HYBRIDOMA Volume 11, Number 4, 1992 Mary Ann Lieben, Inc., Publishers

Purification of MPB70 and Production of Specific Monoclonal Antibodies SHINJI

MASARO NAKAGAWA,2 SADAMU KAORU MIURA,1 and MITSUO HONDA1

HAGA,1

NAGAI,3

'Department of Cellular Immunology and AIDS Research Center, and 2Department of Veterinary Science, National Institute of Health, 2-10-35 Kamiosaki, Shinagawaku, Tokyo 141, Japan Osaka City University Medical School, Toyonaka, Osaka 560, Japan

3Toneyama Institute for Tuberculosis Research,

ABSTRACT MPB70 is a protein secreted into the culture filtrate of Mycobacterium bovis BCG (substrain Tokyo 172), which is able to induce a delayed-type hypersensitivity (DTH) skin reaction in guinea pigs immunized with BCG-Tokyo. By high-pressure chromatofocusing and size-exclusion high performance liquid chromatography, a further purified MPB70 protein was obtained, which was visualized as a single band with a molecular mass of 22 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis. A series of hybridoma cell lines that produced monoclonal antibodies (MAbs) against the purified MPB70 protein was prepared, and three MAbs, Bov-1, Bov-2, and Bov-3, with strong antigen-binding capacities were established. Bov-1 was the most potent MAb among them and binds to only a 22 kDa protein band in culture filtrates of M. bovis, but not to bands in those of M. tuberculosis by Western immunoblotting analysis, suggesting that Bov-1 recognize different epitope of MPB70 from MAbs that have been shown previously to recognize several species of molecules in culture filtrates of M. bovis. The purified MPB70 protein elicited a strong DTH skin reaction in guinea pigs sensitized with BCG-Tokyo vaccine. Bov-1 had no inhibitory effect on generation of the DTH skin reaction, showing that MAb bound to an epitope distinct from that inducing the skin reaction. All of the three MAbs were specific to MPB70 and each recognized a different epitope on MPB70. MPB70 was

detected in the culture filtrate of M. tuberculosis H37Rv. Thus, these MAbs may be useful for detecting MPB70 in studies on discriminating infection with M. bovis in domestic animals or in distinguishing vaccination with BCG-Tokyo from other mycobacterial infections in humans. not

INTRODUCTION Human tuberculosis is an infectious disease caused by Mycobacterium tuberculosis and is a worldwide serious problem, especially in developing countries. The delayed-type hypersensitivity (DTH) skin reaction to the purified protein derivative of tuberculin (PPD) has been used for diagnosis of M. tuberculosis infection. BCG has been used as a vaccine, because of its cross-immunity with and less virulence than M. tuberculosis. Skin reactive proteins from the culture filtrate of M. bovis BCG were studied in place of PPD. MPB70, one of the secreted proteins of M. bovis BCG (BCG-Tokyo), has been isolated and its unique species-specificity and considerable productions in a limited number of

483

BCG substrains have been reported (1,2,3,10). Furthermore, MPB70 was able to elicit a DTH skin reaction in guinea pigs sensitized with both Tokyo and Moreau (Brazil) substrains of BCG ( 1,2). Interest in this species-specific antigen has been focused on further characterization of the molecule by antibody based techniques (4) and characterization of the genetic structure of MPB70 (5,6,7). Wood et al. (4) isolated monoclonal antibodies (MAbs) to MPB70 by immunization with UVkilled M. bovis. Furthermore, they characterized antigenic determinants that the MAbs recognized as at least four major proteins including MPB70 (8,9). Hasl0v et al. (12) described an anti-MPB70 MAb which recognized several bands in addition to the main 22-kDa band. In this study, we further purified the MPB70 protein to homogeneity and developed a series of mouse monoclonal antibodies to the purified protein. Bov-1, one of our MAbs, recognized only MPB70 molecule specifically, among the secreted proteins in the culture filtrate. The availability of such a MAb might be of potential usefullness in the study of MPB70 in the infections of M. bovis to animals and humans.

MATERIALS AND METHODS Animals Female BALB/c mice 6 to 8 weeks of age were maintained in safety cabinets at the Experimental Animal Facility Center, National Institute of Health, Tokyo, Japan. Female guinea pigs of Hartley strain, 6 to 7 weeks of age, weighing 300 to 350 g, were obtained from Shizuoka Laboratory Animal Center (Shizuoka, Japan). Growth

of Organisms and Preparation of Culture Filtrates

Substrain Tokyo-172 of M. bovis BCG and M. tuberculosis H37Rv were grown as described The culture filtrates were obtained after 2 to 4 weeks of incubation in Sauton liquid medium at 37°C by filtration through a membrane filter (Millipore Corp., Bedford, MA) (1).

previously (1).

