As to better illustrate how ELISA test works, this section took a deeper insight into: a definition of the 'sensitivity', standard curve calculation, and control samples in ELISA test, followed by a list of diseases in which ELISA test could be applied.
ELISA Test-Sensitivity
The factors that determine the ultimate sensitivity of a competitive ELISA test are the antibody affinity constant and the experimental errors. The detection limit of the substrate is not typically limiting. It has been calculated theoretically that with a K = 1012 M-1 (an extraordinarily high equilibrium constant for an antigen-antibody interaction) and a 1% co-efficient of variation (CV) for the response at zero dose, the lowest detection limit possible would be 10-14 M. A more easily achievable limit would be 10-9 - 10-10 M. The factors limiting the sensitivity of a sandwich ELISA test are the affinity of the antibody, the experimental errors and the nonspecific binding of the labeled antibody, expressed as a percentage of the total antibody. It has been estimated that with a K = 1012 M-1, 1% CV of the response at zero dose, and a 1% nonspecific binding of the labeled antibody, the detection limit can be as low as 10-16 M. In addition, this can be enhanced further by using more sensitive detection substrates.
ELISA Test-Calculation from the standard curve
Create a standard curve by plotting the mean absorbance for each standard concentration (x axis) against the target protein concentration (Y axis). Draw a best fit curve through the points in the graph (A suitable computer program is suggested for this). We recommend including a standard on each ELISA test plate to provide a standard curve for each plate used. To determine the concentration of target protein concentration in each sample:
First, find the mean absorbance value of the sample. From the X axis of the standard curve graph, extend a horizontal line from this absorbance value to the standard curve. E.g if the absorbance reading is 1, extend the line from this absorbance point on the X axis. If the samples that have an absorbance value falling out of the range of the standard curve, these samples should be diluted before proceeding with the ELISA test staining. For these samples, the concentration obtained from the standard curve when analyzing the results must be multiplied by the dilution factor.
The coefficient of variation (CV) is the ratio of the standard deviation σ to the mean µ:
This is expressed as a percentage of variance to the mean and therefore indicates any inconsistencies and inaccuracies in the results. This sets a standard for the quality of the validated results. Computer programs can be used to calculate the CV values from ELISA test results.
ELISA Test-Control samples
• Positive control
As for the positive control in an ELISA test, use either an endogenous soluble sample known to contain the protein you are detecting, or a purified protein or peptide known to contain the immunogen sequence for the antibody you are using. A positive result from the positive control, even if the samples are negative, will indicate the procedure is optimized and working. It will verify that any negative results are valid. Any tissues, cells or lysates that have been used successfully can be considered a suitable positive control. Try looking at the Swiss-Prot or Omnigene database links on the datasheet. These databases will often have a list of tissues that the protein is expressed in. These can also be considered suitable positive controls. Check the GeneCards entry for the protein. This will usually provide you with relative levels of expression in various tissues. If you still have difficulty finding a suitable control, we recommend doing a quick literature search on PubMed to see which tissues and cells express the protein of interest.
• Negative control
The negative control in an ELISA test means a sample known not to express the protein you are detecting. This is to check for non-specific binding and false positive results. Each plate you use should contain a negative control sample in order to validate the results.
• Standard
A sample containing a known concentration of the target protein from which the standard curve can be obtained.
• Standard in sample matrix (spike control)
When testing serum samples in ELISA tests, include a standard in normal diluent buffer as usual. But we recommend to also include a standard diluted in serum from the species you are testing. The two can then be compared to ensure there is no effect on the standard curve from other proteins in the serum. This is known as a spike control.
• Endogenous positive control
We recommend including an endogenous positive control if you are testing a recombinant protein sample. This should be an essential component of your experiment.
There are inherent difficulties with antibody detection of recombinant proteins that need to be considered. Folding of the recombinant protein may be different from the endogenous native form, and may prevent antibody access to the epitope. This is particularly the case with tagged proteins. Always ensure tags are placed on the N or C terminal end of the recombinant protein.
Most importantly, always ensure the recombinant protein includes the immunogen sequence of the antibody you are using. An endogenous positive control is important to validate the results, as well as to indicate how well the reagents (eg antibodies) and procedure are working.
ELISA Test Related Diseases
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Influenza Influenza (flu) is a respiratory infection in mammals and birds. This virus is divided into three main types (A, B and C). Influenza A is found in a wide variety of bird and mammal species. Influenza B is largely confined to humans and is an important cause of morbidity. Influenza C infects humans, dogs and...Learn more » |
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AIDS HIV (human immunodeficiency virus) is the cause of AIDS (acquired immunodeficiency syndrome). HIV is a type of virus called a retrovirus, which infects humans when it comes in contact with tissues such as those that line the vagina, anal area, mouth, or eyes, or through a break in the skin...Learn more » |
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Hemolytic Anemia Hemolytic anemia is a condition of an inadequate number of circulating red blood cells (anemia), caused by premature destruction of red blood cells. Hemolytic anemia occurs when the bone marrow is unable to compensate for premature destruction of red blood cells by increasing their production ...Learn more » |
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Lyme disease Lyme disease is a bacterial illness caused by a bacterium called a "spirochete." In the United States, the actual name of the bacterium is Borrelia burgdorferi. In Europe, another bacterium, Borrelia afzelii, also causes Lyme disease. Certain ticks found on deer harbor the bacterium in their...Learn more » |
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Food Allergy The most common food allergies are to eggs, tree nuts, milk, peanuts, fish, shellfish, soybeans and wheat. These allergens are responsible for over 90% of the documented food allergen-related cases. To prevent food allergy, tests must be done to detect these allergens in finished products at very low levels...Learn more » |
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RMSF Rocky Mountain spotted fever (RMSF) is transmitted by the bite of an infected tick. The American dog tick (Dermacentor variabilis) and Rocky Mountain wood tick (Dermacentor andersoni) are the primary arthropods (vectors) which transmit RMSF bacteria in the United States. ...Learn more » |
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Chagas disease Chagas disease is named after the Brazilian physician Carlos Chagas, who discovered the disease in 1909. It is caused by the parasite Trypanosoma cruzi, which is transmitted to animals and people by insect vectors and is found only in the Americas (mainly, in rural areas of Latin America where poverty is widespread)...Learn more » |
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Leishmaniasis Leishmaniasis includes two major diseases, cutaneous leishmaniasis and visceral leishmaniasis, caused by more than 20 different leishmanial species. Cutaneous leishmaniasis, the most common form of the disease, causes skin ulcers. Visceral leishmaniasis causes a...Learn more » |
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Ebola HF Ebola hemorrhagic fever (Ebola HF) is a severe, often-fatal disease in humans and nonhuman primates (monkeys, gorillas, and chimpanzees) that has appeared sporadically since its initial recognition in 1976. The disease is caused by infection with Ebola virus, named after a river in the Democratic Republic of the Congo (formerly Zaire) in Africa...Learn more » |
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Monkeypox Monkeypox is a poxvirus that was identified in Cynomolgus monkeys in 1958 and then in humans in 1970. Monkeypox is zoonotic, passing from animals (such as rodents) to humans and vice versa. Human infections are often caused by animal bites or from direct contact with infected bodily fluids...Learn more » |