What is the ELISA?
The ELISA is an important, high-throughput immunoassay that replaced the lower-throughput complement fixation and hemagglutination inhibition as primary assays in the 1980s. In the ELISA, tissue samples (such as urine and especially blood serum) are assayed to detect the presence of antibodies. The presence of antibodies indicates that the sample source has been exposed to an antigen, that is, a substance which elicits an immune response upon exposure, usually a collection of proteins. Antibodies serve as the antigen's fingerprints, indirectly indicating to a diagnostician its presence in a host. Thus, the ELISA is an indirect immunoassay. Several other indirect immunoassays are also commonly used, especially the indirect fluorescent antibody test, or IFA.
The ELISA typically works by binding antigens to a solid surface and then adding tissue samples, secondary antibody, and a reporter label, with wash steps in between. Antibodies in the tissue will bind to both the antigen and the antibody (or another antigen if it's a sandwich assay). The secondary antibody will bind to the label, which fluoresces. After washing, the only fluorescence will be due to antibodies, since they will anchor the secondary antibody and labels to the antigen, which is bound to the solid surface, which is also known as the solid phase.
Though the ELISA can be used to demonstrate pharmacokinetic or hormonal effects, its chief use is in diagnostics, in which capacity it detects antibodies to antigens expressed by infectious agents, including viruses, bacteria, fungi, and protozoa, as well as parasites. The most common use of the ELISA in humans is the detection of HIV-1 in serum samples. The ELISA is also used for many different animal species. For this writeup, I will focus on the ELISA's capacity to detect infectious agents in sera.
Before Starting the Test....
...several things have to be accomplished. First, antigens must be propagated. There are typically two types of antigens: conventional and recombinant. Conventional antigens are prepared by inoculating a host or a tissue culture with an infectious agent seed, that is, a sample of the infectious agent that has been assayed by quality control and possibly secondarily prepared. The inoculated host or culture is incubated and the target tissue or harvest extracted following incubation. Recombinant antigens are prepared by first reviewing the infectious agent's genome to isolate the gene of interest (GOI) which encodes for the antigen. The GOI is then spliced into a vector and inoculated into a host or culture. Recombinant antigens have several advantages, namely, additional purity and the safety of the people working with the infectious agent. It's easier to work with a harmless vector that contains the Ebola virus antigen then to work with live Ebola virus, after all. After harvest, antigens are purified by different treatments to remove cell debris or other extraneous substances and assayed for potency and purity.
Secondly, if positive and negative control sera are to be used they must be prepared; the former by infecting a host with the infectious agent and extracting immune serum; the latter by using non-immune serum. Field positive and Field negative sera, that is, serum samples known to be positive or negative from historical data, may also be used.
Lastly, the antigen must be coated onto the solid phase. Typically, a 96-well microtiter plate is used. Antigen is coated onto the plates. Additionally, tissue control may be coated. Tissue control is an uninfected sample of the kind of host or tissue culture used to propagate the antigen. For recombinant antigens, this may simply be the kind of vector into which the GOI was spliced.
The antigen- and tissue-control-coated plates are removed from storage. Serum samples, including control sera, are pre-diluted and dispensed onto the plate. The plate is then incubated for a certain amount of time and washed.
Following the first incubation and wash, secondary antibody is dispensed onto the plate; the plate is incubated and washed again. The secondary antibody, also called conjugate, may consist of horseradish peroxidase-conjuagted anti-immunoglobulin (Ig), and binds to serum from the species in question.
Following the second incubation and wash, the label is dispensed onto the plate; the plate is incubated and washed again. There are different types of labels: some fluoresce, others are radioactive.
Following the third incubation and wash, the
plate is read; this may be done manually, which subjective quantification, or through a reader, which yields objective quantitative results. The data is normalized. Typically, serum contains different retroviruses that may react in some small way with the antigen whether or not there are antibodies present in the serum sample. Fluorescence due to this is known as background, or simply "noise", as opposed to "signal", the antigen score. This problem is commonly solved by subtracting the tissue control score from the antigen score. Based on control or historical data, the sample is then qualified as either immune (positive) or non-immune (negative) for the infectious agent.
The ELISA does have considerable advantages, as outlined in the immunoassay writeup: namely, high-throughput processing, specificity, and reliability of data. Of these, the specificity is also its great weakness: each well can only have one type of antigen or tissue control. In large testing facilities, plates are usually coated with alternating rows of one antigne and its tissue control. The great disadvantage of this is that for running a serum against a panel of viruses, you need one type of plate for each antigen. Thus, to check one sample against three antigens takes three plates, which can be a huge waste of time and money. Lastly, though the test itself is cheap, the production of antigens, tissue controls, sera, and other reagents, if done in-house, is considerable. The next generation of immunoassays will future suspension microarray testing, which will allow more than one type of antigen or tissue control in a well.