Purification of modified mycobacterial A60 antigen by affinity chromatography and its use for rapid diagnostic tuberculosis infection

Journal of Microbiological Methods 87 (2011) 184–188

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Purification of modified mycobacterial A60 antigen by affinity chromatography and its use for rapid diagnostic tuberculosis infection Sh. Yari a, A. Hadizadeh Tasbiti a,⁎, A. Fateh a, A Karimi a, F. Yari b, F. Sakhai a, M. Ghazanfari a, A. Bahrmand a,c a b c

Mycobacteriology Dept, Pasteur Institute of Iran, Tehran -13164, Iran Research Center, Iranian Blood Transfusion Organization, Tehran, Iran Molecular Pathology Dept, Methodist Hospital, Houston, Texas 77030, USA

a r t i c l e

i n f o

Article history: Received 29 June 2011 Received in revised form 12 August 2011 Accepted 12 August 2011 Available online 27 August 2011 Keywords: A60 antigen Tuberculosis infection Absorbed antibodies

a b s t r a c t Tuberculosis has been declared a global emergency. The mainstay for its control is the rapid and accurate identification of infected individual. Antibodies to A60, one of the macromolecular antigen complexes of mycobacteria were commonly used in the rapid detection of Mycobacterium tuberculosis. The aim of this study was to prepare specific antibodies against A60 for detection of tuberculosis infection. Specific polyclonal antibodies against A60, (A60-Ab) were prepared in rabbits using 2 boosted injections of the antigen (A60). The antibodies were purified and treated with normal oral flora to remove any non-specific and crossreactive antibodies. These antibodies were conjugated to CNBr-activated Sepharose 4B and used to isolate subunits of A60 with more specificity for M. tuberculosis. A new affinity column was designed to prepare modified (purified) A60 antigen. Purified A60 antigen (PA60-Ag) was used to develop antibody production by Immunoaffinity chromatography. 113 patients with a confirmed diagnosis of pulmonary TB at Pasteur Institute were selected for the study. The specificity of the results was analyzed with TB-rapid test by using PA60-antibodies. TB-rapid test revealed that normal oral flora-absorbed antibodies could lead to more specific results than that of the non-absorbed antibodies. The developed, modified A60 antibodies, (PA60-Ab)rapid test showed higher sensitivity, specificity, Positive Predictive Value (PPV), Negative Predictive Value (NPV) and overall efficiency (93.0%, 86.0%, 90.0%, 91.0%, and 90.0% respectively) for the detection of the Mycobacterium antigen. Moreover, PA60-Ag showed only two protein bands of molecular weight 45 and 66 kDa in SDS-PAGE while untreated A60 showed multiple bands. Thus, our study helped in the purification of a novel and well characterized A60 antigen and good diagnostic potential for detecting tuberculosis infection. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Tuberculosis (TB) is among the leading causes of death worldwide. The World Health Organization (WHO) estimates that 32% of the world population is infected with Mycobacterium tuberculosis, the causative agent of TB (World Health Organization, 2007). Fast and accurate diagnosis of TB is a very important element in global health measures to control the disease (World Health Organization, 2010). Traditionally, diagnosis of TB rests on sputum examination and cultures for acid-fast bacilli (AFB) (Aber et al., 1980). In addition, the direct identification of the mycobacteria and culture requires very skilled personnel and special culture media, and it is also time consuming (Cambiaso et al., 1990). On the other hand, the sensitivity of the sputum smear for AFB is very poor and facilities for mycobacterial culture are often scarce in regions where tuberculosis is common, and even when they are available, culture results are frequently usually ⁎ Corresponding author at: TB Protein chemistry Lab, TB Dept, Pasteur Institute of Iran, Pasteur Ave, Tehran, Iran. Tel.: + 98 2166968853; fax: + 98 2166469871. E-mail addresses: [email protected], [email protected] (A. Hadizadeh Tasbiti). 0167-7012/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2011.08.007

