Use of recombinant pemphigus vulgaris antigen in development of ELISA and IB assays to detect pemphigus vulgaris autoantibodies.
OBJECTIVE
The objectives of this study are: (1) to measure the titers of pemphigus vulgaris (PV) autoantibody in the sera of patients with active disease, using three different assays: (a) Indirect immunofluorescence (IIF) using monkey esophagus as a substrate, (b) immunoblot (IB) and, (c) enzyme-linked immunosorbent assay (ELISA) using recombinant PV antigen (rPVA). (2) To compare the sensitivity of these three assays.


BACKGROUND
The titer of PV autoantibodies and disease severity and extent do not always correlate. This could be due to the lack of consistency and specificity of the substrate. Different results are obtained using different substrates. A standard substrate with uniformly controlled source of antigen would be more useful and clinically beneficial.


METHODS
In this study we studied 25 PV patients, six each with bullous pemphigoid (BP), ocular cicatricial pemphigoid (OCP), mucous membrane pemphigoid (MMP), and herpes gestationis (HG), and sera from 16 normal subjects. IIF was used to determine the PV autoantibody using monkey esophagus. IB assay was used according to standard protocol using normal human epidermis and rPVA as substrates. ELISA was performed using rPVA as antigens expressed in E. coli.


RESULTS
Sera of all 25 PV patients showed binding to the rPVA, normal human sera and the sera from the six BP, six OCP, six MMP, and six HG patients did not show any binding. In addition, we used antisera from rabbits immunized with PVA peptides (Bos-1, Bos-6) which also showed binding to rPVA, whereas normal rabbit sera did not show any reactivity. ELISA and IB titers in all the patients were 2.5 to 160 times higher than with the conventionally used IIF assay. The titers of the PV specific autoantibody measured using the rPVA did not show statistically significant differences between the ELISA and IB assays.


CONCLUSIONS
IB and ELISA are superior to IIF in evaluating the antibody levels in PV patients. ELISA is more practical and is preferable to IB and is recommended for clinical use.
Bhol
K
K
Center for Blood Research, Harvard School of Dental Medicine, Boston, MA 02115, USA.
Tyagi
S
S
Natarajan
K
K
Nagarwalla
N
N
Ahmed
A R
AR
D000818:Animals; D000941:Antigens; D001323:Autoantibodies / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D007074:Immunoglobulin G / Q000276:immunology; D010391:Pemphigoid, Bullous / Q000276:immunology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D012680:Sensitivity and Specificity; D018709:Statistics, Nonparametric
D000941:Antigens; D001323:Autoantibodies; D007074:Immunoglobulin G; D011994:Recombinant Proteins