ELISA reagents - Huaqiang Electronic Network

EL-C1600N100013-B

Kaixin micro test

Test - lowercase jpg

Test probe PH-5H

In clinical tests, the test is generally carried out using a commercial kit. There are three essential reagents in the ELISA: immunosorbent, conjugate and substrate for the enzyme. The complete elisa kit contains the following components:

(1) has been coated with antigen or

antibody

Solid phase carrier (immunoadsorbent);

(2) Enzyme-labeled antigen or

antibody

(combination);

(3) a substrate for the enzyme;

(4) Negative control and positive control (in qualitative determination), reference standard and control

serum

(in quantitative measurement);

(5) a dilute of the enzyme conjugate (conjugate) and the specimen;

(6) washing liquid;

(7) Enzyme reaction stop solution.

1.1 Immunosorbent

The solid phase carrier coated with the antigen or antibody can be generally stored for more than 6 months under conditions of low temperature (2 to 8 ° C) drying. Some incomplete test kits only supply the antigen or antibody for the package, and the tester needs to coat it by himself. The solid phase carrier and coating process are briefly described below.

1.1.1 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, the most common being polystyrene. 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

test tube

. Microtiter plates are most commonly used. Products dedicated to EILSA are called ELISA plates. 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 the same size as the standard ELISA plate after being placed in the mount. The ELISA plate is characterized by the ability to perform a large number of specimens at the same time, and can be specially made.

Colorimeter

Read the results quickly. 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 polystyrene is irradiated by radiation, its adsorption performance is especially

Immunoglobulin

The adsorption performance is increased, and the double antibody sandwich method can increase the amount of antibodies on the solid phase, but the blank value is larger when used for indirect method measurement of antibodies.

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 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 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 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 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.