Triiodothyronine T3 CLIA Kit

Catalog No. ABIN504759

The Human Triiodothyronine T3 ELISA Kit (ABIN504759) is a Chemiluminescent ELISA Kit designed to quantify Human Triiodothyronine T3.

Quick Overview for Triiodothyronine T3 CLIA Kit (ABIN504759)

Target: Triiodothyronine T3 (T3)

Reactivity: Human

Detection Method: Chemiluminescent

Method Type: Competition ELISA

Application: ELISA

Product Details Triiodothyronine T3 CLIA Kit

Purpose: Competitive Enzyme Immunoassay The required components for assessing the binding capacity of human serum are enzyme-T3 conjugate, thyroxine, binding protein (P), and immobilized thyroxine antibody (Ab). Upon mixing the enzyme-conjugate and thyroxine with the specimen, a binding reaction results between the patient's binding proteins and the added thyroxine but not with the enzyme conjugate. This interaction is represented below: T4 + P _x0001_ T4-P (1) T4 = Thyroxine added (constant quantity) P = Specific binding proteins (varying quantity) The added thyroxine (T4) not consumed in reaction 1 then competes with the enzyme-T3 conjugate for a limited number of insolubulized binding sites.

Analytical Method: Quantitative

Characteristics: The Measurement of the Total Amount of Binding Sites Available for the Thyroid Hormones in Human Serum or Plasma by a Microplate Chemiluminescense Immunoassay (CLIA)

Application Details

Application Notes: All products that contain human serum have been found to be non-reactive for Hepatitis B Surface Antigen, HIV 1&2 and HCV Antibodies by FDA required tests. Since no known test can offer complete assurance that infectious agents are absent, all human serum products should be handled as potentially hazardous and capable of transmitting disease. Good laboratory procedures for handling blood products can be found in the Center for Disease Control / National Institute of Health, "Biosafety in Microbiological and Biomedical Laboratories," 2nd Edition, 1988, HHS Publication No. (CDC) 88-8395.

Plate: Pre-coated

Protocol:

Specimien Collection and Preparation:

The specimens shall be blood, serum in type and the usual precautions in the collection of venipuncture samples should be observed. For accurate comparison to established normal values, a fasting morning serum sample should be obtained. The blood should be collected in a plain redtop venipuncture tube without additives or anti-coagulants. Allow the blood to clot. Centrifuge the specimen to separate the serum from the cells.

Reagent Preparation:

1. Working Reagent A - T3U-Enzyme Tracer Solution Dilute the T3U-enzyme Tracer 1:11 with T3 Uptake Tracer Buffer in a suitable container. For example, dilute 160l of conjugate with 1.6ml of buffer for 16 wells (A slight excess of solution is made). This reagent should be used within twenty-four hours for maximum performance of the assay. Store at 2-8(C. General Formula: Amount of Buffer required = Number of wells ( 0.1 Quantity of T3-Enzyme necessary = # of wells * 0.01 i.e. = 16 x 0.1 = 1.6ml for Total T3/T4 Conjugate Buffer 16 x 0.01 = 0.16ml (160l) for T3 enzyme conjugate 2. Wash Buffer Dilute contents of Wash Concentrate to 1000ml with distilled or deionized water in a suitable storage container. Store diluted buffer at room temperature 20-27(C. 3. Working Signal Reagent Solution - Store at 2 - 8(C. Determine the amount of reagent needed and prepare by mixing equal portions of Signal Reagent A and Signal Reagent B in a clean container. For example, add 1 ml of A and 1ml of B per two (2) eight well strips (A slight excess of solution is made). Discard the unused portion if not used within 36 hours after mixing. If complete utilization of the reagents is anticipated, within the above time constraint, pour the contents of Signal Reagent B into Signal Reagent A and label accordingly.

