Catalogue: Equipment-For-Elisa-Kit-Manufacturing
ELISA, the enzyme-linked immunosorbent assay, is a commonly used class of immunoenzymatic techniques. The main method is to adsorb a known antigen or antibody onto the surface of a solid phase carrier, then incubate the enzyme-labeled (coupled) antibody or antigen with it, add a color developer to develop the color, determine the difference between the color of the analyte to be measured and the color of the standard by an enzyme marker, and plot the enzyme activity curve to obtain the concentration of the analyte to be measured.
There are seven steps involved in performing an ELISA:
Direct ELISA is to immobilize the antigen on the ELISA plate and then use the enzyme-labeled antibody to detect the antigen directly.
Compared to other types of ELISA experiments, direct ELISA experiments have fewer steps, faster detection, and no need to use secondary antibodies, to avoid cross-reactivity, the measurement results are not prone to error. However, because the antigen of direct ELISA is not specifically fixed, the target protein and other impurities in the sample will be bound to the ELISA plate, and the experimental background will be higher. And each target protein of direct ELISA needs to prepare a primary antibody that can bind specifically to it, the experiment is less flexible. In addition, because no secondary antibody is used, the signal is not amplified, reducing the sensitivity of the assay.
Indirect ELISA is a two-step process in which an antigen is bound to an ELISA plate, followed by a two-step process in which a detection antibody is added to specifically bind to the antigen, followed by the addition of an enzyme-labeled secondary antibody for detection and color development using the substrate.
Compared to direct ELISA, indirect ELISA uses enzyme-labeled secondary antibodies, which are more sensitive and require fewer labeled antibodies, making it more economical. Indirect ELISA also offers greater flexibility as different primary antibodies can be used with a single labeled secondary antibody. The disadvantages of indirect ELISA experiments are the possibility of cross-reactivity (direct binding of the enzyme-labeled secondary antibody to the antigen), which may increase the background, and the additional step of incubating the secondary antibody compared to direct ELISA, which prolongs the experimental cycle.
In a sandwich ELISA, the capture antibody is first immobilized in the wells of the ELISA plate, and then the sample is added, followed by the detection antibody. If the detection antibody is enzyme-labeled, it can be called direct sandwich ELISA; if the detection antibody is not labeled, it is also necessary to use an enzyme-labeled secondary antibody to bind to the detection antibody, and this is called indirect sandwich ELISA.
The sensitivity of sandwich ELISA is high, it is 2-5 times more sensitive than direct or indirect ELISA; at the same time, sandwich ELISA uses two specific antibodies to bind to the antigen and has high specificity. In addition, the sandwich ELISA can detect by direct or indirect means, which is more flexible. The disadvantage of sandwich ELISA is the high requirement for paired antibodies. If no standardized kits or tested paired antibodies are available, paired antibody customization and optimization are required, as it is important to reduce cross-reactivity between capture and detection antibodies.
The competition ELISA is a pre-encapsulated antigen on a solid-phase carrier with an enzyme-labeled antibody of specificity. During the experiment, the antigen (or antibody) to be tested is added. If the test is an antigen, the antigen to be tested competes with the antigen pre-encapsulated on the solid phase carrier to bind the enzyme-labeled antibody; if the test is an antibody, the antibody to be tested competes with the enzyme-labeled antibody originally present in the system to bind the antigen encapsulated on the solid phase carrier. The competitively bound antibody is washed off by washing and finally, the substrate is added for color development. It is important to note that the color development result is inversely proportional to the amount of antigen (or antibody) to be detected.
The competition ELISA is a little more complex than the three methods described above, but all three of the above ELISA types are applicable in the form of competition ELISA. Its main advantage is that it can detect impure samples and has high reproducibility of data, but has the problem of poor overall sensitivity and specificity.
Positive results for negative controls
a. Sample, the reagent is contaminated, or improper operation when adding samples leads to cross-contamination by spilling solution between adjacent wells - replace reagents and operate carefully.
b. The enzyme plate is not washed thoroughly - pour the antibody solution clean before washing the plate, and then pour the washing solution all over the plate wells to ensure that it can be washed fully.
c. An excessive amount of antibody leads to non-specific binding - use the antibody according to the recommended amount in the instructions and dilute the antibody to the appropriate concentration.
Enzyme plate overall background high
a. Non-specific binding of antibodies - ensure that the wells are closed and that the appropriate closure solution is used to prevent non-specific binding.
b. High substrate binding concentration - dilute the substrate appropriately.
c. The reaction time is too long - terminate the reaction with a termination solution as soon as the plate has developed enough color for absorbance reading and shorten the development time appropriately.
d. Contamination of substrate solution - Normal substrate solution should be clear and transparent, if yellow or other colors appear, it indicates contamination and should be replaced with a new substrate solution.
e .Substrate incubation is not protected from light - Substrate incubation should be performed under light-proof conditions.
Poor repeatability between replicate wells
a. ELISA sample quantity is not neat, and the spiking time is long or short - try to keep the spiking time close to the first time when adding duplicate wells.
b. Inconsistent spiked volume - samples should be well mixed before dilution and the same pipette gun should be used.
c. Inconsistent wash conditions, operators - When repeating samples, the operating conditions and personnel should be as consistent as possible with the last time.
d. Substrate incubation is not protected from light - Substrate incubation should be performed under light-proof conditions.
