Why do some samples not give linear results when diluted (particularly in Celikey IgG)?
The immune response in the human body is polyclonal. There are different B-cells producing slightly different antibodies with different avidity and affinity and which may even react with different epitopes on the same antigen. If you dilute a sample, the different avidities and affinities may affect their frequency of binding. The more the serum contains specific antibodies and the more it is positive for the respective parameter, the more these effects of different antibody types may be pronounced. Thus, a relatively small concentration of high avidity antibodies may give the same result in a diluted sample as a big amount of low avidity antibodies in an undiluted sample will give. Because real samples contain a mixture of such antibodies, they will not necessarily behave linearly.
In some tests such as tTG IgG assays there is an additional reason for possible non-linearity: most sera which are positive in this test are also positive for tTG IgA and quite often these IgA antibodies against the same antigen may be present in high amounts. These IgA antibodies compete with the tTG IgG antibodies for the binding to the antigen and, thus, occupy most of the available binding sites. The higher the avidity of the IgA antibodies is, the more this effect occurs.
If the sample is diluted the tTG IgA antibodies are diluted in the same degree and more binding sites can be accessed by the tTG IgG antibodies. For some samples, this can even lead to higher results for tTG IgG in diluted samples than in non-diluted ones. In samples with very high antibody concentration it may also happen that the antigen coated to the wells is not sufficient to bind all tTG antibodies (IgG and IgA). Even if the sample is diluted there may still be enough antibodies to bind all antigens.
Result in both cases: the sample does not behave linearly.
EliA Celikey IgG is evaluated for a sample dilution of 1:100. If a sample is diluted more with diluent, the matrix of the antibody containing sample will change. The more it is diluted the more it will differ from the original, physiological environment of the antibodies and will be more artificial with every dilution step. This may result in the binding features of the tTG antibodies changing which would lead to non-expected results. Result: the sample does not behave linearly.
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