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Comparison of protocols for depleting anti-E. coli antibody in immunoblotting of recombinant antigens
JOURNALOF IMMUNOLOGICAL METHODS
ELSEVIER
Journal of Immunological Methods 190 ( 1996) 143- 145
Short communication
Comparison of protocols for depleting anti-25 coli antibody in immunoblotting of recombinant antigens ’ Giovanni Covini a*b,Edward K.L. Chan a, Massimo Colombo b, E.M. Tan a** a W.M. Keck Autoimmune Disease Center, Deparhnent of Molecular and Experimental Medicine, The Scripps Research Institute. LA Jolla. CA 9203, USA b Istituto di Medicina Interna. Via Pace 9, 20122 Milan, Italy
Received 3 October 1995; accepted 11 December 1995
Abstract A common problem in immunoblotting for the detection of specific antibodies to recombinant proteins synthesized in E. is the presence of contaminating antibodies to E. cofi proteins. Four protocols for the efficient depletion of anti-E. coli antibodies from human sera are provided and their uses are discussed.
coli
Keywords:
Anti-E. coli antibody depletion; Autoantibody; lmmunoblotting
Immunoblotting (IB) analysis is an important technique for characterizing the specificity of antigen-antibody system. The basic IB method consists of the electrophoretic separation of a protein mixture and the electrotransfer to nitrocellulose (Towbin et al., 1979; Chan and Pollard, 1992). The nitrocellulose is cut into strips which are then incubated with different diluted sera. The reactivities of the sera are detected with radiolabeled probe or enzyme-conjugated secondary antibody and dye or chemiluminant substrates (Ghan and Pollard, 1992).
Abbreviations: PBS, phosphate buffered saline; PBS-T, PBS containing 0.05% Tween 20; IB, immunoblotting; PAGE, polyacrylamide gel electrophoresis; L.SB, Laemmli’s sample buffer. * Corresponding author. Tel.: 619-554-8686; Fax: 619-5546805. ’ This is publication %13-MEM from The Scripps Research Institute. ‘Ihis work is supportedby National Institutes of Health Grants CA 56956 and AR 41803 and by Associazione Italiana COPE!!. 0022-1759/%/S
In the past few years we have analyzed by IB a large number of sera from patients with different diseases to detect antibodies to recombinant proteins expressed in E. coli. IB analysis is also employed to detect the presence of antibodies after immunization with peptides in experimental animals. Using whole E. coli lysate expressing the recombinant protein as antigen source for IB, a common problem was the presence of additional reactivities of the sera with E. coli antigens. Since E. coli is physiologically present in intestinal flora, anti-E. coli antibodies are often responsible for the high background and false positivity in IB. A way to improve the results could be the use of purified recombinant proteins as antigen sources for IB analysis but purification techniques are laborious, expensive and sometimes it is not possible to have a 100% pure protein without contamination of other E. coli proteins. A simple protocol for the complete depletion of E. coli antibodies is essential because it permits IB analysis of sera without using pure recombinant pro-
15.00 0 1996 Elsevier Science B.V. All rights reserved
SSDl 0022-1759(95)00288-X
144
G.
