Question
How well does a cloned, soluble recombinant human monoclonal antibody fragment with the ability to react specifically against G glycoprotein of HSV-2 identify cells infected with HSV clinical isolates previously typed using commercial type-specific monoclonal antibodies in an indirect immunofluorescence assay? 

Design
A low-cost alternative to expensive, mouse derived type-specific antibodies for use in immunofluorescent staining for HSV subtyping is described that is based on construction of a combinatorial phage display library and selection of cloned human monoclonal antibody fragments (Fab) with the ability to react to G glycoprotein in HSV type 2.  

Participants
The antibodies were tested using cells infected with an HSV-1 or HSV-2 reference strains from ATCC, and 50 HSV-1 and 62-HSV-2 clinical isolates previously typed using commercially-available type-specific monoclonal antibodies. 

Description of Tests and Diagnostic Standard
cDNA reverse transcribed from the Fab portion of human antibody mRNA was inserted into bacteriophage genomes, giving rise to live viral particles that displayed antibody fragments on their surface, while packaging the corresponding antibody gene inside.  This combinatorial phage display library of human immunoglobulin G1 (k) recombinant Fab (rFab) was previously constructed using mRNA from a donor who was positive for both HSV-1 and HSV-2.  In this report, the library was used to select bacterial clones producing soluble human monoclonal antibody fragments (rFab) with the ability to react specifically to HSV type 2 glycoprotein G (gG2).  The highest-affinity antibodies were selected from the phage library by four rounds of binding to commercially available gG2 immobilized in wells (DiaSorin, Saluggia, Italy).  

An ELISA was used to test the rFabs for gG2 reactivity.  One antibody was tested in an indirect immunofluorescence assay for its ability to subtype 50 HSV-1 and 62 HSV-2 clinical isolates that had been previously typed using commercial type-specific monoclonal antibodies (Dako Diagnostics, Ltd., Ely, UK).  Competition between the selected antibody and antibodies from human sera was assayed using competition ELISAs in which wells were coated with gG2.  Diluted sera was added and, after two hours incubation, the Fab was added.  Results were expressed as percentages of inhibition.  

Main Outcome Measures
The ability of one rFab clone to subtype HSV isolates previously identified using commercially available type-specific monoclonal antibodies was determined.

Main Results
Of the 20 soluble rFabs obtained from individual bacterial clones, 14 showed specific reactivity for gG2 in an ELISA assay.  All 14 ELISA–positive rFabs produced positive immunofluorescence staining in cells infected with an HSV-2 reference strain but not HSV-1 or uninfected cells. In an indirect immunofluorescence assay, one clone, Hg2, gave strongly positive signals on all cells infected with HSV-2 clinical isolates.  No positive reactions were seen on any cells infected with HSV-1 isolates or on uninfected cells.  Competition ELISAs using serum samples from 4 HSV-2-positive individuals showed that these samples inhibited the binding of Hg2 to gG2.  Serum samples from 4 HSV-1-positive patients failed to prevent binding.  

Authors’ Conclusions
The human monoclonal rFab described in this report successfully discriminated between HSV-1and HSV-2 in clinical specimens and showed potential as a low-cost tool for HSV subtyping in clinical diagnosis.

Source of funding:  In part by a grant from Ministero della Ricerca Scientifica e Tecnologica.  

For correspondence:  Paola Cattani, Istituto di Microbiologia, Universita Cattolica del Sacro Cuore, L. go F. Vito 1, 00168 Rome, Italy.  E-mail address: pcattani@rm.unicatt.it.