ELISA experimental conditions: loading, coating material and its concentration

ELISA experimental conditions: loading, coating material and its concentration

Adding samples: In addition to coating in ELISA, it is generally necessary to add 4-5 samples. In the qualitative measurement, the accuracy of the sample volume is sometimes not emphasized. For example, it is specified as a drop of sample. At this time, a dropper of the same caliber should be used to maintain an accurate posture of liquid addition, so that the volume of each drop is basically the same. In the quantitative determination, the amount of sample added should be accurate. The dilutions of specimens and conjugates should be prepared as specified. When adding the sample, the liquid should be added to the bottom of the hole, avoid adding to the upper part of the hole wall, and pay attention to that no bubbles can appear.

Coating substance and its concentration: In ELISA, the antigen or antibody is fixed on the surface of the carrier, which is called Guxiang coating or embedding. The quality of Guxiang coating is directly related to the results of enzyme immunoassay. The ideal coating should be: the surface of the solid phase should adsorb as many specific antibodies (or pure antigens) as possible; the adsorption should be strong and able to withstand several washings; the non-specific adsorption should be small. The following factors should be considered in the specific operation: coating substance and coating concentration, pH and ionic strength of the coating buffer, coating temperature, time and washing, etc.

Antigen or antibody is adsorbed on the solid phase, and the adsorption capacity is different. There are three mechanisms for solid phase coating: the first is physical adsorption, that is, the antigen or antibody is passively adsorbed on the surface of the hydrophobic solid phase carrier by physical force. The process of physical adsorption is relatively simple, no chemical reaction occurs, no Special conditions are required, suitable for various types and types of carriers, such as polystyrene test tubes and microplate holes, cellulose membranes, etc., currently the most widely used; (Solid phase) Connect a chemically active group, and then cross-link the antigen or antibody with the solid phase carrier through this group. The active group introduced is often glutaraldehyde. The filter paper can also be used as a solid phase. The paper cellulose is activated with cyanogen bromide first, and then cross-linked with the antibody. The solid phase prepared by the chemical cross-linking method has large absorption capacity, small difference between batch numbers, and low price. Cross-linking by covalent method may be the best way to standardize solid-phase coating; the third is cross-linking to protein, with few applications.

The adsorption of the solid phase antibody on the solid phase surface is through physical action. Glycoproteins such as carcinoembryonic antigens, blood group substances, lipoproteins, bacterial lipopolysaccharides, glycolipids and denatured DNA can be adsorbed on the surface of polystyrene plastics, while natural DNA and neutral polysaccharides are used to coat plastic solid phase surfaces The effect is poor or the background is darkly colored. Some people think that polystyrene adsorbs antigens well, and polyethylene adsorbs antibodies well. There are some methods for pretreatment of solid-phase plates to improve adsorption, such as manufacturers using radiation, and users using acetic acid, hydrochloric acid, glutaraldehyde or polylysine to treat the plates. Pretreatment with glutaraldehyde and hydrochloric acid not only promotes adsorption, but also reduces non-specific reactions. Polylysine can enhance the adsorption of certain polysaccharide antigens.