This chapter provides an overview of affinity chromatography. It is a method of fractionation whi... more This chapter provides an overview of affinity chromatography. It is a method of fractionation which exploits the biospecific binding of a particular molecule to a second molecule, often termed the “ligand”. The technique is extremely powerful. Purification factors of 2,000-20,000-fold are often possible, and it is sometimes possible to achieve purification to homogeneity in a single step. Affinity chromatography is a very common method for purification of low-concentration proteins, and the eluted protein is well suited for amino acid sequencing. The advent of monoclonal antibodies has revolutionized affinity chromatography. The presence of a charged linkage tends to cause nonspecific ionexchange effects which could degrade the specificity of the gel when used for affinity chromatography. This ion-exchange effect is most pronounced at very low salt concentrations, and it is overcome by performing the chromatography in buffers of high salt concentration. Affinity chromatography on antibody columns is divided into five equally important phases: sample preparation, precycling, binding, washing, and elution.
Vaccination is one of the most cost-effective means of controlling and eradicating infectious dis... more Vaccination is one of the most cost-effective means of controlling and eradicating infectious disease but at present there is no vaccine against any human parasitic disease. Developments in gene cloning have focused attention on protein antigen vaccines and have opened the way for their large-scale production. There is, however, another class of non-clonable, biologically important molecules, the complex carbohydrates. In this review Emanuela Handman and her colleagues examine the structure and function of carbohydrate antigens of Leishmania, and their possible role in the search for a vaccine against this organism.
7S immunoglobulins of a monotreme mammal, the echidna Tachyglossus aculeatus, bind to Staphylococ... more 7S immunoglobulins of a monotreme mammal, the echidna Tachyglossus aculeatus, bind to Staphylococcus aureus A protein coupled to an insoluble matrix. This binding supports the homology between the previously described echidna IgG and IgG molecules of higher mammals and provides a rapid, simple method for the isolation of this protein from echidna serum. In addition, another echidna 7S immunoglobulin became bound to protein A-Sepharose. The major protein A-binding immunoglobulin has a slow electrophoretic mobility and possesses a heavy chain comparable in mass to typical mammalian γ chains (52,000 daltons). We suggest the designation IgG2 for this immunoglobulin. The minor protein A-binding immunoglobulin is electrophoretically faster than IgG2 and can be resolved from the former by gradient elution from DEAE-Biogel. We propose the nomenclature IgG1 for this molecule although we cannot discount the possibility that it might represent another main class such as IgA. The γ1 heavy chain is distinguishable from the γ2 chain on polyacrylamide gel electrophoresis in sodium dodecylsulphate containing buffers. The γ1 chain possesses a nominal mass of 61,000 daltons. The intact molecule has an apparent mass of 177,000 Daltons and comprises two pairs of light and heavy chains.
International journal of peptide & protein research, Jan 12, 2009
A procedure for the purification of subnanomole levels of polypeptides has been developed. Revers... more A procedure for the purification of subnanomole levels of polypeptides has been developed. Reversed-phase high performance liquid chromatography on short (10 cm or less) microbore (1-2 mm internal diameter) columns has been used to fractionate and purify a number of tryptic peptides generated from approximately 600 pmol of purified murine plasma cell antigen PC-1, a major membrane glycoprotein on all cells secreting immunoglobulins. The use of reversed-phase microbore columns permits the recovery of subnanomole amounts of polypeptides from large volumes in high yield (greater than 90%) and in small eluent volumes (40-60 microL) which can be loaded directly onto the gas-phase sequencer without further concentration. This procedure avoids the severe sample loss which frequently occurs with other concentration procedures such as lyophilization and evaporation. The use of a photodiode-array detector for identifying tryptophan-containing peptides from on-the-fly, ultraviolet spectra is described. This procedure permits the selection of tryptophan-containing peptides from complex tryptic digests for use as candidate peptides for oligonucleotide probe construction. Automated Edman degradation was performed on seven tryptic peptides, yielding 110 unique assignments; this corresponds to approximately 11% of the molecule.
