TSH ELISA Kit

Catalog No. ABIN649052

Product Details TSH ELISA Kit

Target: TSH (Thyroid Stimulating Hormone (TSH))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Purpose: Immunoenzymometric assay (TYPE 3): The essential reagents required for an immunoenzymometric assay include high affinity and specificity antibodies (enzyme conjugated and immobilized), with different and distinct epitope recognition, in excess, and native antigen. In this procedure, the immobilization takes place during the assay at the surface of a microplate well through the interaction of streptavidin coated on the well and exogenously added biotinylated monoclonal anti-TSH antibody. Upon mixing monoclonal biotinylated antibody, the enzyme-labeled antibody and a serum containing the native antigen, reaction results between the native antigen and the antibodies, without competition or steric hindrance, to form a soluble sandwich complex. After equilibrium is attained, the antibodybound fraction is separated from unbound antigen by decantation or aspiration. The enzyme activity in the antibodybound fraction is directly proportional to the native antigen concentration. By utilizing several different serum references of known antigen values, a dose response curve can be generated from which the antigen concentration of an unknown can be ascertained.

Analytical Method: Quantitative

Components: A. Thyrotropin Calibrators (1ml/vial) . Seven vials of references for TSH Antigen at levels of 0 (A), 0. 5 (B), 2. 5 (C), 5. 0 (D), 10 (E), 20 (F) and 40 (G) µIU/ml. Store at 2-8°C. A preservative has been added. Note: The calibrators, human serum based, were calibrated using a reference preparation, which was assayed against the WHO 2nd IRP 80/558. B. TSH Enzyme Reagent (13ml/vial): One vial containing enzyme labeled affinity purified polyclonal goat antibody, biotinylated monoclonal mouse IgG in buffer, dye, and preservative. Store at 2-8°C. C. Streptavidin Coated Plate (96 wells). One 96-well microplate coated with streptavidin and packaged in an aluminum bag with a drying agent. Store at 2-8°C. D. Wash Solution Concentrate (20 ml). One vial containing a surfactant in buffered saline. A preservative has been added. Store at 2-30°C. E. Substrate A (7ml/vial). One bottle containing tetramethylbenzidine (TMB) in buffer. Store at 2-8°C. F. Substrate B (7ml/vial). One bottle containing hydrogen peroxide (H2O2) in buffer. Store at 2-8°C. G. Stop Solution (8ml/vial). One bottle containing a strong acid (1N HCl). Store at 2-30°C. H. Product Instructions: Note 1: Do not use reagents beyond the kit expiration date. Note 2: Opened reagents are stable for 60 days when stored at 2-8°C. Note 3: See end of this product insert for various configurations of reagents by kit size.

Material not included: 1. Pipette(s) capable of delivering 50µl and 100µl volumes with a precision of better than 1. 5%. 2. Dispenser(s) for repetitive deliveries of 0. 100ml and 0. 350ml volumes with a precision of better than 1. 5% (optional). 3. Microplate washer or a squeeze bottle (optional). 4. Microplate Reader with 450nm and 620nm wavelength absorbance capability. 5. Absorbent Paper for blotting the microplate wells. 6. Plastic wrap or microplate cover for incubation steps. 7. Vacuum aspirator (optional) for wash steps. 8. Timer. 9. Storage container for storage of wash buffer. 10. Distilled or deionized water. 11. Quality Control Materials.

Application Details

Application Notes: Precautions: 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

Sample Volume: 50 μL

Plate: Pre-coated

Reagent Preparation:

1. Wash Buffer: Dilute contents of wash concentrate to 1000ml with distilled or deionized water in a suitable storage container. Store at room temperature 20-27 °C for up to 60 days. 2. Working Substrate Solution: Pour the contents of the amber vial labeled Solution A into the clear vial labeled Solution B. Place the yellow cap on the clear vial for easy identification. Mix and label accordingly. Store at 2 - 8 °C. Note: Do not use the working substrate if it looks blue.

Sample Collection: 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 gel barrier. Allow the blood to clot. Centrifuge the specimen to separate the serum from the cells. Samples may be refrigerated at 2-8°C for a maximum period of five days. If the specimen(s) cannot be assayed within this time, the sample(s) may be stored at temperatures of -20 °C for up to 30 days. Avoid repetitive freezing and thawing. When assayed in duplicate, 0. 100 ml of the specimen is required.

Calculation of Results:

A dose response curve is used to ascertain the concentration of thyrotropin in unknown specimens. 1. Record the absorbance obtained from the printout of the microplate reader as outlined in Example 1 2. Plot the absorbance for each duplicate serum reference versus the corresponding TSH concentration in µIU/ml on linear graph paper (do not average the duplicates of the serum references before plotting). 3. Draw the best-fit curve through the plotted points. 4. To determine the concentration of TSH for an unknown, locate the average absorbance of the duplicates for each unknown on the vertical axis of the graph, find the intersecting point on the curve, and read the concentration (in µIU/ml) from the horizontal axis of the graph (the duplicates of the unknown may be averaged as indicated).

