Different varieties of immunological tests have been developed to measure latex antigens/allergens in latex products. These are all based upon the principle that antibodies can recognize and bind to particular proteins. This binding can be measured and is quantitated by comparing similar binding to standards.
Two general configurations of these immunological assays exist. These include the measurement of direct antibody binding and inhibition of antibody binding. In the former, the amount of antibody binding to solid phased latex proteins is measured. In the latter, latex proteins are allowed to react with a specific concentration of antibodies. Then, the amount of remaining antibodies is measured by their attachment to solid phased latex proteins. The amount of latex protein in the original sample is therefore inversely proportional to the level of antibodies measured. In both cases, a second antibody or ligand that is tagged with isotope (RIA) or enzyme (ELISA) is utilized to measure the antibodies that bind.
Both these assay configurations are heavily dependent upon the attachment of proteins in a relevant manner to the solid phase, the quality of the antibody preparations, the detection system, the standard used and other factors that can influence assay performance.
The anti-latex antibodies measured depend upon what they can recognize and bind to on the solid phase. In complex mixtures of proteins like those from NRL, this has not been well characterized. Because binding to solid phases is usually limited, it is highly unlikely that all the proteins bind to the solid phase in sufficient quantity and relevant manner such that all the antibodies present can be measured. Thus, the antibodies being measured represent those binding with the major proteins that initially coated the solid phase in a relevant orientation.
The quality of the antibody preparations also may vary. Those typically used are either natural IgE antibody pools from latex-sensitized individuals or hyperimmune sera raised by immunizing animals. The titer, affinity, and proteins that different humans react to are also variables, so such a pool tends to emphasize those proteins that are the most common sensitizers. Thus, the major reacting proteins that are relevant to human sensitization and that become bound in sufficient quantity are likely to be those measured. Human sera are also likely to contain other antibodies that may interfere with the binding to proteins by the antibodies being measured. Hyperimmune animal sera also depend upon what the animals react most strongly to. The antibodies raised also depend upon what the animals were immunized to and responses vary from animal to animal. Obviously if a protein is missing in these immunizations it will not be measured by the assay. Both the number of different proteins recognized (specificity) and their respective binding affinities can affect these assays.
The least variable portion of these assays is the detection system used. These can affect the assays in a number of different ways but are usually factors that can be predetermined. A standard curve is usually generated using a preparation containing various amounts of latex protein, which can vary from preparation to preparation. This variation can affect the assay results in a number of different ways. For example, if the sample doesn’t contain proteins that antibodies are present for, then these antibodies will attach to the solid phase and a false positive result will result. And, in the direct binding assay, if the standard and proteins in the sample are not well matched in number and content, interpolated values can give widely varying results. Various non-specific factors such as detergents or enzymes can affect these assays, leading to false positives or negatives.
A popular variation of these two assay formats, the LEAP assay, uses extracts of latex products that are allowed to bind to a plastic solid phase. Hyperimmune animal antiserum is then allowed to bind to the protein that is present, but the protein-to-plate binding is variable. Again, measurements are made based on antibody binding to a standard preparation. As before, problems arise with the variability in the animal immunizations/sensitizations, which can ultimately lead to poor correlation between final results and actual product protein content. Also, interfering substances can lessen the amount of protein bound and yield falsely negative results.
In spite of their shortcomings, these immunologic assays are still better measurements of specific latex proteins and their amounts in NRL products than the chemical assays used to determine total protein. The chemical assays have a much greater variability, making it difficult to compare one product with another.
The ideal immunological assays for measuring NRL proteins consist of paired monoclonal antibodies that measure each specific protein one at a time. These assays are highly quantitative and assays for four of these proteins have been developed and marketed by Finnish researchers. These seem to cover the majority of the proteins found in natural latex products but this has not been completely proven to date. A drawback here is that purified proteins are needed for standards and cost. Until these monoclonal assays are fully developed, the status of most polyclonal immunological assays for latex-derived proteins are at best only a first approximation and at worst can be misleading.
