Pub Date : 2000-05-01DOI: 10.1080/01971520009349535
V Kumar
Immunohistochemistry is a very versatile immunopathological tool for the study of distribution and differentiation of antigens and of the presence of in vivo-bound immune complexes. In addition, these methods are invaluable for detection of circulating antibodies to the various antigens. Such methods may be the only ones of choice in certain situations. For the detection and quantitation of these antigens, it is very essential that the immunohistochemical methods for detecting them are properly standardized, with the inclusion of appropriate controls.
{"title":"Immunofluorescence and enzyme immunomicroscopy methods.","authors":"V Kumar","doi":"10.1080/01971520009349535","DOIUrl":"https://doi.org/10.1080/01971520009349535","url":null,"abstract":"<p><p>Immunohistochemistry is a very versatile immunopathological tool for the study of distribution and differentiation of antigens and of the presence of in vivo-bound immune complexes. In addition, these methods are invaluable for detection of circulating antibodies to the various antigens. Such methods may be the only ones of choice in certain situations. For the detection and quantitation of these antigens, it is very essential that the immunohistochemical methods for detecting them are properly standardized, with the inclusion of appropriate controls.</p>","PeriodicalId":16060,"journal":{"name":"Journal of immunoassay","volume":"21 2-3","pages":"235-53"},"PeriodicalIF":0.0,"publicationDate":"2000-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/01971520009349535","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21768559","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2000-05-01DOI: 10.1080/01971520009349536
C C Stewart
Immunophenotyping is an important method for identifying cells, and there is no single field for which it is applied more often than in hematology. Although immunohistochemistry or fluorochrome-labeled antibodies are often used for microscopic identification of cells, flow cytometry has the advantage of identifying cell subsets more rapidly by using multiple antibodies simultaneously, and these subsets can be sorted for further characterization. Microscopic analysis is the method of choice if morphological information is desired, but flow cytometry is the choice for identifying and quantifying cell subsets and evaluating their frequency in a heterogeneous population. Even though polyclonal antibodies are still used occasionally, monoclonal antibodies (MAbs) to epitopes on membrane or internal antigens are the reagents used for identification. We now know that any given protein is often found on several different kinds of cells, and one antibody cannot be used to identify any particular cell lineage. Instead, each cell subpopulation has a very unique repertoire of proteins, and a mixture of antibodies to them can be used for explicit identification. When the function of the protein is known, additional information about the cell is obtained. In this chapter, I will describe the methods for labeling cells with up to four antibodies simultaneously. This number is chosen because it is possible to measure all of them with a single laser. With human lymphocytes as the example, we now recognize over 80 subpopulations and, although these subpopulations are not all mutually exclusive, they do represent specific functional subsets that interact together to produce the hematopoietic system and exemplify the power of flow cytometry for resolving them.
{"title":"Multiparameter flow cytometry.","authors":"C C Stewart","doi":"10.1080/01971520009349536","DOIUrl":"https://doi.org/10.1080/01971520009349536","url":null,"abstract":"Immunophenotyping is an important method for identifying cells, and there is no single field for which it is applied more often than in hematology. Although immunohistochemistry or fluorochrome-labeled antibodies are often used for microscopic identification of cells, flow cytometry has the advantage of identifying cell subsets more rapidly by using multiple antibodies simultaneously, and these subsets can be sorted for further characterization. Microscopic analysis is the method of choice if morphological information is desired, but flow cytometry is the choice for identifying and quantifying cell subsets and evaluating their frequency in a heterogeneous population. Even though polyclonal antibodies are still used occasionally, monoclonal antibodies (MAbs) to epitopes on membrane or internal antigens are the reagents used for identification. We now know that any given protein is often found on several different kinds of cells, and one antibody cannot be used to identify any particular cell lineage. Instead, each cell subpopulation has a very unique repertoire of proteins, and a mixture of antibodies to them can be used for explicit identification. When the function of the protein is known, additional information about the cell is obtained. In this chapter, I will describe the methods for labeling cells with up to four antibodies simultaneously. This number is chosen because it is possible to measure all of them with a single laser. With human lymphocytes as the example, we now recognize over 80 subpopulations and, although these subpopulations are not all mutually exclusive, they do represent specific functional subsets that interact together to produce the hematopoietic system and exemplify the power of flow cytometry for resolving them.","PeriodicalId":16060,"journal":{"name":"Journal of immunoassay","volume":"21 2-3","pages":"255-72"},"PeriodicalIF":0.0,"publicationDate":"2000-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/01971520009349536","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21768562","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2000-05-01DOI: 10.1080/01971520009349534
M H van Regenmortel, A Azimzadeh
(2000). Determination of Antibody Affinity. Journal of Immunoassay: Vol. 21, No. 2-3, pp. 211-234.
