Med Microbiol Immunol (1989) 178:323-335

�9 Springer-Verlag 1989

Development of an enzyme immunoassay for the serodiagnostic of tuberculosis and mycobacterioses

R. Maes 1, J.-P. Homasson 2, M. Kubin 3, and M. Bayer 4

i ANDA Biologicals, 37 rue de la Course, F-67000 Strasbourg, France 2 Centre Hospitalier sp~cialis~ en Pneumologie, 24 rue Albert Thuret, F-94669 Chevilly-Larue, Cedex, France 3 Institut Hygieny a Epidemiologie, CS-10042 Praha 10, Srobarova 48, Czechoslovakia 4 Zentral Krankenhaus Gauting, Zentrallabor, Unterbrunnerstr. 85, D-8035 Gauting, Federal Republic of Germany

Abstract. An enzyme-linked immunosorbent assay (ELISA) test has been developed for the serodiagnosis of mycobacterioses. The test is based on an interspecific antigen, antigen 60 (A60). This antigen belongs to the group of thermostable macromolecular antigens and is the principal constituent of tuberculin RT 23. A60 is composed of proteins, polysaccharides and lipids in a ratio that varies with the growth cycle of the mycobacterium. The test detects antibodies against mycobacteria and is most effective in cases of active infections. We found that the best way to cope with the day-to-day variations in the results obtained was to transform the absorbance values into arbitrary IgG units. The variations observed with IgM antisera were, however, too great to allow more than a qualitative test. Healthy individuals are negative in the IgG and IgM tests and tuberculous patients are usually positive in the IgG test. The presence of IgM antibodies was only rarely observed. For IgG antibodies, the best discriminative power at a 1:100 dilution of the analyzed sera seemed to lie at 125 sero-units. It was found that some sera originating from clinically documented tuberculous cases responded in a poor way in this test.

Introduction

Many attempts have been made to develop a serodiagnostic test for mycobacteria. These efforts, which were at times very thorough [9, 12], relied, by necessity, on mycobacterial extracts of various types such as purified protein derivative (PPD), crude sonicates and saline extracts. Yet, the composit ion of mycobacterial extracts varies widely [6, 10], hence repeated efforts to define the best antigens for such a purpose [13, 18]. The use of whole bacteria as capture antigen [1, 11, 18] resulted in

Offprint requests to: R. Maes

324 R. Maes et al.

difficulties in sensitizing the solid phase and produced an intolerably high background. Purified antigens, such as glycolipids [16], antigen 6 [17] and antigen 5 [3] were applied, of which antigen 5 proved superior to PPD [2]. The difficulty of isolation of this antigen [8], its internal location in the cell and its scarcity are drawbacks to its wide use.

A renewed attempt to develop a serodiagnostic test was initiated when antigen 60 (A60) became available [7]. This antigen is thermostable, easily available in substantial amounts and is a major component of the mycobacterial cytoplasm. It is homologous to antigen 7 from M. leprae and is also the principal component of PPD-RT 23 [10]. This report describes the technical development and the main characteristics of the test. Attention was given to sources of variation [4], hence the development of ready-to-use reagents and the introduction of reference sera.

The diagnostic validity of the developed enzyme-linked immunosorbent assay (ELISA) test will be published elsewhere.

Material and methods

Microtiter plates were purchased from Greiner. They are the Dynatech Immulon type, consisting of bars bearing 16 wells each.

Mycobacteria of the species paratuberculosis, avium, smegmatis and scrofula- ceum were obtained from the National Veterinary Institute, Oslo, Norway through Dr. F. Saxegaard. The A60 were extracted according to Cocito and Vanlinden [7] and were a gift of Professor Cocito.

The antigens were coated on microtiter wells in carbonate buffer pH 9.6 [5] at a concentration of 10 gg/ml, with 0.1 ml/well for 24 h at room temperature (RT). After thorough washing with distilled water, the plates were stored dry at RT. Horseradish Peroxidase (RZ = 3) was purchased from Boehringer (Mannheim, FRG) and Tebu (France). The anti-human IgM coupled to Peroxidase was purchased from Dako (Denmark). Rabbit antibodies to M. tuberculosis were purchased from Dako.

Igy anti-human IgG was obtained from the eggs of vaccinated hens. After purification [ 15], the antibodies were isolated through affinity chromatography on IgG-Sepharose and lyophilized. Peroxidase was conjugated to Igy using the periodate method [14].

