Rabbit Complement Fragment 3B (C3B) ELISA Kit protocol.
1. Experimental principle.
In this study, indirect enzyme-linked immunosorbent assay (indirect ELISA) was used to detect rabbit complement fragment 3b (C3b). The basic principle of indirect ELISA is to use the specific binding of antigen to antibody to amplify the signal through the chromogenic reaction of the enzyme and substrate, so as to achieve quantitative detection of the target protein.
2. Experimental procedures.
1.Sample treatment: Take an appropriate amount of rabbit serum samples to be tested and centrifuge them to remove suspended solids and impurities. Take the supernatant, which is the rabbit complement fragment 3b (C3b) sample to be tested.
2.Coating: The antigen (rabbit complement fragment 3b) is coated in the well of the microplate plate and an appropriate amount of blocking solution (sterile saline or normal saline containing 10% calf serum) is added to block the non-specific binding site. Incubate at 37 h for 1 h to allow the antigen to tightly bind to the plate wells.
3.Washing: Wash the wells of the microplate 3 times, and add the washing solution (005% tween-20) approximately 250 μl to remove unbound antigens and impurities.
4.Loading: Add the rabbit serum sample to be tested in the well of the microplate plate, and set up a negative control group and a positive control group. At the same time, an appropriate amount of enzyme-labeled secondary antibody (anti-rabbit IgG antibody) is added to capture the antigen-antibody complex. Incubate at 37 h for 1 h to allow the antibody to bind to the antigen-antibody complex.
5.Wash: Wash the plate wells 3 times, adding about 250 μl of wash solution each time to remove unbound enzyme-labeled secondary antibodies and impurities.
6.Add substrate: Add an appropriate amount of substrate solution (tetramethylbenzidine) to the microplate wells and incubate at 37 for 15-30 minutes to make the substrate and the enzyme-labeled secondary antibody have a chromogenic reaction.
7.Stop the reaction: Add an appropriate amount of stop solution (2 M sulfuric acid) to abort the chromogenic reaction between the substrate and the enzyme-labeled secondary antibody.
8.Detection: The optical density value (OD value) of each well is measured at a wavelength of 450 nm using a microplate reader, and the data is recorded.
9.Result: The relative OD value of the sample to be tested was calculated according to the OD value of the negative control group and the positive control group. Set a cut-off value, usually 2 of the OD value of the negative control group1 times. A relative OD value greater than the critical value is positive, and vice versa.
10.Data analysis: Statistical analysis of experimental data, corresponding graphs and curves were plotted, and the relationship between the expression level of rabbit complement fragment 3b (C3b) and diseases or other biological processes was analyzed.
3. Precautions.
1.Aseptic operation should be maintained during the experiment to avoid contamination.
2.The kit should be used within the expiration date and operated in strict accordance with the kit instructions.
3.The experimental results are only used as a reference, and the specific application needs to be comprehensively analyzed in combination with other experimental results and clinical data.