ELISA kit 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 commonly used in Zui. 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. The microtiter plate Zui is commonly used, and the product dedicated to EILSA is called ELISA plate. The international standard microtiter plate is 8×12 96-well type. In order to facilitate the detection of a small number of specimens, there are 8 joint strips or 12 joint strips, which are the same size as the standard ELISA plate after being placed in the mount. 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 ELISA assays for microtiter plate types, including loading, washing, incubation, colorimetric, etc., which are highly 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 the 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 ELISA 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. Commonly used test methods are: coating a certain concentration of human IgG (generally 10 ng / ml) in each well of the ELISA plate, after washing, adding appropriate dilution of the enzyme-labeled anti-human IgG antibody to each well, washing after warming, adding substrate Color development, after terminating the enzyme reaction, the absorbance of each well solution was measured. 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 is 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 result differs from the negative result, which is the appropriate 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 well is about 200 mm 2 , and the beads are 1000 mm 2 , which is 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 adsorption of the antigenic determinant or the exposed surface of the antibody binding site is in a good reaction state, 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 allow the beads to 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, while 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, directly placed in the spectrophotometer colorimetric.

Microparticles made of polystyrene latex or other materials are also used as ELISA solid phase carriers. This has the advantage 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.

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