The incubation media for serum and enzyme-antiglobulin conjugates consi- sted of 0.9 % NaC1 in Tris-HC1 buffer, pH 7.2 containing 0.2% Tween 20, 0.05 % merthiolate and 5 % serum.

Human sera positive for tuberculosis were obtained from Professor Feldmann (Gauting, FRG), Dr. Homasson (Chevilly-Larue, France) and Professor Cocito (U.C.L., Brussels, Belgium). Human sera positive for M. avium were obtained from Dr. Wolinsky (General Hospital, Cleveland) and human sera positive for M. xenopi and M. kansasii originated from the Hygiene Institute Praha (Czechoslovakia). Sera from healthy individuals, from blood donors and from hospital attendants were either analyzed separately or were obtained from Dr. Zorn, Centre de Transfusion Sanguine de Strasbourg (C.T.S.S.,), France; Dr. Daftary, Bharhat Serums & Vaccines, Bombay, India; Professor Feldmann, Gauting, FRG;

Development of an enzyme immunoassay 325

Professor Cocito, U.C.L., Brussels, Belgium; Dr. M. Kubin, Institut Hygieny a Epidemiologie, Praha, Czechoslovakia and Dr. Khaled, Institut Pasteur d'Alg6rie, Alger, Algeria. H u m a n sera positive for IgM anti-A60 were received f rom Professors Cocito and Feldmann.

ELISA tests were performed by incubation of 0.1 ml of the sera at a 1 : 100 to 1:1000 dilution for 1 h at RT. The wells were washed with 0.9% NaC1 then incubated for 1 h at RT with 0.1 ml of peroxidase-anti-human IgG or peroxidase- anti-human IgM. After washing, the wells were incubated with 0.1 ml of OPD (Ortho-Pheylene Diamine)(4mg/10ml) in citric-acid-phosphate buffer, pH 5.3, for 15 min. The reaction was blocked by addition of 100 I.tl 2 M H2SO4 and the intensity of the coloration analyzed in a microtiter reader at 492 nm.

Results

Our work bears on two distinct problems. The first concerns the setting up of the test and the analysis of the principal parameters whose optimization is crucial for its good functioning and the second is the unravelling of the features that characterize it. These will determine its usefulness as a diagnostic tool.

Development of the ELISA test

Stability of A60-coated plates

Preliminary experiments demonstrated that 5 ~tg of A60 prote in/ml coating solution was necessary to adequately coat a microtiter well. The stability of the adsorbed material was analyzed by placing dry coated plates or coated lyophilized plates at --20~ 4~ RT, 37~ and 50~ for 28 days. After this time, the wells were incubated in the presence of a serum originating f rom a patient who was found BK (Bacillus Koch) positive in bacilloscopy and with progressive pulmonar lesions. The presence of antibodies was revealed by incubation in the presence of peroxidase-coupled anti-gammaglobulin. Results are given in Table 1.

Table 1. Heat stability of antigen 60 (A60) antigen

50~ 37~ RT 4~

Control dry 0.91 0.71 0.75 0.73 Lyophilised 0.80 0.76 0.96 0.88

A60 antigen from Bacille Calmette-Gu6rin (BCG) was coated on the wells of microtiter plates. The wells were thereafter either lyophilized or dried, and stored at various temperatures for 28 days, before being analyzed in an enzyme-linked immunosorbent assay (ELISA) for IgG antibodies. The antigen remained stable for 28 days at 50~

326 R. Maes et al.

These results demonstrate that the A60-coated plates are stable after storage for 1 month at 50~ and that the stability is not increased by lyophilization. Having established the optimal coating and the stability of the coated wells, we proceeded to determine a reference serum pool from positive sera.

Serum reference pool

Several human sera originating from tuberculous patients were analyzed in serial twofold dilutions and their contents in IgG antibodies assessed in an ELISA. The results are given in Fig. 1.

Three of the analyzed sera (no 1-3) diverge from the norm. They are all low in A60-specific antibodies and the slopes of the response curves are different. Two behave identically but one is very poorly reactive. The four other sera tested are richer in A60-specific antibodies and the slopes of the curves are parallel to each other.

