Dry information: Biotin (B) and avidin (A) amplified ELISA principles, steps and precautions!
1. Principle
BA-ELISA is a detection system established based on the principle of conventional ELISA and combined with the high amplification effect between biotin (B) and avidin (A). Avidin is a basic glycoprotein extracted from ovalbumin, with a molecular weight of 68 kDa, composed of 4 subunits, and has a very high affinity for biotin (binding constant as high as 1015M-1). Biotin is easily covalently bound to proteins (such as antibodies, etc.). In this way, the avidin molecule bound to the enzyme reacts with the biotin molecule bound to the specific antibody, which not only plays a multi-level amplification role, but also develops color due to the catalytic effect of the enzyme when it encounters the corresponding substrate, achieving detection. The purpose of the antigen (or antibody) molecule is unknown.
2. Materials and instruments
Antibody
serum avidin
Plastic plate, detector, injector, dripper, towel, washing bottle, beaker, glass rod, test tube, straw, measuring cylinder, refrigerator, incubator
3. Steps
1) BA-ELISA for detecting unknown antigens
1. Coated antibodies
Dilute the known antibody appropriately with 0.05 M pH9.6 carbonate buffer, add 0.1 ml to the reaction well of each polystyrene enzyme plate, and incubate at 4°C overnight (18-24 hours). The next day, discard the solution in the wells and wash 3 times with washing buffer for 3 minutes each time (referred to as washing, the same below).
2. Add sample
Add the sample to be tested (unknown antigen) or appropriately diluted standard reference material into the reaction well, and make blank wells, negative and positive well controls at the same time. Incubate at 37°C for 1 hour and wash.
3. Add B-Ab
Add 0.1 ml of appropriately diluted biotinylated antibody (B-Ab) to each well and incubate at 37°C for 30 to 60 minutes. washing.
4. Add A-HRP
Add 0.1 ml of appropriately diluted horseradish peroxidase-labeled avidin (A-HRP) to each well and incubate at 37°C for 30 to 45 minutes. Wash 4 times.
5. Substrate color development
Add 0.1 ml of the temporarily prepared TMB (or OPD) substrate application solution to each of the above reaction wells, and incubate at 37°C for 10 to 30 minutes. Add 0.05 ml of 2 M H2SO4 to stop the reaction.
6. Result Judgment
Measure the OD value visually or on an ELISA colorimeter.
2. BA-ELISA procedure for detecting unknown antibodies
1. Coated antigen
Dilute the known antigen appropriately with 0.05 M pH9.6 carbonate buffer, add 0.1 ml to the wells of the polystyrene enzyme plate, and incubate at 4°C for 18-24 hours. Wash 3 times with wash buffer for 3 minutes each time.
2. Add sample
Add appropriately diluted sample to be tested (unknown antibody) into the reaction well, and make blank, negative and positive control wells at the same time. Incubate at 37°C for 1 hour and wash.
3. Add B-Ab2
Add diluted biotinylated anti-antibodies (anti-human or mouse IgG), 0.1 ml per well, incubate at 37°C for 30-60 minutes, and wash.
4. The remaining steps are the same as the BA-ELISA for testing unknown antigens.
4. Things to note
1. Concentration and proportion of reactants
Since this method is highly sensitive and uses less antigen or antibody than the ELISA method, the concentration of the biotin-labeled antibody (or second antibody) should be strictly controlled. Increasing the antibody concentration can improve the sensitivity, but If it is too large, the non-specificity will increase.
2. Stability of biotin
The stability of biotin-labeled antibody conjugates is better than that of enzyme-labeled antibodies and should be stored for a long time. It can be stored for about 2 years by adding an equal amount of 60% glycerol at -20°C. Avoid repeated freezing and thawing during storage, as repeated freezing and thawing will damage the activity of the preparation.
3. Stability of avidin
Avidin is stable under normal conditions and can tolerate heat and a variety of proteins. However, it is sensitive to light and easily deactivated in the presence of certain metal ions. Therefore, it is advisable to use deionized water when preparing avidin solution and store it at 4°C for 2 months or at -30°C for half a year. Its activity will remain unchanged after repeated freezing and thawing.
4. Non-specific adsorption of avidin
Avidin is positively charged in a neutral pH environment and can be non-specifically adsorbed to the solid-phase carrier through electrostatic interaction, which is the main reason for non-specific color development in the test. If the pH and ionic strength of the avidin diluent are increased, the non-specific adsorption of avidin can be significantly reduced, and the specific color development will have little effect. Avidin can also be chemically modified with glutaraldehyde or acetic anhydride to acetylate it and reduce its non-specific adsorption.
5. Action time and temperature of reactants
Due to the high affinity between avidin and biotin, the reaction time of the two markers is much shorter than the antigen-antibody reaction time. In order to reduce non-specific adsorption, the reaction time should not be too long, generally 20-30 minutes at 37°C.
Seal it in a desiccator (with desiccant inside) and keep it in a refrigerator at 0-4 degrees. The following points should be noted when storing liquids:
1). The sample should not be too dilute. It must be concentrated to a certain concentration before it can be packaged and stored. If the sample is too dilute, it will easily cause oxidation. 2) Generally, preservatives and stabilizers need to be added. Commonly used preservatives include toluene, benzoic acid, chloroform, and thymol. wait. Commonly used stabilizers for proteins and enzymes include ammonium sulfate paste, sucrose, glycerin, etc. For enzymes, substrates and coenzymes can also be added to improve their stability. In addition, solutions such as calcium, zinc, and boric acid also have a certain protective effect on certain enzymes. Nucleic acid macromolecules are generally stored in standard buffers containing sodium chloride or sodium citrate.
3) The storage temperature requirements are low, most are stored in refrigerators around 0 degrees, and some require lower temperatures, depending on different substances.
