|
Prinzip
|
Immunoenzymometric assay (TYPE 3): The essential reagents required for an immunoenzymometric assay include high affinity and specificity antibodies (Ab), (enzyme conjugated and immobilized), with different and distinct epitope recognition, in excess, and native antigen (Ag). 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 Insulin antibody. A reaction results upon mixing monoclonal biotinylated antibody, the enzyme-labeled antibody and a serum containing the native antigen, without competition or steric hindrance, to form a soluble sandwich complex. Simultaneously, the complex is deposited to the well through the high affinity reaction of streptavidin and biotinylated antibody. After equilibrium is attained, the antibody-bound fraction is separated from unbound antigen by decantation or aspiration. The enzyme activity in the antibody-bound 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.
|
|
Aufbereitung der Reagenzien
|
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.
|
|
Probennahme
|
The specimens shall be blood, serum or plasma 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 (for serum) or evacuated tube(s) containing EDTA or heparin. . Allow the blood to clot for serum samples. Centrifuge the specimen to separate the serum or plasma 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. 100ml of the specimen is required.
|
|
Ergebnisberechnung
|
A dose response curve is used to ascertain the concentration of Insulin 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 Insulin concentration in µIU/ml on linear graph paper (do not average the duplicates of the serum references before plotting). Draw the best-fit curve through the plotted points. To determine the concentration of Insulin 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). In the following example, the average absorbance (0. 514) intersects the dose response curve at 45. 5 µIU/ml for the Insulin concentration.
|
|
Applikationshinweise
|
Precautions: For In Vitro Diagnostic Use. Not for Internal or External Use in Humans or Animals. 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
|
|
Handhabung
|
Before proceeding with the assay, bring all reagents, serum references and controls to room temperature (20 - 27°C). 1. Format the microplates’ wells for calibrator, 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 calibrators, controls and samples into the assigned wells. 3. Add 0. 100 ml (100µl) of the Insulin Enzyme Reagent to each well. It is very important to dispense all reagents close to the bottom of the microwell. 4. Swirl the microplate gently for 20-30 seconds to mix. Cover with a plastic wrap. 5. Incubate for 60 minutes at room temperature (20-27°C). 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 used, fill each well to the top by squeezing the container. Avoiding air bubbles. Decant the wash and repeat two additional times. 8. Add 0. 100 ml (100µl) of substrate solution to all 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 15 minutes of adding the stop solution.
|
|
Bestandteile
|
A. Insulin Calibrators (2. 0 ml/vial) (Dried). Six vials of references for Insulin antigen at levels of 0(A), 5(B), 25(C), 50(D), 100(E), and 300(F) µIU/ml. Reconstitute each vial with 2ml of distilled or deionized water. The reconstituted calibrators are stable for 60 days 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 1st IRP 66/304. B. Insulin Enzyme Reagent (13ml/vial). One vial containing enzyme labeled affinity purified monoclonal mouse x-insulin IgG, biotinylated monoclonal mouse x-insulin 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 contains a surfactant in buffered saline. A preservative has been added. Store at 2-30°C. E. Substrate Solution (12ml/vial). One bottle contains tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) in buffer. Store at 2-8°C. F. Stop Solution (8ml/vial). One bottle contains a strong acid (1N H2SO4). Store at 2-30°C. G. 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. 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. 300ml 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 (The 620nm filter is optional). 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.
|
|
Lagerung (Langzeit)
|
-20 °C
|
|
Lagerung (Kurzzeit)
|
2-8°C
|
|
Beschränkungen
|
Nur für Forschungszwecke einsetzbar
|
