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Tan Y.-H.,Guangzhou Fenghua Bio engineering Co. | Yu T.,Guangzhou Fenghua Bio engineering Co. | Dong M.,Guangzhou Fenghua Bio engineering Co. | Cen Q.-H.,Guangzhou Fenghua Bio engineering Co. | And 5 more authors.
Chinese Journal of Biologicals | Year: 2014

Objective: To develop a detection kit of biotin-avidin monoclonal antibody capture time-resolved fluoroim-munoassay (BA Mac TrFIA) for IgM antibody to human cytomegalovirus (HCMV IgM) and evaluate its performance. Methods: Mouse anti-human IgM p chain monoclonal antibody (McAb) was coated onto microplates, while the recombinant HCMV antigen labeled with biotin was served as bridging antigen, and streptavidin(SA)-labeled Europium (Eu3+) as a tracer. The mainly component of enhancement solution was β-naphthalene thrifluoroacetone. The developed BA Mac TrFIA kit was used for detection of HCMV IgM. Three consecutive batches of kits were prepared and evaluated for minimum detection limit, reproducibility, coincidence rates with positive and negative references and heat stability. A total of 1 020 serum or plasma samples were detected by the developed kit, and the results were compared with those by diagnostic kit for HCMV IgM, while the consistency was analyzed by Kappa test. Results: The optimal coating concentration of mouse anti-human IgM μ chain McAb was 4 μg/ml, while the optimal working concentration of HCMV-Bio was 1:10 000, and that of SA-Eu3+ was 1:1 500. The reference value of prepared kit was recommend as negative control counter (NCx) x 2.1. The minimum detection limit, reproducibility as well as coincidence rates of negative and positive references of three consecutive batches of kits met the national requirements, while the performance showed no significant change after storage at 37°C for 6 d. The Kappa index of clinical comparative research was 1, indicating a consistency of 100%. Conclusion: The BA Mac TrfIA kit for HCMV IgM was successfully prepared and showed high sensitivity and precision, of which the detection result was highly correlated to those by the kits of same kind. It provided a scientific and reliable diagnostic basis for detection of HCMV IgM in clinic.

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