PC-1 is a membrane glycoprotein, found on the surface of plasma cells and a few types of nonlymph... more PC-1 is a membrane glycoprotein, found on the surface of plasma cells and a few types of nonlymphoid cells, which has recently been found to have 5'-nucleotide phosphodiesterase activity. In this paper, we demonstrate the existence of enzymically active water-soluble forms of PC-1 in ascites from plasmacytoma-bearing mice, normal mouse serum, and in supernatants of cultured mouse plasmacytoma cells and mouse L cells transfected with a cDNA encoding the membrane form of PC-1. The water-soluble enzyme activity can be specifically immunoprecipitated by a monoclonal antibody to an allotypic determinant on the membrane form of PC-1, and resides on a slightly smaller polypeptide than membrane PC-1. Biosynthetic studies revealed a single, monomeric, endoglycosidase-H-sensitive membrane PC-1 precursor, which was gradually converted to a disulphide-bonded, endoglycosidase-H-resistant form over a period of about 2 h. Soluble PC-1 was first detectable in the supernatant after about 2 h. A distinct intracellular form of soluble PC-1 was not seen. The soluble form of PC-1 does not appear to arise by proteolytic cleavage from the cell surface, although cleavage inside the cell remains a possibility. When taken together with the structure of the relevant portions of PC-1 gene exons, the data suggest that the most likely site of cleavage is between Pro152 and Ala153.
Publisher Summary It is noted that much remains unknown concerning the nature of autoimmunity and... more Publisher Summary It is noted that much remains unknown concerning the nature of autoimmunity and its induction, maintenance, and effector mechanisms. Autoimmune processes can be analyzed with much greater clarity if they are divided into initiation, maintenance, and effector mechanisms. The chapter begins by considering autoantigens, which must be common to all three stages. The topic of autoimmunity has no shortage of antigens. In most cases, it is not clear whether any individual autoantigen is involved in the pathogenesis of the associated disease, or is merely an innocent bystander. A “real” autoantigen is the one wherein there is definitive evidence that autoimmune attack against it causes disease in humans, either as an initiator or a final target or both. The technology required to identify antigens that are recognized by autoantibodies is well established and relatively straightforward. In marked contrast, the identification of autoantigens recognized by T cells is very much more of a challenge. However, numerous T-cell antigens that are centrally involved in pathogenesis probably remain to be identified.
This chapter discusses monoclonal antibodies. For decades, immunologists have sought ways of prod... more This chapter discusses monoclonal antibodies. For decades, immunologists have sought ways of producing homogeneous antibodies of defined specificity. Some limited successes were achieved by screening myeloma proteins for antigen binding, but a general method did not become available until 1975, when Kohler and Milstein used cell hybridization to produce continuous cell lines secreting antibody of a hypoxanthine, aminopterin, and thymidine (HAT)-sensitive variant of MOPC-21 myeloma cells with spleen cells from mice immunized with sheep red cells. The fusion was mediated by Sendai virus, and hybrids were selected by growth in medium containing HAT. The chapter discusses some strategies for the production of human monoclonal antibodies. It shows the ways in which old uncertainties of specificity and reproducibility have been replaced by the promise of unlimited supplies of standardized, monospecific antibodies. The chapter also discusses mass and affinity of antibody in a very precise way. However, the successful use of monoclonal antibodies requires a firm understanding of the differences between conventional and monoclonal serology.
The plasma cell membrane antigen PC-1 and the receptor for the iron transport protein transferrin... more The plasma cell membrane antigen PC-1 and the receptor for the iron transport protein transferrin are high m.w., developmentally regulated proteins consisting of two similar or identical disulfide-bonded subunits. In this paper, we report the results of a serologic and biochemical analysis of these proteins in various strains of inbred mice, and in rats and hamsters. A monoclonal antibody against the PC-1a allelic product is shown to detect an antigenic determinant on the PC-1 molecule that has the same strain distribution as the antigen previously detected with polyclonal alloantisera. The mouse PC-1 protein was purified from plasma cells of the PC-1a genotype and was used to generate polyclonal rabbit anti-PC-1 antibodies. These antibodies precipitated a homologous protein from plasmacytoma cells derived from PC-1- congenic mice, demonstrating that PC-1b is not a "null" allele. The PC-1b allelic product had a slightly lower apparent m.w. than the PC-1a product, and had a slightly more basic isoelectric point. Rabbit anti-mouse PC-1 antibodies also precipitated a homologous protein from immunoglobulin-secreting cells of rat and hamster origin, but did not show detectable cross-reaction with the transferrin receptor. Disulfide bonding between chains was conserved in both PC-1 and the transferrin receptor in all species examined, but transferrin receptors from mouse cells had a significantly higher apparent m.w. than those of rat, hamster, or human cells.