Purification ofMPB70 The original MPB70 preparation which was isolated by ammonium sulfate precipitation and fractionation by DEAE anion exchange chromatography followed by G-75 Sephadex chromatography from the culture filtrate of M bovis BCG-Tokyo was prepared as described previously (1). All the following steps of purifying MPB70 were performed by high-performance liquid chromatography (HPLC) using the Pharmacia Fast Protein Liquid Chromatography (FPLC™) system equipped with a Pharmacia High Performance Pump P-3500 (Pharmacia LKB, Uppsala, Sweden). High-pressure chromatofocusing was performed on a Mono P HR 5/20 column (20 X 0.5 cm, Pharmacia) in the pH range of 4.0-5.2. The column was equilibrated with 0.025 M piperazine-HCl, pH 5.2. After injection of the sample, elution was performed with polybuffer 74-HC1, pH 4.0, 0.5 ml/min. Pooled fractions of pH 4.5-4.6 (stippled area, Fig. la) were concentrated by Diaflow* membrane system ( Amicon Inc., Bevery MA) and loaded on a Superóse 12 HR 16/50 size-exclusion HPLC column (Pharmacia, 500 X 16 mm) which had been equilibrated with phosphate buffered saline (PBS) and calibrated with the following molecular weight (m.w.) standards (Pharmacia): Blue-dextran (exclusion marker), bovine serum albumin (BSA) (m.w., 67,000), ovalbumin (OVA) (m.w., 43,000), chymotrypsinogen A (m.w., 25,000), and ribonuclease A (m.w., 13,000). Pooled fractions of m.w. 22 kDa (stippled area, Fig.lb,c) were concentrated and loaded on two consecutive Superóse 12 columns at a flow rate of 1.0 ml/min. By using the further purification procedures, MPB70 could be obtained as 22 kDa molecule which does not show any other band in SDS-PAGE analysis.

484

20

30 40 50 Fraction number

10

60

20 30 40 50 Fraction number

60

kDa —

—

—

94 67 43

—

30

—

20.1

*-

14.4

20 30 40 50 60 70 80 Fraction number

FIGURE 1. Steps of Purification of MPB70. a) Mono P chromatofocusing column chromatography of MPB70. b) Superóse 12 column chromatography of MPB70 in the pooled fractions of a chromatofocusing column (stippled area), c) Two consecutive Superóse 12 column chromatographies of MPB70 obtained after size-exclusion HPLC (Fig. lb), d) Relative molecular mass estimation of further purified MPB70. The original MPB70 fraction (0.2 (ig, lane 1). The purified protein (0.2 fig, lane 2) and protein marker (lane 3) were electrophoresed on an 8-25% gradient SDS polyacrylamide gel under reducing conditions. Silver stained gels are shown. Pharmacia LMW calibration kit (phosphorylase b, 94,000; albumin, 67,000; ovalbumin, 43,000; carbonic anhydrase, 30,000; trypsin inhibitor, 20,100; and a-lactalbumin, 14,400) were used as protein markers.

485

Preparation ofHybridomas Mice were immunized by subcutaneous and intraperitoneal injection of 1 mg of the purified MPB70 protein emulsified with incomplete Freund's adjuvant (DIFCO Laboratories, Detroit MI) and 0.1 mg of B30-MDP (14), and given 5 booster injections 1- to 2-week intervals. Three days after the last injection, the spleens were harvested for cell fusion. Hybridomas were produced by fusion of the spleen cells with P3/X63-Ag8.653 myeloma (15). After selection with hypoxanthine/aminopterin /thymidine (HAT), the cultures were weaned to medium without HAT and the hybridoma supernatants were screened for their ability to react with the MPB70. Hybridoma cells of interest were cloned by the limiting dilution technique. MAb was obtained from ascitic fluid of pristane-pretreated mice growing each hybridoma as a tumor. The antibodies IgG and IgM were purified by protein A-Sepharose affinity chromatography (Bio-Rad Laboratories, Richmond, CA) and Bakerbond ABx™ column (Yamazen Co., Ltd., Osaka, Japan) HPLC chromatography (16), respectively. The purified immunoglobulin was practically free of contaminating proteins as judged by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The isotypes of MAb were determined by using a Mouse-Typer™ subisotyping kit (Bio-Rad). Protein concentration was determined by Warburg-Christian's method (17).