We thus observe that some positive subjects escape detection to a larger extent than others in serological detection by the capture antigen, so that specific antibody will be undervalued. The four sera that were rich in antibodies were pooled and thereafter served to establish reference serounits. First, howe- ver, the need to establish the adequate working dilution of peroxidase-anti-IgG arose.

100

200

(2) I--

.~ 800 c~

ZZ,

1600

3200

640C

12800 OS 10 ABSORBANCE (A~gz.nm)

15

Fig. 1. Human IgG tuberculosis enzyme-linked immunosorbent assay (ELISA). Seven tuberculosis sera were analyzed in serial dilutions extending from 1 : 100 to 1 : 12,800 with one single dilution of peroxidase-anti-IgG. Sera number 1, 2 and 3 are abherant

Development of an enzyme immunoassay 327

Ca l ib r a t i on of p e r o x i d a s e - a n t i - I g G

The a m o u n t of pe rox idase coup led to each an t i -g lobul in molecule and the a m o u n t o f specific an t ibod ies presen t in a p r e p a r a t i o n specify the react iv i ty o f a con juga te [14]. F igure 2 shows the results o b t a i n e d with the poo l o f posi t ive sera ana lyzed at successive twofo ld d i lu t ions , in the presence of va r ious con- cen t ra t ions of a conjugate . At a high concen t r a t i on of 1 : 500, the reac t iv i ty o f the conjugate is great . However , the b a c k g r o u n d is also high. At lower con- cen t ra t ions , its reac t iv i ty is weaker and the b a c k g r o u n d is also reduced. In Fig. 2 a 200-fold d i lu t ion o f the reference serum gave an a b s o r b a n c e o f 1.5 and a 1 : 1, 600 d i lu t ion y ie lded an abso rbance of 0.5 when the p e r o x i d a s e - a n t i - I g G was d i lu ted 1 : 1000.

The t angen t a of the curve ob t a ined with tha t d i lu t ion o f con juga te was 3 [ 14]. This d i lu t ion of p e r o x i d a s e - a n t i - I g G was a d o p t e d to define the posi t ive reference serounits .

I g G reference units

To take into account the da i ly va r ia t ions tha t occur dur ing the p e r f o r m a n c e of an enzyme i m m u n o a s s a y test and also to c o m p a r e results ob t a ined on var ious days , a value o f 2 serouni ts was a t t r i bu t ed to tha t concen t r a t i on of the posi t ive p o o l e d serum tha t y ie lded an a b s o r b a n c e of 0.5.

One se rouni t co r r e sponds to one ha l f o f 2 uni ts and 16 units c o r r e s p o n d to an e ight fo ld concen t ra t ion o f 2 units. Sixteen units will thus ideal ly yield an abso rbance of 1.5 in the op t ima l cond i t ions of the test.

t:20r (161

g ' 1 : / . 0 (

i lo)

t~ 1:80r qt~)

1:160 121

2

, , , i . . . - - i , , . , i o . . . .

/

/ . . . . . i , , . ,

05 1.0 15 2.0 ABSORBANEE ( A~.921

1:100

1:200

l:t, O0

1:800

1:1600

1:3200

1:6~00

3

i , , , I , , , , i , , , i , , , ,

o

i t i i I i i i , , , , , ,

0.5 1.0 1.5 ABSORBANCE (A~s2)

2 . 0

Fig. 2. Calibration of peroxidase-anti-IgG. A pool of tuberculosis-positive sera was analyzed in serial twofold dilutions with successive twofold dilutions of peroxidase-anti-IgG ranging from 1:500 to 1:4,000. The slope of the four curves is characterized by its tangent. (0) 1:500, (O) 1 : 1000, (11) 1:2,000 amd (U3) 1:4,000 dilution

Fig. 3. Human IgM tuberculosis ELISA. Three tuberculosis sera were analyzed by serial dilutions extending from 1 : 100 to 1 : 6.400 with one single dilution of peroxidase-anti-IgM. The tangents of the three curves vary widely from each other

328 R. Maes et al.

The absorbance obtained with negative serums varies between 0.05 and 0.2, while that yielded with 1 unit reference serum varies between 0.15 and 0.35. Assuming ideal conditions, with a negative sample of A492 0.2, the absorbance obtained with an unknown sample will be

A492 unknown = 1.08 X log unit + 0.2

and units = 10 A492 unknown - 0.2

1.08

These formulae are essentially valid for values lying between 2 and 16 serounits.