Cold Spring Harbor Symposia on Quantitative Biology, 1977
In this paper, we review experiments verifying clonal selection through the use of hapten-fractio... more In this paper, we review experiments verifying clonal selection through the use of hapten-fractionated B lymphocytes of such uniformity that up to one cell in three can be induced to form an anti-hapten antibody-forming clone in vitro; the use of such cells in studies on antigen-induced receptor aggregation and modulation using immunogenic or tolerogenic antigen concentrations; application to the tolerance problem of the cloning methods devised for the study of these cells; studies of the IgM- and IgD-like receptors of hapten-specific cells; and the use of an anti-delta allotype serum to study the distribution and function of IgD-like molecules on the B-lymphocyte surface.
Australian journal of experimental biology and medical science, Apr 1, 1977
SummaryHypothymic BALB/c.nu/nu (“hude”) mice were markedly more susceptible than their intact BAL... more SummaryHypothymic BALB/c.nu/nu (“hude”) mice were markedly more susceptible than their intact BALB/c.nu/+ littermates to infection with the murine larval cestode, Mesocestoides corti, and antigens of this parasite induced T cell‐dependent immune responses in intact mice. Defective anti‐dinitrophenol (DNP) antibody responses were induced by DNP‐M. corti larvae and DNP‐human gamma globulin (HGG) in infected (parasitized) intact mice. However, on transfer to uninfected irradiated recipients, lymphoid cells from infected intact mice responded well to DNP‐M. corti larvae.By using washed peritoneal larvae from long term infected intact mice to absorb antisera directed against various Ig classes and IgG subclasses, and by analysis of acid eluates of such living larvae, large amounts of IgG1, lower amounts of IgM, trace amounts of IgG2 and IgG3 and no IgA immunoglobulins were found to be associated with the larvae. In the case of nu/nu derived larvae, IgM immunoglobulins predominated. Chronically infected intact mice contained large amounts of IgG1, immunoglobulins in their serum. The technique of lactoperoxidase‐catalysed radioiodination revealed that very few proteins were available for labelling on peritoneal larvae under conditions used for labelling of lymphoid cell surface molecules. In fact, proteins with Ig chain mobilities accounted for much of the labelled and extracted proteins detected by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Patterns of labelled and extracted proteins from nu/nu derived larvae were even simpler and no clear protein peaks were detected consistently in material from labelled, acid treated, nu/nu derived larvae. Evidence is presented that at least the IgG1 molecules associated with M. corti larvae from long term infected mice include antiparasite antibodies. Immunoglobulins associated with M. corti larvae in long term infected mice are most unlikely to be useful to the mouse in terms of preventing parasite establishment (in secondary murine hosts) or reducing the proliferative rate of larvae. However, no clear cut evidence is as yet available to answer the question of whether the M. corti associated Ig (and, in particular, IgG1 which is reported to be relatively inert with respect to direct pathogenic effects) serves the function of masking parasite antigens and protecting the established parasites from more aggressive T cell‐dependent immunological effector mechanisms.
The monoclonal antibody OKT-9 recognizes a surface protein of human lymphocytes that consists of ... more The monoclonal antibody OKT-9 recognizes a surface protein of human lymphocytes that consists of a disulfide bonded homodimer of m.w. 200,000 intact and 95,000 reduced. A similar protein is precipitated by transferrin-agarose, but not by agarose alone. Peptide mapping by limited proteolysis shows that the proteins precipitated by OKT-9 antibodies and transferrin-agarose are homologous. It is concluded that OKT-9 antibodies recognize the transferrin receptor. Expression of receptors for transferrin may be useful as a marker for activated or dividing cells.