Restrictions: For Research Use only

Handling

Handling Advice: 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. 050 ml (50µl) of the appropriate serum reference, control or specimen into the assigned well. 3. Add 0. 100 ml (100µl) of the TSH Enzyme Reagent to each well. It is very important to dispense all reagents close to the bottom of the coated well. 4. Swirl the microplate gently for 20-30 seconds to mix and cover. 5. Incubate 60 minutes at room temperature. 6. Discard the contents of the microplate by decantation or aspiration. If decanting, tap and blot the plate dry with absorbent paper. 7. Add 350µl of wash buffer (see Reagent Preparation Section) decant (tap and blot) or aspirate. Repeat two additional times for a total of three washes. An automatic or manual plate washer can be used. Follow the manufacturer's 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 two additional times. 8. Add 0. 100 ml (100µl) of working substrate solution to all wells (see Reagent Preparation Section). Always add reagents in the same order to minimize reaction time differences between wells. DO NOT SHAKE THE PLATE AFTER SUBSTRATE ADDITION. 9. Incubate at room temperature for 15 minutes. 10. Add 0. 050ml (50µl) of stop solution to each well and mix gently for 15-20 seconds. Always add reagents in the same order to minimize reaction time differences between wells. 11. Read the absorbance in each well at 450nm (using a reference wavelength of 620-630nm to minimize well imperfections) in a microplate reader. The results should be read within 30 minutes of adding the stop solution. For better low-end sensitivity (lower than 0. 5µIU/ml), incubate 120 minutes at room temperature. The 40µIU/ml calibrator should be excluded since absorbance over 3. 0 units will be experienced. Follow the remaining steps. Note: Dilute samples reading over 40 µIU/ml by 1:5 and 1:10 with TSH 0 Calibrator. Multiply the results by the dilution factor to obtain accurate results.

Storage: 4 °C/-20 °C

Target Details for TSH

Target: TSH (Thyroid Stimulating Hormone (TSH))

Alternative Name: Thyrotropin (TSH) (TSH Products)

Synonyms: CG7665 ELISA Kit, DLGR-1 ELISA Kit, DLGR1 ELISA Kit, Dmel\CG7665 ELISA Kit, FSH ELISA Kit, FSH-TSH ELISA Kit, Fsh ELISA Kit, Fsh/CG7665 ELISA Kit, LGR1 ELISA Kit, LH/CG receptor ELISA Kit, RH44949p ELISA Kit, TSH ELISA Kit, dLGR1 ELISA Kit, Leucine-rich repeat-containing G protein-coupled receptor 1 ELISA Kit, Lgr1 ELISA Kit

Target Type: Hormone

Background: Summary and Explanation of the test: Measurement of the serum concentration of thyrotropin (TSH), a glycoprotein with a molecular weight of 28,000 daltons and secreted from the anterior pituitary, is generally regarded as the most sensitive indicator available for the diagnosis of primary and secondary (pituitary) hypothyroidism (1,2). Increase in serum concentrations of TSH, which is primarily responsible for the synthesis and release of thyroid hormones, is an early and sensitive indicator of decrease thyroid reserve and in conjunction with decreased thyroxine (T4) concentrations is diagnostic of primary hypothyroidism. The expected increase in TSH concentrations demonstrates the classical negative feedback system between the pituitary and thyroid glands. That is, primary thyroid gland failure reduces secretion of the thyroid hormones, which in turn stimulates the release of TSH from the pituitary. Additionally, TSH measurements are equally useful in differentiating secondary and tertiary (hypothalamic) hypothyroidism from the primary thyroid disease. TSH release from the pituitary is regulated by thyrotropin releasing factor (TRH), which is secreted by the hypothalamus, and by direct action of T4 and triiodothyronine (T3), the thyroid hormones, at the pituitary. Increase levels of T3 and T4 reduces the response of the pituitary to the stimulatory effects of TRH. In secondary and tertiary hypothyroidism, concentrations of T4 are usually low and TSH levels are generally low or normal. Either pituitary TSH deficiency (secondary hypothyroidism) or insufficiency of stimulation of the pituitary by TRH (tertiary hypothyroidism) causes this. The TRH stimulation test differentiates these conditions. In secondary hypothyroidism, TSH response to TRH is blunted while a normal or delayed response is obtained in tertiary hypothyroidism. Further, the advent of immunoenzymometric assays has provided the laboratory with sufficient sensitivity to enable the differentiating of hyperthyroidism from euthyroid population and extending the usefulness of TSH measurements. This method is a second-generation assay, which provides the means for discrimination in the hyperthyroid-euthyroid range. The functional sensitivity (lower than20% between assay CV) of the one-hour procedure is 0. 195 µIU/ml while the two-hour procedure has a functional sensitivity of 0. 095µIU/ml (3). In this method, TSH calibrator, patient specimen or control is first added to a streptavidin coated well. Biotinylated monoclonal and enzyme labeled antibodies are added and the reactants mixed. Reaction between the various TSH antibodies and native TSH forms a sandwich complex that binds with the streptavidin coated to the well. After the completion of the required incubation period, the antibody bound enzyme-thyrotropin conjugate is separated from the unbound enzyme-thyrotropin 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 color. The employment of several serum references of known thyrotropin levels permits construction of a dose response curve of activity and concentration. From comparison to the dose response curve, an unknown specimen's activity can be correlated with thyrotropin concentration. Intended Use: The Quantitative Determination of Thyrotropin Concentration in Human Serum by a Microplate Immunoenzymometric assay. Q. C. Parameters: In order for the assay results to be considered valid the following criteria should be met: 1. The absorbance of calibrator -G- (40 µIU/ml) should be greater than 1.3. 2. Four out of six quality control pools should be within the established ranges.