too late to affect initial management (Parry, 1993). Therefore, a rapid diagnostic tool with both high sensitivity and specificity is needed to improve the conventional diagnostic methods. Several new techniques have been developed to improve the diagnosis of tuberculosis, including newer radiometric methods, DNA probes, chromatography of mycolic acid, PCR, and serologic tests. These diagnostic approaches have had a dramatic effect on the ability to diagnose disease accurately and expeditiously (Kallenius et al., 1994). Antigen 60 is one of the best known antigens used in the serologic tests. The clinical usefulness of detection of serum immunoglobulin A (IgA), IgG, and IgM antibodies raised against the mycobacterial A60 antigen for the diagnosis and discrimination of active tuberculosis (TB) from other pulmonary diseases have been analyzed and showed that the clinical use of the A60-based serodiagnostic IgG assay is of great value for the rapid diagnosis and discrimination between active TB and pulmonary non-TB diseases (Ben-selma et al., 2010). It has been claimed that the modified Anda-TB enzyme immunoassay test offers a good and reliable test for diagnosis of tuberculosis in suspected cases of active pulmonary tuberculosis (Al-Hajjaj et al., 1999]). Newer antigens such as 38 kDa (with identity with

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antigen 5) (Harboe and Wiker, 1992) and Kp90 (Kreatech Diagnostics, Madrid, Spain) have recently been introduced as commercially available kits, claiming even higher specificity (Bothamley and Rudd, 1994; Kadival et al., 1994). In the present study, we introduced 45 and 66 kDa antigens and evaluated Tuberculosis (TB) Rapid Test (developed by PA60-Ab) to determine its performance with sputum and compared to that of the conventional methods of smear microscopy and culturing on solid media. 2. Material and methods 2.1. Rabbits Animals were supplied by (ALF) Animal Lab Facility, Pasteur Institute of Iran, Karaj, Iran. Groups of 3 to 5 male rabbits (weighing 1200– 1500 g at the beginning of the experiments) were immunized with 250 μg of A60 purified antigen (Anda-Biologicals, Strasbourg, France) emulsified in Freund's incomplete adjuvant (FIA). The purified antigen was injected subcutaneously (SC) into two sites on the flank. To aid in the stimulation of the immune response, booster immunization with (FIA) was used. The booster injection was carried out by subcutaneous (SC) route in Freund's incomplete adjuvant (FIA). The maximum amount to inject into one site was 0.2 ml. Re-immunization injection sites were on the opposite side of the back from the initial immunization. The rabbits were re-immunized (boosted) at 21-day intervals until peak antibody titers were reached. Freund's incomplete adjuvant (FIA) was used, again at 1:1 ratio with antigen (Harboe et al., 1977; Canadian council on animal care, 2002).

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Table 1 Demographic data of patients studied. Characteristic Age

Age group 0–20 21–40 41–60 61≤

Mean ± SD (Yr)

Range (Yr)

Minimum / maximum (Yr)

52 ± 21.9

87.0

3 /90

Frequency N (%) 12(10.6) 26(23.0) 26(23.0) 49(43.4)

Total N = 113

Gender N(%) cases Male 58(51.3) Female 55(48.7)

Patient group TB-cases Non-TBN (%) cases N (%) 64(56.6) 49(43.4)

Total N = 113

Total N = 113

TB-cases Non-TBN (%) cases N (%) Age(mean ± SD) 50.5 54.1 ± 22.5 ± 21.0

TB-cases N (%) Gender n (%) Male 31 (48.4) Female 33 (51.6)

Non-TBcases N (%) Male 27 (55.1) Female 22 (44.9)

Decontamination is an essential step for killing other microorganisms, which may hamper MTB detection and denaturing sputum components. Ziehl-Neelsen acid-fast staining was used to confirm the presence of acid-fast bacilli (Kent and Kubica, 1985).