Test Procedure:

Before proceeding with the assay, bring all reagents, serum references and controls to room temperature (20 - 27(C). 1. Format the microplates wells for each serum reference, control and patient specimen to be assayed in duplicate. Replace any unused microwell strips back into the aluminum bag, seal and store at 2-8(C. 2. Pipette 0.025 ml (25l) of the appropriate serum reference, control or specimen into the assigned well. 3. Add 0.100 ml (100l) of Working Reagent 1, T3U-enzyme tracer solution to all wells. 4. Swirl the microplate gently for 20-30 seconds to mix and cover. 5. Incubate 45 minutes at room temperature. 6. Discard the contents of the microplate by decantation or aspiration. If decanting, blot the plate dry with absorbent paper. 7. Add 350l of wash buffer (see Reagent Preparation Section), decant (tap and blot) or aspirate. Repeat four (4) additional times for a total of five (5) washes. An automatic or manual plate washer can be used. Follow the manufacturers instruction for proper usage. If a squeeze bottle is employed, fill each well by depressing the container (avoiding air bubbles) to dispense the wash. Decant the wash and repeat four (4) additional times. 8. Add 0.100 ml (100l) of working Signal Reagent to all wells (see Reagent Preparation Section). Always add reagents in the same order to minimize reaction time differences between wells. 9. Incubate for five (5) minutes in the dark. 10. Read the Relative Light Units (RLUs) in each well using a microplate luminometer. The results should be read within thirty (30) minutes of adding the stop solution.

Restrictions: For Research Use only

Handling

Target Details

Target: Triiodothyronine T3 (T3)

Alternative Name: Triiodothyronine

Target Type: Amino Acid

Background: The thyroid gland under the regulatory control of thyrotropin hor_x001F_mone secretes thyroxine (T4) and triiodothyronine (T3) into the general circulation. The released hormones do not circulate as free molecules but are almost entirely (99.9%) bound to specific serum proteins. Three protein fractions with varying affinities and capacities for interaction with T3 and T4 have been identified by reverse flow paper electrophoresis (1). Thyroxine binding globulin (TBG) carries 65~75% of the total circulating concentration. Thyroxine binding pre-albumin (TBPA) has an intermediate avidity for thyroxine (carries approx.15~25%) but little if any avidity for triiodothyronine. Albumin with a low affinity but high capacity carries 10% of thyroxine and 30% of the available triiodothyronine (1, 2, 3). Since the metabolic processes are regulated entirely by the concentration of the free thyroid hormones, which are inversely related to the levels of the binding proteins, an assessment of the binding capacity of human serum was developed in 1957 by Hamolsky (4). In this early method, radioactive T3 was added to a specimen of whole blood. After an incubation period, the mixture was centrifuged and the red cells washed. The radioactivity uptake of the red cells was inversely related to the binding capacity of the serum. Although this method had severe limitations, it proved to be a valuable diagnostic tool. Further technical improvements in the assay methodology of the T3-uptake test resulted as various separation agents such as coated charcoal (5), ion-exchange resins (6), denatured albumin (7), silicates (8), antibodies and organic polymers were employed in place of the red cells. This microplate chemiluminescence immunoassay methodology provides the technician with optimum sensitivity while requiring few technical manipulations. In this method, serum reference, patient specimen, or control is first added to a microplate well. Enzyme-T3 conjugate and thyroxine (T4) are added, and then the reactants are mixed. The endogenous binding proteins of the sample react with the thyroxine, but not with the enzyme conjugate. This leads to a higher binding of the enzyme conjugate to the antibody combining sites, reactive for triiodothyronine and thyroxine, immobilized on the well as the binding capacity of the specimen increases. After the completion of the required incubation period, the antibody bound enzyme conjugate is separated from the unbound enzyme conjugate by aspiration or decantation. The activity of the enzyme present on the surface of the well is quantitated by reaction with a suitable substrate to produce light. The employment of several serum references of known unsaturated thyroid hormone binding capacity permits construction of a graph of light and concentration. From comparison to the dose response curve, an unknown specimen's absorbance can be correlated with thyroid hormone binding capacity