This chapter provides an overview of affinity chromatography. It is a method of fractionation whi... more This chapter provides an overview of affinity chromatography. It is a method of fractionation which exploits the biospecific binding of a particular molecule to a second molecule, often termed the “ligand”. The technique is extremely powerful. Purification factors of 2,000-20,000-fold are often possible, and it is sometimes possible to achieve purification to homogeneity in a single step. Affinity chromatography is a very common method for purification of low-concentration proteins, and the eluted protein is well suited for amino acid sequencing. The advent of monoclonal antibodies has revolutionized affinity chromatography. The presence of a charged linkage tends to cause nonspecific ionexchange effects which could degrade the specificity of the gel when used for affinity chromatography. This ion-exchange effect is most pronounced at very low salt concentrations, and it is overcome by performing the chromatography in buffers of high salt concentration. Affinity chromatography on antibody columns is divided into five equally important phases: sample preparation, precycling, binding, washing, and elution.
Vaccination is one of the most cost-effective means of controlling and eradicating infectious dis... more Vaccination is one of the most cost-effective means of controlling and eradicating infectious disease but at present there is no vaccine against any human parasitic disease. Developments in gene cloning have focused attention on protein antigen vaccines and have opened the way for their large-scale production. There is, however, another class of non-clonable, biologically important molecules, the complex carbohydrates. In this review Emanuela Handman and her colleagues examine the structure and function of carbohydrate antigens of Leishmania, and their possible role in the search for a vaccine against this organism.
7S immunoglobulins of a monotreme mammal, the echidna Tachyglossus aculeatus, bind to Staphylococ... more 7S immunoglobulins of a monotreme mammal, the echidna Tachyglossus aculeatus, bind to Staphylococcus aureus A protein coupled to an insoluble matrix. This binding supports the homology between the previously described echidna IgG and IgG molecules of higher mammals and provides a rapid, simple method for the isolation of this protein from echidna serum. In addition, another echidna 7S immunoglobulin became bound to protein A-Sepharose. The major protein A-binding immunoglobulin has a slow electrophoretic mobility and possesses a heavy chain comparable in mass to typical mammalian γ chains (52,000 daltons). We suggest the designation IgG2 for this immunoglobulin. The minor protein A-binding immunoglobulin is electrophoretically faster than IgG2 and can be resolved from the former by gradient elution from DEAE-Biogel. We propose the nomenclature IgG1 for this molecule although we cannot discount the possibility that it might represent another main class such as IgA. The γ1 heavy chain is distinguishable from the γ2 chain on polyacrylamide gel electrophoresis in sodium dodecylsulphate containing buffers. The γ1 chain possesses a nominal mass of 61,000 daltons. The intact molecule has an apparent mass of 177,000 Daltons and comprises two pairs of light and heavy chains.
International journal of peptide & protein research, Jan 12, 2009
A procedure for the purification of subnanomole levels of polypeptides has been developed. Revers... more A procedure for the purification of subnanomole levels of polypeptides has been developed. Reversed-phase high performance liquid chromatography on short (10 cm or less) microbore (1-2 mm internal diameter) columns has been used to fractionate and purify a number of tryptic peptides generated from approximately 600 pmol of purified murine plasma cell antigen PC-1, a major membrane glycoprotein on all cells secreting immunoglobulins. The use of reversed-phase microbore columns permits the recovery of subnanomole amounts of polypeptides from large volumes in high yield (greater than 90%) and in small eluent volumes (40-60 microL) which can be loaded directly onto the gas-phase sequencer without further concentration. This procedure avoids the severe sample loss which frequently occurs with other concentration procedures such as lyophilization and evaporation. The use of a photodiode-array detector for identifying tryptophan-containing peptides from on-the-fly, ultraviolet spectra is described. This procedure permits the selection of tryptophan-containing peptides from complex tryptic digests for use as candidate peptides for oligonucleotide probe construction. Automated Edman degradation was performed on seven tryptic peptides, yielding 110 unique assignments; this corresponds to approximately 11% of the molecule.
