Production of Puumala Virus Antigen for Use in Diagnostic Assays

Abstract

Puumala virus is a member of the Hantavirus genus, and is found predominantly in Europe. It is transmitted to humans via aerosolized virus present in the excreta of voles. Puumala virus causes a mortality rate of 0.1-1.0% in humans, and death is usually by renal failure and hemostatic imbalance. Effective diagnosis of Puumala virus-induced disease is important for studying the epidemiology of the virus and may play a key role in the effective treatment of the disease. Because of the difficulty in producing quality diagnostic reagents for antibody detection assays, Puumala virus diagnostics are typically only available in a very limited number of laboratories. This paper reports on the production of nucleocapsid (N) protein from Puumala virus using a genetically engineered, truncated S gene. This protein is a key component of diagnostic assays and is difficult to produce in large quantities by conventional methods. Growth conditions of bacteria containing a plasmid engineered with the truncated S segment were optimized to ensure that the maximum amount of protein was produced. The pH of the Luria-Bertani (LB) broth, Miller method, was tested between 6.0 and 8.0 in increments of 0.5 pH units. It was found that pH 7.0-7.5 yielded the best results. The expression induction point was monitored from ODA600 of 0.2 to 1.1. The best yield of recombinant N protein was found when the cells were induced at an OD A600 of 0.4-0.5. After induction, the optimal expression time period was found to be two hours. Cells were also grown at various temperatures (25°C, 30°, 37°C, 45°C, and 50°C). The best growth temperature was found to be 37°C. The concentration of isopropyl-B-D-thiogalactopyranoside (IPTG) used to induce expression was tested, and 0.1 ug/ml was found to be optimal. Yields of N protein were found to be further improved by providing T7 polymerase using bacteriophage CE6 instead of by induction with IPTG. N protein was purified using immobilized metal affinity chromatography (IMAC) plus reverse phase and cation exchange chromatography. The N protein concentration was optimized in both an immunoglobulin M (IgM) and an immunoglobulin G (IgG) direct enzyme-linked immunosorbent assay (ELISA). The best concentration to be used in the assays was found to be 600 ng/ml. Due to high background problems with the direct IV ELISA, several other ELISA formats were attempted. However, none of these formats proved useful. An attempt was also made to develop a Western blot (WB) to detect IgM antibodies. However, when the WB was probed with an IgM positive human sera, no reaction was observed. A direct IgG ELISA was developed which had no background problems. Development of an IgG WB was attempted, but due to inconsistent results, was abandoned. A panel of 62 human sera was tested by ELISA for IgM and IgG antibodies using both native antigen and the purified recombinant antigen with the results confirmed by WB. The ELISAs using native antigen had a sensitivity of 38.3% and a specificity of 100%. The ELISAs using the recombinant antigen had a sensitivity of 95.2% and a specificity of 84.1%. Based on these observations, the recombinant antigen appears to be more useful than native antigens in certain diagnostic assays.