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Immunoblotting and related techniques
THE JOURNAL OF
ALLERGY AND
CLINICAL
IMMUNOLOGY
VOLUME 76
NUMBER 4
Editorial lmmunoblotting
and related techniques
In this issue of THE JOURNALOF ALLERGY AND CLINICALIMMUNOLOGY, page 537,anarticleby Dewair et al. describes the detection of IgG and IgE binding to specific antigenic componentsamong mixtures of silk proteins. They used a technique called immunoblotting in which the mixture of proteins was resolved by SDS-polyactylamide gel electrophoresis, and then the separatedproteins were transferred to nitrocellulose. Patient seracontaining antibodieswere allowed to react with the nitrocellulose-boundproteins andthe presenceand isotype of reacting antibody were revealed by radioiodinated anti-isotype antiglobulin. This powerful technique is one of the several “blotting” methods now finding use across the face of bioscience. “Blotting” came into the current lexicon of scientific methodology with the 1975 article of E. M. Southern.’ This article described a method of identifying specific base sequencesin DNA fragments by hybridization with radiolabeledRNA. In the technique fragmentswere madeby digestion of large DNA molecules such as Escherichia coli genomic DNA, and the fragments were separatedby electrophoresis in agarosegels. The fragmentswere then transferredto nitrocellulose paper by capillarity. The flow of buffer was directed by placing several layers of dry filter paper on one side of the nitrocellulose, whereasthe gel with the DNA fragments was in contact with the other side. Thus, the fragments were drawn by the flow of the buffer from the gel onto the nitrocellulose where they were immobilized asa result of the surface properties of the paper. The secondstep of the technique was a hybridization in which radiolabeled RNA specieswere reacted with the transferred and immobilized DNA, and finally, the position of the RNADNA hybrids was revealedby autoradiographyor fluorography.
The techniquerapidly found wide application in the mapping of DNA molecules. The article by Alwine et al.* described a method of covalently attaching RNA (or DNA) molecules to diazobenzyloxymethyl paper to detect complementary sequencesof DNA. This constituted an opposite configuration from the Southern procedure in which the transferred DNA fragments were immobilized. To emphasizethe opposite nature of the technique, the procedure was called a “Northern” blot. The two methodshave become major factors in the rapid progressin molecular biology and genetic engineering. Bumette3 extended the geographic metaphor by calling the electrophoretic transfer of proteins from SDS-polyacrylamide gels to nitrocellulose with subsequent immunologic localization of the transferred species“Western” blotting. The Bumette article described the extension of a method applied by Towbin et a1.4to the analysis of protein speciesseparatedby electrophoresisin polyacrylamide-urea gels. Towbin et al. transferred separatedproteins to nitrocellulose by migration in an electric field applied perpendicular to the long surface of the separating gel, thus generating a replica of the gel pattern. They used direct protein staining methods as well as immunologic methods to visualize the transferred species on the nitrocellulose. Bumette’s work was the development of the method as applied to SDS-polyacrylamide gel separations.The latter procedure is very widely used and allows the separationof proteins on the basis of molecular weight.5 In his article he documentedthat the time required for electrophoretic transfer of the 527
528
J. ALLERGY
Flanagan
SDS-protein complexes to the nitrocellulose is dependent on the molecular weight. Large proteins are transferred more slowly than small proteins under conditions of constant voltage. Bumette also applied the method to analysis of two-dimensional separations in which complex protein mixtures were subjected to isoelectric focusing, and the resulting separated species were further resolved by SDS-polyacrylamide gel electrophoresis. He used immunologic methods to identify transferred proteins on the nitrocellulose paper as had Towbin et al. However, Bumette used radiolabeled antiglobulins and staphylococcal protein A to localize the antibody-antigen reactions; whereas Towbin et al. had used fluorescein and peroxidaseconjugated antiglobulins. The immunoblot technique developed from several antecedent procedures. Immunoelectrophoresis described by Grabar and Williams6 in 1953 was perhaps the “grandparent” method. It made use of the principle of electrophoretic separation of protein molecules and subsequent localization of different antigenie species by immune precipitation in gel. Showe et al.’ implemented electrophoretic migration in SDSpolyacrylamide gel to separate proteins and then allowed them to diffuse into