{"title":"Determination of antibody affinity.","authors":"M H van Regenmortel, A Azimzadeh","doi":"10.1080/01971520009349534","DOIUrl":"https://doi.org/10.1080/01971520009349534","url":null,"abstract":"(2000). Determination of Antibody Affinity. Journal of Immunoassay: Vol. 21, No. 2-3, pp. 211-234.","PeriodicalId":16060,"journal":{"name":"Journal of immunoassay","volume":"21 2-3","pages":"211-34"},"PeriodicalIF":0.0,"publicationDate":"2000-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/01971520009349534","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21768557","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2000-05-01DOI: 10.1080/01971520009349531
C J van Oss
The binding forces involved in the specific interactions between antigens (Ag) and antibodies (Ab), lectins and carbohydrates, ligands and their receptors and, in most cases, enzymes and their substrates, are of a noncovalent, purely physicochemical nature. The same (attractive) physicochemical forces that govern their binding also constitute the (repulsive) forces which prevent i t [e.g., freely suspended peripheral blood cells to approach each other more closely than a given minimum distance (van Oss, 1989, 1990a)l and which allow proteins and other biopolymers to remain in aqueous solution (van Oss et al., 1986b). The three primary physical forces that play a role in Ag-Ab bonds are Lifshitz-van der Waals (LW), electrostatic (EL), and Lewis acid-base (AB), or electron-acceptor-electron-donor interactions. While it is usually possible to distinguish between LW, EL, and AB contributions to Ag-Ab bonding, for example, by observing the effect of changes in ionic strength (which influence EL, but not AB or LW forces), or of the admixture of polar organic solvents (which mainly influence AB forces), it remains difficult to discriminate between general AB interactions (which, in water, usually are mainly due to hydrogen-bonding), and direct hydrogen bond formation. It is important to be able to distinguish between the three primary forces. The difference in the rate of decay as a function of distance of the three primary physical forces involved in cell interactions as well as in Ag-Ab bonds, is a major reason for treating the three forces individually. The rate of decay with distance of the three forces involved in specific bonds plays an important role in the study of these interactions, because Ags and Abs and other ligands and receptors must be able to attract each other from a distance, which can be of the order of 3-8 nm. It should also be realized that various physicochemical changes one can make in the conditions under which Ags and Abs (and other receptors and ligands) interact (such as ionic strength, pH, temperature, content of organic solvent or other solute), can have a different influence on LW, or EL, or AB interaction forces.
{"title":"Nature of specific ligand-receptor bonds, in particular the antigen-antibody bond.","authors":"C J van Oss","doi":"10.1080/01971520009349531","DOIUrl":"https://doi.org/10.1080/01971520009349531","url":null,"abstract":"The binding forces involved in the specific interactions between antigens (Ag) and antibodies (Ab), lectins and carbohydrates, ligands and their receptors and, in most cases, enzymes and their substrates, are of a noncovalent, purely physicochemical nature. The same (attractive) physicochemical forces that govern their binding also constitute the (repulsive) forces which prevent i t [e.g., freely suspended peripheral blood cells to approach each other more closely than a given minimum distance (van Oss, 1989, 1990a)l and which allow proteins and other biopolymers to remain in aqueous solution (van Oss et al., 1986b). The three primary physical forces that play a role in Ag-Ab bonds are Lifshitz-van der Waals (LW), electrostatic (EL), and Lewis acid-base (AB), or electron-acceptor-electron-donor interactions. While it is usually possible to distinguish between LW, EL, and AB contributions to Ag-Ab bonding, for example, by observing the effect of changes in ionic strength (which influence EL, but not AB or LW forces), or of the admixture of polar organic solvents (which mainly influence AB forces), it remains difficult to discriminate between general AB interactions (which, in water, usually are mainly due to hydrogen-bonding), and direct hydrogen bond formation. It is important to be able to distinguish between the three primary forces. The difference in the rate of decay as a function of distance of the three primary physical forces involved in cell interactions as well as in Ag-Ab bonds, is a major reason for treating the three forces individually. The rate of decay with distance of the three forces involved in specific bonds plays an important role in the study of these interactions, because Ags and Abs and other ligands and receptors must be able to attract each other from a distance, which can be of the order of 3-8 nm. It should also be realized that various physicochemical changes one can make in the conditions under which Ags and Abs (and other receptors and ligands) interact (such as ionic strength, pH, temperature, content of organic solvent or other solute), can have a different influence on LW, or EL, or AB interaction forces.","PeriodicalId":16060,"journal":{"name":"Journal of immunoassay","volume":"21 2-3","pages":"109-42"},"PeriodicalIF":0.0,"publicationDate":"2000-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/01971520009349531","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21768638","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2000-05-01DOI: 10.1080/01971520009349533