2.2. Antibody purification procedure 2.4. Rapid test procedure Affinity chromatography was used to purify specific polyclonal antibodies against A60, (A60-Ab). This step was carried out on an affinity column based on the use of A60 covalently coupled to CNBr-activated Sepharose 4B (Cuatrecasas, 1970). The antibodies were treated with normal oral flora to remove any non-specificity and cross-reactivity. In our experiments, cross-reactive Immunoglobulin was removed by incubation of antibodies (A60-Ab), for 12 h at 4 °C with tested sputum. Sputum samples (three samples per patients) were obtained from patients who have clinically confirmed cases or suspected TB, are referred to the Pasteur Institute (Tehran) for culture and susceptibility testing. Acid-fast microscopy, to confirm the presence of acid-fast bacilli, was done on each sputum samples. Cultures were done on LowensteinJensen (LJ) slopes by the N-acetyl-L-cysteine-sodium hydroxide method, described by Kent and Kubica (1985). Sputum samples that were identified as positive culture or positive smear (acid-fast bacilli) were not selected for antibody preabsorption. Antibodies (A60-Ab) were incubated overnight at 4 °C in the presence of negative sputum (Coetsier et al., 1994). The efficiency of preabsorption was controlled by rapid test procedure. 2.3. Specimens and study groups Sputum from 113 patients (58 male and 55 female, mean age 52), who had been admitted to Pasteur institute (Tehran) were obtained before anti-TB chemotherapy was started. Tuberculosis group consisted of 64 patients with active tuberculosis. The inclusion criteria required both of smear and culture positive (S+, Cul+). Non-tuberculosis group (S−, Cul−) consisted of 49 patients with chest problems other than tuberculosis. The disease included bacterial pneumonia, bronchial asthma, bronchiectasis and pneumoconiosis. Only 11 patients had been vaccinated with M. bovis BCG and were positive in purified protein derivative. The demographic data of the patients are shown in Table 1. The sputum sample (three samples per patients) was digested and decontaminated with 4% NAOH, 2.9% sodium citrate and 0.5% N-acetyl cysteine.

The rapid test, provided by Anda-Biologicals (Strasbourg, France), detects antigen-antibody (Ag-Ab) complexes if the sample contained TB bacilli. A total of 200 μl of processed sputum is added to the reaction funnel and was allowed to absorb completely. A funnel is used to concentrate the sample in the middle of the filter. After washing with the buffer, 150 μl TB antibody solution, (A60-Ab) was added to the test funnel. After another wash with the buffer, 150 μl protein gold conjugate was added to the test funnel. Finally 150 μl of wash buffer was added to remove all unbound conjugate. A red spot development was observed immediately when the patient sputum contained mycobacterial antigens. The rapid test was carried out using a TB antibody solution (A60-Ab) and modified A60 antibodies (PA60-Ab) to detect MTB antigen in the sputa of patients with pulmonary tuberculosis. The sputum samples were screened for the presence of Mycobacterium sp. Specific antigens. Three groups of sputa were tested. The positive test is a central red-pink color that differentiates well from the surrounding area. Negative test yielded a white central region. A red or pink ring without a red-pink center is an artifact and should be considered negative. Fig. 1 shows test results for the three groups. The treated positive sputum samples (A1 and A2), negative sputum samples (B3 and B4) and the control groups included H2O (C5) and BCG (C6) as negative and positive controls, respectively. 2.5. Purification of modified A60 antibodies (PA60-Ab) A new affinity chromatography column was designed to prepare subunits of A60 that were more specific to M. tuberculosis. For this purpose, affinity columns were prepared by covalent linkage of specific polyclonal antibodies against A60, (A60-Ab), which had been already treated with normal flora of throat, to sepharose beads (CNBractivated sepharose 4B, Pharmacia, Uppsala, Sweden) as described by Cuatrecasas(1970). A60 purified antigen was exposed to A60-Ab beads overnight with constant shaking at 4 °C and the unbound

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loaded on 12% vertical polyacrylamide slab gels (Bio-Rad Laboratories, Hercules, CA) according to the method of Laemmli (1970). The gel was stained with Coomassie Brilliant Blue (Wilson, 1983). Molecular weight marker in the range 14.4–116.0 kDa was used (Fermentas, Ontario, Canada).

A

2.7. Statistical analysis

B

C

Fig. 1. The results of rapid test-(PA60-Ab) on the filtering cartridge. The presence of TB bacilli in the sample is revealed by the presence of a red-pink dot in the middle of the filter. The negative control and negative samples yielded a white central region. The treated positive sputum samples (A1 and A2), negative sputum samples (B3 and B4), negative control (C5)⁎ and positive control (C6) ⁎⁎ on the filtering cartridge by Modified A60 antibodies (PA60-Ab). *Negative Control (C5): Distilled water **Positive Control (C6): BCG.