Covini
et al./Journd
of Immunological
teins. Initially, we tried to deplete the sera with commercial bacteria lysate without satisfactory results because anti-E. coli protein reactivities were still present. Different methods for the preparation of E. coli lysates have been described, and the most common protocols use a combination of lysis buffers with lysozyme treatment, sonication, or freeze-tbawing (Sambrook et al., 1989). We decided to compare the depletion of anti-E. coli antibodies from sera using bacterial lysates obtained by these standardized lysis protocols. The efficiency in the lysis of E. coli was monitored by light microscopy. In this report we describe the comparison of two preparations of bacterial lysates and our methods for depleting anti-E. co/i antibodies in a representative human serum from a patient with liver cancer containing antibodies to cyclin Bl to demonstrate that we have established protocols for the successful depletion of anti-e. coli antibodies. Antigen preparation for IB. Antigen source for IB was obtained using E. cofi BL21 (DE3) lysate carrying plasmid pK171 encoding cyclin B 1 (kindly provided by Steve Reed, The Scripps Research Institute). Bacteria were grown with standard culture technique. Briefly, 5 ml of LB medium (IO g tryptone, 5 g yeast extract, 5 g NaCl in 1 L of distilled water) were inoculated with a single bacterial colony and grown overnight at 37°C with vigorous agitation (200 rpm). Cells were collected by centrifugation and resuspended in 50 ml of LB medium for continuous culture. When the culture OD,, was about 0.6, isopropyl-P-thiogalactopyranoside was added to a final concentration of 5 mM. The cells were further cultured for 4 h. Cells were collected by centrifugation and resuspended in 3 ml of TG buffer (10 mM Tris pH 6.9, 10% glycerol) and solubilized in 3.5 ml 2 X Laemmli’s sample buffer (LSB) (Laemmli, 1970) for every 500 ~1 of E. coli pellet. The lysate was boiled for 3 min. and centrifuged at 10000 X g for 30 min. to remove insoluble debris. For the detection of antibody to cyclin Bl, a 15% slab gel of 15 X 15 X 0.15 cm (h X 1 X w) was loaded with 0.5 mg of protein supematant and IB was performed as described (Ghan and Pollard, 1992). Antigen sources for absorption. E. coli BL21 (DE3) without plasmid pK171 was grown in LB medium overnight for the preparation of four different lysates as bacterial antigens for absorption.
Methods
Lysate
190
#l
(1996)
143-145
Cells were harvested by centrifugation and washed twice in cold phosphatebuffered saline (PBS), resuspended in 20 volumes of the cell pellet in E. coli lysis buffer A (20 mM phosphate pH 8.0, 150 mM NaCl, 1% Triton X-100) containing 1 mg/ml lysozyme for 20 min on ice, and sonicated six times for 10 s. The extract was centrifuged at loo00 X g for 30 min to remove insoluble debris. Lysate #2 Cells were harvested by centrifugation and washed twice in cold PBS. The pellet was resuspended in 20 volumes of E. coli lysis buffer B (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA, 0.05% Nonidet P-40, 1 mM dithiothreitol, and 0.25 mM phenylmethylsulfonyl fluoride) and the E. coli preparation was freeze-thawed five times and followed by sonication (six times for 10 s). The extract was centrifuged at 10000 X g for 30 min to remove insoluble debris. Lysate #3 The lysate was prepared as described above for the preparation of IB antigens, except after the final centrifugation step, the supematant was dialyzed overnight at 4°C in distilled water using cellulose membrane with a 12-14 kDa molecular mass cut off (Spectra/Par, Houston, TX) to reduce the concentration of SDS and mercaptoethanol. Lysate #4 The lysate was performed as described in antigen preparation for IB. Depletion protocols. Human serum (4 ~1) was diluted in 180 ~1 of PBS containing 0.05% Tween 20 (PBS-T) and mixed with 20 ,ul lysate # 1, #2, or #3. Each mixture was incubated with a gentle rotation at 4°C for 2 h and subsequently centrifuged at 10 000 X g for 5 min; 100 ~1 of supematant was diluted l/2 (v/v) with 10% non-fat milk in PBS-T and used in IB analysis at a final serum dilution of l/100. The depletion protocol using lysate #4 was performed using nitrocellulose strips to which the E. coli lysate was separated by a 15% gel and electrotransferred as described above. The gel was overloaded with ten-fold higher amount (50 mg) of E. coli lysate proteins in order to increase the efficiency of antibody depletion when the corresponding nitro-
G. Cooini et al./Journal
A kD
of Immunological
12345
Fig. 1. A: immunoblotting analysis of a human serum containing antibody to cyclin Bl using different bacterial lysates to deplete anti-E. coli antibodies. Whole bacterial lysate expressing cyclin B 1 was used as antigen substrate in IB. The arrow indicates the 62 kDa cyclin Bl reactivity. Nitrocellulose strips were probed with unabsorbed serum (lane I), lysate #I absorbed serum (lane 21, lysatc #2 absorbed serum (lane 3). lysate #3 absorbed serum (lane 4). or serum absorbed with E. coli proteins transferred to nitrocellulose (lane 5). The efficiency of bacterial lysis was different for the different E. coli lysates; intact bacteria were present in lysate #l (C) and lysate #2 CD), and complete E. coli solubilization was obtained only for lysate #3 (D) and #4 (data not shown). In B, untreated E. coli are shown.
