C1q Measurement Protocol by ELISA
GUIDELINE
This experiment describes the procedure for the enzyme-linked immunosorbent assay C1q assay. The binding of solid-phase condensed IgG encapsulated on a plastic plate to enzyme-linked C1q is inhibited by immune complexes in the specimen, making it a competitive inhibition test. To avoid interference from endogenous C1q in the specimen, it should be removed beforehand with IgG immunosorbent.
METHODS
- Encapsulated cohesive IgG is taken from cohesive IgG (100 μg/ml) dialyzed overnight by 0.02 mol/L Tris-HCl buffer (pH 9.0) and added into the wells of polystyrene plates. 100 μl per well, after being encapsulated for 30 min at 37°C. The wells are washed three times with buffer, the 1st and 3rd times with 0.2 mol/L Tris-HCl buffer (pH 7.4), and the 2nd time with the same buffer containing 0.05% Tween20). Vacuum dry and store at 4°C.
- Take 50 μl of serum specimen, add 50 μl of 0.2 mol/L EDTA (pH 7.6), and act for 10 min at room temperature to free endogenous C1q. Then add 500 μl of IgG immunosorbent, oscillate at room temperature for 20 min, and then centrifuge at 500 r/min for 5 min to obtain 1:10 diluted supernatant.
- Take the plastic reaction plate that has been coated with coagulated IgG, add 90 μl of the above supernatant and 10 μl of enzyme-linked C1q (1:100, 200 dilution) to each well, and react at room temperature for 20 min. Then, wash 3 times with 0.2 mol/L Tris-HCl buffer (pH 7.4) containing 0.2 mol/L NaCl, 0.05% Tween20, and then add 100 μl of substrate solution (pH 6.0, 0.1 mol/L phosphate citrate buffer in 50 ml, add o-phenylenediamine 20 mg, 30% (W/V) hydrogen peroxide 10 μl), let it rest for 30 min at room temperature, add 2 mol/L sulfuric acid 50 μl to abort the reaction, and read the absorbance value at 492 nm. For quantification, a standard curve can be made with coagulated IgG to measure the relative content of immune complexes.
NOTES
- Ensure proper coating of the ELISA plate with the capture antibody specific for C1q. The coating buffer, incubation time, and temperature should be carefully followed as per the protocol to achieve optimal capture of the C1q protein.
- Thoroughly block the coated wells to prevent nonspecific binding of other proteins to the plate. Common blocking agents include BSA (bovine serum albumin) or non-fat dry milk. Follow the recommended blocking buffer, incubation time, and temperature stated in the protocol.
- Prepare a precise standard curve using known concentrations of the C1q standard. This is essential for accurate quantification of C1q levels in the samples. Handle the standard curve samples carefully to avoid introducing variability.
- Thorough and consistent washing of the plate between steps is essential to remove unbound substances and reduce background noise. Follow the recommended wash buffer, wash times, and agitation methods to minimize variability.
