Molecular and Biochemical Parasitology, 44 (1991) 149-152 Elsevier
149
MOLBIO 01461
Short C o m m u n i c a t i o n
Cross-linking of a monoclonal antibody-antigen complex enables detection of parasite antigen in immunoblots and in an expression library Marc Awobuluyi, Claude V. Maina and Clotilde K.S. Carlow Molecular Parasitology Group, New England Biolabs, Beverly, MA, U.S.A. (Received 13 September 1990; accepted 17 September 1990)
Key words: Low-affinity; Immunoblotting; Glutaraldehyde; Surface antigen; Expression library; Brugia malayi
The generation of low affinity antibodies to parasite antigens, traditionally of the IgM isotype, frequently occurs as a consequence of either the immunization procedure used to generate the antibodies or the nature of the antigens involved. Following a brief immunization scheme designed to optimize the production of monoclonal antibodies against surface antigens with few parasites, an IgM monoclonal antibody (D1E5), reactive with a surface antigenic determinant of Brugia malayi infective stage larvae, was generated [ 1]. In an effort to produce IgG antibodies to the same surface determinant, anti-idiotypic antibodies containing an 'internal image' of the surface epitope were employed as antigen. Despite the use of an extended immunization protocol in this case, only IgM antibodies were produced suggesting an isotype restricted response to the D1E5 epitope [2]. Using immunofluorescence (IFAT) the epitope was found to be species-specific, expressed in a developmentally regulated manner, coinciding with the transmission period [3], and rapidly shed from the parasite surface [4]. These interesting biological characteristics suggest an important role for the epitope in the host-parasite interaction. Thus, numerous attempts were made to identify and isolate the antigen, including immunoblotting and Correspondence address: Clotilde K.S. Carlow, Molecular Parasitology Group, New England Biolabs, 32 Tozer Road, Beverly MA 01915, U.S.A.
screening of a B. malayi genomic expression library [2,3], all of which were unsuccessful. We have developed a simple modification of the general procedure for immunoblotting and antibody screening of expression libraries that is applicable to proteins recognized by lowaffinity antibodies. By chemically cross-linking the antigen/monoclonal antibody complex with glutaraldehyde [5,6], we were able to identify antigens bearing the epitope recognized by the D1E5 monoclonal antibody and screen a genomic expression library of B. malayi. The D1E5 monoclonal antibody used in this study was purified from ascitic fluid by precipitation in 7% polyethylene glycol (PEG 6000, Sigma) followed by gel filtration on Sephacryl S-200 (Pharmacia). Purified normal mouse IgM (Chemicon, Temecula, CA) served as a control. B. malayi infective larvae were purchased from TRS Laboratories, Athens, Georgia. A larval extract was prepared by boiling 1000 parasites for 10 min in 150/zl SDS-PAGE sample buffer (60 mM TrisHC1, pH 7.5/1.0% SDS). Samples were then electrophoresed [7] on 10% Tris-Glycine gels (Novex, Encinatas, CA) and transferred to nitrocellulose paper [8]. A portion of the gel was silver stained (Gelcode, Pierce, Rockford, IL) using a kit as described by the manufacturer. After electroblotting, the membranes were processed in two ways: using either a 'traditional' method or the 'modified' procedure described below. In the 'traditional'
0166-6851/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
150
method, strips were blocked with 3% non-fat powdered milk for 1 h at room temperature and then probed with appropriate concentrations of primary antibody diluted in Tris-buffered saline (20 mM Tris-HC1, pH 7.5/150 mM NaCI) containing 0.03% Tween 20/0.05% Triton X-100 (TBSTT). After incubation for 1 h at room temperature, the strips were washed three times using TBSTT and incubated with an appropriate dilution (in TBSTT) of biotinylated anti-mouse IgM antibody (Vector). After washing as above, an avidin/peroxidasebiotin complex, prepared (in TBSTT) according to the manufacturer's instructions (Vectastain ABC system, Vector), was added. The chromogen used was 4-chloro-l-naphthol. The 'modified' procedure for antigen detection involving cross-linking of the primary antibody/antigen complex with glutaraldehyde and the absence of detergents, was as follows: nitrocellulose strips were washed three times in PBS and incubated for 30 min at room temperature in 2% bovine serum albumin/2% Fico11-400/2% polyvinylpyrolidone in 50 mM Tris-HCl, pH 8.0. The blocking solution was removed and membranes were then incubated overnight at 4°C in primary antibody diluted in PBS. After one wash in PBS, fixation of immune complexes was