Discuss the evaluation of monoclonal antibody-based commercial kits

Immune reagents have been the principal tools for the identification and diagnosis of viral diseases. The most common tests used are enzyme immunoassay, immunofluorescence and neutralization. The most important criterion for these tests is the specificity of the antisera used. Optimal sensitivity and specificity are attained with reagents that give strong reactions with the homologous virus and do not cross react with unrelated viruses.

Conventional polyclonal sera are produced by injecting animals with viral antigens usually in the from of virus-infected cells. The resultant antiserum contain antibodies to both viral and host cell proteins. The antibodies to the host cell proteins are then usually removed by absorbing the antiserum with host tissue before use. Other disadvantages of polyvalent antisera are the variability of the immune response of the animals and the limited quantities of antisera produced.

Monoclonal antibodies are produced from hybridomas of mouse B lymphocytes and cultured myeloma cells which inherit the property of immortalization from the myeloma parent and the immunoglobulin producing property of the B cell parent. Monoclonal antibodies are now extensively in the diagnosis of microbial diseases by the detection of microbial antigen. Examples include the DEAFF and antigenaemia test for CMV, detection of rotavirus in faeces, and hepatitis B surface antigen. Monoclonal antibodies have the advantage of being available in unlimited quantities. Being a homogeneous product, they do not need purification. They are also highly specific. However, the high specificity of monoclonal antibodies may pose problems in the detection of viruses with extensive antigenic variation, such as influenza A.

Evaluation

Where possible, the evaluated assays should be compared with recognized reference methods eg. EIA for the detection of rotavirus and adenovirus antigens with electron microscopy, or the CMV antigenaemia test with cell culture and the DEAFF test. When reference methods are not available as in the case of many serological tests, the assays should be compared to the one in current use in the laboratory.

Specimens known to have given positive, borderline and negative results by the reference method should be selected and tested by the assay to be evaluated. The more specimens used the better as the evaluation result will be more statistically significant. At the end of the evaluation, one would then be able to calculate a sensitivity and specificity of the new assay compared to the reference method. Statistical analysis should be performed to assess whether differences between the evaluated assay and the reference method can be accounted for by random variation or not. Any discrepant results should be investigated with the clinical picture of the patient assessed.

Where quantification is possible, such as in the case of HBsAg, the results of the assay being assessed and the one which the assay is being compared to should be assessed for the degree of correlation by performing linear regression analysis. The degree of fit and also the gradient of the line should be looked at, both figures should be near the figure of one. If the gradient is considerably less than one, that means that the evaluated assay consistently produces results of a lower quantity than that of the reference method and vice-versa if the gradient is greater than one.

In the evaluation of monoclonal antibody based commercial kits, one must be able to show that the kit is sensitive for all of the common strains of the virus involved. It is important to make sure that all the strains that are prevalent locally are covered. For example, in African countries where HIV subtype O is common, it is essential that serum taken from patients with HIV subtype O be tested. The kit should be tested against available reference strains of viruses for which it is claimed to be sensitive for. Similarly, the specificity of the kit should be confirmed by testing with various related and unrelated viruses eg. HSV and VZV in the case of CMV. Different known concentrations of virus should be tested in order to assess the sensitivity of the assay compared to the reference method and the presence of a pro-zone effect.

The assay to be evaluated should then be put to the test in the field so that it is run in parallel with the reference method. Individual specimens are tested as they come in so that how the test performs under actual working conditions is assessed. This may be the only way in which some tests could be assessed eg. with the CMV antigenaemia test, one cannot use stored specimens as these specimens lose their antigenicity very quickly. Only after careful evaluation should be assay be used routinely in the laboratory. It is important to write a detailed report and analysis on the evaluation that led to the adoption of the assay which can be easily traced and referenced back in future should the need arise.