In clinical tests, the test is generally carried out using a commercial kit. As mentioned in (2.2) above, there are three essential reagents in the ELISA: immunosorbent, conjugate and substrate for the enzyme.
The complete ELISA kit contains the following components:
(1) a solid phase carrier (immunoadsorbent) that has been coated with an antigen or an antibody;
(2) an enzyme-labeled antigen or antibody (conjugate);
(3) a substrate for the enzyme;
(4) Negative control and positive control (in qualitative determination), reference standard and control serum (quantitative determination);
(5) a dilution of the combination and the specimen;
(6) washing liquid;
(7) Enzyme reaction stop solution.
3.1 Immunosorbent
The solid phase carrier coated with the antigen or antibody can be generally stored for 6 months under conditions of low temperature (2 to 8 ° C) drying. Some incomplete test kits are only used to supply antigens or antibodies, and the testers need to coat them themselves. The solid phase carrier and coating process are briefly described below.
3.1.1 Solid phase carrier
The solid phase carrier acts as an adsorbent and container during the ELISA assay and does not participate in the chemical reaction. There are many materials that can be used as carriers in ELISA, and polystyrene is the most commonly used. Polystyrene has a strong ability to adsorb proteins, and the antibody or protein antigen retains its original immunological activity after being adsorbed thereon, and its price is low, so it is widely used. Polystyrene is plastic and can be made in various forms.
There are three main types of ELISA vectors: microtiter plates, beads, and small tubes. Microtiter plates are most commonly used. The products dedicated to EILSA are called ELISA plates. The international standard microtiter plates are 8×12 96-well. In order to facilitate the detection of a small number of specimens, there are 8 joint strips or 12 joint strips, which are placed in the same size as the standard ELISA plate. The ELISA plate is characterized by the ability to simultaneously perform a large number of specimens and quickly read the results on a special colorimeter. A variety of automated instruments are now available for microtiter plate type ELISA testing, including loading, washing, incubation, colorimetric, etc., which is extremely advantageous for standardization of operations. After the irradiation of polystyrene, the adsorption performance of the polystyrene increases especially for the immunoglobulin. The double antibody sandwich method can increase the amount of antibody on the solid phase, but the blank value is larger when used for indirect method.
A good ELISA plate should have good adsorption performance, low blank value, high transparency at the bottom of the hole, and similar performance between the plates, between the holes of the same plate, and between the holes of the same plate. Polystyrene ELIS A plates vary greatly in quality due to differences in raw materials and manufacturing processes. Therefore, each batch of ELISA plates must be inspected beforehand for performance. The commonly used test method is: coating the wells of the ELISA plate with a certain concentration of human IgG (generally 10 ng/ml), adding appropriate enzyme-labeled anti-human IgG antibody to each well after washing, washing after warming, adding substrate Color development, after terminating the enzyme reaction, the absorbance of each well solution was measured separately. The reaction conditions were controlled so that the readings of each well were at an absorbance of about 0.8. Calculate the average of all readings. The difference between the mean of all individual readings and all readings should be less than 10%.
A plastic similar to polystyrene is polyvinyl chloride. As an ELISA solid phase carrier, polyvinyl chloride is characterized by a thin, soft board that can be cut and inexpensive, but the finish is not as good as that of polystyrene, and the bottom of the hole is not as flat as polystyrene. Polyvinyl chloride has higher adsorption properties for proteins than polystyrene, but the blank value is also slightly higher.
In order to compare the advantages and disadvantages of different solid phases in an ELISA assay, the following test can be applied: a typical positive and negative specimens are selected by other immunological methods, and they are subjected to a series of dilutions at different solids. The phase carrier was assayed according to a predetermined ELISA procedure and the results were compared. On which carrier the positive and the negative results differ the most, and this vector is the most suitable solid phase carrier for this ELISA assay.
In the ELISA, the beads used as the solid phase carrier are generally 0.6 cm in diameter, and the adsorption area is greatly increased after the surface is frosted. The adsorption area of ​​the ELISA plate was about 200 mm 2 , and the beads were 1000 mm 2 , which was 5 times that of the ELISA plate. An increase in the adsorption area means an increase in the amount of solid phase antigen or antibody. Furthermore, the surface curvature of the spherical beads is more favorable for the optimal reaction state of the exposed antigenic determinant or the exposed surface of the antibody binding site, so the reaction of the bead ELISA is often more sensitive. Another feature of the beads is that it is easier to wash thoroughly, using a special scrubber to make the beads roll and rinse during the washing process, and the washing effect is much better than the immersion of the plate holes. However, due to the difficulty of the sanding process, the uniformity of the beads is poor.
The small test tube as a solid phase carrier also has a large adsorption surface, and the reaction amount of the specimen also increases accordingly. The sample size of the plate and bead ELISA is generally 100-200 ul, and the small test tube can increase the reaction volume as needed, and the increase of the sample reaction amount contributes to the improvement of the test sensitivity. The small test tube can also be used as a cuvette, and finally placed directly into the colorimeter in the spectrophotometer.
Microparticles made of polystyrene latex or other materials are also used as ELISA solid phase carriers. The advantage is that the surface area is extremely large and the reaction proceeds in a suspension at a rate similar to that of the liquid phase. The magnetic particles containing iron are used as the ELISA solid phase carrier, and the reaction is separated by magnet attraction after the reaction, and the washing is convenient, and the kit is generally equipped with a special instrument.
3.1.2 Way of coating
Fixing an antigen or antibody in a process is called coating. In other words, coating is the process by which an antigen or antibody binds to the surface of a solid support. Protein and polystyrene solid phase carrier are through
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