achieved by the addition of 0.25% glutaraldehyde in cold PBS for 15 min. The blots were washed once in PBS and reblocked with 1% non-fat milk in PBS for 20 min at room temperature. After further washing with PBS, the strips were processed with the above reagents diluted in PBS. As a consequence of a few simple changes in the immunoblotting procedure we have succeeded in identifying antigens recognized by the D1E5 monoclonal antibody. A reproducible pattern of antigen recognition was observed (Fig. 1), which was consistent with the species- and stage-specific reactivity observed in IFAT (data not shown). In agreement with previous experiments [3], D1E5 did not react with any components when immunoblotting was performed by the traditional method. The absence of detergents also contributed to the detection of the antigens, since a reduced signal was obtained when detergents were employed even with cross-linking of the immune complex (data not shown). These results suggest that, in addition to dissociation of the monoclonal
o
bc
d e
MW
-67
II
-45 -51 -21
Fig. 1. Immunoblot detection of antigens recognized by an IgM zmonoclonal antibody (D1E5), reactive with the surface of Brugia malayi infective larvae, following glutaraldehyde fixation. Silver-stained gel containing the extract of B. malayi infective larvae used as antigen (a). Nitrocellulose strips containing L3 antigens were probed with monoclonal antibody (lanes b and d) or with normal mouse IgM (lanes c and e). Primary antibody/antigen complexes were cross-linked with glutaraldehyde (lanes b and c).
antibody/antigen complex, the target antigen is probably removed from nitrocellulose by detergents during the immunoblotting procedure. As a result of the two modifications described we have been able to detect as few as 7 larval equivalents using this method while previous attempts using as many as 100-fold more parasites failed. Efficient screening of antigen producing clones in a recombinant DNA library depends on the quality of the antibody used [9] and purified antibodies of high affinity are ideal in this respect. The results of the immunoblotting experiments described above indicate that the D1E5 monoclonal antibody binds weakly to its target antigen bound to nitrocellulose. Accordingly, this may also explain our previous inability to isolate the antigen from a B. malayi genomic library, constructed in the bacteriophage vector Agtll [10]. Using the glutaraldehyde procedure described above for immunoblotting, we rescreened approximately 340 000 recombinant phage. Low signal to noise ratios were observed (Fig. 2A) and 4 phage
151
A
B XDIE5.1 >,gt11 D1E5
Modified ~
"~. \.
\
~
o~-~ cjalactosidase
\ D1E5 Traditional
oL-]] galactosidase
Fig. 2. (A) Nitrocellulose filter containing approximately 30000 plaques of a B. malayi genomic library probed with monoclonal antibody DIE5. (B) 1 id of a concentrated phage solution of either Agtl I or ADIE5.1 was spotted onto a freshly poured lawn of Escherichia coli, incubated at 43°C until large plaques appeared. Isopropyl-fl-D-thiogalactopyranoside (IPTG) soaked filters were placed on top and were then probed with monoclonal antibody D1E5 or anti-fl-galactosidase antibodies, using either the 'traditional' or 'modified' methods described.
clones were detected, two of which reacted very strongly. The method-dependency of the signal was confirmed by processing duplicate filters in the two ways described. Nitrocellulose containing products from one positive plaque purified clone (AD1E5.1) or from Agtl 1 were then probed with D1E5, or control antibodies (Fig. 2B). In accordance with the results of the immunoblots, D1E5 bound specifically to AD1E5.1 and not to the Agtll control only after chemical crosslinking. Polyclonal rabbit anti-fl-galactosidase antibodies (Cappel, Malvern, PA) bound both to AD1E5.1 and to Agtll but the signal was greatly enhanced when the 'modified' method was employed. This increased sensitivity of immunoblot assays following glutaraldehyde fixation has previously been reported [5]. In addition, we have extended the method described in this paper to screening expression libraries with polyclonal sera, pre-absorbed against Escherichia coli antigens, and have observed greater sensitivity than 'traditional' methods. Antibodies that fail to react in immunoblotting are often considered inappropriate for screening
expression libraries. We have described an immunoblotting method, applicable to the screening of a expression libraries, which functions with such antibodies. This technical advance could enable the identification and isolation of many important antigens, not only of parasite origin, which have hitherto eluded biochemical and molecular analysis.
