| Principle | The enzyme-linked immunosorbent assay (ELISA), Utilizing labeling technology to mark antibodies(antigens) with enzyme and then make antibodies and antigens in the samples to be tested have a specific response with Enzyme-labeled antibodies (antigens). When meet with enzyme substrate, enzyme can catalyze and decompose substrate efficiently and exclusively and then it will produce colored products. According to the intensity of color, the specific antigens(antibodies) and its quantum in the samples can be tested by the intensity of color. The assay can be used in analysis of qualitative and quantification of sample to be tested. In addition, it is tracing, specific, efficient, economical, and easy, so it has become a trace measurement techniques which has a wide application in biology and medicine. There are a variety of ELISA methods now and they can be classified by the test purpose. Indirect ELISA - for the determination of antibodies; Sandwich ELISA - mainly used to determine macromolecular antigen; Competitive ELISA - mainly for measurement of small molecule antigen; Using serum IgE detection as an example, the double antibody Sandwich ELISA can be learned in this experiment. |
| Experimental Materials | Serum to be tested |
| Reagents, kits | Horse anti-human; IgE antibody horseradish peroxidase- labeled anti-human IgE antibodies; horse carbonate - sodium bicarbonate solution; BSA solution; H2SO4 |
| Equipment, supplies | Microtiter plates |
| Experimental Procedure | 1, Coats microtiter plate. Add 1:500 Horse anti-human IgE antibody (IgG) and incubate it at 37℃. After 3 hours, wash it for three times every 10 minutes. 2, The closure. Adds 1% BSA, 0.3 ml / hole, make it stay for whole night at 4℃. Remove, wash. 3. The serum to be tested Adds serum to be tested of different dilutions, 0.1 ml / hole. Incubates it at 37℃ for 40 min , wash. 4, Enzyme-labeled antibody Add the Enzyme-labeled antibody of appropriate dilution, 0.1 ml / hole. Incubates it at 37 ℃ for 40 mins, wash. 5. Substrate Adds OPD ( o-phenylenediamine ) solution , 0.15 ml / hole. keep it at room temperature for 30 minutes. 6. Stop solution Adds 5N HCI of stop solution, 0.1 ml / hole. 7, measure OD of every holes with 495 nm measurement wavelength of Microplate reader within 20 minutes. |
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| Tips | 1. To make sure the accuracy of the experimental results, positive control and negative control wells need to be set up and every sample to be tested needs to be divided in to two pieces. 2, Uses sheep serum, rabbit serum or BSA blocking to prevent non-specific reactions in this experiment. Others: The results of ELISA: This result needs to be expressed by a group ratio (P / N) which is conducted by the value of the test specimen with the absorption hole and a group of negative samples’ mean absorption value of measured hole. When the P / N is greater than a certain value, it is positive; the value depends on the specific detection requirements. |
| Others | The results of ELISA: This result needs to be expressed by a group ratio (P / N) which is conducted by the value of the test specimen with the absorption hole and a group of negative samples’ mean absorption value of measured hole. When the P / N is greater than a certain value, it is positive; the value depends on the specific detection requirements. |
Application