(90%) patients had levels above the upper control mark.

In an attempt to obtain a better sero-diagnostic method for tuberculosis, tests based on the inhibition of monoclonal antibodies (MABs) have been tried. This test is based on the inhibition of binding of labelled MABs to antigen by antibodies present in the sera (Hewitt et a l . 1982).

Five MABs which bind to distinct epitopes of M.

tuberculosis have been used to measure antibody titres in patients with tuberculosis and control subjects. These are TB78, TB71, TB23, TB68, TB72 and of these MAB TB72 shows a marked specificity for M. tuberculosis i.e. it binds to an antigenic determinant which is immunodominant in humans with active tuberculoisis (Ivanyi et a l . 1985; Young et a l . 1986). Using MAB TB72 Ivanyi and colleagues

(1983) reported 25 sera out of 34 (74%) as positive from tuberculosis patients and only 4 out of 51 (8%) control sera being positive giving a sensitivity of 74% and

specificity of 90.5%. One of the current limitations of MABs for diagnosis of tuberculosis is that other epitopes

for which MABs are not yet available may be

immunodominant and potentially important in establishing a sero-diagnostic tool capable of detecting tuberculosis

(Abou-Zeid et a l . 1986; Ranadive et a l , 1986).

anticipated using antigenic products produced by molecular techniques (Matsuo et a l . 19881) or synthesized

carbohydrate antigens (Chanteau et a l . 1988) such hopes have not been realized.

In most studies done so far ELISA has been sensitive enough to show antibodies produced against mycobacterial antigens in many infected individuals. The problem is not the sensitivity of the technique, but it is the specificity of the assay that limits its use in clinical practice for diagnosis of tuberculosis. The reason for the low specificity of the sérodiagnostic tests currently at use when applied to tuberculosis may be due to either one of two points (Grange, 1989).

Humoral immune response in disease caused by mycobacteria is usually weak despite the fact that the organisms are good adjuvants. The other reason is cross-reactivity of antigens amongst the mycobacteria and other related

bacterial genera. A further reason is that many people are infected with tubercle bacilli and have latent

disease. Such persons do not require treatment, and they must be distinguished from those with progressive disease who do require treatment. Although many studies have used purified antigens there is still an unacceptable overlap between antibody levels in diseased and healthy individuals (Grange, 1984; Grange and Laszlo, 1990). Healthy infants whose subsequent tuberculin tests prove

negative have been shown to possess antibodies which bind to soluble mycobacterial extracts (Bardana et a^. 1973).

Mycobacteria contain shared and species specific antigens (Stanford and Grange, 1974). Any tuberculosis patient's sera therefore contains antibodies raised against shared and species specific antigens. Sero- diagnostic assays for tuberculosis would be most

important if one could measure the level of antibodies specific to the species sought,and there was a major

difference in titre between diseased persons, and healthy but infected persons.

In this experiment an attempt is made to improve the diagnostic accuracy of ELISA by use of a simple

absorption technique to remove antibodies raised against common mycobacterial antigens. This modified ELISA

technique is compared with two other ELISA systems on serum samples obtained from individuals living in high prevalence areas for tuberculosis.

Materials and Methods

Sera

Sera used in this study were obtained from

tuberculosis patients and persons in Nigeria not known to have the disease. 53 sera were obtained from

microscopically confirmed TB patients and 30 were taken from subjects who were either healthy or had non-

mycobacterial diseases .These sera were supplied as a randomized numbered series, and at the time of their use I did not know which was which.

Sonicate antigen ELISA and "andaelisa" kit u s e d .

Antigen used for coating the microtiter plates(other than the "andaelisa" plates which were pre-coated) was sonicate antigen prepared from M. tuberculosis provided by Dr Stanford. The antigen was used at 5 ugm/ml to coat the ELISA plates.Vaccin,sonicate antigen prepared

from M. vaccae ,was used to absorb the sera and was obtained from the same source.

ELISA using "andaelisa" kit from anda biologicals was done according to the manufacturer's instructions with minor modifications as explained below.

Enzyme and substrate

Peroxidase-conjugated rabbit immunoglobulins to

human (IgA, IgG, IgM Kappa lambda were purchased from Dako Immunoglobulins Ltd., Copenhagen, Denmark. The substrate was 2,2'-azino-di-(3-ethyI)-benzothiazoIine-6'-sulphonic acid (ÀBTS, Sigma) (Saunders and Bartlett, 1977). The substrate was prepared by dissolving 50mg of ABTS in lOOml of citrate phosphate buffer 0.05M, pH 4.0. Immediately before use 35ul of 20-voIume hydrogen peroxide was added to lOOml of substrate solution.

Vaccin absorption technique for the sera

Before determining the use of M.vaccae for the absorption of the sera antibody profiles of some sera were determined. In addition to Vaccin, in sonicate antigens of M .tuberculosis,M .avi u m ,M .duv a l i , M .kansassi and M .scrofulaceum were tried.

For some series of tests, sera were absorbed with Vaccin antigen, in order to remove some of the antibodies against common mycobacterial antigens. For this,

different concentrations of Vaccin ranging from 0.001-100 ugm were made in phosphate buffered saline with Tween 20

(PBSTV). Dilutions of the sera were made in the PBSTV and kept at 37°C for I hour.The antigen concentration providing optimal results in ELISA was chosen(for

details see results) and the rest of the tests were done using a single absorption antigen concentration.

Absorption of the sera for the" andaelisa" test was done in the same way using the serum dilution buffer provided in the kit.

ELISA tests

The dilution of sera used in this study was 1:500 made in PBST, PBSTV,and in the "andeliza" buffer with and without absorbing antigen . The M. tuberculosis sonicate used for coating plates was diluted to 5 ugm/ml in 0.05M sodium carbonate buffer pH 9.6. lOOul volumes of the antigen were added to multiwell microtitre plates. For each serum two wells with antigen and two wells

without antigen were used. The plates were incubated in a humid chamber at 4°C overnight after which they were

washed 3 times in PBST. At each wash the buffer was left