Immunological detection of mycobacterial antigens in infected fluids, cells and tissues by latex agglutination

Journal of Immunological Methods, 129 (1990) 9-14

9

Elsevier JIM05534

Immunological detection of mycobacterial antigens in infected fluids, cells and tissues by latex agglutination Animal model and clinical application C.L. C a m b i a s o 1, j.p. Van V o o r e n 2, a n d C.M. F a r b e r 3 i Unit of Experimental Medicine, Unioersitd Catholique de Louoain, Bruxelles, Belgium, and 2 Chest Department, and J Department of Immunology, H@ital Erasme, Unioersit~Libre de Bruxelles, Bruxelles, Belgium

(Received 3 April 1989, revisedreceived2 October 1989, accepted 18 December1989)

We devised an immunoassay for the detection of mycobacterial antigens in cell lysates and in tissue extracts which is based on the agglutination of latex particles coated with anti-Mycobacterium boois F(ab')2, followed by counting of non-agglutinated particles. Mycobacterium boris cell lysates were tested and a reference curve was established, having a lower limit of detection of 15-20 Mycobacteria. W e were able to detect mycobacterial antigens in cell lysates from bronchoalveolar washings and in spleen and liver lysates obtained from experimentally infected rabbits. Antigens were also detected in ten out of 11 samples obtained from patients with proven tuberculous infection. These samples were readily distinguished from 32 negative control samples after pepsin treatment. In contrast, periodate treatment of samples to destroy carbohydrate, abolished all reactivity. Following gel filtration chromatography we identified three peaks with antigenic properties in samples of all types. The detection of mycobacterial carbohydrate antigens by latex agglutination and particle counting should be a useful adjunct in the diagnosis of tuberculosis. Key words: ~'uberculosis;Latex agglutination;Antigen detection

• Introduction

Tuberculous disease still occurs at a high incidence in developing countries (Lowell, 1984), and there has been an increase m the number of cases of tuberculosis as well as other mycobacterial diseases in Western countries partly due to the advent of AIDS (Chaisson et al., 1987). The diagnosis of tuberculosis is still based on bacteriological

Correspondence to: C.L. Cambiaso, Unit of Experimental Medicine, UCL, Av. Hippocrate, 74, 1200-Bruxelles, Belgium. Abbreviations: BCG, baciUe de Calmette et Gu~rin; BAL, bronchoalveolarlavage; CFU, colony formingunit.

techniques. The direct identification of the mycobacteria (by the Auramine or Ziehl Nilsen techniques) is seldom possible except in severe disease and culture requires very skilled personnel and special culture media. Moreover, results are usually obtained after 6 weeks of culture (Glassroth et al., 1980). Previous attempts to isolate mycobacterial antigens from infected samples have utilized the liquid, supernatant phase (Ruiz Palacios and Sada, 1986). Since mycobacteria are usually observed within cells it seemed logical to attempt the detection of mycobacterial antigens inside cells or inside tissues thought to be infected. Latex agglutination and particle counting was chosen because it has been shown previously to be

0022-1759/90/$03.50 © 1990 ElsevierScience Publishers B.V. (BiomedicalDivision)

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both sensitive and specific (Cambiaso et al., 1977a; Masson et al., 1981). The present assay has been evaluated in an experimental model of primary mycobacterial infection in rabbits using the strain Mycobacterium boris (BCG) which induces a selflimiting disease. The assay has also been applied to samples obtained from human patients presenting with respiratory complaints and in whom bronchoalveolar lavage or pleural drainage was necessary.

Materials and methods

Preparation of F(ab')., fragments of anti-BCG IgG F(ab')2 fragments of rabbit anti-BCG IgG (Dakopatts, Copenhagen, Denmark) were prepared by peptic digestion using 2 mg of pepsin (Sigma, St. Louis, MO) per 100 mg of protein dissolved in 0.1 M acetate buffer pH 4.5 at 370C. After a 20 h incubation, the reaction was stopped by increasing the pH to 8 using 1 M Tris solution. F(ab')2 fragments were purified by chromatography on Ultrogel AcA 4-4. The IgG fraction from rabbit antisera directed against group B Streptococcus, Streptococcus pneumoniae, Haemophilus influenzae type b and Staphylococcus aureus were isolated by treatment with acridine lactate (Rivanol) followed by precipitation with half-saturated ammonium sulfate.

