Four kinds of ELISA here are here illustrated as you may concern: Direct ELISA
(1) Direct ELISAs involve attachment of the antigen to the solid phase, followed by an enzyme-labeled antibody. This type of assay generally makes measurement of crude samples difficult, since contaminating proteins compete for plastic binding sites. Indirect ELISA
(2) Indirect ELISAs also involve attachment of the antigen to a solid phase, but in this case, the primary antibody is not labeled. An enzyme-conjugated secondary antibody, directed at the first antibody, is then added. This format is used most often to detect specific antibodies in sera. Competitive ELISA
(3) The third type of ELISA is the Competition Assay, which involves the simultaneous addition of 'competing' antibodies or proteins. The decrease in signal of samples where the second antibody or protein is added gives a highly specific result. Sandwich ELISA
(4) The last type of assay is the sandwich ELISA. Sandwich ELISAs involve attachment of a capture antibody to a solid phase support. Samples containing known or unknown antigen are then added in a matrix or buffer that will minimize attachment to the solid phase. An enzyme-labeled antibody is then added for detection. The ELISA method is a benchmark for quantitation of pathological antigens and there are indeed many variations to this method. ELISAs are adaptable to high-throughput screening because results are rapid, consistent and relatively easy to analyze. The best results have been obtained with the sandwich format, utilizing highly purified, prematched capture and detector antibodies. The resulting signal provides data which is very sensitive and highly specific.
Detailed information of specified ELISA types:
Indirect ELISA, conventional but efficient
Figure of Indirect ELISA
Indirect ELISA is a two-step ELISA which involves two binding process of primary antibody and labeled secondary antibody. The primary antibody is incubated with the antigen followed by the incubation with the secondary antibody. However, this may lead to nonspecific signals because of cross-reaction that the secondary antibody may bring about. 1. Micro-well plates are incubated with antigens, washed up and blocked with BSA. 2. Samples with antibodies are added and washed.
3. Enzyme linked secondary antibody are added and washed.
4. A substrate is added, and enzymes on the antibody elicit a chromogenic or fluorescent signal. » Learn more about indirect ELISA protocol
Indirect ELISA advantages
High sensitivity: More than one labeled antibody is bound per antigen molecule; Flexible: Different primary detection antibodies can be used with a single labeled secondary antibody; Cost-saving: Fewer labeled antibodies are required.
In the indirect ELISA test, the sample antibody is sandwiched between the antigen coated on the plate and an enzyme-labeled, anti-species globulin conjugate. The addition of an enzyme substrate-chromogen reagent causes color to develop. This color is directly proportional to the amount of bound sample antibody. The more antibody present in the sample, the stronger the color development in the test wells. This format of indirect ELISA is suitable for determining total antibody level in samples (Newcastle disease virus, B. abortus, etc.). Detailed information about indirect ELISA application in the determination of antibody titer and procedures of antibody concentration determination are discussed in the following section of ELISA applications.
Direct ELISA, Simple and Time-Saving
Initially in a direct ELISA test which is considered to be the simplest type of ELISA the antigen is adsorbed to a plastic plate, then an excess of another protein (normally bovine serum albumin) is added to block all the other binding sites. While an enzyme is linked to an antibody in a separate reaction, the enzyme-antibody complex is applied to adsorb to the antigen. After excess enzyme-antibody complex is...
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