ELISA

Enzyme Linked Immunosorbent Assay (ELISA) denotes an antibody-based detection methods ( assay). Such as radioimmunoassay ( RIA ) is also the group of the ELISA immunoassay method, but is not based on a radioactivity measurement, but in an enzymatic color reaction, and thus is one of the enzymatic immunoadsorption (EIA). The antigen was originally bound and enriched by adsorption on a first antibody to a microtiter plate, an enzyme-linked secondary antibody ( synonym: detection antibody ) resulted in the reaction of a dye substrate.

By means of ELISA, proteins (e.g., antibodies), and viruses, as well as low molecular weight compounds such as hormones, toxins and pesticides in a sample ( blood serum, milk, urine, etc.) can be detected. Particularly convenient is the property of specific antibodies advantage that bind to the substance to be detected ( antigen). An antibody is previously labeled with an enzyme. The reaction catalyzed by the reporter enzyme reaction serves as evidence for the presence of the antigen. The so-called substrate is converted by the enzyme, the reaction product can usually by a color change, may be also detected by chemiluminescence. The signal strength is measured using a photometer very precisely definable function of the antigen concentration, it can also be used for quantitative evidence that ELISA performed as multiple measurements. As a reporter enzymes and glucose oxidase ( GOD) are usually the horseradish peroxidase (HRP, of Engl. Horseradish peroxidase ), alkaline phosphatase (AP ) or rarely used. In the case of alkaline phosphatase is used as a dye substrate ( synonym: chromogen ) is added, while the peroxidase is used in most of o-phenylenediamine (OPD) for example, p -nitrophenyl phosphate ( pNPP ). Alkaline phosphatase, cleaves the phosphate moiety from the colorless nitrophenyl phosphate and p-nitrophenol is formed which is slightly yellow. The change in concentration of the dye formed by the enzymatic reaction can be monitored according to the Lambert - Beer's law using a photometer. The intensity of color increases with the concentration of the resultant nitrophenol and hence the concentration of the antigen to be determined in the sample in comparison with a dilution series of known concentrations ( standard series).

History

The precursor of the ELISA was the radioimmunoassay since 1960. For an enzymatic detection of the direct coupling of proteins was necessary for even paired the reporter signal occurs with the specific binding antibodies. The chemical coupling of proteins was developed simultaneously by Stratis Avrameas and GB Pierce. The adsorption of proteins on surfaces was investigated in 1966 by Jerker Porath. The ELISA was developed simultaneously in 1971 by two research groups, including Peter Perlmann and Eva Engvall in Sweden.

Signal amplification

Instead of an enzyme -coupled detection antibody can be used for signal amplification and the combination of a non-coupled detection antibody and an additional (third) secondary antibody ( secondarily because it is an antibody against antibodies Engl. Secondary antibody ), to which an enzyme is bound, is used are (see figure). This still requires a further incubation and washing step. Although more involved, the use of a secondary antibody conjugate has the advantage that the costly preparation of many different enzyme-linked primary antibodies which are specific for only one antigen, can be avoided. Enzyme-linked secondary antibody used, which can at the same time as the polyclonal antibodies to different epitopes in the constant region of binding ( Fc region ) of all Erstantikörpern a species are widely used, and lead to a signal amplification. In addition, the secondary antibody -enzyme conjugates can be used from a species for a variety of immunoassays due to the specificity for Fc regions of an antibody subtype, so that it is the secondary antibody is a cost-effective industrial mass production product. Another common signal amplification may be performed by the binding of streptavidin or avidin conjugates to biotinylated detection antibody in the final incubation. The detection with ( strept) avidin-enzyme conjugates lead due to several biotinylations the primary and the subsequent binding of several reporter molecules to a signal amplification.

Modern reporter systems allow the use of fluorescence or the polymerase chain reaction partially higher sensitivities ( eg immuno-PCR ), or parallel provisions in one approach (multiplex ), but are in the stricter sense no ELISAs.

Antibody ELISA

In this enzyme-linked immunosorbent assay (EIA), the antigen is adsorbed onto the polystyrene surface of a microtiter plate, directly and without a coating antibody, which subsequently antibody concentrations can be measured in comparison to a standard curve.

