Automated, Quantitative Microhemagglutination Assay for Treponema pallidum Antibodies

Abstract

An automated, quantitative microhemagglutination assay for antibodies to Treponema pallidum was developed by using T. pallidum-sensitized erythrocytes and an automatic serial-dilution instrument. Reactivity was found in sera from 54 rabbits and 6 chimpanzees infected with T. pallidum. Reactivity was also found in sera from animals infected with T. pertenue, T. carateum, and T. cuniculi. No reactivity was found in sera from 75 normal rabbits or from 129 rabbits immunized with cultivatable treponemes or a variety of other bacteria. In approximately 3 min, 13 twofold serial dilutions of each of 8 preabsorbed sera and the addition of sensitized erythrocytes to each dilution were accomplished automatically. The automated assay can serve as a research tool in quantitating antibodies to pathogenic treponemes, and evaluation of its clinical usefulness seems warranted.

Laboratory diagnosis and interpretation of tests for syphilis

“…A reactive result is reported, without reference to titer, if (i) the hemagglutination with sensitized cells is at least two doubling dilutions (four times) greater than with unsensitized cells, and (ii) the first dilution showing no hem- Because the test is based on agglutination, quantitation of treponemal antibody is possible but has not proven to be worthwhile. Most studies demonstrate no practical relationship between the titer and either the progression of the disease or the clinical stage of syphilis diagnosed, and unlike the quantitative nontreponemal tests, the quantitative MHA-TP test does not seem to be useful in posttreatment evaluation (11,28,93). The sources for error with the MHA-TP are usually associated with the use of dusty or improper plates, pipetting errors, and vibrations in the laboratory.…”

“…The hemagglutination procedure was first a tube test known as the T. pallidum hemagglutination test. Subsequently, reagents for a microvolume (27) hemagglutination test, the microhemagglutination assay for antibodies to T. pallidum (MHA-TP), became commercially available. Another hemagglutination test for syphilis (HATTS) (210), based on the use of sensitized turkey cells and performed in microtiter plates, was considered a standard test for syphilis from the mid-1970s to late 1980s, when the product was withdrawn from the American market because of a lack of reproducible results between batches of reagents.…”

Evaluation of the Qualitative and Automated Quantitative Microhemagglutination Assay for Antibodies to Treponema pallidum

“…The BFP group included sera from nine narcotic users; all were nonreactive in the MHA-TP test. An additional 22 sera from VDRL reactive, FTA-ABS nonreactive narcotic users were tested, and 21 were nonreactive in the MHA-TP test; valid MHA-TP results could not be obtained with 1 The "normal" group consisted of 425 VDRL nonreactive sera obtained from a local blood bank. These sera were tested by the MHA-TP test, and all reactors were further tested by the FTA-ABS test.…”

“…In the evaluation reported here, the MHA-TP test compared favorably with the TPI and FTA-ABS tests in all stages of syphilis except primary syphilis, in which it was less sensitive than the FTA-ABS test and possibly less sensitive than the TPI test. Other investigators have also noted a lack of sensitivity in the hemagglutination test in primary syphilis (2,3,8). In a recent field study of the MHA-TP test, the three participating laboratories reported MHA-TP reactivities in primary syphilis of 92.5% on 134 sera, 75% on 32 sera, and 94.7% on 19 sera (2).…”

“…A passive hemagglutination test for syphilis in which an antigen derived from the virulent Nichols strain of Treponema pallidum is used has been described by Rathlev (6) and by Tomizawa and associates (7,8). This test was adapted to an automated microhemagglutination procedure by Cox et al (1), and in evaluations to date the automated procedure has yielded results comparable to those obtained with the fluorescent treponemal antibody-absorption (FTA-ABS) tests for syphilis (2,4). Because a reliable hemagglutination test would possess a considerable advantage in time and cost over the manually performed FTA-ABS test or the T. pallidum immobilization (TPI) test, an evaluation of the manual and automated microhemagglutination assay for T. pallidum antibodies (MHA-TP) was undertaken and is reported here.…”