an antiserum-containing agar gel overlay wherein immune precipitation occurred. They termed the technique “immune replication. ” The technique, although it was elegant in concept, suffered from problems of distortion, making analysis of complex mixtures very difficult. Towbin et al. recognized a need to immobilize the separated species in a way to preserve the pattern of the electrophoregram. They cited the work of Southern’ for the immobilization of nucleic acid and that of Broome and Gilbert* for methods involving immobilization of protein antigens. The latter workers described a method of adsorbing IgG antibodies to polyvinyl disks and using the disks to detect translation products in lysed bacterial colonies. The procedure was essentially a sandwich technique in which the captured antibody selected the antigen from the lysate, and, after washing, the presence of the antigen was demonstrated by reaction with a radiolabeled antibody and autoradiography. In principle, it immobilized antigen on a solid substrate followed by localization by immunologic means. Most published articles demonstrating the technique used radiolabeled antiglobulins, often isotypespecific, and/or staphylococcal protein A. The isotope lZ51is most frequently used as the radiolabel. Several authors have commented on the advantages of an enzyme-labeling technique in place of radioisotopes. In this regard efforts have been made to adapt this
CLIN. IMMUNOL. OCTOBER 1985
method to the immunoblot procedure. Knecht and Dimond’ used alkaline phosphatase conjugated to antiglobulin in conjunction with the histochemical substrate 5bromo-4 chloro-3 indolyl phosphate that was incorporated into agar gel that was overlayed on the nitrocellulose after reaction with the conjugate. The blue reaction product formed in the gel and could be preserved by drying of the gel. This method was exquisitely sensitive, detecting antigen in nanogram amounts. Blake et al.‘” using the same enzyme-substrate system described a method of directly staining the nitrocellulose strips. The same degree of sensitivity was achieved. More recently, a number of improvements and special applications of the technique have been reported. Among these are amplification systems such as the avidin-biotin system. In conclusion, a thought or two on the appropriateness of carrying on the jargon of “geographic” designations. One could envisage the determination of the antibody specificities in oligoclonal IgG in cerebrospinal fluid by their ability to bind antigen after electrophoretic separation in gel and transfer to nitrocellulose. Would such a test configuration be an “Eastern” blot? Or, worse yet, would detection of anti-DNA antibodies on immobilized DNA be a “Southwestern” blot? In my opinion, the needs for clear unambiguous communication in science are paramount. Although we may enjoy the game that has developed around the naming of these techniques, it is still encumbent on the scientific establishment to maintain the literature as free as possible from jargon and the ambiguity it inevitably engenders. The responsibility primarily lies with each investigator not to allow the current “buzz word” of the day to enter into his or her articles. Moreover, it is the responstbility of editors and reviewers to be vigilant in protecting the written record of science from this possible source of error. Thomas D. Flanagan Department of Microbiology State University of New York at Buffalo Buffalo, NY 14214 REFERENCES 1. Southern EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol98503, 1975 2. Alwine JC, Kemp DJ, Stark GR: Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Nat1 Acad Sci USA 74:5350, 1977 3. Burnette WN: “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to
VOLUME NUMBER
76 4
unmodified nitrocellulose and radiographic detection with antibody and radioiodinated Protein A. Anal Biochem 112: 195, 1981 4. Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Nat1 Acad Sci USA 764350. 1979 5. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680, 1970 6. Grabar P. Williams CJ Jr: Methods permettant 1‘etude conjugee des proprietes Clectrophoretiques et immunochimiques d’un melange des proteines: application au serum sanguin. Biochim Biophys Acta 10:193, 1953
lmmunoblotting
and related
techniques
529
7. Showe MK, Isobe E, Onorato L: Bacteriophage T4 prehead proteinase. II. Its cleavage from the product of gene 21 and regulation in phage-infected cells. J Mol Biol 107:55, 1976 8. Broome S, Gilbert W: Immunological screening method to detect specific translation products. Proc Nat1 Acad Sci USA 7512746. 1978 9. Knecht DA, Dimond RL: Visualization of antigenic proteins on western blots. Anal Biochem 136:180, 1984 10. Blake MS, Johnston KH, Russell-Jones GJ, Gotschlich EC: A rapid. sensitive method for the detection of alkaline phosphatase-conjugated anti-antibody on western blots. Anal Biochem 136:175, 1984