J E Butler
The acronym for enzyme-linked immunosorbent assay (ELISA) has become a standard abbreviation in many scientific journals. ELISAs are immunoassays in which one reactant is immobilized on a solid phase and the “signal generator” or “reporter,” is an enzyme. This definition includes enzyme immunoassay (EIA) which like classical radioimmunoassay (RIA), is competitive in design, but employs an enzyme reporter instead of a radionuclide. ELISAs and EIAs, which consist of both a solid and a fluid phase, are designated heterogeneous EIAs (HetEIA). Homogeneous enzyme immunoussuys (HomEIA) do not involve a solid phase and are often not included under the ELISA umbrella, although they will be briefly discussed in this chapter. The HetEIA is often treated under the category of solid-phase irnrnunoassuy (SPI) because assays done on a solid phase have more in common with each other than they do with assays that share a common reporter system (i.e., an enzyme). ELISA and SPI are performed on a variety of solid phases ranging from smooth polystyrene to membranous nitrocellulose (NC) and nylon. The term ELISA was coined by Engvall and Perlmann in 1971 for a noncompetitive HetEIA configuration, whereas EIA historically denoted a competitive one. These acronyms, as well as EM1 (enzymemediated immunoassay) and others, are often used interchangeably in the literature. Solid-phase immunoassays and ELISA have technically simplified antigen quantitation and antibody detection. They are the basis for most modern immunodiagnostic tests for acquired immunodeficiency syndrome (AIDS), hepatitis, allergies, and many other human and animal diseases. Before 1970, nearly all immunodiagnostic tests were based either on fluid phase or cellular interactions of antigens and antibodies (e.g., agglutination, precipitation, and complement fixation). Also before that time, immunological tests for quantifying the levels of small molecules used radioisotopes as reporters and involved tedious procedures for the separation of the bound and free reactants. The observation that proteins are spontaneously adsorbed to hydrophobic surfaces (Catt and Tregear, 1967), led to the development of contemporary SPI, which technically simplified immunoassays and permitted them to be easily automated. Add to this development the use of enzymes as alternatives to radioisotopes, and you describe “immunoassay for the common man” (i.e., ELISA).
{"title":"Enzyme-linked immunosorbent assay.","authors":"J E Butler","doi":"10.1080/01971520009349533","DOIUrl":"https://doi.org/10.1080/01971520009349533","url":null,"abstract":"The acronym for enzyme-linked immunosorbent assay (ELISA) has become a standard abbreviation in many scientific journals. ELISAs are immunoassays in which one reactant is immobilized on a solid phase and the “signal generator” or “reporter,” is an enzyme. This definition includes enzyme immunoassay (EIA) which like classical radioimmunoassay (RIA), is competitive in design, but employs an enzyme reporter instead of a radionuclide. ELISAs and EIAs, which consist of both a solid and a fluid phase, are designated heterogeneous EIAs (HetEIA). Homogeneous enzyme immunoussuys (HomEIA) do not involve a solid phase and are often not included under the ELISA umbrella, although they will be briefly discussed in this chapter. The HetEIA is often treated under the category of solid-phase irnrnunoassuy (SPI) because assays done on a solid phase have more in common with each other than they do with assays that share a common reporter system (i.e., an enzyme). ELISA and SPI are performed on a variety of solid phases ranging from smooth polystyrene to membranous nitrocellulose (NC) and nylon. The term ELISA was coined by Engvall and Perlmann in 1971 for a noncompetitive HetEIA configuration, whereas EIA historically denoted a competitive one. These acronyms, as well as EM1 (enzymemediated immunoassay) and others, are often used interchangeably in the literature. Solid-phase immunoassays and ELISA have technically simplified antigen quantitation and antibody detection. They are the basis for most modern immunodiagnostic tests for acquired immunodeficiency syndrome (AIDS), hepatitis, allergies, and many other human and animal diseases. Before 1970, nearly all immunodiagnostic tests were based either on fluid phase or cellular interactions of antigens and antibodies (e.g., agglutination, precipitation, and complement fixation). Also before that time, immunological tests for quantifying the levels of small molecules used radioisotopes as reporters and involved tedious