Purpose
1. Familiar with the basic principles and methods of enzyme immunotechnology and the basic principles of immunofluorescence technology.
2. Understand the basic process and role of enzyme-linked immunosorbent assay (indirect method).
At present, the most widely used immunoenzyme technology is enzyme-linked immunosorbent assay (ELISA), which adsorbs antigen or antibody to a solid-phase carrier, so that subsequent antigen-antibody reactions are carried out on the surface of the carrier, thereby simplifying the separation step and improving Sensitivity can detect both antigen and antibody. Experimental methods include indirect method, sandwich method and competition method.
In order to detect the antigen, a sandwich method can be used. The specific antibody is adsorbed on the solid phase carrier (small tube, small plate or small hole made of polystyrene), and then the test solution is added. If there is a corresponding antigen in the sample, it forms a complex with the antibody on the surface of the carrier. After washing, an enzyme-labeled specific antibody is added, which is also bound to the surface of the carrier through the antigen. The excess labeled antibody is washed away, the substrate of the enzyme is added, and the amount of colored product catalyzed by the enzyme within a certain period of time is proportional to the antigen content in the solution, which can be determined by visual observation or spectrophotometer.
In order to detect antibodies, an indirect method can be used. The antigen is adsorbed on the carrier, and then the test serum is added. If there is an antibody, it forms a complex with the antigen on the carrier. After washing, enzyme-labeled antiglobulin (antibody) reacts with it. After washing, a substrate is added for color development, and the amount of colored product is proportional to the amount of antibody. (See Figure 5-1)
Commonly used enzymes are horseradish peroxidase (HRP) and alkaline phosphatase (AP), the corresponding substrates are o-phenylenediamine (OPD) and p-nitrophenyl phosphate, the former color reaction is brownish yellow , The latter is blue. It can be measured visually, or the microplate reader can be used to determine the optical density (OD) value to reflect the antigen content.
Experiment 6 Enzyme-linked immunosorbent assay (ELASA)
ELASA is a method of labeling antigens or antibodies with enzymes and performing antigen-antibody reactions on a solid-phase reaction plate. It is commonly used to detect trace antigens and antibodies in body fluids, and has the advantages of high sensitivity, strong specificity, simple operation and easy judgment. For the detection method, precautions and result analysis, please refer to the instruction manual of the detection reagent. In this experiment, the double antibody sandwich method is used as an example to detect hepatitis B virus surface antigen as follows:
Purpose
It is required to understand the test principle, test method and result analysis.
Experimental Materials
1. Specimen: Serum to be tested
2. Reagents: enzyme-labeled antibody (anti-HBs), HBsAg positive control serum, negative control, washing solution, developer A, B, stop solution
3. Equipment: Micro reaction plate (48 wells) coated with antibody, micro pipette, microplate reader, etc.
Experimental content and methods
1. Add 50μl of the specimen to be tested to each well of the micro-reaction plate, set 2 wells for the positive and negative controls, add 1 drop of each positive (or negative) control to each well, and set 1 well for the blank control.
2. Add 1 drop of enzyme conjugate to each well (except the blank control), mix well, seal the plate, and incubate at 37 ° C for 30 minutes.
3. Discard the liquid in the wells, fill the wells with the washing solution, let stand for 5 seconds, spin dry, pat dry after repeating 5 times.
4. Add 1 drop of each of developer A and B to each well, mix well, seal the plate, and incubate at 37 ° C for 15 minutes.
5. Add 1 drop of stop solution to each well and mix well.
6. Use a microplate reader to read, take a wavelength of 450nm, first use a blank hole to zero, and then read the OD value of each well.
Experimental results and evaluation
The OD value of the sample ≥ 2.1 average OD value of the negative control is judged as positive, otherwise it is negative. Negative control OD value is less than 0.05 for 0.05 calculation, higher than 0.05 is calculated based on actual OD value.
Silicone Bread Form,Bread Form Moulds,Cake Molds,Bread Mold Forms
Changshu Xinneng Silicone Products Co., Ltd. , https://www.xnmat.com