Use of a fragment of glycoprotein G-2 produced in the baculovirus expression system for detecting herpes simplex virus type 2-specific antibodies

Abstract

Fragments of glycoprotein G (gG-2(281-594His)), comprising residues 281 to 594 of herpes simplex virus type 2 (HSV-2), glycoprotein G of HSV-1 (gG-1(t26-189His)), and glycoprotein D of HSV-1 (gD-1(1-313)), were expressed in the baculovirus expression system to develop an assay for the detection of HSV-1 and HSV-2 type-specific antibodies. The expression of the gG-1(t26-189His) and gG-2(281-594His) fragments was analyzed by Western blotting using monoclonal antibodies LP10 and AP1, respectively. The molecular masses of the major products of gG-1(t26-189His) and the fragment of gG-2(281-594His) were 36 to 39 kDa and 64 to 72 kDa, respectively. Human sera positive for HSV-1 reacted with gG-1(t26-189His), sera positive for HSV-2 reacted with the gG-2(281-594His) fragment, and sera positive for both types reacted with gG-1(t26-189His) and gG-2(281-594His) in Western blotting. The human sera recognized polypeptides of gG-2(281-594His) with molecular masses of 57 to 67 and 120 to 150 kDa and additional faint bands of 21, 29, and 45 kDa. The recombinant gG-1(t26-189His) and the recombinant gG-2(281-594His) fragment were used as type-specific antigens for the detection of HSV-1- and HSV-2-specific antibody responses in human sera, respectively. As type-common antigens, an extract of HSV-1-infected Vero cells and recombinant gD-1(1-313) were used. An enzyme-linked immunosorbent assay to detect type-specific antibodies was developed, and the sensitivity and specificity were evaluated by comparison with commercial tests by using sera obtained from different sources. The sensitivity and specificity were 91.5 and 95.5%, respectively, compared to the Gull assay. The gG-2(281-594His) fragment can be obtained in relatively large quantities at low cost.

FIG. 1.

Reactivities of human sera and MAbs with concentrated medium of insect cells expressing gG-1_t26-189His and gG-2_281-594His. Insect cells were infected with recombinant baculovirus expressing gG-1_t26-189His or gG-2_281-594His. Medium of the infected cells was collected 3 days after infection, dialyzed against demineralized water, and lyophilized. Samples were heated prior to electrophoresis in SDS sample buffer. Electrophoresis of samples of reconstituted medium of insect cells expressing gG-1_t26-189His (A) and gG-2_281-594His (B) was performed on 12% polyacrylamide gels. The polypeptides were transferred to PVDF membranes and incubated with the antisera. Binding of antibodies was visualized by chemiluminescence (ECL). Lanes: 1, MAb A16, specific for gD; 2, MAb AP1, specific for gG-2; 3, MAb LP10, specific for gG-1; 4, HSV-negative human serum; 5, HSV-1-positive human serum; 6, HSV-2-positive human serum; 7, HSV-1- and HSV-2-positive human serum. Molecular mass markers are indicated.