IgM antibodies

The frequency of patients with circulating IgM antibodies against A60 was very low; here n ----- 3. Two were high in IgM antibodies alone, whereas the other was high in IgM and IgG antibodies.

Figure 3 shows that the slopes obtained with one single dilution of peroxidase- anti-IgM (1 : 1600) with these three antisera differ widely, from tangent c~ = 4.87 to tangent ct = 2.1. The anti-IgM antibody used was very specific for IgM, since 16 IgG serounits of tuberculosis yielded an A492 of only 0.32.

Characteristics of the test

A60 vs a sonicate extract

Plates coated with A60 were used in parallel with plates coated with a saline sonicate extract (SSE). Sera from confirmed tuberculosis cases as well as from healthy subjects and blood donors were analyzed. The results are shown in Table 2.

This experiment demonstrates initially that a sonicate extract produces a high frequency of false positives among the blood donors control population. With A60, two blood donors were found positive (A492 0.52 and 0.37), which were also positive with the SSE antigen. None of the healthy subjects was positive. The anti- IgM test was uniformly negative among these control subjects.

Among the tuberculosis patients, two sera responded poorly to A60-IgG (A492 0.24 ans 0.26). With A60-IgM, the level of antibodies detected was sometimes high, but with a very low frequency.

This preliminary experiment establishes the superiority of a purified antigen over a sonicate extract; we will now proceed to define more accurately the limits of sensitivity of the test and the possible significance of the positive cases found among the control subjects.

Development of an enzyme immunoassay

Table 2. Comparison of two different capture antigens

329

Tuberculosis Controls

A60 Antigen SSC A60

Anti-IgG Anti-IgM Anti-IgG Anti-IgG Anti-IgM

Antigen SSC

Anti-IgG

1.02 1.44 1.36 0.52 0.07 0.86 0.98 0.05 0.78 0.00 0.01 0.04 0.41 0.09 0.52 0.17 0.05 0.40 0.36 0.13 0.50 0.09 0.04 0.95 0.55 0.06 1.07 0.03 0.07 0.18 0.43 0.04 1.36 0.04 0.07 0.36 0.78 0.05 1.09 0.01 0.05 0.16 0.46 0.21 1.15 0.05 0.11 0.86 0.90 0.30 2 0.09 0.05 0.23 1.11 0.05 1.20 0.05 - 0.68 0.35 0.09 0.75 0.15 - 1.28 0.54 0.09 1.02 0.26 - 1.18 0.24 0.05 0.42 0.37 - 1.42 0.39 0.05 1.27 0.05 - 0.33 0.62 0.20 2 0.10 - 0.42 0.26 0.08 0.78 0.14 0.07 0.94 0.52 0.18 1.35 0.13 0.06 1.16

A60 and sonicate extract SSC were used with tuberculosis-positive and -negative human sera. The presence of antibodies of the IgG and IgM type were analyzed. The number of false positive in the control group is much reduced when A60 is used SSC = saline sonicate extract

Table 3. Limits of sensitivity of IgG test

Units Negative sera Positive sera 130 cases 63 cases

Number Percentage Number Percentage of cases of total of cases of total

Neg: 100 107 82.3 6 9.3 Latent: 100-124 14 10.8 6 9.3 Weakly positive: 125-199 7 5.3 17 27 Positive: 200-299 1 0.7 12 19 Evolutive: 300 1 0.7 22 35

Neg.+latent: 93% Neg.+latent: 18.6% Positive: 6.8 % Positive: 81%

One hundred and thirty hospital attendants sera and 63 positive sera were analyzed at a 1 : 100 dilution in an IgG ELISA and the absorbances translated into units. The greatest discriminating power in the test was obtained by setting the limit of sensitivity of the test at 125 units 1

i More recent studies made us set the limit of negativity at 160 units

330 R. Maes et al.

L imi t s o f sens i t iv i ty

IgG antibodies. N o n - v a c c i n a t e d h e a l t h y in fan t subjec ts a n d h e a l t h y v a c c i n a t e d adu l t sujects ( c u t i - r e a c t i o n pos i t ive ) were ana lyzed . N o pos i t ive s e ro log i ca l r e s p o n s e was f o u n d a m o n g the 94 cases a n a l y z e d (Dr . H o m a s s o n , P r o f e s s o r C o c i t o

