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AFRO-NETS> Field Immunodiagnostic Kit for Chancroid

Field Immunodiagnostic Kit for Chancroid

My laboratory is working both on diagnostic and vaccine candidates 
for the etiologic agent of chancroid, Haemophilus ducreyi. As you 
know, Chancroid is endemic in Africa, where it has been found to be 
an important risk factor for the heterosexual transmission of HIV. I 
am writing you to ask if you know a funding agency, which might be 
relevant for assembling and field testing a "quickie" test for chan-
croid. We have done the "hard part" in making the kit-Monoclonal an-
tibodies (Mabs) to H. ducreyi - see summary report below.

Thanks for your time.

Christopher Elkins Ph.D.
Research Assistant Professor
Departments of Medicine and Microbiology and Immunology
School of Medicine
Room 521 Burnett-Womack
Campus Box 7030
Univ. of North Carolina
Chapel Hill, NC 27599, USA
Tel: +1-919-966-3661
Fax: +1-919-966-6714

Direct detection of Haemophilus ducreyi using Mabs to the conserved 
Hemoglobin Receptor Protein

Christopher Elkins, PhD 
Division of Infectious Diseases and 
Department of Microbiology and Immunology 
University of North Carolina at Chapel Hill 
Chapel Hill, NC 27599-7030, USA

Abstract of Progress 1-1-99

The objective of this project is to develop a non-cultural, mono-
clonal antibody-based, diagnostic field test for Chancroid. Success-
ful antigen detection tests, similar to the one proposed here, in-
clude kits for diagnosis of Streptococcal pharyngitis and pregnancy 
test kits. The monoclonal antibodies (Mabs) used are to the Hemoglo-
bin-binding protein (HgbA) of Haemophilus ducreyi. The rationale for 
choosing HgbA for detection of H. ducreyi is that HgbA is a major 
protein found in all tested isolates, is immunologically and func-
tionally conserved, small amounts can be readily purified in a single 
step, and it is highly immunogenic in laboratory animals. Isogenic 
mutants of hgbA are avirulent in the human model of H.ducreyi infec-
tion (unpublished data) suggesting that this gene must be expressed 
to cause disease and naturally occurring mutants do not exist. There-
fore HgbA would be a good target for immunologic detection. 

Native (nHgbA, from H. ducreyi) and recombinant HgbA (rHgbA, over-
expressed in E. coli) were purified, mice immunized, and several 
thousand hybridoma supernatants screened for reactivity. Several hy-
bridoma cell lines were cloned by limiting dilution and clones prov-
ing to be stable were used to produce 1/2 liter amounts of cell su-
pernatant for further characterization. We have purified analytical 
amounts of IgG from each. We have developed a simple capture ELISA 
assay using each purified Mab as the capture antibody and a rabbit 
anti-peptide sera as the detection antibody. Mabs have been tested 
for cross reactivity against a panel of geographically diverse of 
strains which contain a variety of ribotypes (P. A. Totten, manu-
script in preparation). 

Several Mabs were isolated, characterized and purified. We have grown 
the above strains and prepared the cellular extracts for testing. In 
our format, H. ducreyi are detergent solubilized and all Mabs recog-
nize this form of HgbA. Among the several tested, we have identified 
3 unique Mabs which are non-competitive and recognize the HgbA pro-
tein from all strains of H. ducreyi tested. We estimate that in a 
simple non-optimized capture ELISA, we can readily detect 50 ng of 
purified HgbA mixed with a cell extract from a mutant H.ducreyi un-
able to make HgbA. This corresponds roughly to 100,000 bacteria (de-
pending on strain, amount of heme starvation and other variables). We 
have not tried to optimize this ELISA system, but have used it only 
for strain surveys. Thus, we have 3 Mabs, suitable for capture and 
detection Mabs in hand.

A second hybridoma laboratory is independently producing additional 
Mabs to rHgbA, should current Mabs prove unsuitable. We have been un-
successful in garnering industry support for this project. Therefore, 
we have established a collaboration with Dr. Stephen Morse at the 
Centers Disease Control (CDC). Hybridoma clones for these Mabs have 
been sent to the CDC and a "prototype immunochromatography kit" will 
be assembled for testing of mock specimens to determine sensitivity 
and specificity. Our STD group here at UNC, lead by Dr. Myron Cohen, 
has set up several labs where Chancroid is endemic and will be freez-
ing specimens in liquid nitrogen for shipment back to UNC for analy-
sis by culture, PCR and immunodetection. These sites represent an in-
valuable tool for extensive field evaluation of the kit. Should these 
tests indicate the kit has potential we will need to obtain a corpo-
rate partner to manufacture kits and further funding for field test-
ing of these kits.

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