Development of an immunochromatography strip test based on truncated nucleocapsid antigens of three representative hantaviruses

Abstract

Background: Hantaviruses are causative agents of hemorrhagic fever with renal syndrome (HFRS) and nephropathia epidemica (NE) in the Old World and hantavirus pulmonary syndrome (HPS) in the New World. There is a need for time-saving diagnostic methods. In the present study, recombinant N antigens were used as antigens in an immunochromatography strip (ICG) test to detect specific IgG antibodies.

Methods: The N-terminal 103 amino acids (aa) of Hantaan virus (HTNV), Puumala virus (PUUV) and Andes virus (ANDV) nucleocapsid (N) protein were expressed in E. coli as representative antigens of three groups (HFRS, NE and HPS-causing viruses) of hantavirus. Five different types of ICG test strips, one antigen line on one strip for each of the three selected hantaviruses (HTNV, PUUV and ANDV), three antigen lines on one strip and a mixed antigen line on one strip, were developed and sensitivities were compared.

Results: A total of 87 convalescent-phase patient sera, including sera from 35 HFRS patients, 36 NE patients and 16 HPS patients, and 25 sera from healthy seronegative people as negative controls were used to evaluate the ICG test. Sensitivities of the three-line strip and mixed-line strip were similar to those of the single antigen strip (97.2 to 100%). On the other hand, all of the ICG test strips showed high specificities to healthy donors.

Conclusion: These results indicated that the ICG test with the three representative antigens is an effective serodiagnostic tool for screening and typing of hantavirus infection in humans.

Figure 1

ICG test lines of the HTNV, PUUV and ANDV strips. A: Test lines of the HTNV strip with sera from HFRS patients infected with HTNV and SEOV. B: Test lines of the PUUV strips with sera from an NE patient infected with PUUV and a seronegative sera from healthy donor. C: Test lines of the ANDV strip with sera from HPS patients infected with SNV, ANDV and LANV.

Figure 2

ICG test lines of the three-line strip and mixed-line strip. A: Test lines of the three-line strip with sera from HFRS, NE and HPS patients and a seronegative healthy donor. B: Test lines of the mixed-line strip with sera from sera from HFRS, NE and HPS patients and a seronegative healthy donor.

Figure 3

Detection method and scheme of ICG strip. A: Structure of ICG strip. N-terminal 103 amino acids of N protein and rabbit IgG were placed at the test line and control line, respectively. The ICG strip consisted of 4 membrane pads: sample pad, conjugate pad, nitrocellulose membrane and absorbent pad. Protein A-colloidal gold conjugate was kept in the conjugate pad. B: Detection method. Two μl of serum was diluted with 150 μl of PBS and then placed in a microtiter well. The strip was dipped in the solution. After standing still for 15 min, the test line and/or control line appeared.