Sensitivity, specificity, positive predictive value (PPV), Negative predictive value (NPV) and efficiency were calculated according to Grange and Laszlo method (1990). Sensitivity is the proportion of patients with disease who test positive [number true positive / (number true positive + number false negative)], specificity is the proportion of patients without disease who test negative [number true negative / (number true negative + number false positive], the positive predictive value (PPV) is the proportion of patients with positive test results who are correctly diagnosed [number true positive / (number true positive + number false positive], the negative predictive value (NPV) is the probability that a test negative is a true negative [number true negative / (number true negative + number false negative] and efficiency is the proportion of number true positive + number true negative) / total number. 3. Results 3.1. Rapid test

material was removed. After washing with PBS pH 7.2, the bound material was eluted with 0.1 glycine buffer pH 2.8 and then dialyzed against PBS pH 7.2 for 24 h, Fig. 2 (Cuatrecasas, 1970; El-Masry et al., 2008). The protein content, Affinity-Purified Antigen (AP-A60) was measured by Bradford's method (Bradford, 1976) and stored at −20 °C until used. Modified A60 antibodies (PA60-Ab) was obtained by immunizing rabbits repeatedly with 250 μg of protein from new affinity chromatography column according to our technique as described previously (Harboe et al., 1977).

2.6. SDS-PAGE Two hundred microgram per lane of intact A60 Anda-Biologicals (Strasbourg, France) and Affinity-Purified Antigen, (AP-A60) were

The results of rapid-test-(PA60-Ab) on the filtering cartridge, are displayed in Fig. 1. A central red-pink color that differentiates well from the surrounding area signifies presence of mycobacteria in the sample. The color intensity is proportional to the number of antigens in the sample. The appearance of a red-pink ring around the red-pink central area is an artifact that does not influence the results. The grade of a positive test color dot varied from a dark reddish purple to scanty light pink. The negative control and negative samples yielded a white central region. The results of rapid test performance using modified A60 antibodies (PA60-Ab) in comparison to culture in combination with smear examination are summarized in Table 2. Out of the 113 sputum samples processed, 67 (59.2) were positive by rapid test and 64 (56.6%) were positive by smear and culture, 46 (40.8%) were negative

Fig. 2. Flow sheet showing purification procedure for isolation of purified A60 (PA60) antigen by affinity chromatography method.

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by rapid test and 49 (43.4%) tested negative by the culture test. The results of the smear examination and culture were combined and then compared to those of rapid test-(PA60-Ab) method. In this combination, if both smear and culture was positive (S +, Cul +) then the combined test was regarded as positive. The combined test was regarded as negative if both smear and culture results were negative(S −, Cul −). Compared to combined test (Smear and Culture together), the rapid test-(PA60-Ab) gave a sensitivity (true positive rate) of 93%, specificity (true negative rate) of 86%, positive predictive value (PPV) of 90%, Negative predictive value (NPV) of 91% and efficiency of 90%. There were 7(6.2%) false positive and 4(3.6%) false negative results in this group. 3.2. SDS-PAGE Fig. 3 is showing the results obtained from the SDS-PAGE to compare the intact A60 (Anda) and (AP-A60). The gel was stained with Coomassie blue. Lanes 1, 3 and 5 contained purified A60 antigen (45 and 66 kDa). Lanes 2 and 6 contained A60 Antigen (Anda). Lane 4 contained the standard molecular weight markers β-galactosidase (116.0 kDa), Bovine serum albumin (66.2 kDa), Ovalbumin (45.0 kDa), Lactate dehydrogenase (35.0 kDa), REase Bsp981 (25.0 kDa), β-lactoglobulin (18.4 kDa), and lysozyme (14.4 kDa). 4. Discussion Because the conventional methods are low in sensitivity, time consuming or both, presumptive diagnosis of Mycobacterium diseases is usually based on clinical findings such as persistent cough, fever and weight loss. Although tuberculin skin tests and radiologic findings may help, the diagnosis must be confirmed by isolation and identification of the etiologic agent (Shinnick and Good, 1995). The Mycobacterium antigen detection assays are promising in this regard. Any test which is to replace direct microscopy or bacterial culture must offer advantages in terms of speed and ease of use and preferably have a higher sensitivity (Bothamley and Rudd, 1994). The diagnostic value of a given test in clinical practice depends on its positive and negative predictive values (Chiang et al., 1997). These values vary markedly with the prevalence of the disease in a given community (Daniel and Debanne, 1987). The high positive predictive values of tests would make a positive test result useful in strengthening the clinical suspicion, but a negative result would be less useful (Pottumarthy et al., 2000). We established modified A60 antibodies, (PA60-Ab) as a fast, sensitive, specific, immunodiagnostic assay for detection of pulmonary TB patients. The developed, modified A60 antibodies, (PA60-Ab)-rapid test showed higher sensitivity, specificity, Positive Predictive Value (PPV), Negative Predictive Value (NPV) and overall efficiency (93.0%, 86.0%, 90.0%, 91.0%, and 90.0% respectively) for the detection of the Mycobacterium antigen. The test provides a good positive predictive value of 90% which is very useful for Table 2 The results of rapid test performance using modified A60 antibodies (PA60-Ab) in comparison to culture in combination with smear examination. Assay Rapid test