procedures for the separation of the bound and free reactants. The observation that proteins are spontaneously adsorbed to hydrophobic surfaces (Catt and Tregear, 1967), led to the development of contemporary SPI, which technically simplified immunoassays and permitted them to be easily automated. Add to this development the use of enzymes as alternatives to radioisotopes, and you describe “immunoassay for the common man” (i.e., ELISA).","PeriodicalId":16060,"journal":{"name":"Journal of immunoassay","volume":"21 2-3","pages":"165-209"},"PeriodicalIF":0.0,"publicationDate":"2000-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/01971520009349533","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21768555","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2000-05-01DOI: 10.1080/01971520009349537
D I Stott
Immunoblotting (or Western blotting) involves the transfer of proteins that have been separated by electrophoresis or isoelectric focusing (IEF) from the electrophoresis gel to a membrane, to which they become bound. The bound molecules are then detected by a specific probe, usually an antibody. The advantage of immunoblotting is that it combines the resolution obtained by electrophoretic separation of mixtures of proteins in a gel with the specificity of antibodies used as probes to identify individual antigens. Proteins can be separated by any of the electrophoretic techniques available by using agarose, polyacrylamide in the presence or absence of sodium dodecyl sulfate (SDS), isoelectric focusing (IEF), or two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Direct overlay of the gel with antibody (immunofixation) has been used to identify antigens of interest, but such methods suffer from the disadvantages of prolonged incubation times, resulting in diffusion of the bands, and consequent loss of resolution. Immunofixation is also primarily limited to agarose gel systems, since antibody molecules cannot readily penetrate polyacrylamide gels, owing to the gels’ small pore size. Because of these difficulties, the idea of transferring electrophoretically separated proteins from a gel to a membrane, where they are readily accessible to high relative molecular mass (M,) probes, such as antibody molecules, opened up a new vista for ,electrophoretic analysis of proteins. The method of transfer is termed blotting, since the pattern of bands on the membrane is an exact replica of the pattern in the original gel. The Southern-blotting method for analysis of DNA was named after Ed Southern; hence, analysis of RNA molecules by a similar technique was called Northern blotting, and analysis of proteins by transfer to a membrane and detection by antibody became known as Western blotting or immunoblotting. Dot-blotting, slot-blotting, and line-blotting are variants of this in which antigens are applied directly to a membrane without prior separation and identified in the same way. The amount of antigen present in the mixture can be measured quantitatively. ELISPOT assays use similar principles to measure the number of cells secreting specific antibody or other proteins. Many adaptations have been developed and applied to a wide variety of fields of research and clinical applications. In this chapter I shall discuss the basic techniques and principles of immunoblotting, dot-blotting, and the ELISPOT assay, and describe a selection of some of their
{"title":"Immunoblotting, dot-blotting, and ELISPOT assays: methods and applications.","authors":"D I Stott","doi":"10.1080/01971520009349537","DOIUrl":"https://doi.org/10.1080/01971520009349537","url":null,"abstract":"Immunoblotting (or Western blotting) involves the transfer of proteins that have been separated by electrophoresis or isoelectric focusing (IEF) from the electrophoresis gel to a membrane, to which they become bound. The bound molecules are then detected by a specific probe, usually an antibody. The advantage of immunoblotting is that it combines the resolution obtained by electrophoretic separation of mixtures of proteins in a gel with the specificity of antibodies used as probes to identify individual antigens. Proteins can be separated by any of the electrophoretic techniques available by using agarose, polyacrylamide in the presence or absence of sodium dodecyl sulfate (SDS), isoelectric focusing (IEF), or two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Direct overlay of the gel with antibody (immunofixation) has been used to identify antigens of interest, but such methods suffer from the disadvantages of prolonged incubation times, resulting in diffusion of the bands, and consequent loss of resolution. Immunofixation is also primarily limited to agarose gel systems, since antibody molecules cannot readily penetrate polyacrylamide gels, owing to the gels’ small pore size. Because of these difficulties, the idea of transferring electrophoretically separated proteins from a gel to a membrane, where they are readily accessible to high relative molecular mass (M,) probes, such as antibody molecules, opened up a new vista for ,electrophoretic analysis of proteins. The