Interfering factors and countermeasures of enzyme immunoassay

The interference factors and countermeasures of enzyme immunoassay are the most widely used, but this solid-phase measurement technique is not perfect, grasp every key problem that may occur during the experiment, and take corresponding measures to make the experiment wrong. (Missing) diagnosis is reduced to a minimum. 1. The surface effect must first be clearly pointed out that: "solid phase" ELISA is the biggest compared with traditional "liquid phase" serological tests. The most essential difference is that there is a step of pre-solid phase antigen or antibody onto the carrier surface, and The antigen-antibody binding reaction moves from the liquid environment to the surface of the solid phase carrier. In the adsorption process of protein molecules, in order to overcome the repulsive force with the solid phase carrier, it is necessary to redistribute the functional genes on its surface to fully expose the hydrophobic genes. Then, the dipole molecules in the local contact area are dehydrogenated and then passed The van der Waals force attracts and solidifies the surface of the carrier. Surface effects can directly affect the conformation and function of antigens and antibodies. In addition, surface effects also affect the kinetic process of antigen-antibody binding reactions. Antigen changes due to solid phase To determine antibody levels, the solid phase of the antigen needs to be pre-coated (coated) onto polar and aqueous polystyrene (PS) or polyvinyl chloride (PVC) microplates. Direct physical adsorption methods such as solid-phase protein antigens and DNA cause changes in many aspects, which can cause changes in molecular conformation and antigenicity. The enzyme activity can disappear when measured. After the bovine serum albumin solid phase, its antigen value can be reduced from 5 to 1 price. In addition, it has been found that the solid phase ferritin in the passive adsorption method is clustered and unevenly distributed randomly. This situation of affecting quality control is universal. Finally, most small molecule haptens are not easily adsorbed directly on the carrier surface. The solution to this problem is generally to use small molecule antigens, first coupled with dextran, gelatin and other arms before solid phase coating. For macromolecular antigens with multiple expression epitopes, the antibody bridge coating method can avoid the effects of surface effects. Adsorption of protein antigen on colloidal AI (OH) 3 and solid phase can also avoid protein denaturation. Irradiating (400GY) PS plates with Y rays can not only increase the adsorption capacity of protein antigens, but also reduce the background of antibody measurement. The effect of solid relative antibody directly adsorbs solid phase antibody (Igs) molecules. In addition to the general disadvantages such as clusters, uneven distribution and easy desorption, Igs molecules spread on the surface of the carrier, not only the conformation changes, but also affects The activity of the antibody, such as the reduction of the binding value of IgG, can be changed from 2 to 1 value, or even completely inactivated. The experimental results show that monoclonal antibodies are more likely to be inactivated than polyclonal antibodies, and only a few molecules that can maintain binding ability after solid phase are 3% and 5% -10%, respectively. This is not only a waste of antibody reagents, but unfortunately, there is a limited surface area of ​​micropores. The N-terminus of the antibody molecule is Fab, the C-terminus is Fc, the arbitrariness of direct passive adsorption causes part of the Fab, and the C-terminus is Fc, the arbitrariness of the direct passive adsorption causes part of the Fab binding site to face the carrier side, or due to excessive dosage As a result of the overlapping adsorption, part of the Fab binding site is covered, which can lead to a decrease in its overall binding capacity. For this reason, there has been a covalent binding method that can "anchor" the F. segment on the surface of the carrier and make the Fab face outward, that is, introduce an active gene such as an aminohydrazine group on the carrier, and then under the action of water-soluble carbodiamine, Covalently bonded to the carboxyl group of Igs, or directly bonded to the aldehyde group generated by hydroxylated sugar groups: PS plates containing bromovinyl groups can be covalently coupled to proteins with the help of bifunctional crosslinking agents such as glutaraldehyde Combine. At present, related enzyme plate products (patents) have been supplied abroad. The bridge method is a solid-phase method that avoids direct contact between Igs and the carrier. There are several different types: (1) anti-antibody method: (2) SPA method: (3) streptavidin biotin method, which One method is the most successful, as long as the first capture antibody is biotinylated in advance: (4) Anti-hapten antibody method, which requires the capture body to be