Smear and culture Positive (n)% Negative (n)% Total (n)%

Positive (n)% (60) 53.0% (4) 3.6% (64) 56.6%

Negative (n)% (7) 6.2% (42) 37.2% (49) 43.4%

Total (n)% (67) 59.2% (46) 40.8% (113) 100.0%

Assay

Sensitivity

Specificity

PPV

NPV

Efficiency

Rapid test

93 (%)

86 (%)

90 (%)

91 (%)

90 (%)

Sensitivity [true positive rate], Specificity [true negative rate], (PPV) Positive predicative value, (NPV) Negative predicative value, Number false positive (7) 6.2%, Number false negative (4) 3.6%, Efficiency (number true positive + number true negative) / total number.

Fig. 3. SDS-PAGE results to compare the intact A60 (Anda-Biologicals) and AffinityPurified Antigen (AP-A60). The antigens were separated by SDS-PAGE and then analyzed by Coomassie blue staining. Lanes 2 and 6, A60 antigens (Anda). Lanes 1, 3 and 5, purified 66 and 45 kDa proteins, respectively, products from different stages of purification, after affinity chromatography procedure. Lane 4, Horizontal lines on the left indicate molecular mass markers (116.0, 66.2, 45.0, 35.0, 25.0, 18.4 and 14.4 kDa).

diagnosis of symptomatic patients suspected of having tuberculosis. The diagnostic values of this test depend on the context of its use. While a negative result would be useful in excluding disease in a population with a low prevalence of tuberculosis, a positive result could potentially aid in clinical decision making when used with sputa from a group of selected symptomatic patients when there is moderate to high degree of clinical suspicion of tuberculosis (Pottumarthy et al., 2000). Our findings may improve the results obtained in previous studies. There is a report of the use of a single antigen, the 38 kDa antigen, as a very good diagnostic marker (Bothamley and Rudd, 1994) but it still lacks sufficient sensitivity, especially for smear-negative individuals, where sensitivity is considerably lower (Stavri et al., 2003). Antigen 60, a thermo stable component of PPD, has also been used in the serodiagnosis of TB (Gupta et al., 1995; Cocito, 1991; Charpin et al., 1990). The sensitivity rate of antigen 60 for adults with active pulmonary TB is approximately 60 to 89%, with the rate lower for children and patients with extra-pulmonary TB (Charpin et al., 1990; Turneer et al., 1994). Another report on a serological test based on anti A60 IgG that utilized serum from pulmonary smear positive TB patients yielded a sensitivity and specificity of 78% and 86%, respectively (Adjei et al., 2003). Results of a rapid direct sputum and antibody assay in Bucharest, Romania showed a sensitivity, specificity, PPV, and NPV of 86.1%, 92.9%, 91.6%, and 88.2%, respectively (Stavri et al., 2003). These rates are approximately similar to our findings. The discrepancy in findings between the present and an earlier study (Stavri et al., 2003; Gupta et al., 1995; Cocito, 1991; Charpin et al., 1990; Turneer et al., 1994 and Adjei et al., 2003) can be explained by the difference in the number of patients and difference based on the detection of antigenic fractions. Although our study has a small number of patients, it still highlights the usefulness of rapid and accurate diagnostic assay for detection of TB. In addition, changes in antigen conformation that may occur as a result of passive coating of the antigens to solid supports may cause technical artifacts resulting in false positive and false negative reactions (Pereira Arias-Bouda et al., 2003). On the other hand, many serological assays have been developed for specific antibody detection in TB patients (Pottumarthy et al., 2000; Stavri et al., 2003; Khomenko

