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Common Modes of Clinical Fully Automated ELISA

Common Modes of Clinical Fully Automated ELISA

Depending on the difference in the determination of antigens and antibodies, there are many automatic ELISA measurement modes, such as direct method, indirect method, double antibody sandwich method (direct, indirect), competitive inhibition method (direct, sandwich, etc.), capture method, etc. In clinical laboratories, commonly used ELISA measurement modes include double antibody sandwich method, indirect method, double antigen sandwich method, competitive inhibition method and capture method.

In the determination of antigens, the most commonly used mode for the determination of protein macromolecule antigens is the double antibody sandwich method. For small molecules with only a single epitope, competitive inhibition method is used; antibody determination usually uses indirect method, double antigen sandwich method, competitive inhibition method and capture method.

1. Double antibody sandwich method of ELISA workstation

For macromolecular proteins containing multiple epitopes, the ELISA workstation uses the double antibody sandwich method to determine it is quite simple. The existing commercial kits for measuring protein antigens basically all adopt this measuring mode.

The first full-automatic ELISA kits for double-antibody sandwich detection of antigens all adopted the "two-step method." Later, in order to shorten the detection time and simplify the operation steps, reagent manufacturers gradually introduced the "one-step" medical test kit.

At present, in our clinical laboratory, the determination of macromolecular antigens such as HBsAg, alpha-fetoprotein (α-fetoprotein, αFP), prostate specific antigen (PSA), CA series markers, hCG and hormones, etc. basically use a one-step method. Compared with the two-step method, the one-step method is simple to operate, but has its inherent shortcomings. If it is not handled well, the double-antibody sandwich immunoassay results will be false negative or low quantitative results due to the existence of the hook effect.

2. Competitive law test of ELISA workstation

Small molecule haptens such as digoxin, theophylline and other drugs and hormones such as T3, T4, and testosterone may have only one epitope. Or because the molecule is too small, after binding to one antibody, it cannot bind to another antibody due to steric hindrance, so the double-antibody sandwich method cannot be used for measurement. The ELISA workstation can only use the competitive inhibition method.

In this assay mode, a combination of small molecules and enzymes needs to be obtained. The preparation of small-molecule enzyme conjugates is not as simple as that of antibody enzyme conjugates, and its purification is also quite difficult. The molecular weight difference between the small molecule-bound enzyme and the free enzyme is small, and it is difficult to separate using general molecular sieve methods. Therefore, some people try to establish a double-antibody sandwich competition ELISA method for the determination of small molecules based on the synthesis of di- or multimer small molecules. The sensitivity and specificity of the determination have been improved.

3. Indirect method of antibody determination in ELISA workstation

Measure the antibodies produced by the body against pathogen antigens of infectious diseases, and the same is true for the antibody test kit. Before it is difficult to obtain high-purity and well-defined antigens, or when the antigens are more complicated, the indirect method is generally used.

Nowadays, the commonly used clinical test items of hepatitis C virus antibody (anti-HCV) are all indirect methods, and the indirect method is used to detect antibodies. Strictly speaking, only the IgG class of antibodies is measured, and IgM and IgA classes are not involved. This is determined by the enzyme-labeled secondary antibody. At present, some specific IgⅢ antibody detection kits adopt the indirect method.

4. Double antigen sandwich method of ELISA workstation

The antibodies measured by the double antigen sandwich method include all types of specific antibodies, and are not interfered by non-specific IgG. Therefore, the sensitivity and specificity of the double antigen sandwich method to detect antibodies are higher than the indirect method. At present, in order to improve the sensitivity of antibody determination, the kit of the ELISA workstation has basically adopted the double-antigen sandwich method, except that anti-HCV is more complicated due to its antigen.

5. Competition law of ELISA workstation

The determination of antibodies generally does not use a competition method. When the impurities in the antigen are difficult to remove or the binding specificity of the antigen is unstable, the ELISA workstation can use this mode to determine antibodies. The most typical example is the determination of hepatitis B virus core antibody (HBcAb) and hepatitis B virus e antibody (HBeAb).