A Small Guide to Understand Enzyme-Linked Immunosorbent Assay

The technique which is used to quantify and detect the specific antibodies in samples which may enclose the mixtures of proteins, it is known as ELISA (the enzyme-linked Immunosorbent assay). Specific proteins which are immobilized on the microplate wells surface are detected through the use of antibodies. ELISA plate coating protocol technique facilitated fast throughput analysis and high volume which is ideal for evaluating large numbers of samples.

ELISA Formats and Methods:

Sandwich ELISA troubleshooting is done when there some error occurs. However, the ELISA test can be performed in a number of ways. The most common way is the capture or sandwich assay. Also, labeled streptavidin is also used to strengthen analyte signal from the biotinylated antibody.

Blocking for ELISA:

In ELISA, blocking reagents are important in which a serum is used for the species of the labeled antibody in order to block the unsaturated binding sites on the microplates which will avoid background from non-specific binding.

Secondary Antibody Conjugates for ELISA:

The conjugation of horseradish peroxides and alkaline phosphatase is used to bother colorimetric assay by using a chromogenic substrate. To detect the chemiluminescent, a luminol-based substance is used with peroxidase conjugates for highly sensitive detection. Sandwich ELISA troubleshooting can be done with the use of fluorescent conjugates which allow concurrent detection of various antigens through the use of primary antibodies resulting from different species. Each antigen can be distinguished by using labeled secondary antibodies specifically by the individual fluorescent signal. However, the detection limit is lower than chemiluminescent or colorimetric discovery using a reporter enzyme.

Labeled Streptavidin with Biotinylated Primary Antibodies for Enhanced Sensitivity:

To detect a biotinylated antibody using labeled streptavidin, enhancement of signal can be achieved (primary or secondary antibody for enhanced amplification).

Each antibody can present itself as multiple biotin molecules that bind with multiple streptavidin molecules. These collective factors mean that several probe molecules are accessible to generate fluorescent emission or to catalyze the finding substrate to its end product in order to achieve a brighter signal and better sensitivity.

Sandwich ELISA troubleshooting should be done by experts. All you need to consult with an expert who knows sandwich Elisa protocol in detail. So, if you want to conduct ELISA test, consult with the best Enzyme-Linked Immunosorbent Assay expert. Get the accurate results with a professional lab technician help.