Acknowledgements We gratefully acknowledge the support of Dr. D.G. Comb. We thank Dr. M. Philipp for stimulating discussions, D. Hough for technical assistance, and Dr. P. Arasu and Dr. F. Perler for kindly providing the B. malayi genomic library used in this study.
References 1 Carlow, C.K.S., Edwards, M.K., James, E.R. and Philipp, M. (1987) Monoclonal antibodies to parasite antigens: a rapid immunization protocol requiring small numbers of parasites. J. Parasitol. 73, 1054-1057. 2 Carlow, C.K.S., Busto, P., Storey, N. and Philipp, M. (1990) Anti-idiotypic antibodies function as a surrogate surface epitope of Brugia malayi infective larvae. Acta
152 Trop. 47, 391-397. 3 Carlow, C.K.S., Franke, E.D., Lowrie, R.C., Partono, F. and Philipp, M. (1987) Monoclonal antibody to a unique surface epitope of the human filaria Brugia malayi identifies infective larvae in mosquito vectors. Proc. Natl. Acad. Sci. USA. 84, 6914-45918. 4 Philipp, M., Davis, T.B., Storey, N. and Carlow, C.K.S. (1988) Immunity in filariasis: perspectives for vaccine development. Annu. Rev. Microbiol. 42, 685-716. 5 Ikegaki, N. and Kennett, R.H. (1989) Glutaraldehyde fixation of the primary antibody-antigen complex on nitrocellulose paper increases the overall sensitivity of immunoblot assay. J. Immunol. Methods 124, 205-210. 6 March, J. (1968) Advanced Organic Chemistry: Reactions,
Mechanisms, and Structure, McGraw-Hill, New York. 7 Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 593-596. 8 Towbin, M., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA. 76, 4350-4354. 9 Young, R.A. and Davis, R.W. (1983) Efficient isolation of genes by using antibody probes. Proc. Natl. Acad. Sci. USA. 80, 1194-1198. 10 Arasu, P., Philipp, M. and Perler, F. (1987) Brugia malayi: recombinant antigens expressed by genomic DNA clones. Exp. Parasitol. 64, 2281-291.
149
MOLBIO 01461
Short C o m m u n i c a t i o n
Cross-linking of a monoclonal antibody-antigen complex enables detection of parasite antigen in immunoblots and in an expression library Marc Awobuluyi, Claude V. Maina and Clotilde K.S. Carlow Molecular Parasitology Group, New England Biolabs, Beverly, MA, U.S.A. (Received 13 September 1990; accepted 17 September 1990)
Key words: Low-affinity; Immunoblotting; Glutaraldehyde; Surface antigen; Expression library; Brugia malayi
The generation of low affinity antibodies to parasite antigens, traditionally of the IgM isotype, frequently occurs as a consequence of either the immunization procedure used to generate the antibodies or the nature of the antigens involved. Following a brief immunization scheme designed to optimize the production of monoclonal antibodies against surface antigens with few parasites, an IgM monoclonal antibody (D1E5), reactive with a surface antigenic determinant of Brugia malayi infective stage larvae, was generated [ 1]. In an effort to produce IgG antibodies to the same surface determinant, anti-idiotypic antibodies containing an 'internal image' of the surface epitope were employed as antigen. Despite the use of an extended immunization protocol in this case, only IgM antibodies were produced suggesting an isotype restricted response to the D1E5 epitope [2]. Using immunofluorescence (IFAT) the epitope was found to be species-specific, expressed in a developmentally regulated manner, coinciding with the transmission period [3], and rapidly shed from the parasite surface [4]. These interesting biological characteristics suggest an important role for the epitope in the host-parasite interaction. Thus, numerous attempts were made to identify and isolate the antigen, including immunoblotting and Correspondence address: Clotilde K.S. Carlow, Molecular Parasitology Group, New England Biolabs, 32 Tozer Road, Beverly MA 01915, U.S.A.