Latex agglutination assay 25 #1 of samples were mixed with 25 #1 of F(ab')2-coated latex and 25 #1 of additive. The mixture was vortexed in a specially designed incubator-agitator at a fixed temperature of 37°C for 45 min. The reaction was stopped by the addition of 2 ml of GBS and unagglutinated particles were counted in an optical counter which discriminated the unagglutinated from the agglutinated latex particles (Masson et al., 1981). The concentration of antigen was inversely proportional to the number of unagglutinated particles. The anti-bacterial IgG antibodies, including anti-BCG were used in an inhibition assay to test the specificity of agglutination.

Mycobacteria Animals were infected with the BCG strain 1173 P 2 (Institut Pasteur du Brabant, IPB) used at a concentration of 80 mg/ml (1 mg containing 5 x 106 CFUs or 6 x 106 mycobacteria).

BCG cellular extract The standard mycobacterial extract was prepared from the same BCG strain that was injected into the rabbits. Mycobacteria were submitted to three cycles of freezing and thawing. After each cycle, the preparation was sonicated for 10 min and unsolubilized fragments eliminated by centrifugation at 12,000 × g for 20 min.

Pepsin and periodate treatment Latex F(ab')2 fragments from anti-BCG IgG were covalently coupled to carboxylated latex beads measuring 0.8 #m in diameter (Estapor K150, Rhrne Poulenc, Courbevoie, France) by the carbodiimide method (Galanti et al., 1987). The coated particles were stored at - 2 0 ° C in 100 #1 aliquots. Before use, the latex particles were sonicated for 10 s and diluted 200-fold with GBS-BSA (0.1 M glycine, 0.17 M NaCI, pH 9.2, containing 10 g of bovine serum albumin and 40 mg sodium azide per liter).

Additive The additive comprised a solution of 30 g dextran T500 (Pharmacia, Uppsala, Sweden) and 1,25 g of rabbit glutaraldehyde aggregated F(ab')2 per liter in GBS (Collet-Cassart et al., 1983).

To order to destroy the protein fraction of the antigenic preparations pepsin treatment was used as follows: 100 #1 of 0.9% NaCI and 100 #1 of a solution of 10 mg/ml of pepsin in 0.3 M HC1 were added to 50 #1 of sample. After a 10 rain incubation at 37 ° C, the reaction was stopped with 50 #I of a 1 M "Iris solution. Samples were treated in parallel with 0.075 M periodate in phosphatebuffered saline (10 mM phosphate buffer, 150 mM NaC1, pH 7.4 (PBS)) for 20 h at 4 ° C in order to oxidize carbohydrates. Since pepsin treatment involved a six-fold dilution of the samples, all samples, whether treated or not, were tested at this dilution.

Gel filtration chromatography Some cellular extracts were fractionated by gel filtration on a column of Ultrogel AcA 3-4 (2.5 ×

11 90 cm), equilibrated in 0.01 M phosphate buffer pH 7.25 containing 1 M NaCI. The presence of mycobacterial antigens was monitored by antiBCG latex agglutination of all fractions.

Rabbits Six New Zealand White rabbits free of common pathogens were injected intravenously with 10 mg (5 × 107 CFUs) BCG suspended in normal saline. This induced a self-limiting infection. Three control animals were injected with 0.9% NaCI alone. The three uninfected rabbits and three infected rabbits were killed after 7 days. The three other infected rabbits were killed 85 days after the injection. The animals were anaesthetised using Pyp-butyrophenon and piperidin (Hypnorm, Duphar) used out 1 rrd/kg and exsanguinated by intracardiac puncture. The lungs were removed from the thoracic cavity and perfused with cold 0.9% NaCI through the pulmonary arteries in order to remove blood cells. They were then lavaged repeatedly through the trachea with a total of 100 ml of cold 0.9% NaCI. The lavage yielded 85 + 5 ml. Cells were enumerated and their concentration adjusted to 106/1111. Cellular pellets were separated from the supernatant by centrifuging the samples for 15 rnin at 3800 x g. The cells were then resuspended in distilled water and submitted to three cycles of freezing-thawing and sonication. Samples of the liver, spleen and lung were obtained and were also maintained at 4 ° C in 0.9% NaC1 until used for antigen detection. 100 mg aliquots of the tissue samples were first dissociated with a loose-fitting homogenizer and were then treated as described above for the cell pellets.

patients: 20 BAL: from three patients with neoplasms, two patients with heart failure, three cases of sarcoidosis, one case of pulmonary fibrosis, eight cases of anthracosilicosis and three individuals with opportunistic infections (one aspergilhis, two pneumocystis carinii); 11 pleural fluids: from three patients with neoplasms, five patients with heart failure, two individuals with bacterial pleural effusions (one with Streptococcus pneumoniae, one with pseudomonas aeruginosa), one individual with pulmonary embolism; one ascitic fluid (from a patient with ethylic liver cirrhosis). Cellular pellets and supernatants were separated by centrifugation and the cellular pellets processed as described above.