PC-1 is a membrane glycoprotein, found on the surface of plasma cells and a few types of nonlymph... more PC-1 is a membrane glycoprotein, found on the surface of plasma cells and a few types of nonlymphoid cells, which has recently been found to have 5'-nucleotide phosphodiesterase activity. In this paper, we demonstrate the existence of enzymically active water-soluble forms of PC-1 in ascites from plasmacytoma-bearing mice, normal mouse serum, and in supernatants of cultured mouse plasmacytoma cells and mouse L cells transfected with a cDNA encoding the membrane form of PC-1. The water-soluble enzyme activity can be specifically immunoprecipitated by a monoclonal antibody to an allotypic determinant on the membrane form of PC-1, and resides on a slightly smaller polypeptide than membrane PC-1. Biosynthetic studies revealed a single, monomeric, endoglycosidase-H-sensitive membrane PC-1 precursor, which was gradually converted to a disulphide-bonded, endoglycosidase-H-resistant form over a period of about 2 h. Soluble PC-1 was first detectable in the supernatant after about 2 h. A distinct intracellular form of soluble PC-1 was not seen. The soluble form of PC-1 does not appear to arise by proteolytic cleavage from the cell surface, although cleavage inside the cell remains a possibility. When taken together with the structure of the relevant portions of PC-1 gene exons, the data suggest that the most likely site of cleavage is between Pro152 and Ala153.
Publisher Summary It is noted that much remains unknown concerning the nature of autoimmunity and... more Publisher Summary It is noted that much remains unknown concerning the nature of autoimmunity and its induction, maintenance, and effector mechanisms. Autoimmune processes can be analyzed with much greater clarity if they are divided into initiation, maintenance, and effector mechanisms. The chapter begins by considering autoantigens, which must be common to all three stages. The topic of autoimmunity has no shortage of antigens. In most cases, it is not clear whether any individual autoantigen is involved in the pathogenesis of the associated disease, or is merely an innocent bystander. A “real” autoantigen is the one wherein there is definitive evidence that autoimmune attack against it causes disease in humans, either as an initiator or a final target or both. The technology required to identify antigens that are recognized by autoantibodies is well established and relatively straightforward. In marked contrast, the identification of autoantigens recognized by T cells is very much more of a challenge. However, numerous T-cell antigens that are centrally involved in pathogenesis probably remain to be identified.
This chapter discusses monoclonal antibodies. For decades, immunologists have sought ways of prod... more This chapter discusses monoclonal antibodies. For decades, immunologists have sought ways of producing homogeneous antibodies of defined specificity. Some limited successes were achieved by screening myeloma proteins for antigen binding, but a general method did not become available until 1975, when Kohler and Milstein used cell hybridization to produce continuous cell lines secreting antibody of a hypoxanthine, aminopterin, and thymidine (HAT)-sensitive variant of MOPC-21 myeloma cells with spleen cells from mice immunized with sheep red cells. The fusion was mediated by Sendai virus, and hybrids were selected by growth in medium containing HAT. The chapter discusses some strategies for the production of human monoclonal antibodies. It shows the ways in which old uncertainties of specificity and reproducibility have been replaced by the promise of unlimited supplies of standardized, monospecific antibodies. The chapter also discusses mass and affinity of antibody in a very precise way. However, the successful use of monoclonal antibodies requires a firm understanding of the differences between conventional and monoclonal serology.
The plasma cell membrane antigen PC-1 and the receptor for the iron transport protein transferrin... more The plasma cell membrane antigen PC-1 and the receptor for the iron transport protein transferrin are high m.w., developmentally regulated proteins consisting of two similar or identical disulfide-bonded subunits. In this paper, we report the results of a serologic and biochemical analysis of these proteins in various strains of inbred mice, and in rats and hamsters. A monoclonal antibody against the PC-1a allelic product is shown to detect an antigenic determinant on the PC-1 molecule that has the same strain distribution as the antigen previously detected with polyclonal alloantisera. The mouse PC-1 protein was purified from plasma cells of the PC-1a genotype and was used to generate polyclonal rabbit anti-PC-1 antibodies. These antibodies precipitated a homologous protein from plasmacytoma cells derived from PC-1- congenic mice, demonstrating that PC-1b is not a "null" allele. The PC-1b allelic product had a slightly lower apparent m.w. than the PC-1a product, and had a slightly more basic isoelectric point. Rabbit anti-mouse PC-1 antibodies also precipitated a homologous protein from immunoglobulin-secreting cells of rat and hamster origin, but did not show detectable cross-reaction with the transferrin receptor. Disulfide bonding between chains was conserved in both PC-1 and the transferrin receptor in all species examined, but transferrin receptors from mouse cells had a significantly higher apparent m.w. than those of rat, hamster, or human cells.