a n d Dr . K h a l e d , p e r s o n a l c o m m u n i c a t i o n s ) . Th is i m p o r t a n t o b s e r v a t i o n , to be p u b l i s h e d e l sewhere , c o r r o b o r a t e s the o b s e r v a t i o n s desc r ibed in Tab le 2 a n d m u s t

be pu t in a m o r e gene ra l c o n t e x t in the use o f the test. W e a n a l y z e d 130 hosp i t a l pa t i en t s o r i g i n a t i n g f r o m the C.T .S .S . , S t r a s b o u r g ,

e x c l u d i n g those in the P n e u m o l o g y d e p a r t m e n t a n d 63 c o n f i r m e d tube rcu los i s p o s i t i v e cases. A d i l u t i on o f 1:100 o f the sera was app l i ed a n d the A492 va lues were t r a n s f o r m e d in to se roun i t s . T a b l e 3 s u m m a r i z e s the results . C o n t r a r y to the n e g a t i v e resul ts o b t a i n e d wi th hea l t hy subjec ts , a ce r t a in n u m b e r o f the sera f r o m

h o s p i t a l a t t e n d a n t s was n o w f o u n d pos i t ive . A l so , the sera f r o m a smal l n u m b e r o f t u b e r c u l o s i s p a t i e n t s s h o w e d nega t ive . Th i s o b s e r v a t i o n c o n f i r m s the resul ts o f T a b l e 2. T h e best d i s c r i m i n a t i v e p o w e r o f the test was f o u n d to lie at 125 se roun i t s ,

Table 4. Incidence of tuberculosis endemy on IgG ELISA

1 unit 2 units 16 units

0.23 0.37 1.29 0.27 0.44 1.35

Serum no. Serum dilution applied in the test

1/100 1/200 1/400 1/800

36 0.74 0.46 0.28 0.17 99 0.48 0.27 0.17 0.12

136 0.21 0.16 0.11 0.09 181 0.23 0.16 0.10 (0.12) 210 1.14 0.78 0.52 0.29 219 0.28 0.17 0.15 0.11 221 0.23 0.15 0.12 0.11 257 0.23 0.17 0.13 0.10 270 0.36 0.18 0.11 0.09 277 0.52 0.24 0.15 0.10 278 0.44 0.20 0.13 0.09 283 0.53 0.35 0.23 0.15 313 0.22 0.13 0.08 0.08 352 0.27 0.24 0.20 0.13 353 0.18 0.11 0.09 0.07 370 0.31 0.22 0.14 0.11 376 0.40 0.34 0.15 0.08 379 0.43 0.27 0.17 0.t 1

Blood donor sera originating from Bombay were analyzed at dilution ! : I00 to 1 : 800 in an IgG ELISA test. About half of these sera were positive at a 1 : 100 dilution. Effective erasing of these "false positives" occurs only at dilution of 1:400, where two sera are still strongly positive

Development of an enzyme immunoassay 331

which separated best the positive and the negative cases with the least misclassification (today, it has been set at 160 serounits). This value being established it remained to be seen if it held true in an environment where the prevalence of the disease was high.

When the test was applied to 18 sera of blood donors originating from Bombay (India), it appeared that 44% were less than 125 serounits and 44% were greater than 200 units. Only 2 sera out of the 18 analyzed were between 125 and 200 units. It was clear (Table 4) that the dilutions necessary to avoid positive results among a seemingly healthy population should be four- to five-fold greater than the dilution used in Europe.

IgM antibodies. For IgM, we chose from the hospital attendants, a few sera, including four cases that were positive for IgG (marked in Table 5 by an asterisk), and compared their response in an IgG and IgM test.

It is seen that most of them were negative for IgM antibodies, and that those sera which were positive for IgG antibodies were not necessarily positive for IgM.

Most blood donor sera originating from Bombay contained antibodies of the IgM class when analyzed at a 1 : 100 dilution (Table 6). Even at a dilution 1:400, one case is very positive.

Table 5. Comparison between IgG and IgM antibodies in a control group

A492 IgG A492 IgM

1 0.13 0.13 2 0.15 0.08 3 0.33 0.13 4 0.27 0.29 5 0.32 0.08 6 0.50 (+) 0.08 7 0.85 (+) 0.26 8 0.36 (+) 0.32 (+) 9 0.57 (+) 0.17

10 0.17 0.28

A control group composed of sera positive and negative for IgG antibodies were analyzed for IgM; most were negative. There is no correlation between IgG and IgM positivity

Having established the possibility of positive and negative misclassification and the existence of different levels of antibodies (mean adult norm) in regions of different tuberculosis prevalence, the extent of specificity of the capture antigen still had to be investigated.