Results

BCG cellular extract Serial dilutions of the BCG extract were tested with the anti-BCG coated latex and significant agglutination ( > 5%) was still present at a dilution of 1/750,000 of the initial sample, which corresponded to the antigenic material extracted from 15-20 Mycobacteria. The agglutination activity of the BCG cellular extract was reduced by 15% after pepsin treatment (Fig. 1) and this residual activity was totally abolished by periodate treatment.

70 6O z 5o _o

Patients BALs were performed with a bronchofibroscope in the diseased area (as identified by chest X-ray). A total of 150 ml of sterile NaC1 0.9% was injected and aspirated with a yield of about 50%. A 10 ml fraction was used for our studies. Pleural and ascitic fluids were obtained by sterile puncture. Samples were stored at 4 °C until use. Ten patients had tuberculosis as defined by positive culture of the following fluids: six BAL fluids; two synovial fluid samples obtained from one patient; three samples from pleural effusions. 32 samples were obtained from non-tuberculous

F-

4o ao

< N 20 lO 0

I

I

I

104 105 106 BCG EXTRACT DILUTION (l/X)

Fig. ]. And-BCG latex agglutination (~) obtained during

sequential dilutionsof BCG extract (mean of three determinations -61 standard deviation),e: before; and o: after pepsin treatment.

12 80

~/~

,05

,0,

/L

60 40 20

A

SAMPLES FROM NON-TUBERCULOUS PATIENTS: PERCENTAGE OF LATEX AGGLUTINATION (MEAN ± SD) (n = NUMBER OF SAMPLES TESTED)

0 Z

TABLE I

B

80

Q eo

C

Before pepsin treatment a

Supernatant 12.4+ 1190 (n = 16)

Cellularextract 18.2:t: 13~ (n - 8)

After pepsin treatment

< 590 (n = 32)

< 59o (n = 16)

20 0

D

80 6(1 40

" Samples producing an agglutination of < 590 of latex partides are included; a value of 590 has been arbitrarily attributed.

20 ! 0 30

40

50

60

70

80

TUBE NUMBER

Fig. 2. Gel filtration chromatography on Uhrogel AcA 3-4 of various preparations. The peaks represent mycobacterial antigens as detected by anti-BCG latex agglutination (expressed as percentage of agglutination). A: BCG extrr :t; B: BCG extract after pepsin digestion; C: infected rabbit bronchoalveolar cell extract after pepsin digestion; D human pleural cell extract after pepsin digestion. The appare.lt molecular weight scale is indicated by arrows. Gel filtration chromatography of the native extract produced three peaks of agglutination activity having molecular weights of 10 6, 3 × l0 s and 2 × 10 4 (Fig. 2A). Pepsin treatment of samples before chromatography did not abolish any of the peaks, but induced a slight decrease in their apparent molecular weights (Fig. 2B).

Animals Mycobacteria were detected by direct examination of BAL and tissue samples 7 days after mycobacterial inoculation (diseased rabbits). Histological examination revealed numerous granulomatous lesions. Mycobacteria and histological lesions always disappeared after 85 days (healed rabbits). BAL samples (supernatants and cellular pellets) from control and healed rabbits did not agglutinate latex beads coupled to anti-BCG F(ab')2 (less than 5% of latex particles were agglutinated) (Table I). When cellular pellets from the BAL of infected rabbits were tested under the same conditions, an agglutinating activity greater than 85% was observed and an attempt was made to titrate this activity. However, it was too strong to permit the determination of the 5% agglutina-

tion point on the linear portion of the titration curve and we chose to determine the sample titres that showed 50% agglutinating activity (Median titre 1 : 145, (n = 3) range 1 : 35 - 1 : 300). Supernatants from the BAL of the infected rabbits has a much weaker agglutinating activity (Median 8.6%, ( n = 3) range: 6-11%), and they were therefore tested at the standard 1 / 6 dilution. These levels of activity were retained after pepsin treatment but were abolished after periodate treatment. Tissue samples from control and healed rabbits all showed less than 5% agglutination after pepsin treatment whereas before pepsin treatment these samples had displayed weak (6-9%) agglutinating activity. Liver, lung and spleen samples from an infected rabbit showed very strong agglutination. Samples had to be diluted 15, 60 and 90 fold respectively to obtain 50% agglutination. These samples retained their activity after pepsin treatment whereas periodate treatment abolished it. Gel filtration chromatography of the extract from infected BAL cells showed an extra peak of activity in a fraction of apparent molecular weight lower than 10 4 Daltons (Fig. 2C).