Cold Spring Harbor Symposia on Quantitative Biology, 1977
In this paper, we review experiments verifying clonal selection through the use of hapten-fractio... more In this paper, we review experiments verifying clonal selection through the use of hapten-fractionated B lymphocytes of such uniformity that up to one cell in three can be induced to form an anti-hapten antibody-forming clone in vitro; the use of such cells in studies on antigen-induced receptor aggregation and modulation using immunogenic or tolerogenic antigen concentrations; application to the tolerance problem of the cloning methods devised for the study of these cells; studies of the IgM- and IgD-like receptors of hapten-specific cells; and the use of an anti-delta allotype serum to study the distribution and function of IgD-like molecules on the B-lymphocyte surface.
Australian journal of experimental biology and medical science, Apr 1, 1977
SummaryHypothymic BALB/c.nu/nu (“hude”) mice were markedly more susceptible than their intact BAL... more SummaryHypothymic BALB/c.nu/nu (“hude”) mice were markedly more susceptible than their intact BALB/c.nu/+ littermates to infection with the murine larval cestode, Mesocestoides corti, and antigens of this parasite induced T cell‐dependent immune responses in intact mice. Defective anti‐dinitrophenol (DNP) antibody responses were induced by DNP‐M. corti larvae and DNP‐human gamma globulin (HGG) in infected (parasitized) intact mice. However, on transfer to uninfected irradiated recipients, lymphoid cells from infected intact mice responded well to DNP‐M. corti larvae.By using washed peritoneal larvae from long term infected intact mice to absorb antisera directed against various Ig classes and IgG subclasses, and by analysis of acid eluates of such living larvae, large amounts of IgG1, lower amounts of IgM, trace amounts of IgG2 and IgG3 and no IgA immunoglobulins were found to be associated with the larvae. In the case of nu/nu derived larvae, IgM immunoglobulins predominated. Chronically infected intact mice contained large amounts of IgG1, immunoglobulins in their serum. The technique of lactoperoxidase‐catalysed radioiodination revealed that very few proteins were available for labelling on peritoneal larvae under conditions used for labelling of lymphoid cell surface molecules. In fact, proteins with Ig chain mobilities accounted for much of the labelled and extracted proteins detected by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Patterns of labelled and extracted proteins from nu/nu derived larvae were even simpler and no clear protein peaks were detected consistently in material from labelled, acid treated, nu/nu derived larvae. Evidence is presented that at least the IgG1 molecules associated with M. corti larvae from long term infected mice include antiparasite antibodies. Immunoglobulins associated with M. corti larvae in long term infected mice are most unlikely to be useful to the mouse in terms of preventing parasite establishment (in secondary murine hosts) or reducing the proliferative rate of larvae. However, no clear cut evidence is as yet available to answer the question of whether the M. corti associated Ig (and, in particular, IgG1 which is reported to be relatively inert with respect to direct pathogenic effects) serves the function of masking parasite antigens and protecting the established parasites from more aggressive T cell‐dependent immunological effector mechanisms.
The monoclonal antibody OKT-9 recognizes a surface protein of human lymphocytes that consists of ... more The monoclonal antibody OKT-9 recognizes a surface protein of human lymphocytes that consists of a disulfide bonded homodimer of m.w. 200,000 intact and 95,000 reduced. A similar protein is precipitated by transferrin-agarose, but not by agarose alone. Peptide mapping by limited proteolysis shows that the proteins precipitated by OKT-9 antibodies and transferrin-agarose are homologous. It is concluded that OKT-9 antibodies recognize the transferrin receptor. Expression of receptors for transferrin may be useful as a marker for activated or dividing cells.
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Papers by James Goding