromogen, wash buffer and stop solution may be stored at ambient temperature. Expiration date printed on the kit indicates limits of stability.

The T3 ANTIBODY COATED WELLS are supplied in a resealable bag containing a desiccant and must be stored with the bag sealed to protect from moisture. Wells can be stored at 2°- 30°C.

Performance of the Free T₃ test requires use of a precision microtiter plate reader at a wavelength of 450 ± 20 nm:

SPECIMEN COLLECTION:Serum samples are used in the EIA Free T3 Kit procedure. No special preparation of the patient is necessary; fasting is not required. Repeated freezing and thawing of specimens should be avoided. No additives or preservatives are necessary.

STORAGE: Specimens may be stored in a tightly stoppered tube at 2°- 8°C for two days. If the serum is not assayed within 2 days, store frozen (- 20°C) in a tightly sealed tube for up to 3 weeks. Specimens should be allowed to come to room temperature and should be mixed thoroughly by gentle inversion before assaying.

Do not use grossly lipemic specimens. Moderately lipemic, hemolyzed and icteric specimens should not interfere with the assay.

1. Pipet 50 μl of Free T3 standards into the appropriate well. (Only the 0 pg/ml standard need be run if using the previously stored curve).
2. Pipet 50 μl of each control and patient serum into the appropriate well.
3. Pipet 100 μl (0.1 ml) of Free T3-enzyme conjugate into all wells and mix gently.
4. Incubate at room temperature (18°- 30°C) for 60 minutes ± 5 minutes.
5. Decant or aspirate and discard liquid contents of all wells.
6. Fill each well with diluted WASH BUFFER. Decant or aspirate liquid contents of all wells. Do not use tap water.

WARNING: WASHING THE WELLS IS OF CRITICAL IMPORTANCE. Fill the wells to overflowing, you CANNOT cause any carryover between wells. You CANNOT over wash the wells. Completely decant or aspirate all of the liquid out of the wells. SLAP the inverted wells on a FRESH clean piece of absorbent paper AFTER EACH WASH. YOU CANNOT SLAP TOO HARD, REMOVE ALL OF THE LIQUID FROM THE WELLS.

7. Repeat step 6 twice more (for a total of 3 washes). Tap wells free of any liquid or aspirate thoroughly.
8. Pipet or dispense 100 μl (0.1 ml) of SUBSTRATE / CHROMOGEN REAGENT into each well.
9. Mix thoroughly and incubate 15 minutes at room temperature (18°-30°C).
10. Pipet or dispense 100 μl (0.1 ml) of STOPPING REAGENT into each well and mix thoroughly.
11. Read the absorbance of each well at 450 ± 20 nm against water.

REAGENTS:Dilute the entire contents of the WASH BUFFER to 1,000 ml with deionized water. Expiration date is the same as the concentrate. Store at 2°-8°C.

Patient sample may contain pathogens: treat all samples as potentially infectious.
CAUTION: Source material used to prepare Standards was derived from human material. The material was tested using FDA-approved methods and found non-reactive for Hepatitis B Surface Antigen (HBsAg) by ELISA and non-reactive for HIV by ELISA. No known test method can offer total assurance that infectious agents are absent. HANDLE THESE REAGENTS AS IF THEY ARE POTENTIALLY INFECTIOUS. Information on handling human serum is provided in the CDC/NIH manual "Bio-safety in Microbiological and Biomedical Laboratories" (1984).

1. When pipeting reagents, maintain a consistent order of addition from well to well. This will ensure equal incubation times for all wells. Carry out each addition step without pausing. The timing sequence in the addition of each reagent should be the same for all wells.
2. Samples should be pipetted to the bottom of the coated wells.
3. SINGLE POINT CALIBRATION USING A STORED CURVE.

The following optional procedure may be used: 1. For each new kit lot, run one complete standard curve. This standard curve may be used for up to 30 days.
2. For all subsequent sample runs, only the 0 pg/ml standard and controls need to be run with the patient serum samples as described in the Assay Procedure.
3. See the Results Section to calculate patient sample Free T3 values using the single point calibration method.

CAUTION: If control values deviate from their established range, then the assay should be re-calibrated with a new standard curve.

NOTE: It is important in using a single point calibration that:
1. The assay procedure should be the same from run to run.
2. The same spectrophotometer or instrument should be used.
3. The spectrophotometer and all pipettes should be calibrated for accuracy and precision.
4. All test kit components used in an assay must be of the same master lot number. Materials should not be used after the expiration date shown on the package label. Components and test specimens should be at room temperature (18°- 30°C) before testing.

As with all diagnostic tests, a definite clinical diagnosis should not be based on the results of a single test, but should only be made by the physician after all clinical and laboratory findings have been evaluated. In patients receiving drug therapy, the equilibrium between Free T₃ and TBG bound T₃ may be affected. Heparin therapy is known to increase the concentration of non-esterified fatty acids which may displace T₃ on serum binding proteins and thus cause an elevation of Free T₃ values.

Good laboratory practice requires that quality control specimens be run with each patient sample run to check the assay performance. Three controls with normal, low and elevated values should be used. Pooled human serum or commercially available control sera are suitable. Any material used should be assayed repeatedly to establish mean values and acceptable ranges to assure proper performance.

Do not mix or interchange reagent from kits with different lot numbers. Pool and mix reagents from different bottles before use.

Do not use reagents beyond the expiration date printed on each vial or bottle.

Specificity of this test system was proven by determining the interference of the following cross-reactants in the Free T3 assay. Results are expressed as the ratio of free T3 concentration to the concentration of the cross-reactant that will displace 50% of the T3 enzyme conjugate.
l-Triiodothyronine (T3) (100% Cross Reactivity)
l-Thyroxin (< 0.01% Cross Reactivity)
Diiodothyronine (< 0.01% Cross Reactivity)
Diiodotyrosine (< 0.01% Cross Reactivity)
Iodotyrosine (< 0.01% Cross Reactivity)

Intra assay reproducibility was determined by measurement of 10 replicates of three serum pools in a single run.
Serum A: 1.42 (Mean fT₃ (pg/ml); 0.15 (SD); 10.3 (%CV)
Serum B : 2.51 (Mean fT₃ (pg/ml); 2.51 (SD); 5.1 (%CV)
Serum C: 5.11 (Mean fT₃ (pg/ml); 0.20 (SD); 4.0 (%CV)

The interassay reproducibility was determined by duplicate measurement of three serum pools in twelve separate runs.
Serum A: 1.37 (Mean fT₃ (pg/ml); 0.13 (SD); 9.8 (%CV)
Serum B : 2.47 (Mean fT₃ (pg/ml); 0.15 (SD); 6.0 (%CV)
Serum C: 4.97 (Mean fT₃ (pg/ml); 0.17 (SD); 3.5 (%CV)

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