Analysis of interference

M. kansasii sera. Two sera positive for M kansasii were analyzed with A60 originating from M bovis, strain BCG, M. paratuberculosis and Mavium. The results

332

Table 6. Incidence of tuberculosis endemy on IgM ELISA

R. Maes et al.

1/100 1/200 1/400

0.46 0.26 0.13 0.35 0.21 0.12 0.42 0.23 0.12 0.58 0.35 0.18 0.75 0.50 0.29 0.40 0.23 0.12 0.32 0.16 0.11 0.26 0.15 0.10 0.16 0.09 0.07 0.31 0.17 0.09 0.37 0.22 0.12 0.26 0.15 0.11 0.25 0.15 0.08 0.31 0.19 0.13 0.48 0.27 0.16 0.24 0.14 0.14 0.18 0.11 0.08 1.73 1.57 1.32 +

Blood donor sera originating from Bombay were analyzed at dilution 1:100 to 1:400 for IgM antibodies. Most of the sera are positive at a 1 : 100 dilution. Sera must be diluted to 1:400 to erase the "false positive". Even at this high dilution, two cases remain positive (A492 1.32 and 0.29)

a lOO

F- 20O _i

40(

80C

1600

I A60-Avium i A60- ~ 0 .~

/ . ~ A60-PARA

~.0 S

I I I f I I

0.5 1.0 1.5

b z 100

20C

~" 40(

80C

160(

i i , i i i i i d B [ ]

0.2 0.4 0.6 0.8 ABSORBANCF (Atmnm)

Fig. 4a, b. IgG analysis of M. kansasii sera with three different A60. Two human sera positive for M. kansasii were analyzed in serial serum dilutions in an lgG ELISA with A60 originating from M. avium, M. BCG and M. paratuberculosis. The interference due to A60-avium is less pronounced than that due to paratuberculosis. (�9 A60 from M. paratuberculosis, (0) A60 from BCG and (IS]) A60 extracted from M. avium

s h o w n in Fig. 4 d e m o n s t r a t e t h a t the t w o se r a r e a c t less w i t h A 6 0 a v i u m t h a n w i t h

t h e t w o o t h e r a n t i g e n s . C o m p a r i n g A 6 0 p a r a t u b e r c u l o s i s a n d b o v i s , it is c l ea r t h a t

t h e i n t e r f e r e n c e is n o t 1 0 0 % w i t h t h e s e se ra b u t s l igh t ly d i s f a v o r B C G vs

p a r a t u b e r c u l o s i s . T h e s e s u b t l e d i f f e r e n c e s in r e a c t i v i t y o f i n d i v i d u a l se ra we re m o r e t h o r o u g h l y a n a l y z e d w i t h M. av ium sera .

M. a v i u m sera. Five s e r a o r i g i n a t i n g f r o m p a t i e n t s i n f e c t e d w i t h M. av ium b u t n o t

s e r o p o s i t i v e f o r h u m a n i m m u n o d e f i c i e n c y v i r u s ( H I V ) were a n a l y z e d a t se r ia l

Development of an enzyme immunoassay 333

100

200

c )

_ J 123

g00

tad t / I

800

op p .up .o o . . e , ' l l , , "" , ," l /

" / ," / i"

I / ' / / / ' / ' / ABSORBANCE (A~.,z,~ ,} 1, ' /

,' ,:7 / / , , , " . . . .

, .i ':1 / , ' / I " r # ~'u o / o , ; / ~ 1 ,///,':'/~

- - ~ ~,~,,'1 �9 / z 6: j~,L/ I /'S.l" ,, I c~ ' ~'7 / , , ,,, / ~ ,. i , / , I 1 2 200 l

J/// 2 3 ,'1, h' i ,,;,/,:// ePl I ;

H. AVIUH ANTISERA ,oolJ~ 6

...... I I I I

O.S 1.0 1.5 2.0 ABSORBANCE (A49zn m)

Fig. 5. IgG analysis of M. avium sera with three different A60. Five sera positive for M. avium were analyzed in serial twofold dilutions on an ELISA with A60 originating from M. avium (0), M. paratuberculosis ([]) and M. BCG (O). In all cases, A60 BCG is the most effective as capture antigen

twofold dilutions with A60 originating from M. avium, M. paratuberculosis and M. BCG. The results are summarized in the Fig. 5.