Sandwich ELISA

One of the ELISA techniques ( sandwich ELISA or antigen ELISA ) using two antibodies (Ak), which both bind specifically to the antigen. It is important that both antibodies bind to different sites (epitopes ) on the antigen bind, since they would otherwise interfere with each other. The first antibody (german coat antibody or capture antibody ) is applied to a solid phase ( usually microtiter plates with 96 wells mentioned wells) bound. The sample with the antigen to be detected is then added to the wells and incubated for a period of time. During this time, the bound antibody will bind to the plate, the antigen present in the sample. After expiry of the incubation period, the plate is washed: The unbound constituents of the sample are removed, and what remains is only bound to the coat antibody antigen. In the next step, a second antibody for detection (English detection antibody ) is added that recognizes a different epitope than the capture antibody, and at the end of a reporter enzyme is bound. This second antibody also binds to the antigen, and the result of antibody-antigen -antibody complex. And derives its name, sandwich ELISA ', because the antigen is packed between the two antibodies as in a sandwich. By again washing the plate, the excess detection antibody is washed away. Only now, the antigen can be detected and quantified: An enzyme for suitable chromogenic substrate was added. This is converted by the enzyme to a reaction product, the detection can be carried out by a color change, fluorescence or chemiluminescence. For quantitative determinations with a series of known antigen concentrations ( standard range ) is usually carried out in order to obtain a calibration curve for the measured signal (optical absorbance, intensity emitted ).

Competitive immunoassay

However, the competitive immunoassay ( enzyme linked immunosorbent assay, EIA ) is frequently used. In this case, no second, labeled antibody is used for detection, but a labeled competitor antigen ( a synthetic compound which is structurally similar to the analyte, and also binds to the antibody ) were used. So it comes to competition ( competition) between the analyte and competitor for a binding site on the antibody. The signal here is inversely related to analyte concentration: low analyte = almost all antibody binding sites are occupied by labeled competitor = strong color reaction; much analyte = weak color reaction. The detection systems employed (enzymes / substrates ) are generally the same as for ELISA.

Evaluation of the ELISA using the logit-log plot

A sigmoid waveform occurs when the x- axis the logarithm of the concentration, and the y - axis plots the absorbance ( OD = optical density = absorption). This form of representation is the semi - logarithmic graph.

In order to calculate a linear regression, one must linearize these sigmoid before. To this end, maintains the dimension of the x -axis, and converts the Y- axis into logit values ​​. It should create a straight line. This representation is called a log - logit plot, logit-log plot or logit plot.

To calculate logit values ​​from the extinction, one must first read the absorbance (w) normalize (s ) so that they cover a range from 0 to 1. This requires the bottom (U) and the upper (O) asymptote of the sigmoid curve.

Inverse function:

These normalized absorbance values ​​( n ) then go into the logit equation (L):

Inverse function:

The pairs of values ​​of the logit-log plots of the x value = the natural logarithm of the concentration, and the y value = logit of the normalized absorbance values ​​(L ) then go into the calculation of the linear regression. This then provides the height (a) and the slope ( b ) of the linear equation:

Inverse function:

For the interpolation of unknown measurement values ​​on the calibration curve thus created, falsely often referred to as a calibration curve, one then needs the inverse functions. The high precision is achieved in the vicinity of the in the center of the sigmoid inflection point, because at this point, its slope is greatest. The least accurate is being built near their asymptotes.

If you can calculate a plot of several different measurements using the asymptotes of the curve, then it is most accurate when comparing the turning point concentration of the calibration curve with the inflection point dilution of the trace. The asymptotes of the measured curves are ignored, because you have to refer all measurements to the turning point of the calibration curve, which can be found in the y-values ​​n = 0.5 in the semi-logarithmic diagram identical with L = 0 in the logit-log plot. In the semi-log plot of the plot of the natural logarithm of the reciprocal of the dilution is used as the x- axis, because the measured values ​​of the concentration before the calculation is not yet known.

Basically, it is also possible, instead of the natural logarithms ln the decadic logarithms or those with the base of 2 to use. Instead the reciprocal of the dilution can also be only the dilution (Dilution ) itself can be used. It is also acceptable to calculate the value of N from 0 to 100 percent as possible, provided that the modified equations are correct. Not really but it would be to calculate the logit directly from the non-normalized absorbance values ​​of w.