Patients When control patients were evaluated a positive reaction was observed in most supernatants and cellular extracts but was abrogated after pepsin digestion (Table I). All further samples were treated with pepsin, including samples from tuberculous patients. In the latter group, 10 out of 11 supernatants showed agglutination with antiBCG latex as well as six out of seven cellular

13 T A B L E I1 SAMPLES F R O M T U B E R C U L O U S P A T I E N T S (ALL T R E A T E D W I T H PEPSIN); P E R C E N T A G E O F L A T E X AGGLUTINATION No. 4 and no. 5 are two synovial fluid samples from the same patient. Means including the negative sample, equated to 5% (see Table I). Sample

Supernatant

Cellular extract

no.

(%)

(%)

1 2 3 4 5 6 7 8 9 10 11

39 27 19 <5 37 53 43 42 41 50 66

43 <5 47 61 56 52 77 -

Mean ± SD

38.4 ± 15.9%

48.7 ± 20%

extracts tested. The one negative sample was one of the two synovial fluid samples obtained from the same patient (Table II). No inhibition at all was observed using IgG from antisera raised against group B Streptococcus, Streptococcus pneumoniae, Haemophilus influenzae type b and Staphylococcus aureus. However, agglutination was completely inhibited by anti-BCG IgG, demonstrating the specificity of the reaction. Gel filtration chromatography of a pleural fluid cellular extract produced two of the three peaks with agglutinating activity previously observed with BCG extract. An extra peak having a molecular weight less than 104 was also detected (Fig. 2D).

Discussion

Latex agglutination has been used previously to detect mycobacterial antigens (Krambovitis et al., 1984). These authors used latex coated with noncovalently bound IgG and agglutination was estimated visually. We have used an instrument to evaluate latex particle agglutination and this appears to have improved the precision and the sensitivity of the test (Cambiaso et al., 1977b). The use of F(ab')2 fragments of IgG prevents interac-

tions with rheumatoid factors or other molecules recognizing the Fc fragment. Covalent binding of F(ab') 2 fragment to latex particles ensures better stability and aliquots can be stored at - 2 0 0 C for months with no loss of activity (Fagnart et al., 1985). The antiserum used to prepare the anti-BCG F(ab')2 is well known and standardized (Closs et al., 1980); it is directed against BCG, an attenuated strain of Mycobacterium boris, which is antigenically very close to Mycobacterium hominis. Previously most authors have used polyclonal antibodies to mycobacteria in either ELISA procedures or radioimmunoassays. The results obtained with a monoclonal antibody to one antigen (Chandramukhi et al., 1985) or with a polyclonal antiserum directed against a single mycobacterial antigen (Raja et al., 1988) are still fragmentary. Most authors define the sensitivity of their assays in terms of the quantity of protein detected (Yanez et al., 1986; Kadival et al., 1987; Watt et al., 1988). The antigens we detect are clearly carbohydrates and we prefer to estimate the amount of Mycobacteria yielding the minimal amount of antigen we can still detect (about 10-15 Mycobacteria). Unlike other authors, we have looked for mycobacterial antigens in cell lysates as well as in supernatants, since mycobacteria are intracellular organisms in the host (Ruiz Palacios and Sada, 1986). Pepsin treatment of the samples was observed to abrogate anti-mycobacterial antibody activity and probably modified the three-dimensional structure of the antigens by exposing hidden determinants (Galanti et al., 1987). The low levels of reactivity in samples obtained from non-tuberculous patients were abolished by pepsin treatment and, unlike Dhand (1988), we observed no false positive results. It would be of interest to identify the major components reacting with anti-BCG and isolated by gel filtration chromatography. It is possible that the agglutination activity that disappears following pepsin treatment is due to the destruction of heat shock proteins, which are increasingly recognised to be ubiquitous in bacteria (Shinnick et al., 1988). We believe that the present technique could prove to be a helpful adjunct in the diagnosis of tuberculosis, especially in cases where direct examination and culture are difficult,

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e.g., in assessing dissemination into lymph nodes, liver and spleen or other organs. However, it is important to bear in mind that, a positive result corresponds to the presence of antigens, and does not necessarily prove that living mycobacteria are present.

Acknowledgements We thank M. Van Dam for her technical assistance in the preparation of this manuscript. The excellent technical assistance of Marta Perrone and Jean Luc Guarin was greatly appreciated.

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