It is seen that the sera ofM. avium patients are most reactive, in all five cases, with A60-BCG. The amount of bound antibody to A60 avium and paratuber- culosis varies for each antiserum analyzed. Some antisera showed almost 100% interference between A60 avium and paratuberculosis (no. 4), or else paratuber- culosis and BCG (no. 1), avium and BCG (no. 6) or else among the three antigens (no. 2).

Mycobacterial sera. All in all, A60 from M. smegmatis, M. bovis BCG, M. avium, M. scrofulaceum, M. paratuberculosis and M. leprae were assayed and shown to react with sera positive for tuberculosis, avium, xenopi, kansasii and leprae. These results will be published in more detail elsewhere.

Discussion

No healthy subject, vaccinated or not, has been found positive with this test. We attribute this specificity to the use of a purified antigen. This result is obtained despite a positive cellular immune response (intra-dermal reaction) and demonstrates that serology and cellular immunity are distinct. Upon revaccination, however, the circulating antibody level rises spectacularly (investigations in progress).

334 R. Maes et al.

Among less-stringently selected control groups, be these supposedly healthy blood donors or hospital patients, positive cases are repeatedly found. The frequency of these cases and the level of circulating antibodies increase in regions of important endemy. This observation is by no means new [1, 2, 9, 12] and we attribute these positive cases to clinically inapparent booster infections. Since the capture antigen used here is interspecific, any mycobacterial infection may be at the origin of the observed raised antibody level.

As a result, the necessity to adapt the test according to local conditions is obvious. However, it seems to us significant that the analysis of blood donors sera in Bombay yielded 44% with levels below the 125 serounits mark, while another 44% were above the 200 units mark. This observation runs counter to the prevalent opinion that the mean adult norm is higher in regions of high endemy. In fact, healthy individuals in Bombay have levels of antibodies as low as in developed countries. What increases is the number of people suffering from inapparent infections.

We also speculate that pregnant women, due to the lowered capacity of their immune defense, will show a higher frequency of weakly positive cases than the expected norm. People who have leishmaniose may also show positive (Professor Petithory, H6pital de Gonesse, France and Dr. Khaled, Institut Pasteur, Algeria; personal communications).

Using positive sera, A60 has been shown in this study to be interspecific. The interference has consistently been high, sometimes, reaching 100% with tuberculosis sera reacting equally well with antigen 60-BCG and A60 paratuberculosis.

In some cases, however, the antisera obtained against one mycobacterium type show a marked preference for a particular antigen, not necessarily the homologous one, as observed when M. avium sera were tested, which reacted preferentially with A60-BCG. One should note that a difference in coating, or a difference in the quality or quantity of the antigen applied are unlikely, since the experiments were all performed together and the differences observed with A60-paratuberculosis and A60-avium vary in an unforseeable way.

In addition, different individual sera demonstrate a very different reactivity with the same capture antigen. Sometimes, the reactivity is very poor. This was obvious with three tuberculosis sera that were discarded from inclusion into a tuberculosis reference pool and this phenomenon was more evident when IgM- tuberculosis sera were analyzed.

The choice of A60 in such a serodiagnostic test has been of benefit in the reduction of these "false negatives" to an acceptable level because the antigen is a potent and interspecific immunogen, allowing the easy recognition of any active mycobacterial infection, including leprosy.

Since vaccinated adult subjects are devoid of specific antibodies, one must assume that the level of these antibodies recedes in the course of time, but this aspect of the immune response has not been yet investigated.

Development of an enzyme immunoassay 335

Acknowledgements. The reader will have noticed that this work was made possible through a collegial and collaborative approach. Besides my co-authors, my most sincere thanks for material for discussions and interest go to: Dr. Grange, Professor Feldmann, Dr. Subramaniam, Dr. Raheman, Professor Prignot, Dr. Vanderelst, Dr. Zorn, Professor Portaels, Professor Petithory, Dr. Saxegaard and Professor Cocito. I should not forget the secretary who kept track of everybody.

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Received July 5, 1989