ALLERGY AND
CLINICAL
IMMUNOLOGY
VOLUME 76
NUMBER 4
Editorial lmmunoblotting
and related techniques
In this issue of THE JOURNALOF ALLERGY AND CLINICALIMMUNOLOGY, page 537,anarticleby Dewair et al. describes the detection of IgG and IgE binding to specific antigenic componentsamong mixtures of silk proteins. They used a technique called immunoblotting in which the mixture of proteins was resolved by SDS-polyactylamide gel electrophoresis, and then the separatedproteins were transferred to nitrocellulose. Patient seracontaining antibodieswere allowed to react with the nitrocellulose-boundproteins andthe presenceand isotype of reacting antibody were revealed by radioiodinated anti-isotype antiglobulin. This powerful technique is one of the several “blotting” methods now finding use across the face of bioscience. “Blotting” came into the current lexicon of scientific methodology with the 1975 article of E. M. Southern.’ This article described a method of identifying specific base sequencesin DNA fragments by hybridization with radiolabeledRNA. In the technique fragmentswere madeby digestion of large DNA molecules such as Escherichia coli genomic DNA, and the fragments were separatedby electrophoresis in agarosegels. The fragmentswere then transferredto nitrocellulose paper by capillarity. The flow of buffer was directed by placing several layers of dry filter paper on one side of the nitrocellulose, whereasthe gel with the DNA fragments was in contact with the other side. Thus, the fragments were drawn by the flow of the buffer from the gel onto the nitrocellulose where they were immobilized asa result of the surface properties of the paper. The secondstep of the technique was a hybridization in which radiolabeled RNA specieswere reacted with the transferred and immobilized DNA, and finally, the position of the RNADNA hybrids was revealedby autoradiographyor fluorography.
The techniquerapidly found wide application in the mapping of DNA molecules. The article by Alwine et al.* described a method of covalently attaching RNA (or DNA) molecules to diazobenzyloxymethyl paper to detect complementary sequencesof DNA. This constituted an opposite configuration from the Southern procedure in which the transferred DNA fragments were immobilized. To emphasizethe opposite nature of the technique, the procedure was called a “Northern” blot. The two methodshave become major factors in the rapid progressin molecular biology and genetic engineering. Bumette3 extended the geographic metaphor by calling the electrophoretic transfer of proteins from SDS-polyacrylamide gels to nitrocellulose with subsequent immunologic localization of the transferred species“Western” blotting. The Bumette article described the extension of a method applied by Towbin et a1.4to the analysis of protein speciesseparatedby electrophoresisin polyacrylamide-urea gels. Towbin et al. transferred separatedproteins to nitrocellulose by migration in an electric field applied perpendicular to the long surface of the separating gel, thus generating a replica of the gel pattern. They used direct protein staining methods as well as immunologic methods to visualize the transferred species on the nitrocellulose. Bumette’s work was the development of the method as applied to SDS-polyacrylamide gel separations.The latter procedure is very widely used and allows the separationof proteins on the basis of molecular weight.5 In his article he documentedthat the time required for electrophoretic transfer of the 527
528
J. ALLERGY
Flanagan
SDS-protein complexes to the nitrocellulose is dependent on the molecular weight. Large proteins are transferred more slowly than small proteins under conditions of constant voltage. Bumette also applied the method to analysis of two-dimensional separations in which complex protein mixtures were subjected to isoelectric focusing, and the resulting separated species were further resolved by SDS-polyacrylamide gel electrophoresis. He used immunologic methods to identify transferred proteins on the nitrocellulose paper as had Towbin et al. However, Bumette used radiolabeled antiglobulins and staphylococcal protein A to localize the antibody-antigen reactions; whereas Towbin et al. had used fluorescein and peroxidaseconjugated antiglobulins. The immunoblot technique developed from several antecedent procedures. Immunoelectrophoresis described by Grabar and Williams6 in 1953 was perhaps the “grandparent” method. It made use of the principle of electrophoretic separation of protein molecules and subsequent localization of different antigenie species by immune precipitation in gel. Showe et al.’ implemented electrophoretic migration in SDSpolyacrylamide gel to separate proteins and then allowed them to diffuse into an antiserum-containing agar gel overlay wherein