screening of a B. malayi genomic expression library [2,3], all of which were unsuccessful. We have developed a simple modification of the general procedure for immunoblotting and antibody screening of expression libraries that is applicable to proteins recognized by lowaffinity antibodies. By chemically cross-linking the antigen/monoclonal antibody complex with glutaraldehyde [5,6], we were able to identify antigens bearing the epitope recognized by the D1E5 monoclonal antibody and screen a genomic expression library of B. malayi. The D1E5 monoclonal antibody used in this study was purified from ascitic fluid by precipitation in 7% polyethylene glycol (PEG 6000, Sigma) followed by gel filtration on Sephacryl S-200 (Pharmacia). Purified normal mouse IgM (Chemicon, Temecula, CA) served as a control. B. malayi infective larvae were purchased from TRS Laboratories, Athens, Georgia. A larval extract was prepared by boiling 1000 parasites for 10 min in 150/zl SDS-PAGE sample buffer (60 mM TrisHC1, pH 7.5/1.0% SDS). Samples were then electrophoresed [7] on 10% Tris-Glycine gels (Novex, Encinatas, CA) and transferred to nitrocellulose paper [8]. A portion of the gel was silver stained (Gelcode, Pierce, Rockford, IL) using a kit as described by the manufacturer. After electroblotting, the membranes were processed in two ways: using either a 'traditional' method or the 'modified' procedure described below. In the 'traditional'
0166-6851/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
150
method, strips were blocked with 3% non-fat powdered milk for 1 h at room temperature and then probed with appropriate concentrations of primary antibody diluted in Tris-buffered saline (20 mM Tris-HC1, pH 7.5/150 mM NaCI) containing 0.03% Tween 20/0.05% Triton X-100 (TBSTT). After incubation for 1 h at room temperature, the strips were washed three times using TBSTT and incubated with an appropriate dilution (in TBSTT) of biotinylated anti-mouse IgM antibody (Vector). After washing as above, an avidin/peroxidasebiotin complex, prepared (in TBSTT) according to the manufacturer's instructions (Vectastain ABC system, Vector), was added. The chromogen used was 4-chloro-l-naphthol. The 'modified' procedure for antigen detection involving cross-linking of the primary antibody/antigen complex with glutaraldehyde and the absence of detergents, was as follows: nitrocellulose strips were washed three times in PBS and incubated for 30 min at room temperature in 2% bovine serum albumin/2% Fico11-400/2% polyvinylpyrolidone in 50 mM Tris-HCl, pH 8.0. The blocking solution was removed and membranes were then incubated overnight at 4°C in primary antibody diluted in PBS. After one wash in PBS, fixation of immune complexes was achieved by the addition of 0.25% glutaraldehyde in cold PBS for 15 min. The blots were washed once in PBS and reblocked with 1% non-fat milk in PBS for 20 min at room temperature. After further washing with PBS, the strips were processed with the above reagents diluted in PBS. As a consequence of a few simple changes in the immunoblotting procedure we have succeeded in identifying antigens recognized by the D1E5 monoclonal antibody. A reproducible pattern of antigen recognition was observed (Fig. 1), which was consistent with the species- and stage-specific reactivity observed in IFAT (data not shown). In agreement with previous experiments [3], D1E5 did not react with any components when immunoblotting was performed by the traditional method. The absence of detergents also contributed to the detection of the antigens, since a reduced signal was obtained when detergents were employed even with cross-linking of the immune complex (data not shown). These results suggest that, in addition to dissociation of the monoclonal
o
bc
d e
MW
-67
II
-45 -51 -21
Fig. 1. Immunoblot detection of antigens recognized by an IgM zmonoclonal antibody (D1E5), reactive with the surface of Brugia malayi infective larvae, following glutaraldehyde fixation. Silver-stained gel containing the extract of B. malayi infective larvae used as antigen (a). Nitrocellulose strips containing L3 antigens were probed with monoclonal antibody (lanes b and d) or with normal mouse IgM (lanes c and e). Primary antibody/antigen complexes were cross-linked with glutaraldehyde (lanes b and c).
antibody/antigen complex, the target antigen is probably removed from nitrocellulose by detergents during the immunoblotting procedure. As a result of the two modifications described we have been able to detect as few as 7 larval equivalents using this method while previous attempts using as many as 100-fold more parasites failed. Efficient screening of antigen producing clones in a recombinant DNA library depends on the quality of the antibody used [9] and purified antibodies of high affinity are ideal in this respect. The results of the immunoblotting experiments described above indicate that the D1E5 monoclonal antibody binds weakly to its target antigen bound to nitrocellulose. Accordingly, this may also explain our previous inability to isolate the antigen from a B. malayi genomic library, constructed in the bacteriophage vector Agtll [10]. Using the glutaraldehyde procedure described above for immunoblotting, we rescreened approximately 340 000 recombinant phage. Low signal to noise ratios were observed (Fig. 2A) and 4 phage
151
A
B XDIE5.1 >,gt11 D1E5
Modified ~
"~. \.