Enzyme Linked Immunosorbent Assay (ELISA) The further purified MPB70 antigen preparation, containing 5 |ig of protein per ml in 0.1 M carbonate buffer, pH 9.0, was allowed to bind to polystyrene microwell plates (Nunc, Roskilde, Denmark) overnight at 4 °C. The peripheral wells were omitted to avoid edge effects. The plates were washed with 0.15 M PBS containing 0.05% Tween 20 by using a microwell plate washer (Nunc) and all washing steps were performed in quadruplicate. Unbound sites were saturated by incubation with PBS containing 1% BSA, fraction V (Sigma Chemical Co., St. Louis, MO) for at least 30 min at room temperature. After the washing, 100 |il of various dilutions of the hybridoma culture supernatants or purified immunoglobulin fractions which were obtained from the ascitic fluids of pristane-pretreated mice in which a hybridoma was growing as a tumor, were added and then incubated for 60 min at room temperature. The plates were washed and incubated with biotinylated anti-mouse Ig (Zymed, San Francisco, CA) for 60 min at room temperature. The plates were then washed and incubated with streptoavidin-horseradish peroxidase (Bethesda Research Laboratories, Inc., Gaithersburg, MD). After the plates were washed, 1,2-phenylendiamine dihydrocloride (Sigma) was added to each well and absorbance was measured with a microplate photometer (Bio-Rad). For the inhibition test, 100 u.1 of MAb was incubated in each well before the addition of the biotinylated monoclonal antibody in fluorescence sandwich ELISA (13,18). SDS-PAGE Analysis and Western

Immunoblotting

Culture filtrates of M. bovis BCG-Tokyo and M. tuberculosis H37Rv, and the purified MPB70 heated at 100°C for 3 min in an equal volume of Laemmli sample buffer and were electrophoresed through 8-25% gradient SDS-polyacrylamide gels according to the method of Laemmli (19). Proteins were visualized with silver stain. In other runs the separated proteins in SDS-PAGE gels were transferred to nitrocellulose (20) by Pharmacia Phast System™ /Phast Transfer™ (Pharmacia). The nitrocellulose was blocked with 1% BSA and incubated with anti-MPB70 MAb. The blots were incubated with alkaline phosphatase-conjugated anti-mouse Ig and developed with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate ( Promega Co., Ltd., Madison, WI). The proving was carried out various conditions to detect every reactive molecules. were

486

Sensitization of Guinea Pigs with BCG-Tokyo and Skin Tests The skin tests used to induce DTH in guinea pigs were conducted as described previously (2). In brief, 0.5 mg of lyophilized BCG living cells (Japan BCG Laboratory, Tokyo) were suspended in 1 ml of PBS and injected subcutaneously into the lower abdomen of guinea pigs. Seven weeks later, these animals were skin tested on a shaved flank by intradermal injection of PPD (Japan BCG Laboratory) or the purified MPB70 in the presence or absence of anti MPB70 MAb. The mean diameter of induration was measured 24 h after injection. RESULTS

Purification ofMPB70 To produce MAb of definite property against MPB70 antigen we initially performed a further purification of the original MPB70 preparation (1). The lyophilized MPB70 preparation which was purified by ammonium sulfate precipitation of the culture filtrate of M. bovis BCG-Tokyo, anion exchange chromatography and gel filtration was used as the source of MPB70 protein. The MPB70 was first isolated by high-pressure chromatofocusing and eluted as one major protein peak at a pH of 4.5-4.6 (Fig. la). The MPB70-containing fractions were concentrated by a Diaflow® membrane system and were further isolated by size-exclusion HPLC using Superóse 12 columns (Fig. lb). Two protein peaks were eluted and the major peak in fractions corresponding to approximately 22 kDa was purified by two consecutive Superóse 12 columns (Fig. Ic). The MPB70 fraction was eluted as four protein peaks and the third major peaks were pooled and used as further purified MPB70 preparation. Silver staining after SDS-PAGE showed that the purified product had migrated as a single band at apparently 22 kDa under reducing conditions in 8-25% gradient gels (Fig. Id, lane 2). The final yield of isolated MPB70 was calculated as approximately 20%.

Isolation and Characterization of MAb The further purified MPB70 preparation was used as antigen for immunization of mice and a total of 410 hybridomas were derived from fusions. One hundred eighty-seven wells were initially antimouse Ig-positive in the ELISA but this number was decreased to 57 after successive culture. We selected 7 anti-MPB70 positive wells which showed high titers of ELISA. We cloned 3 hybridomas by limiting dilution and coded the MAbs as Bov-1 to Bov-3 (Table 1). These 3 MAbs demonstrated higher binding activity than the other MAbs. The hybridomas which produced Bov-1, -2 and -3 were TABLE 1 Monoclonal Antibodies Specific for MPB70

Original No.

Code

Isotype

5F12/4013

Bov-1

IgG2b

2C11/1 lb

Bov-2

IgM

2E10/26

Bov-3

IgM

487

2.0

h

1.0 0.8 0.7 E 0.6

Bov-2

antibody Bov-1

antibody

0.5

8c

0.4

.Q o

CO

0.3

-Q