title: Assessment of assay sensitivity and precision in a malaria antibody ELISA.
abstract: Many types of ELISA-based immunodiagnostic test kits are commercially available in the market for specific indications. These kits provide necessary assay components, reagents, and guidelines to perform the assay under designated optimal conditions. By using these kits, any unknown or test sample can be assessed as negative or positive based on the results of referral calibrator (Ref+ve and Ref-ve) samples. It is essential to provide reliable test kits to end-users with adequate quality control analysis. Therefore, it is necessary to check the kit for any variations in its performance. While developing a malaria antibody ELISA test-kit, we optimized assay conditions with chequer-board analyses and developed an assay protocol. We have taken out kits randomly from the assembly line and had them evaluated by operators who are new to the test-kits. Assays are performed as per the test guidelines provided. Sera, diluted serially, have shown a clear discriminatory signal between a negative vs. positive sample. A COV is determined by evaluating the Ref-ve calibrator in replicate antigen-coated wells from 6 different plates. This COV is used as a tool to determine S/N ratio of test samples. Besides Ref-ve and Ref+ve calibrators, additional field serum samples are tested with the test kit. Several performance indices, such as mean, standard deviation, %CV are calculated, and the inter- and intra-assay variations determined. The assay precision is determined with large and small replicate samples. In addition, assays are performed concurrently in triplicate-, duplicate-, and single-wells, and the results are analyzed for any assay variations. Different plate areas are identified in antigen-coated 96-well plates and tested blind to detect any variations. The S/N ratio is found to be a very effective tool in determining the assay sensitivity. The %CV was within 10-15%. Variations seen in the assays are found to be due to operator errors and not due to kit reagents. These observations, although, are based upon one type; however, it may as well apply to other line of kits. This is obviously valuable to the end-users of ELISA kits. The operator related error has to be ascertained before lodging any complaint on the kit performance. Based on this data, the test kit has shown acceptable sensitivity and precision and offers compliance on the way the test kits is manufactured. With this, it is concluded that the test kits are suitable for detecting malaria antibody in clinical sample analysis.
authors.affiliation: Cellabs Pty. Ltd., Brookvale, NSW, Australia. raj@cellabs.com.au
authors.forename: G Halli R
authors.initials: GH
authors.lastname: Rajasekariah
authors.forename: Graeme E
authors.initials: GE
authors.lastname: Kay
authors.forename: Natrice V
authors.initials: NV
authors.lastname: Russell
authors.forename: Anthony M
authors.initials: AM
authors.lastname: Smithyman
mesh_terms: D000704:Analysis of Variance; D000913:Antibodies, Protozoan / Q000032:analysis* / Q000097:blood; D002138:Calibration; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D005069:Evaluation Studies as Topic; D005188:False Negative Reactions; D006801:Humans; D008288:Malaria / Q000097:blood / Q000175:diagnosis* / Q000469:parasitology; D011933:Reagent Kits, Diagnostic / Q000592:standards; D012680:Sensitivity and Specificity
chemical_list: D000913:Antibodies, Protozoan; D011933:Reagent Kits, Diagnostic