ELISA (enzyme linked immunosorbant assay)

ELISA is an antibody-based method for detection of small concentrations of substances such as proteins, hormones or metabolites in samples. Furthermore, it is used to detect and identify bacteria in microbiology. The method is common in clinical assays, clinical microbiology, environmental and food microbiology screening, western blots, immunostaining of cell tissue and biochemical research.

ELISA utilizes specific antibodies as a probe. Hence, the method is limited to detecting molecules and bacteria for which specific antibodies have been generated. As with any antibody based procedure, quality is directly linked to the specificity of the antibody. Background signals, false positives and negatives or systematic errors in quantitative assays will result, if specificity is low.

Here we describe two experimental set-ups, the direct ELISA and the indirect ELISA.

ELISA_principles

Direct ELISA

Direct ELISAs use biotinylated antibodies or antibodies with a conjugated marker enzyme such as HRP or AP. Biotinylated (or digitoxigenin-labeled) antibodies can be detected using avidin enzyme conjugates. Antibodies with marker enzymes can be detected using appropriate chromogenic, fluorogenic or luminogenic HRP enzyme substrates such as TMB, 4-chloro-1-naphthol, or luminol: the effect of the enzyme is to amplify the signal. A few antibody marker enzyme conjugates can produce very strong signals.

Selection of Biosynth's HRP Substrates

Cat.No. - Product Name
D-2000 - 3,5-Dichloro-2-hydroxybenzenesulfonic acid, sodium salt
D-2050 - 3,5-Dichloro-2-hydroxybenzenesulfonic acid, disodium salt
T-2100 - 3,3',5,5'-Tetramethylbenzidine, free base
T-2150 - 3,3',5,5'-Tetramethylbenzidine dihydrochloride

AP is often detected using chromogenic indoxyl phosphates in concert with chromogenic tetrazolium oxidants.

Selection of Biosynth's Substrates for Alkaline Phosphatase
Cat.No. - Product Name
B-7450 - 5-Bromo-4-chloro-3-indoxyl phosphate, disodium salt sesquihydrate
B-7452 - 5-Bromo-6-chloro-3-indoxyl phosphate, disodium salt monohydrate
B-7453 - 5-Bromo-6-chloro-3-indoxyl phosphate, disodium salt trihydrate
B-7500 - 5-Bromo-4-chloro-3-indoxyl phosphate, p-toluidine salt
B-7550 - 5-Bromo-6-chloro-3-indoxyl phosphate, p-toluidine salt
C-5100 - 6-Chloro-3-indoxyl phosphate, p-toluidine salt
I-6200 - 3-Indoxyl phosphate, bis(2-amino-2-methyl-1,3-propanediol) salt
I-6250 - 3-Indoxyl phosphate, disodium salt
I-6300 - 3-Indoxyl phosphate, p-toluidine salt
M-5800 - 4-Methylumbelliferyl phosphate, free acid
M-5850 - 4-Methylumbelliferyl phosphate, bis(2-amino-2-methyl-1,3-propanediol) salt
M-5900 - 4-Methylumbelliferyl phosphate, bis(cyclohexylammonium) salt
M-5920 - 4-Methylumbelliferyl phosphate, dilithium salt
M-5950 - 4-Methylumbelliferyl phosphate, disodium salt trihydrate

Selection of Biosynth's Tetrazolium Products
Cat.No. - Product Name
I-7650 - 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride
N-8100 - Nitrotetrazolium blue chloride
T-2300 - Tetranitro blue tetrazolium chloride
T-2590 - Tetrazolium blue chloride
T-2600 - Tetrazolium violet
T-6940 - 2,3,5-Triphenyltetrazolium chloride reagent-grade
X-5000 - XTT, sodium salt


Assay workflow:

1. Apply the sample to a sticky substrate, usually a plate with wells.

2. Apply the enzyme-linked antibodies and let them bind to the substance.

3. Wash the plate so that unbound antibodies are removed.

4. Apply a chemical which is converted to a fluorescent signal by the enzyme.

5. View the result: if it fluoresces, then the sample contained the substance.

Direct ELISAs are limited in their practical use by the availability of specific antibodies. The production of suitably labeled antibodies is difficult, expensive and time consuming. For this reason the indirect ELISA is commonly used in research labs.


Indirect ELISA

This variant of the assay uses two types of antibodies. The primary antibody provides specificity by binding to the target antigen. The primary antibody carries no label and cannot be detected directly. For this purpose an unspecific antibody binding to a whole range of primary antibodies is applied to the purified primary antibody – antigen complex. This second antibody is either biotinylated or enzyme labeled.


Typical assay workflow:

1. Prepare a plate on which the antigen is bound.

2. Apply the human serum to be tested.

3. Wash the plate so that unbound antibodies are removed.

4. Apply the enzyme-linked antibodies which specifically bind to the human antibodies. If biotinylated antibodies are used, the avidin HRP conjugate protein is applied in an additional step.

5. Wash the plate so that unbound enzyme-linked antibodies are removed.

6. Apply a chemical which is converted by the enzyme into a fluorescent signal.

7. View the result: if it fluoresces, then the serum sample contained antibodies against the antigen.



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