Antipurified-Protein-Derivative Antibody in Tuberculous Pleural Effusions

Сommon etiologies of exudative pleural effusions are tuberculous and carcinomatous. Although the ultimate diagnosis of pleurisy depends on pleural biopsy or cytologic or bacteriologic examination, the diagnostic problem is often great. Since the develop­ment of the ELISA technique in 1972, many authors have attempted to use it to estimate antibodies in serum for diagnosis of tuberculosis. However, only a few reports have described anti-PPD antibodies in pleural eflusions. Moreover, the nature of the antigen recognized by pleural IgG antibody is not well known. Therefore, we assessed anti-PPD antibodies in pleural effusions by ELISA and studied the nature of the antigen by immunoblot technique.

Materials and Methods

Pleural Effusions

With informed consent, fluids were tapped by thoracentesis from 31 patients with tuberculous pleurisy and 39 patients with malignant pleural eflusions. Their diagnoses depended on pleural biopsy or on positive cytologic or bacteriologic study. The malignancies included 32 primary lung cancers (19 adeno, four squamous cell, seven small cell, one large cell, and one unclassified carcinomas) and seven metastatic, none of which was colonized or infected with any mycobacterial pathogen. Mean age of tuberculous patients was 53.5 years (range 17 to 86) and of patients with malignancy, 64.8 years (range 32 to 88). All efiusion samples were frozen at — 20°C until analysis.

Antigens

The PPD was purified from culture filtrate of Mycobacterium bovis BCG strain by the method of Seibert et al.5 The sediment of culture fluid of M bovis was sonicated for a total of 30 minutes in an ice bath with one-minute resting at five-minute intervals. The supernatant was collected after centrifugation at 10,000 xg for 30 minutes at 4°C and used for BCG whole cell fraction antigen.
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ELISA for Anti-PPD Antibody

ELISA was performed according to the method of Bullock and Walls with a slight modification. The plate was coated with 100 fil of PPD antigen solution (30 jig/ml) in each well overnight at 4°C. Then it was filled with 200 ul of 2 percent BSA overnight at 4°C to block free adsorption sites. Fifty microliters of efiusion sample diluted 1:20 with PBS was charged and incubated for 30 minutes at 37°C. After washing with PBS-T, 50 ul of peroxidase-conjugated goat anti-human IgG, IgA or IgM antibody, diluted 1:4000 with PBS, was charged and incubated for 30 minutes at 37°C. After washing with PBS-T again, the peroxidase enzymatic activity was assayed by adding 100 ji! of 0.04 percent o-phenylenediamine in phosphate citrate buffer saline solution with 0.01 percent hydrogen peroxide for 15 minutes. The reaction was terminated by adding 50 ul of 4N H2S04. The OD at 490 nm was measured with a minireader. For standardization, we assayed each specimen in duplicate with the standard one that had a high anti-PDD IgG antibody value and corrected OD to ODI in every experiment. A linear relationship between ODI and dilutions of standard antibody was provided.

SDS-PAGE and Immunoblot Analysis

A subgroup of 17 tuberculous and 18 carcinomatous effusions was selected at random for immunoblot analysis from 70 specimens that were assessed by ELISA. Mean ODI value of the subgroup was not significantly different from that of original specimens in each disease. The PAGE was performed in 1.0-mm thick gel containing 13 percent polyacrylamide. Twenty microliters of PPD or BCG whole cell fraction antigen (5 mg protein /ml) was run. The sample buffer either contained or did not contain 1 percent 2-mercapto- ethanol. Electrophoresis was conducted for four hours at 100 V. Transfer of antigen from gel to nitrocellulose paper was based on Burnettes modification of Towbins procedure. After the transfer for two hours at 4°C, 25 V, the blotted paper was placed in TBS containing 2 percent BSA overnight at 4°C. It was removed and immersed in the efiusion at a 1:50 dilution in TBS with 1 percent BSA for one hour with gentle agitation. Then, we washed it for at least one hour in multiple changes of TBS-T. It was incubated with peroxidase conjugated goat anti-human-IgG antibody that was diluted 1:200 with TBS containing 1 percent BSA for 30 minutes and then washed with TBS-T. The peroxidase reaction was com­pleted by placing the blotted paper in TBS containing 0.025 percent 3,3’diaminobenzidine-4HCl and 0.015 percent hydrogen peroxide for 15 minutes. The reaction was stopped by washing in distilled water.
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PAS Staining

Following the electrophoresis with 13 percent acrylamide, the gel was placed in 1 percent НЮ4 and 3 percent CH3COOH at 4°C for two hours. After washing with distilled water overnight, it was immersed in Schiff reagent at 4°C for one hour in the dark and placed in 7 percent CH3COOH for rinsing.

Statistical Analysis

Students f-test or Mann-Whitney test was used for statistical analysis.