method of transfer is termed blotting, since the pattern of bands on the membrane is an exact replica of the pattern in the original gel. The Southern-blotting method for analysis of DNA was named after Ed Southern; hence, analysis of RNA molecules by a similar technique was called Northern blotting, and analysis of proteins by transfer to a membrane and detection by antibody became known as Western blotting or immunoblotting. Dot-blotting, slot-blotting, and line-blotting are variants of this in which antigens are applied directly to a membrane without prior separation and identified in the same way. The amount of antigen present in the mixture can be measured quantitatively. ELISPOT assays use similar principles to measure the number of cells secreting specific antibody or other proteins. Many adaptations have been developed and applied to a wide variety of fields of research and clinical applications. In this chapter I shall discuss the basic techniques and principles of immunoblotting, dot-blotting, and the ELISPOT assay, and describe a selection of some of their","PeriodicalId":16060,"journal":{"name":"Journal of immunoassay","volume":"21 2-3","pages":"273-96"},"PeriodicalIF":0.0,"publicationDate":"2000-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/01971520009349537","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21768564","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2000-05-01DOI: 10.1080/01971520009349530
M H van Regenmortel
Immunological recognition is mediated by T and B lymphocytes and by immunoglobulin molecules secreted by plasmocytes. The T cells recognize protein antigens by means of their T-cell receptors (TCRs) after the antigen has been processed into peptide fragments, a topic discussed in Chapter 13. The B cells recognize antigens by means of B-cell immunoglobulin receptors, which are actually antibody molecules anchored in the B-cell membrane. Unlike T-cell receptors, B-cell receptors are able to recognize the native tertiary structure of a protein antigen. The antibody molecules that are subsequently released by the B cell, after its differentiation into a plasmocyte. possess the same specificity as the receptors of the triggered B cell; therefore, they are also able to recognize the native conformation of the protein antigen. The first selective theory of antibody diversity was developed at the beginning of the century by Paul Ehrlich, who proposed his “side chain” theory to explain the appearance of specific antibodies. According to this theory, lymphocytes possess on their surface a variety of side chain groups or receptors that are able to combine in a specific manner with different antigens. The interaction of the antigen with one of the side chains was believed to result in the release of that side chain group from the cell surface and to trigger the subsequent synthesis and release of large numbers of the same side chains. According to Ehrlich, the antigen selects, on the surface of the lymphocyte, a receptor with a complementary shape, and it induces the cells to excrete large numbers of this receptor in the form of specific antibodies. This side chain theory is remarkably modern, since it is similar to the currently accepted clonal selection mechanism, which links the specificity of antibodies appearing in the serum with the presence of preexisting clones of cells possessing the same antibodies immobilized at the cell surface. The molecular mechanisms of antigen recognition by an antibody anchored in the B-cell membrane or by a free antibody molecule are the same. However, it is much easier to study the binding of antigens to free antibodies than to antibodies attached to B cells, and most of our knowledge concerning antigen-antibody binding has been derived from studies with free antibody molecules. The term antigen refers to any entity that is able to generate an immune response in higher vertebrates and to be recognized by the products of the immune response. The ability of antigens to react specifically with complementary antibodies is known as antigenic reactivity, and their
{"title":"The recognition of proteins and peptides by antibodies.","authors":"M H van Regenmortel","doi":"10.1080/01971520009349530","DOIUrl":"https://doi.org/10.1080/01971520009349530","url":null,"abstract":"Immunological recognition is mediated by T and B lymphocytes and by immunoglobulin molecules secreted by plasmocytes. The T cells recognize protein antigens by means of their T-cell receptors (TCRs) after the antigen has been processed into peptide fragments, a topic discussed in Chapter 13. The B cells recognize antigens by means of B-cell immunoglobulin receptors, which are actually antibody molecules anchored in the B-cell membrane. Unlike T-cell receptors, B-cell receptors are able to recognize the native tertiary structure of a protein antigen. The antibody molecules that are subsequently released by the B cell, after its differentiation into a plasmocyte. possess the same specificity as the receptors of the triggered B cell; therefore, they are also able to recognize the native conformation