cellulose strips were used as absorption substrate. Nitrocellulose strip was blocked with 5% milk in PBS-T for 30 min and incubated with the serum diluted at l/ 100 in the same blocking buffer for 1 h. The absorbed serum was recovered and used in IB. Fig. 1 shows the comparison of reactivities between unabsorbed serum and sera absorbed or depleted by the four described protocols. Untreated serum (lane 1) showed reactivities to different E. coli proteins, making it difficult to interpret the reactivity to cyclin Bl (arrow). When compared with the whole serum, each protocol showed different degrees of reduction in reactivity with bacterial antigens. Using lysate #I (lane 2) and #2 (lane 3), only some E. coli antibodies were depleted because the serum strongly reacted with a number of E. coli polypeptides. Significant reduction of reactivities to other bacterial proteins were observed using lysate #3 (lane 4). However, only by using lysate #4 transferred to nitrocellulose strips was it possible to achieve a 100% absorption (lane 5). It was important to show that the absorption of serum with nitrocellulose did not affect the reactivity with recombinant cyclin B 1, showing the high efficiency of this method
Methods 190 (1996) 143-145
145
in depleting anti-E. coli antibody. The differences among the 4 protocols could be explained by the presence of different bacterial protein concentrations or conformations in each lysate. In fact, when the lysis of E. coli was monitored in light microscopy, many bacteria remained in lysates #l (Fig. lC> and #2 (Fig. 1D) compared to untreated control (Fig. lB), whereas complete solubilization of E. cofi proteins was observed in lysates #3 and 4 (Fig. 1E). Using nitrocellulose strips as absorbent, depletion was complete because E. coli antigens were presented in the same conditions as for IB. Protocol #4 represents a highly complete depletion of anti-E. coli antibodies while protocols using lysates # 1, #2, and #3 only generated competition for antigen binding. This important consideration explains also the difference in results obtained with protocol #3 and #4 in which the bacterial antigens were prepared with the same technique but presented in two different ways - protocol #3 for antigen binding competition and protocol #4 for depletion. In summary, protocol #3 is recommended for the depletion of anti-E. coli antibodies from human sera since it is relatively easy, effective, inexpensive and could be suitable for the majority of sera. If the depletion is incomplete, repeat experiment using protocol #4 (depletion with nitrocellulose strips) is recommended. Each antiserum has its characteristic reactivities to bacterial components; for sera with high level of anti-E. cob antibodies, a second depletion cycle using protocol #4 may become necessary to remove all anti-E. coli reactivities completely.
References than, E.K.L. and Pollard, K.M. (1992) Autoantibodies
to ribonocleoprotein particles by immunoblotting. In: N.R. Rose, E.C. de Macario, J.L. Fahey, H. Friedman and G.M. Penn (Eds.), Manual of Clinical Laboratory Immunology, Vol. 4. American Society for Microbiology. Washington, DC, p. 755. Laemmli. U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680. Sambrook, J.. Fritsch, E.F. and Maniatis, T. (1989) Plasmid vectors. In: N. Ford, C. Nolan and M. Ferguson (Eds.), Molecular Cloning, Vol. II. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p. 1.1. Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Prcc. Natl. Acad. Sci. USA 76, 4350.