pre-recorded with a hapten. (3) Antibody-mediated antigen molecules Allosteric antigens and antibody molecules move freely in three-dimensional space, collide with each other, and specifically bind to each other. This combination is not like the simple "lock-key" relationship in the early similarity, but A flexible induction fit. Antigenic molecular determinants can protrude into the deep bottom of the Fab. In turn, in order to perform its inherent biological function, the Fab actively undergoes variable-angle bending, conical rotation and swinging of the "ball hole" of the most N-terminal variable region to fit Antigenic determinants, Fab can also penetrate into deeper regions of macromolecular antigens. After the antigen body molecules in the liquid state have completed the first-level binding reaction, the complex can be further bound by specific binding between the antigen and antibody, or can be further cross-linked by non-specific binding of the Fc-Fc segment of the antibody, forming a crystal visible to the naked eye The lattice network precipitates agglutination antigen-antibody secondary reaction complexes. Throughout the entire process, the antigen molecule can always maintain its inherent conformation, while the antibody conformation in the complex changes significantly. On the contrary, during the determination of antigens by solid-phase antibodies, due to the relationship of surface effects, antibody-mediated allosteric antigen molecules have special significance. 2. HD-HOOK effect This special effect that occurs during the solid phase test and can cause "false low" or even "false negative" false results is called the "high-dose hook sickle (HD-HOOK) effect". Its production conditions, molecular basis, and resulting consequences are different from the "zone phenomenon" in traditional liquid-phase precipitation and agglutination reactions. In the one-step two-site sandwich ELISA, it is mainly because the total amount of antigen to be tested is too high, competing with the limited labeled secondary antibody in the reaction system, so that the more antigens, the HD-HOOK effect with lower final response signal, such as False negatives can occur with HBsAg. Antigen "quantity" is the deciding factor. It is generally believed that the two-step two-site sandwich solid-phase assay does not produce the "overcast phenomenon" of excess antigen. It has long been rejected by Miles' practice, but not all two-step sandwich methods will Produce HD-HOOK effect. To date, there are only a few macromolecular protein antigens with multiple epitopes, such as ferritin, prostate specific antigen (PSA), human chorionic gonadotropin (HCG), alpha-fetoprotein (Afp), cytolysin-D, etc. , An antigen with a molecular allosteric internal cause, and the HD-HOOK effect was found during the measurement. The characteristic of antigen "quality" is the decisive factor, and labelled secondary antibody-mediated antigen molecule allosteric is an important external factor. Of course, the number of antigens is also a necessary related factor. Taking ferritin as an example, after the captured ferritin molecule cross-over overlaps with the labeled secondary antibody with a greater affinity than the primary antibody, due to the stereo effect, the ferritin molecule can be destructured to dissolve the primary antibody Separated to form a complex of labeled secondary antibody and ferritin molecule, and washed away by the subsequent washing process, the final signal drops. In the high-dose (HD) section of the dose-response curve, the linear direction is not flat and infinitely delayed, but is curved downward, like a hook or a sickle (HOOK). If simply from the dose response The hook-shaped graph of the curve is very similar to the "post-band phenomenon" of the antigen-excess complex in the zone phenomenon. However, the molecular basis is completely different. The "lattice theory" can only explain the zone phenomenon, and the interpretation of the HD-HOOK effect of the solid-phase two-step sandwich ELISA currently holds that the "antigen molecular allosteric theory" is more appropriate. To overcome the HD-HOOK effect, mainly rely on the manufacturer of the kit. First of all, the determinants of the target antigen should be as clear as possible. On this basis, select only one determinant with only two repeated expressions, or two determinants with only one expression each, and select the corresponding One monoclonal antibody, or two corresponding monoclonal antibodies to form a kit, it is possible to fundamentally solve the drawbacks of HD-HOOK effect. As an user, the dilution assay can be used to solve this problem. When measuring the aFP value of the unknown specimen, the original specimen and the 10-fold diluted specimen were measured at the same time, and it was found that the original A-well value was lower than the diluted well, confirming the existence of HD-HOOK, thereby avoiding experimental errors (missing). Consultation

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