NTSH ELISA Kit

Produktnummer ABIN649055

Produktdetails

Target: NTSH

Reaktivität: Human

Nachweismethode: Colorimetric

Methodentyp: Competition ELISA

Applikation: ELISA

Verwendungszweck: Sequential immunoenzymometric assay Type 4: The essential reagents required for a sequential 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 elution buffer with monoclonal biotinylated antibody added, and a dried blood spot containing the native antigen, reaction results between the eluted antigen and the antibody to form an antigen-antibody complex. After a suitable incubation period, the antibody-antigen bound fraction is separated by decantation or aspiration. Another antibody (directed at a different epitope) labeled with an enzyme is added. Another interaction occurs to form an enzyme labeled antibody-antigen-biotinylated-antibody complex on the surface of the wells. Excess enzyme is washed off via a wash step. A suitable substrate is added to produce color measurable with the use of a microplate spectrophotometer. The enzyme activity on the well is directly proportional to the TSH concentration in the dried blood spot. By utilizing several different dried spots of known antigen concentration, a dose response curve can be generated from which the antigen concentration of an unknown can be ascertained.

Analytische Methode: Quantitative

Bestandteile: A. Neo-TSH Calibrators - Dried Blood Spots (Two rows by six dots levels - 2 x 6). Six levels of TSH Antigen in dried blood spots at approximate concentrations of 0(A), 7 (B), 18(C), 45(D), 110(E) and 250(F) µIU/ml placed on S&S type 903 filter paper. Store at 2-8°C. A preservative has been added. Note 1: The Lot Specific calibrators, whole human blood based, were calibrated using a reference preparation, which was assayed against the WHO 2nd, IRP 80/558. Note 2: The exact values are printed on the outside of the aluminum pouch. B. Neo-TSH Controls - Dried Blood Spots (Two rows by three dots - 2 x 3). Three levels of whole human blood controls with different concentrations of TSH antigen placed on S&S type 903 filter paper. Store at 2-8°C. A preservative has been added. Note 1: The controls, whole human blood based, were manufactured to fall within significant clinical ranges using the same WHO reference preparation as the calibrators (see above). Note 2: The exact values are printed on the outside of the aluminum pouch. C. NTSH Enzyme Reagent (13ml/vial). One vial containing enzyme labeled affinity purified polyclonal goat x-TSH IgG in buffer, and preservative. Store at 2-8°C. D. NTSH Biotin Reagent (13ml vial). Anti-TSH monoclonal IgG labeled with biotin in buffer, green dye and preservative. Store at 2-8°C. E. 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. F. Wash Solution Concentrate (20 ml). One vial containing a surfactant in buffered saline. A preservative has been added. Store at 2-30°C. G. Substrate Solutions (12ml/vial). One bottle containing tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) in buffer. Store at 2-8°C. H. Stop Solution (8ml/vial). One bottle containing a strong acid (1N H2SO4). Store at 2-30°C. I. 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: Above reagents are for a single 96-well microplate.

Benötigtes Material: 1. Laboratory Shaker capable of 150rpm rotatio. 2. Dispenser(s) for repetitive deliveries of 0. 050ml, 0. 100ml and 0. 350ml volumes with a precision of better than 1. 5% (optional). 3. Microplate washer or a squeeze bottle. 4. Microplate Reader with 450nm and 620nm wavelength absorbance capability (The 620nm filter is optional). 5. 1/8” Paper punch for dispensing dried blood spots. 6. Absorbent Paper for blotting the microplate wells. 7. Plastic wrap or microplate cover for incubation steps. 8. Vacuum aspirator (optional) for wash steps. 9. Time 10. Storage container for storage of wash buffer. 11. Distilled or deionized water. 12. Quality Control Materials.

Anwendungsinformationen

Applikationshinweise: 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 licensed reagents. 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.

Kommentare:

Sample Volume: DBS two Step (Sequential)

Plattentyp: Pre-coated

Aufbereitung der Reagenzien:

1. Wash Buffer: Dilute contents of wash solution to 1000ml with distilled or deionized water in a suitable storage container. Store at room temperature 20-27°C for up to 60 days.

Probennahme: The sampling from neonates is performed by lancing the heels of the infants and then spotting enough whole blood on S&S filter paper card (TypeNumber 903) to fill the marked circle. Allow the filter paper to dry at room temperature overnight away from heat and moisture. Enclose the dry blood specimen (DBS) in a moisture barrier plastic bag with desiccant and send to the laboratory. The specimen should be collected 3-7 days post partum, Physical data including age and weight of the infant, whether a multiple birth, or a premature birth etc should accompany the sample. It is important for the clinician to know these facts in order to properly assess the thyroid status of the infant. The dried blood samples are stable at 2-8°C for 2-3 weeks if stored in zip-lock, moisture resistant bags with desiccants.