ELSEVIER
Journal of Immunological Methods 190 ( 1996) 143- 145
Short communication
Comparison of protocols for depleting anti-25 coli antibody in immunoblotting of recombinant antigens ’ Giovanni Covini a*b,Edward K.L. Chan a, Massimo Colombo b, E.M. Tan a** a W.M. Keck Autoimmune Disease Center, Deparhnent of Molecular and Experimental Medicine, The Scripps Research Institute. LA Jolla. CA 9203, USA b Istituto di Medicina Interna. Via Pace 9, 20122 Milan, Italy
Received 3 October 1995; accepted 11 December 1995
Abstract A common problem in immunoblotting for the detection of specific antibodies to recombinant proteins synthesized in E. is the presence of contaminating antibodies to E. cofi proteins. Four protocols for the efficient depletion of anti-E. coli antibodies from human sera are provided and their uses are discussed.
coli
Keywords:
Anti-E. coli antibody depletion; Autoantibody; lmmunoblotting
Immunoblotting (IB) analysis is an important technique for characterizing the specificity of antigen-antibody system. The basic IB method consists of the electrophoretic separation of a protein mixture and the electrotransfer to nitrocellulose (Towbin et al., 1979; Chan and Pollard, 1992). The nitrocellulose is cut into strips which are then incubated with different diluted sera. The reactivities of the sera are detected with radiolabeled probe or enzyme-conjugated secondary antibody and dye or chemiluminant substrates (Ghan and Pollard, 1992).
Abbreviations: PBS, phosphate buffered saline; PBS-T, PBS containing 0.05% Tween 20; IB, immunoblotting; PAGE, polyacrylamide gel electrophoresis; L.SB, Laemmli’s sample buffer. * Corresponding author. Tel.: 619-554-8686; Fax: 619-5546805. ’ This is publication %13-MEM from The Scripps Research Institute. ‘Ihis work is supportedby National Institutes of Health Grants CA 56956 and AR 41803 and by Associazione Italiana COPE!!. 0022-1759/%/S
In the past few years we have analyzed by IB a large number of sera from patients with different diseases to detect antibodies to recombinant proteins expressed in E. coli. IB analysis is also employed to detect the presence of antibodies after immunization with peptides in experimental animals. Using whole E. coli lysate expressing the recombinant protein as antigen source for IB, a common problem was the presence of additional reactivities of the sera with E. coli antigens. Since E. coli is physiologically present in intestinal flora, anti-E. coli antibodies are often responsible for the high background and false positivity in IB. A way to improve the results could be the use of purified recombinant proteins as antigen sources for IB analysis but purification techniques are laborious, expensive and sometimes it is not possible to have a 100% pure protein without contamination of other E. coli proteins. A simple protocol for the complete depletion of E. coli antibodies is essential because it permits IB analysis of sera without using pure recombinant pro-
15.00 0 1996 Elsevier Science B.V. All rights reserved
SSDl 0022-1759(95)00288-X
144
G.
