1、 Principle

ELISA stands for Enzyme Linked Immunosorbent Assay. The basis of ELISA is solid-phase preparation of antigens or antibodies and enzyme labeling of antigens or antibodies. The antigen or antibody bound to the surface of the solid-phase carrier still maintains its immunological activity, while the enzyme labeled antigen or antibody retains both its immunological activity and enzyme activity. During the measurement, the tested specimen (the antibody or antigen being measured) reacts with the antigen or antibody on the surface of the solid-phase carrier. Separate the antigen antibody complex formed on the solid-phase carrier from other substances in the liquid by washing. Adding enzyme labeled antigens or antibodies also binds to the solid-phase carrier through reaction. At this point, the amount of enzyme on the solid phase is proportional to the amount of the test substance in the specimen. After adding the substrate to the enzyme reaction, the substrate is catalyzed by the enzyme to become a colored product, and the amount of the product is directly related to the amount of the analyte in the specimen. Therefore, qualitative or quantitative analysis can be performed based on the depth of the color. Due to the high catalytic efficiency of enzymes, they indirectly amplify the results of immune reactions, making the measurement method highly sensitive.

2、 Result judgment

1. Qualitative determination

The result judgment of qualitative determination is to give a simple answer of "yes" or "no" to whether the tested antigen or antibody is present in the tested specimen, represented by "positive" and "negative" respectively. Positive "indicates that the specimen has a reaction in the measurement system. Negative "indicates no response. The qualitative judgment method can also obtain semi quantitative results, that is, using the titer to represent the strength of the reaction, which is still essentially a qualitative experiment. In this semi quantitative assay, a series of dilutions of the specimen are used for testing, and the highest dilution that yields a positive reaction is the titer. Based on the titer, the strength of the reactivity of the specimen can be determined, which is more quantitative than observing the color intensity of undiluted specimens to determine strong or weak positivity. In indirect and sandwich ELISA, positive wells appear darker in color than negative wells. In competitive ELISA, the opposite is true, with negative wells showing darker colors than positive wells.

2. Quantitative measurement

The operation steps of ELISA are complex, and there are many factors that affect the reaction, especially the coating of solid-phase carriers, which is difficult to achieve consistency among individuals. Therefore, in quantitative determination, a series of reference standards with different concentrations must be used to prepare standard curves under the same conditions for each batch of tests. The sandwich ELISA method for determining high molecular weight substances generally has a wide range of standard curves, and the absorbance at the highest point of the curve can be close to 2.0. Half logarithmic values are commonly used when plotting, with the concentration of the analyte as the horizontal axis and absorbance as the vertical axis. The values of each concentration are connected point by point, and the resulting curve is generally S-shaped. The head and tail curves tend to be flat, and the central part with a straight line is the most ideal detection area.

The commonly used competitive method for determining small molecular weight substances shows a negative correlation between the absorbance in the standard curve and the concentration of the substance being tested. The shape of the standard curve varies slightly depending on the mode used in the reagent kit. The standard curve for ELISA measurement should be noted that the horizontal axis in the graph represents a logarithmic relationship, which is more conducive to the expression of the measurement system.

3、 Main production processes

（1） Preparation of enzyme-linked immunosorbent assay (ELISA) plates:

1. Process flow

(A) Preparation of coating solution → (B) Preparation of sealing solution → (C) Coating of enzyme-linked immunosorbent assay (ELISA) plates → (D) ELISA plate sealing → (E) Drying and packaging of ELISA plates (some products require washing the plates before sealing)

2. Key point control

(1) Package:

1) Confirm the relevant information of the coating solution (name, concentration, batch number, batch size, etc.);

2) Control the amount of coating liquid within the required range;

3) The temperature and time for incubation or cold incubation are within the required range. Key equipment: coating machine, balance, sampler.

(2) Closed:

1) Confirm the frequency of board washing (if any);

2) Confirm the relevant information for preparing the sealing solution (name, concentration, batch number, batch size, etc.);

3) Control the sealing liquid volume within the required range;

4) The temperature and time for incubation or cold incubation are within the required range. Key equipment: coating machine, washing machine, balance, sampler.

(3) Drying and packaging:

1) Dry temperature and humidity are within the required range;

2) After drying, each enzyme-linked immunosorbent assay (ELISA) plate should be packed into an aluminum foil bag and a bag of desiccant should be placed. During bagging, any plates with unclear labeling or damaged holes should be removed and scrapped. Key equipment: packaging machine.

（2） Preparation of enzyme-linked immunosorbent assay:

1. Process flow (A) Liquid preparation → (B) Liquid packaging

2. Key point control

(1) Accurate material measurement during liquid preparation process;

(2) Confirm that the appearance of the prepared solution meets the requirements;

(3) Confirm the relevant information of the prepared reagents (name, concentration, batch number, batch size, etc.);

(4) The range of liquid dispensing volume should meet the requirements. Key equipment: balance, packaging machine.

（3） Preparation of negative and positive controls:

1. Process flow (A) Inactivation of raw materials (if necessary) → (B) Preparation of liquids → (C) Packaging of liquids

2. Key point control

(1) Control the inactivation temperature and time (such as inactivation);

(2) Accurate material measurement during liquid preparation process;

(3) Confirm the relevant information (name, concentration, batch number, batch size, etc.) for preparing the positive and negative control;

(4) The range of liquid dispensing volume should meet the requirements. Key equipment: water bath (such as inactivation), balance, packaging machine.

（4） Preparation of other components:

1. Process flow (A) Liquid preparation → (B) Liquid packaging

2. Key point control

(1) Accurate material measurement during liquid preparation process;

(2) Confirm the relevant information of the prepared reagents (name, batch number, batch size, etc.);

(3) The range of liquid dispensing volume should meet the requirements. Key equipment: balance, packaging machine.

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