Ergebnisberechnung:

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. 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.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Handhabung: Before proceeding with the assay, bring all reagents and patient samples to room temperature (20 - 27°C). 1. Obtain the required number of microwells for each calibrator, control and patient sample to be assayed in duplicate. Replace any unused microwell strips back into the aluminum bag, seal and Store at 2-8°C. 2. Punch out a 1/8” blood dot out of each calibrator, control and specimens into the assigned wells. (NOTE: Do not punch blood dots from areas that are printed or that are near the edge of the blood spot). 3. Add 0. 100 ml (100µl) of NTSH Biotin Reagent to all the wells. 4. Shake the microplate gently for 20-30 seconds to mix. (NOTE: Make sure that all blood dots are fully submerged in the liquid and not stuck to the walls of the microwells). 5. Cover with a microplate cover and rotate for 90 minutes at ambient temperature using a laboratory rotator set @ 150rpm. (Note: see alternative overnight incubation). 6. Discard the contents of the microplate by decantation or aspiration. If decanting, blot the plate dry with absorbent paper. NOTE: Make sure all the blood dots are removed at this point. There should be no dots left in the microwells. 7. Add 350µl of wash buffer (see Reagent Preparation Section), decant (tap and blot) or aspirate. Repeat four additional times for a total of Five 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 four additional times. 8. Add 100 µl of NTSH Enzyme Reagent to each well. 9. Cover the microplate and rotate for 45 minutes at ambient temperature using a laboratory rotator set @ 150rpm. (Note: see alternative overnight incubation). 10. Repeat wash step Number7. 11. Add 0. 100 ml (100µl) of substrate solution to each well. 12. Cover the microplate and rotate for 45 minutes at ambient temperature using a laboratory rotator set @ 150rpm. (Note: see alternative overnight incubation). 13. Add 0. 050ml (50µl) of stop solution to each well and gently mix for 15-20 seconds. NOTE: Always add reagents in the same order to minimize reaction time differences between wells. 14. 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 15 minutes of adding the stop solution. Alternative overnight procedure: 1. Substitute overnight incubation (12-16hrs) for the 90 minutes with rotation (Step 5). No rotator is required. Seal the plate(s) with plastic wrap. 2. All other steps remain the same.

Lagerung: 4 °C/-20 °C

Antigendetails

Target: NTSH

Hintergrund: Summary and Explanation of the Test: Determination of hypothyroidism within the first few days of birth has been recognized as the single most important diagnostic test in neonates by the American Thyroid Association. The need for its early detection and treatment has resulted in the establishment of screening centers by federal and state health departments. A program of early screening of neonates for congenital hypothyroidism was started in Quebec, Canada in the early seventies. They used dry blood spots on filter paper as the sampling device. Very soon the program was followed by other major public health institutions in Canada and the US. By 1978, almost one million infants had been screened and an incidence rate of congenital hypothyroidism was established to be approximately 1 in 7000 births. Congenital hypothyroidism is probably the single most common preventable cause of mental retardation. Diagnosis and treatment of congenital hypothyroidism within the first 1-2 months after birth appears to be necessary in order to prevent severe mental retardation. 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. In this method, TSH dried whole blood calibrator, patient specimen or control is first added to a streptavidin coated well. Elution buffer containing biotinylated monoclonal antibodies are added and the reactants mixed. Reaction between the biotinylated x-TSH and the TSH in the dried blood spot forms a complex that binds with the streptavidin coated to the well. After the completion of the first elution/incubation period, the enzyme conjugate is added to the Ag-Ab complex deposited on the plastic surface. The enzyme labeled x-TSH antibody binds to the TSH making a sandwich complex with two antibodies bound to the antigen during a second incubation. The microplate is washed to remove unreacted enzyme. Finally, the activity of the enzyme present on the surface of the well is quantitated by reaction with a suitable substrate to produce color. Intended Use: The Quantitative Determination of Thyrotropin Concentration in Human Whole Blood by a Microplate Immunoenzymometric assay. Q. C. Paramters: In order for the assay results to be considered valid the following criteria should be met. The absorbance (OD) of calibrator F should be greater than 1. 22. Four out of six quality control pools should be within the established ranges.