Are ELISAs considered "semi-quantitative" due to the nature of antibodies being able to bind more than one antigen at a time? Since it is not a true 1:1 ratio, is it rational to assume that the concentration values reported in ELISAs are less than their actual/true values? I am talking about ELISAs in general, although I use sandwich ELISAs the most.
For example, Primary ab binds to two antigens - secondary binds to the one antigen - conjugate reaction strep/tmb produces the colorimetric change. This reaction does not measure whether or not the antibody sandwich has one or two antigens bound. What are the reasons ELISAs are considered semi-quantitative?
The ELISA has been considered the gold standard of immunoassays. ELISA is very sensitive and used to detect & quantify analytes including antibodies, antigens, proteins, glycoproteins, hormones etc.
Normally four major types of ELISA are commonly used i.e. 1) Direct ELISA (antigen-coated plate; screening antibody); 2) Indirect ELISA (antigen-coated plate; screening antigen/antibody); 3) Sandwich ELISA (antibody-coated plate; screening antigen) and 4) Competitive ELISA (screening antibody).
The ELISA is quantitative, qualitative and semiquantitative depending up on the experimental target you set. For instance, the quantitative concentration results are plotted and compared to a standard curve. The qualitative results confirm or deny the presence of a particular antigen/antibody in a sample, while semiquantitative results compare the intensity of the signals, which can compare relative antigen levels in a sample.
Its very important to note that the very sensitive ELISA has been developed that efficiently detect the analytes accurately in the scale of picograms. Hence, considering ELISA as "semi-quantitative" may not be appropriate. Of course, if you target to analyze the relative antigen level in your sample using ELISA then it is semiquantitative.
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Hello Erick Bardalez
ELISA is a versatile technique with different option to fit various analysis. Each version of the assay has different stages and its own benefits and drawbacks. So, whenever you choose ELISA, you should consider these points and it should be based on the sample you test and the results you need.
The two critical elements required for a robust ELISA are sensitivity and specificity.
Sandwich ELISA measures the quantity of antigen caught between two different antibody layers (capture and detection antibodies). Both need to be optimally reactive with the antigen of interest. The advantage of sandwich ELISA is its high sensitivity. It is 2-5 times more sensitive than direct or indirect ELISA. Sandwich ELISA also delivers high specificity as two antibodies are used to detect the antigen.
The example that you have mentioned in the question relates to sensitivity. The sensitivity in ELISA is the lowest detection level of the marker that the antibody pair used in ELISA can detect. Therefore, the use of a high affinity antibody would improve sensitivity. Also, as you mentioned, the concentration values reported in ELISAs are less than their actual/true values. This will actually depend on the antibody used in the ELISA assay. On the other hand, “semi-quantitative” ELISA relates to something different. Semi-quantitative ELISA can be used to compare the relative levels of antigen in assay samples, since the intensity of signal will vary directly with antigen concentration.
Hope this will help.
Best Wishes.
The responses by the first 2 scientists are spot on. What makes an elisa quantitative is the use of a known standard. From this, you can generate the binding curve and the equation of the curve from which you can determine the concentration of analyte in a sample. Semi quantitative in my experience is quite different.
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