\
~
o~-~ cjalactosidase
\ D1E5 Traditional
oL-]] galactosidase
Fig. 2. (A) Nitrocellulose filter containing approximately 30000 plaques of a B. malayi genomic library probed with monoclonal antibody DIE5. (B) 1 id of a concentrated phage solution of either Agtl I or ADIE5.1 was spotted onto a freshly poured lawn of Escherichia coli, incubated at 43°C until large plaques appeared. Isopropyl-fl-D-thiogalactopyranoside (IPTG) soaked filters were placed on top and were then probed with monoclonal antibody D1E5 or anti-fl-galactosidase antibodies, using either the 'traditional' or 'modified' methods described.
clones were detected, two of which reacted very strongly. The method-dependency of the signal was confirmed by processing duplicate filters in the two ways described. Nitrocellulose containing products from one positive plaque purified clone (AD1E5.1) or from Agtl 1 were then probed with D1E5, or control antibodies (Fig. 2B). In accordance with the results of the immunoblots, D1E5 bound specifically to AD1E5.1 and not to the Agtll control only after chemical crosslinking. Polyclonal rabbit anti-fl-galactosidase antibodies (Cappel, Malvern, PA) bound both to AD1E5.1 and to Agtll but the signal was greatly enhanced when the 'modified' method was employed. This increased sensitivity of immunoblot assays following glutaraldehyde fixation has previously been reported [5]. In addition, we have extended the method described in this paper to screening expression libraries with polyclonal sera, pre-absorbed against Escherichia coli antigens, and have observed greater sensitivity than 'traditional' methods. Antibodies that fail to react in immunoblotting are often considered inappropriate for screening
expression libraries. We have described an immunoblotting method, applicable to the screening of a expression libraries, which functions with such antibodies. This technical advance could enable the identification and isolation of many important antigens, not only of parasite origin, which have hitherto eluded biochemical and molecular analysis.
Acknowledgements We gratefully acknowledge the support of Dr. D.G. Comb. We thank Dr. M. Philipp for stimulating discussions, D. Hough for technical assistance, and Dr. P. Arasu and Dr. F. Perler for kindly providing the B. malayi genomic library used in this study.
References 1 Carlow, C.K.S., Edwards, M.K., James, E.R. and Philipp, M. (1987) Monoclonal antibodies to parasite antigens: a rapid immunization protocol requiring small numbers of parasites. J. Parasitol. 73, 1054-1057. 2 Carlow, C.K.S., Busto, P., Storey, N. and Philipp, M. (1990) Anti-idiotypic antibodies function as a surrogate surface epitope of Brugia malayi infective larvae. Acta
152 Trop. 47, 391-397. 3 Carlow, C.K.S., Franke, E.D., Lowrie, R.C., Partono, F. and Philipp, M. (1987) Monoclonal antibody to a unique surface epitope of the human filaria Brugia malayi identifies infective larvae in mosquito vectors. Proc. Natl. Acad. Sci. USA. 84, 6914-45918. 4 Philipp, M., Davis, T.B., Storey, N. and Carlow, C.K.S. (1988) Immunity in filariasis: perspectives for vaccine development. Annu. Rev. Microbiol. 42, 685-716. 5 Ikegaki, N. and Kennett, R.H. (1989) Glutaraldehyde fixation of the primary antibody-antigen complex on nitrocellulose paper increases the overall sensitivity of immunoblot assay. J. Immunol. Methods 124, 205-210. 6 March, J. (1968) Advanced Organic Chemistry: Reactions,
Mechanisms, and Structure, McGraw-Hill, New York. 7 Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 593-596. 8 Towbin, M., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA. 76, 4350-4354. 9 Young, R.A. and Davis, R.W. (1983) Efficient isolation of genes by using antibody probes. Proc. Natl. Acad. Sci. USA. 80, 1194-1198. 10 Arasu, P., Philipp, M. and Perler, F. (1987) Brugia malayi: recombinant antigens expressed by genomic DNA clones. Exp. Parasitol. 64, 2281-291.