Covini
et al./Journd
of Immunological
teins. Initially, we tried to deplete the sera with commercial bacteria lysate without satisfactory results because anti-E. coli protein reactivities were still present. Different methods for the preparation of E. coli lysates have been described, and the most common protocols use a combination of lysis buffers with lysozyme treatment, sonication, or freeze-tbawing (Sambrook et al., 1989). We decided to compare the depletion of anti-E. coli antibodies from sera using bacterial lysates obtained by these standardized lysis protocols. The efficiency in the lysis of E. coli was monitored by light microscopy. In this report we describe the comparison of two preparations of bacterial lysates and our methods for depleting anti-E. co/i antibodies in a representative human serum from a patient with liver cancer containing antibodies to cyclin Bl to demonstrate that we have established protocols for the successful depletion of anti-e. coli antibodies. Antigen preparation for IB. Antigen source for IB was obtained using E. cofi BL21 (DE3) lysate carrying plasmid pK171 encoding cyclin B 1 (kindly provided by Steve Reed, The Scripps Research Institute). Bacteria were grown with standard culture technique. Briefly, 5 ml of LB medium (IO g tryptone, 5 g yeast extract, 5 g NaCl in 1 L of distilled water) were inoculated with a single bacterial colony and grown overnight at 37°C with vigorous agitation (200 rpm). Cells were collected by centrifugation and resuspended in 50 ml of LB medium for continuous culture. When the culture OD,, was about 0.6, isopropyl-P-thiogalactopyranoside was added to a final concentration of 5 mM. The cells were further cultured for 4 h. Cells were collected by centrifugation and resuspended in 3 ml of TG buffer (10 mM Tris pH 6.9, 10% glycerol) and solubilized in 3.5 ml 2 X Laemmli’s sample buffer (LSB) (Laemmli, 1970) for every 500 ~1 of E. coli pellet. The lysate was boiled for 3 min. and centrifuged at 10000 X g for 30 min. to remove insoluble debris. For the detection of antibody to cyclin Bl, a 15% slab gel of 15 X 15 X 0.15 cm (h X 1 X w) was loaded with 0.5 mg of protein supematant and IB was performed as described (Ghan and Pollard, 1992). Antigen sources for absorption. E. coli BL21 (DE3) without plasmid pK171 was grown in LB medium overnight for the preparation of four different lysates as bacterial antigens for absorption.
Methods
Lysate
190
#l
(1996)
143-145
Cells were harvested by centrifugation and washed twice in cold phosphatebuffered saline (PBS), resuspended in 20 volumes of the cell pellet in E. coli lysis buffer A (20 mM phosphate pH 8.0, 150 mM NaCl, 1% Triton X-100) containing 1 mg/ml lysozyme for 20 min on ice, and sonicated six times for 10 s. The extract was centrifuged at loo00 X g for 30 min to remove insoluble debris. Lysate #2 Cells were harvested by centrifugation and washed twice in cold PBS. The pellet was resuspended in 20 volumes of E. coli lysis buffer B (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA, 0.05% Nonidet P-40, 1 mM dithiothreitol, and 0.25 mM phenylmethylsulfonyl fluoride) and the E. coli preparation was freeze-thawed five times and followed by sonication (six times for 10 s). The extract was centrifuged at 10000 X g for 30 min to remove insoluble debris. Lysate #3 The lysate was prepared as described above for the preparation of IB antigens, except after the final centrifugation step, the supematant was dialyzed overnight at 4°C in distilled water using cellulose membrane with a 12-14 kDa molecular mass cut off (Spectra/Par, Houston, TX) to reduce the concentration of SDS and mercaptoethanol. Lysate #4 The lysate was performed as described in antigen preparation for IB. Depletion protocols. Human serum (4 ~1) was diluted in 180 ~1 of PBS containing 0.05% Tween 20 (PBS-T) and mixed with 20 ,ul lysate # 1, #2, or #3. Each mixture was incubated with a gentle rotation at 4°C for 2 h and subsequently centrifuged at 10 000 X g for 5 min; 100 ~1 of supematant was diluted l/2 (v/v) with 10% non-fat milk in PBS-T and used in IB analysis at a final serum dilution of l/100. The depletion protocol using lysate #4 was performed using nitrocellulose strips to which the E. coli lysate was separated by a 15% gel and electrotransferred as described above. The gel was overloaded with ten-fold higher amount (50 mg) of E. coli lysate proteins in order to increase the efficiency of antibody depletion when the corresponding nitro-
G. Cooini et al./Journal
A kD
of Immunological
12345
Fig. 1. A: immunoblotting analysis of a human serum containing antibody to cyclin Bl using different bacterial lysates to deplete anti-E. coli antibodies. Whole bacterial lysate expressing cyclin B 1 was used as antigen substrate in IB. The arrow indicates the 62 kDa cyclin Bl reactivity. Nitrocellulose strips were probed with unabsorbed serum (lane I), lysate #I absorbed serum (lane 21, lysatc #2 absorbed serum (lane 3). lysate #3 absorbed serum (lane 4). or serum absorbed with E. coli proteins transferred to nitrocellulose (lane 5). The efficiency of bacterial lysis was different for the different E. coli lysates; intact bacteria were present in lysate #l (C) and lysate #2 CD), and complete E. coli solubilization was obtained only for lysate #3 (D) and #4 (data not shown). In B, untreated E. coli are shown.