immune precipitation occurred. They termed the technique “immune replication. ” The technique, although it was elegant in concept, suffered from problems of distortion, making analysis of complex mixtures very difficult. Towbin et al. recognized a need to immobilize the separated species in a way to preserve the pattern of the electrophoregram. They cited the work of Southern’ for the immobilization of nucleic acid and that of Broome and Gilbert* for methods involving immobilization of protein antigens. The latter workers described a method of adsorbing IgG antibodies to polyvinyl disks and using the disks to detect translation products in lysed bacterial colonies. The procedure was essentially a sandwich technique in which the captured antibody selected the antigen from the lysate, and, after washing, the presence of the antigen was demonstrated by reaction with a radiolabeled antibody and autoradiography. In principle, it immobilized antigen on a solid substrate followed by localization by immunologic means. Most published articles demonstrating the technique used radiolabeled antiglobulins, often isotypespecific, and/or staphylococcal protein A. The isotope lZ51is most frequently used as the radiolabel. Several authors have commented on the advantages of an enzyme-labeling technique in place of radioisotopes. In this regard efforts have been made to adapt this
CLIN. IMMUNOL. OCTOBER 1985
method to the immunoblot procedure. Knecht and Dimond’ used alkaline phosphatase conjugated to antiglobulin in conjunction with the histochemical substrate 5bromo-4 chloro-3 indolyl phosphate that was incorporated into agar gel that was overlayed on the nitrocellulose after reaction with the conjugate. The blue reaction product formed in the gel and could be preserved by drying of the gel. This method was exquisitely sensitive, detecting antigen in nanogram amounts. Blake et al.‘” using the same enzyme-substrate system described a method of directly staining the nitrocellulose strips. The same degree of sensitivity was achieved. More recently, a number of improvements and special applications of the technique have been reported. Among these are amplification systems such as the avidin-biotin system. In conclusion, a thought or two on the appropriateness of carrying on the jargon of “geographic” designations. One could envisage the determination of the antibody specificities in oligoclonal IgG in cerebrospinal fluid by their ability to bind antigen after electrophoretic separation in gel and transfer to nitrocellulose. Would such a test configuration be an “Eastern” blot? Or, worse yet, would detection of anti-DNA antibodies on immobilized DNA be a “Southwestern” blot? In my opinion, the needs for clear unambiguous communication in science are paramount. Although we may enjoy the game that has developed around the naming of these techniques, it is still encumbent on the scientific establishment to maintain the literature as free as possible from jargon and the ambiguity it inevitably engenders. The responsibility primarily lies with each investigator not to allow the current “buzz word” of the day to enter into his or her articles. Moreover, it is the responstbility of editors and reviewers to be vigilant in protecting the written record of science from this possible source of error. Thomas D. Flanagan Department of Microbiology State University of New York at Buffalo Buffalo, NY 14214 REFERENCES 1. Southern EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol98503, 1975 2. Alwine JC, Kemp DJ, Stark GR: Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Nat1 Acad Sci USA 74:5350, 1977 3. Burnette WN: “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to
VOLUME NUMBER
76 4
unmodified nitrocellulose and radiographic detection with antibody and radioiodinated Protein A. Anal Biochem 112: 195, 1981 4. Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Nat1 Acad Sci USA 764350. 1979 5. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680, 1970 6. Grabar P. Williams CJ Jr: Methods permettant 1‘etude conjugee des proprietes Clectrophoretiques et immunochimiques d’un melange des proteines: application au serum sanguin. Biochim Biophys Acta 10:193, 1953
lmmunoblotting
and related
techniques
529
7. Showe MK, Isobe E, Onorato L: Bacteriophage T4 prehead proteinase. II. Its cleavage from the product of gene 21 and regulation in phage-infected cells. J Mol Biol 107:55, 1976 8. Broome S, Gilbert W: Immunological screening method to detect specific translation products. Proc Nat1 Acad Sci USA 7512746. 1978 9. Knecht DA, Dimond RL: Visualization of antigenic proteins on western blots. Anal Biochem 136:180, 1984 10. Blake MS, Johnston KH, Russell-Jones GJ, Gotschlich EC: A rapid. sensitive method for the detection of alkaline phosphatase-conjugated anti-antibody on western blots. Anal Biochem 136:175, 1984