After the antigen and antibody specifically bind, although the hydrophilic colloid changes into the hydrophobic colloid, if there is no electrolyte in the solution, no visible reaction will occur. In order to promote the formation of precipitates or agglomerates, 0.85% sodium chloride or various buffers are commonly used as dilutions of antigens and antibodies. Since sodium chloride dissociates into Na + and C1- in the aqueous solution, it can neutralize the charge on the colloidal particles, respectively, so that the potential of the colloidal particles decreases. When the potential drops below the critical potential (12 ~ 15mV), it can promote the precipitation of the antigen-antibody complex from the solution, forming visible precipitates or agglomerates.
(1) pH
The antigen-antibody reaction must be carried out in a suitable pH environment. Proteins have amphoteric ionization properties, so each protein has a fixed isoelectric point. The antigen-antibody reaction is generally carried out at a pH of 6-8. If the pH is too high or too low, it will affect the physical and chemical properties of the antigen and antibody. For example, when the pH reaches or is close to the isoelectric point of the antigen, even if there is no corresponding antibody, it will cause non-specific aggregation of the particulate antigen, causing a false positive reaction.
(2) Temperature
Within a certain range, the increase in temperature can accelerate molecular motion, the chance of collision between antigen and antibody increases, and the reaction accelerates. But if the temperature is higher than 56 ℃, it can cause the bound antigen antibody to dissociate again, or even denature or destroy; at 40 ℃, the binding speed is slow, but the binding is firm and it is easier to observe. The commonly used antigen-antibody reaction temperature is 37 ℃. Each test has its own unique optimal reaction temperature. For example, the cold agglutinin binds to red blood cells at about 4 and dissociates above 20 ℃. In addition, proper vibration can also promote the contact of antigen and antibody molecules and accelerate the reaction.
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