Xinfan Bio: Intrinsic factors and solutions affecting ELISA experiments - Database & Sql Blog Articles

Xinfan Bio: The endogenous factors and solutions that affect the ELISA experiment!

Endogenous material interference solution:

Some people think that about 40% of human serum samples contain non-specific interfering substances, which can affect the test results to varying degrees. Common interfering substances are: rheumatoid factor, complement, heterophilic antibody, target antigen autoantibodies, iatrogenic induced anti-mouse Ig (s) antibodies, cross-reactive substances and other substances.

(1)

Rheumatoid factor

IgM and IgG-type rheumatoid factor (RF) in human serum can directly bind to the capture antibody and the FC segment of the enzyme-labeled secondary antibody in the ELISA system, resulting in false positives. The solution is to replace the complete IgG with F(ab)2, and the specimen is treated with a solid phase adsorbent with thermal denaturation (63 ° C, 10 min) IgG (addition of heat-denatured IgG to the sample dilution is also effective) When the antigen is detected, it can be added to the sample diluent to degrade the RF.

(2) in the process of solid phase primary antibody and labeled secondary antibody in the complement ELISA system,

Antibody molecule

The complement C1q molecular binding site of the FC segment is exposed, allowing C1q to join the two, resulting in a false positive. The solution is to dilute the specimen with EDTA and heat the serum at 53 ° C, 10 min or 56 ° C for 30 min to inactivate C1q.

(3)

Heterophilic antibody

Human serum contains natural heterophilic antibodies that bind to rodent (eg, murine, etc.) Ig(s), which can link primary and secondary antibodies in an ELISA system and can also cause false positives. The solution is to add an excess of animal Ig (s) to the sample dilution, but it is not effective when the amount is insufficient or the subclass is different.

(4)

Autoantibodies

Autoantibodies such as anti-thyroglobulin and anti-insulin target antigens sometimes bind to a target antigen to form a complex, which can interfere with antigen-antibody assay results in an ELISA method. In order to avoid the above situation, the solution is: before the measurement, it needs to be dissociated by physical and chemical methods before measurement.

(5)

Iatrogenically induced anti-mouse Ig (s) antibody

Clinically, the use of monoclonal antibodies such as murine CD3, new techniques such as imaging diagnosis and targeted therapy using radiolabeled murine antibodies may result in the production of anti-mouse antibodies in these patients; Anti-mouse Ig(s) antibodies can also be produced in patients with rodent bites. These patients can produce false positives when tested by ELISA. The solution is to add a sufficient amount of normal mouse Ig (s) to the specimen to determine the false positive caused by the above reasons.

(6)

Cross-reactive substances such as AFP-like substances

Is a substance that cross-reacts with a target antigen. When the antigen is measured by the polyclonal antibody, the measurement results are not greatly affected, but when the antigen is measured by the monoclonal antibody, a false positive result may occur if the cross-antigenic determinant is exactly the target determinant corresponding to the monoclonal antibody used.

(7)

Effects of other components in the specimen

Excessive serum lipids, bilirubin, hemoglobin and excessive blood viscosity have an interference effect on ELISA results.