of the protein antigen. The first selective theory of antibody diversity was developed at the beginning of the century by Paul Ehrlich, who proposed his “side chain” theory to explain the appearance of specific antibodies. According to this theory, lymphocytes possess on their surface a variety of side chain groups or receptors that are able to combine in a specific manner with different antigens. The interaction of the antigen with one of the side chains was believed to result in the release of that side chain group from the cell surface and to trigger the subsequent synthesis and release of large numbers of the same side chains. According to Ehrlich, the antigen selects, on the surface of the lymphocyte, a receptor with a complementary shape, and it induces the cells to excrete large numbers of this receptor in the form of specific antibodies. This side chain theory is remarkably modern, since it is similar to the currently accepted clonal selection mechanism, which links the specificity of antibodies appearing in the serum with the presence of preexisting clones of cells possessing the same antibodies immobilized at the cell surface. The molecular mechanisms of antigen recognition by an antibody anchored in the B-cell membrane or by a free antibody molecule are the same. However, it is much easier to study the binding of antigens to free antibodies than to antibodies attached to B cells, and most of our knowledge concerning antigen-antibody binding has been derived from studies with free antibody molecules. The term antigen refers to any entity that is able to generate an immune response in higher vertebrates and to be recognized by the products of the immune response. The ability of antigens to react specifically with complementary antibodies is known as antigenic reactivity, and their","PeriodicalId":16060,"journal":{"name":"Journal of immunoassay","volume":"21 2-3","pages":"85-108"},"PeriodicalIF":0.0,"publicationDate":"2000-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/01971520009349530","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21768637","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2000-02-01DOI: 10.1080/01971520009349500
G Lefévre, M Duval, A Poncin
A solid phase method for direct radioimmunoassay in plasma of the new renin inhibitor CGP 60536 has been developed which does not require the extraction of the parent drug with organic solvents. The assay showed a good reproducibility down to plasma concentrations of 0.15 ng/ml (LOQ) with intra- and inter-assay coefficients of variation < or = 20%. The procedure, which requires only small volumes of plasma (25 microl), is simple to use and well suited for routine analysis. The method allows the investigation of the pharmacokinetics of CGP 60536 in animals and man given low oral doses of the drug.
{"title":"Direct micro-radioimmunoassay of the new renin inhibitor CGP 60536.","authors":"G Lefévre, M Duval, A Poncin","doi":"10.1080/01971520009349500","DOIUrl":"https://doi.org/10.1080/01971520009349500","url":null,"abstract":"<p><p>A solid phase method for direct radioimmunoassay in plasma of the new renin inhibitor CGP 60536 has been developed which does not require the extraction of the parent drug with organic solvents. The assay showed a good reproducibility down to plasma concentrations of 0.15 ng/ml (LOQ) with intra- and inter-assay coefficients of variation < or = 20%. The procedure, which requires only small volumes of plasma (25 microl), is simple to use and well suited for routine analysis. The method allows the investigation of the pharmacokinetics of CGP 60536 in animals and man given low oral doses of the drug.</p>","PeriodicalId":16060,"journal":{"name":"Journal of immunoassay","volume":"21 1","pages":"65-84"},"PeriodicalIF":0.0,"publicationDate":"2000-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/01971520009349500","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21651879","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2000-02-01DOI: 10.1080/01971520009349496
W L Shelver, D J Smith
Antibody generated from ractopamine-hemiglutarate-KLH was used to develop a ractopamine ELISA. The antibody showed good sensitivity in phosphate buffer, with an IC50 of 4.2 ng/ml (ppb) toward ractopamine and 16.2 ng/ml toward glucuronides of ractopamine conjugated to the phenethanolamine phenol of ractopamine. Phenylbutylamine phenol glucuronides of the (RS, SR) ractopamine diastereoisomers showed about 4% cross-reactivity, but the glucuronide of the (RR, SS) diastereoisomers conjugated at the same phenolic group showed no detectable reactivity with the antibody. The antibody generally had cross-reactivity towards compounds with bis-phenylalkyl amine structures rather than compounds with simple branched N-alkyl substituents. For example, the antibody showed little or no cross reactivity towards clenbuterol, isoproterenol, metaproterenol, and salbutamol, but cross-reacted with dobutamine. The system demonstrated a matrix effect similar to other enzyme immunoassays, dilution of urine decreased but did not eliminate the matrix effect.