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et al., 1996). However, people living in the tropical areas are in contact with various pathogens and developed cross-reacting antibodies responsible for poor specificity (Rasolofo and Chanteau, 1999). Moreover, the predictive values of positivity in high endemic areas may be achieved by increasing the specificity, i.e. by modifying the cutoff point separating positive from negative test result. However, increased specificity can only be gained at the expense of the sensitivity (Stavri et al., 2003). Coetsier and others have reported that A60 contains several antigenic determinants that react with the monoclonal antibodies. The 19-, 35-, 38-, 40-, and 65-kDa proteins and LAM were identified by Western blot. The majority (87.5%) of antimycobacterial antibodies in sera from tuberculosis patients was directed against A60. The 45 and 66 kDa protein bands that are reported in this study are likely to be the same as those two antigenic components, 40 and 65 kDa respectively, as reported previously by Coetsier et al. (1994). However, to our knowledge this is the first report on a rapid test based on the detection of a 45 and 66 kDa, antigenic fractions from Iran. In addition, our results demonstrate that TB-rapid test using modified-A60 antibody, reduce the false positive results and improve the detection of TB. Based on this study, we report that this test is useful in diagnosis of active tuberculosis in hospital population suspected of having tuberculosis. The modified A60 antibodies (PA60-Ab) can be used as a probe for serodiagnosis of pulmonary TB infected patients, because the test was user friendly, and the results were obtained within 3 h. In conclusion, the modified A60 antibodies (PA60-Ab)-rapid test, when interpreted by combining both smear and culture readings offers a good and reliable test for diagnosis of tuberculosis. It is particularly helpful in suspected, but not proven, cases of active pulmonary tuberculosis. Acknowledgements We thank the staff and patients who made this study possible. This study received funding from Pasteur Institute of Iran. We thank Shiva Panahpour and Abolfazl Jamshidian for their valuable type and edit of the manuscript. References Aber, V.R., Allen, B.W., Mitchison, D.A., Ayuma, P., Edwards, E.A., Keyes, A.B., 1980. Quality control in tuberculosis: I, Laboratory studies on isolated positive cultures and the efficiency of direct smear examination. Tubercle 61, 123–133. Adjei, A.A., Armah, H., Achaw, O., Adiku, T., Hesse, I.F.A., 2003. Evaluation of a rapid serological chromatographic immunoassay for the diagnosis of pulmonary tuberculosis in Accara. Ghana. Jjid. 56, 161–164. Al-Hajjaj, M.S., Gad-el-Rab, M.O., al-Orainey, I.O., al-Kassimi, F.A., 1999. Improved sensitivity for detection of tuberculosis cases by a modified Anda-TB ELISA test. Tuber. Lung Dis. 79 (3), 181–185. Ben-selma, W., Harizi, H., Marzouk, M., Ben Kahla, I., Ben, Lazreg F., Ferjeni, A., Boukadida, J., 2010. Evaluation of the diagnostic value of measuring IgG, IgM, and IgA antibodies to mycobacterial A60 antigen in active tuberculosis. Diagn. Microbiol. Infect. Dis. 68 (1), 55–59. Bothamley, G.H., Rudd, R.M., 1994. Clinical evaluation of a serological assay using a monoclonal antibody (TB72) to the 38 KDa antigen of Mycobacterium tuberculosis. Eur. Respir. J. 7 (2), 240–246. Bradford, M.M., 1976. A rapid and sensitive for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Cambiaso, C.L., Van Vooren, J.P., Farber, C.M., 1990. Immunological detection of mycobacterial antigens in infected fluids, cells and tissues by Latex agglutination. J. Immunol. Methods 129, 9–14.

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