cellulose strips were used as absorption substrate. Nitrocellulose strip was blocked with 5% milk in PBS-T for 30 min and incubated with the serum diluted at l/ 100 in the same blocking buffer for 1 h. The absorbed serum was recovered and used in IB. Fig. 1 shows the comparison of reactivities between unabsorbed serum and sera absorbed or depleted by the four described protocols. Untreated serum (lane 1) showed reactivities to different E. coli proteins, making it difficult to interpret the reactivity to cyclin Bl (arrow). When compared with the whole serum, each protocol showed different degrees of reduction in reactivity with bacterial antigens. Using lysate #I (lane 2) and #2 (lane 3), only some E. coli antibodies were depleted because the serum strongly reacted with a number of E. coli polypeptides. Significant reduction of reactivities to other bacterial proteins were observed using lysate #3 (lane 4). However, only by using lysate #4 transferred to nitrocellulose strips was it possible to achieve a 100% absorption (lane 5). It was important to show that the absorption of serum with nitrocellulose did not affect the reactivity with recombinant cyclin B 1, showing the high efficiency of this method
Methods 190 (1996) 143-145
145
in depleting anti-E. coli antibody. The differences among the 4 protocols could be explained by the presence of different bacterial protein concentrations or conformations in each lysate. In fact, when the lysis of E. coli was monitored in light microscopy, many bacteria remained in lysates #l (Fig. lC> and #2 (Fig. 1D) compared to untreated control (Fig. lB), whereas complete solubilization of E. cofi proteins was observed in lysates #3 and 4 (Fig. 1E). Using nitrocellulose strips as absorbent, depletion was complete because E. coli antigens were presented in the same conditions as for IB. Protocol #4 represents a highly complete depletion of anti-E. coli antibodies while protocols using lysates # 1, #2, and #3 only generated competition for antigen binding. This important consideration explains also the difference in results obtained with protocol #3 and #4 in which the bacterial antigens were prepared with the same technique but presented in two different ways - protocol #3 for antigen binding competition and protocol #4 for depletion. In summary, protocol #3 is recommended for the depletion of anti-E. coli antibodies from human sera since it is relatively easy, effective, inexpensive and could be suitable for the majority of sera. If the depletion is incomplete, repeat experiment using protocol #4 (depletion with nitrocellulose strips) is recommended. Each antiserum has its characteristic reactivities to bacterial components; for sera with high level of anti-E. cob antibodies, a second depletion cycle using protocol #4 may become necessary to remove all anti-E. coli reactivities completely.
References than, E.K.L. and Pollard, K.M. (1992) Autoantibodies
to ribonocleoprotein particles by immunoblotting. In: N.R. Rose, E.C. de Macario, J.L. Fahey, H. Friedman and G.M. Penn (Eds.), Manual of Clinical Laboratory Immunology, Vol. 4. American Society for Microbiology. Washington, DC, p. 755. Laemmli. U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680. Sambrook, J.. Fritsch, E.F. and Maniatis, T. (1989) Plasmid vectors. In: N. Ford, C. Nolan and M. Ferguson (Eds.), Molecular Cloning, Vol. II. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p. 1.1. Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Prcc. Natl. Acad. Sci. USA 76, 4350.