{"title":"Development of an immunoassay for the beta-adrenergic agonist ractopamine.","authors":"W L Shelver, D J Smith","doi":"10.1080/01971520009349496","DOIUrl":"https://doi.org/10.1080/01971520009349496","url":null,"abstract":"<p><p>Antibody generated from ractopamine-hemiglutarate-KLH was used to develop a ractopamine ELISA. The antibody showed good sensitivity in phosphate buffer, with an IC50 of 4.2 ng/ml (ppb) toward ractopamine and 16.2 ng/ml toward glucuronides of ractopamine conjugated to the phenethanolamine phenol of ractopamine. Phenylbutylamine phenol glucuronides of the (RS, SR) ractopamine diastereoisomers showed about 4% cross-reactivity, but the glucuronide of the (RR, SS) diastereoisomers conjugated at the same phenolic group showed no detectable reactivity with the antibody. The antibody generally had cross-reactivity towards compounds with bis-phenylalkyl amine structures rather than compounds with simple branched N-alkyl substituents. For example, the antibody showed little or no cross reactivity towards clenbuterol, isoproterenol, metaproterenol, and salbutamol, but cross-reacted with dobutamine. The system demonstrated a matrix effect similar to other enzyme immunoassays, dilution of urine decreased but did not eliminate the matrix effect.</p>","PeriodicalId":16060,"journal":{"name":"Journal of immunoassay","volume":"21 1","pages":"1-23"},"PeriodicalIF":0.0,"publicationDate":"2000-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/01971520009349496","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21651875","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2000-02-01DOI: 10.1080/01971520009349498
A Basu, T G Shrivastav
One step competitive enzyme linked immunosorbent assay (ELISA) for direct estimation of cortisol in human serum is described. Cortisol-3-O-carboxymethyl-oxime-bovine serum albumin (cortisol-3-O-CMO-BSA) was used as an immunogen and cortisol-21-hemisuccinate-horse radish peroxidase (cortisol-21-HS-HRP) was used as a tracer. To the cortisol antibody coated microtiter wells, standards or serum samples (25 microl) along with cortisol-HRP conjugate (100 microl) were incubated for 2 hours at 37 degrees C. Bound enzyme activity was measured by, using TMB/H2O2 as a substrate. In this new strategy, chilled acetone stripped pooled human serum and sodium salicylate were used for preparing the standards and blocking the cortisol binding globulin (CBG), respectively. The sensitivity of the assay was .28 microg/100ml. The intraassay and interassay coefficient of variations (CVs) were ranged from 1.3% to 9.3% and 6.8% to 12.3 %, respectively. The analytical recoveries were 94% to 101.5%. The serum cortisol values, obtained by this method were correlated well with those, obtained by radioimmunoassay; r=0.95 (n=52).
{"title":"One step enzyme linked immunosorbent assay for direct estimation of serum cortisol.","authors":"A Basu, T G Shrivastav","doi":"10.1080/01971520009349498","DOIUrl":"https://doi.org/10.1080/01971520009349498","url":null,"abstract":"<p><p>One step competitive enzyme linked immunosorbent assay (ELISA) for direct estimation of cortisol in human serum is described. Cortisol-3-O-carboxymethyl-oxime-bovine serum albumin (cortisol-3-O-CMO-BSA) was used as an immunogen and cortisol-21-hemisuccinate-horse radish peroxidase (cortisol-21-HS-HRP) was used as a tracer. To the cortisol antibody coated microtiter wells, standards or serum samples (25 microl) along with cortisol-HRP conjugate (100 microl) were incubated for 2 hours at 37 degrees C. Bound enzyme activity was measured by, using TMB/H2O2 as a substrate. In this new strategy, chilled acetone stripped pooled human serum and sodium salicylate were used for preparing the standards and blocking the cortisol binding globulin (CBG), respectively. The sensitivity of the assay was .28 microg/100ml. The intraassay and interassay coefficient of variations (CVs) were ranged from 1.3% to 9.3% and 6.8% to 12.3 %, respectively. The analytical recoveries were 94% to 101.5%. The serum cortisol values, obtained by this method were correlated well with those, obtained by radioimmunoassay; r=0.95 (n=52).</p>","PeriodicalId":16060,"journal":{"name":"Journal of immunoassay","volume":"21 1","pages":"39-50"},"PeriodicalIF":0.0,"publicationDate":"2000-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1080/01971520009349498","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"21651877","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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