Immunoassay That T. Ngo BioProbe International Incorporation, Tustin, California, USA Recent developments in immunoassay are reviewed with emphasis on the chemistry of label conjugation, new solid phases, enzyme substrates for enzyme-linked immunosorbent assays, new assay techniques and detection systems. Current Opinion in Biotechnology 1991, 2:102-109
Introduction Immunoassay is a quantitative analytical technique that is characterized by a very high degree of sensitivity and specificity. It is based on the interaction between an antibody and its complementary antigen or hapten. Immunoassays can be classified into six groups [1o]. Group one includes the classic 'competitive' immunoassays of antigens or haptens, using limiting amounts of both antibody and labeled analyte. Group two is similar to Group one in that all reagents are present in limiting amounts. The difference is that the labeled reagent is the antibody rather than the analyte, and the immobilized reagent is a derivative of the analyte instead of the antibody. Nephelometric, turibidimetric and gravimetric precipitation assays comprise Group three. These assays involve a quantification of the sizes of molecular complexes and aggregates formed through the antibody-antigen interactions. Group four includes assays in which all reagents are used in excess. One example is a two-site sandwich immunoassay in which both the immobilized antibody and labeled antibody are used in excess. Similarly, in the one-site immunoenzymometric assay for hapten, a calculated excess of labeled antibody is first incubated with the analyte. Any labeled antibody that is not occupied by analyte is removed by the addition of an excess of immobilized analyte to which it binds. The amount of labeled antibody with bound analyte is then quantified. Group five includes assays for specific antibodies using immobiliT.ed antigens and labeled secondary antibodies. Finally, group six consists of labeled reagent immunoassays that do not require separation of bound from unbound reagents before the intensity of the signal is measured. These assays are generally known as separation-free or homogeneous assays, and involve the signal from the labeled reagent being modulated by the immunochemical binding reaction [2,3].
All immunoassays that require a separation of either antibody or antigen-bound labeled reagents from the unbound labeled reagents will have, at some points during the assay, a heterogeneous phase to facilitate the separation process. Accordingly, such assays are termed heterogeneous assays. With the possible exception of assays in Group three, all immunoassays require, in addition to antibody, labeled reagents. Additionally, heterogeneous assays require a solid phase. This review will focus on recent developments in novel detection systems and new assay principles as well as methods for preparing labeled reagents and solid phases.
Label conjugation A direct covalent attachment of monomeric labels to the amino groups of either antibody or antigen can be achieved for labels carrying any of the following groups: aldehyde, carboxyiate, isocyanate, imidate, epoxide, aziridine, activated halogen, sulfonyi chloride or 2-alkoxy N-methylpyridiniu [4]. For example, labels which carry maleimide groups can be reacted with the thiol groups of a protein. Similarly, the histidine or tyrosine groups of a protein can be linked to labels carrying/>aminophenyl groups. The advent of hybridoma technology has enabled the production of large quantities of monoclonal antibodies with defined specificities. This has created an opportunity for using monoclonal antibodies in vivo for either radio-immunotherapeutic or tumor imaging applications [5]. For instance, monodonal antibodies reactive with human glioma were successfully labeled with the 0t-emitting nuclide astatine 211At [6]. Although At is a halogen, proteins that are labeled with 211At using the procedure commonly used for protein iodination do not give a stable product. A new procedure of labeling employs N-succinimidyt, 31211At] astatobenzoate to acyiate the protein to give a stable product of high specific activ-
Abbreviations AMPPD--3•(2••spir•adamantane)•4-meth•xy•4-(3•-ph•sph•ry••xy)pheny•-1•2-di•xetane; CEA-carcinoembryonic antigen; DAB---p-dimethylaminobenzene; DNP~dinitrophenyl; EA~nzyme-acceptor; ED enzyme-donor; ELISA--enzyme-linked immunosorbent assay; HRP--horseradish peroxidase;Ig--immunoglobulin; Q ~ u a r t z crystal microbalance; SERRS---surface-enhanced resonance Raman scattering; SMCC---succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate; TSH--thyroid-stimulating hormone. 102
~) Current Biology Ltd ISSN 0958-1669
ImmunoassayNgo 103 ity (4 mCi/mg protein), within the time-frame consistent with the half-life of the radionuclide. The label, N-succinimidyl 31211At]astatobenzoate, was synthesized by reacting N-succ'mimidyl 3-(trimethylstannyl)benzoate with 211At in the presence of t-butyl hydroperoxide [6]. The radio-labeling of antibody with metal ion can be carried out with the aid of a bifunctional reagent which reacts with the antibody at one end whilst allowing a metal ion to chelate to the other end. A series of heterobifunctional reagents have been prepared. The new cross-linking reagents are capable of reacting at one end of the reagent with thiol groups of a protein, whilst the thiol groups at the other end of the reagent are protected. Upon deprotection, metal ions such as technetium-99m can chelate to these thiol groups [plo]. The labeling of antibody or antigen with polymeric and poly-functional molecules, such as enzymes or phycobiliproteins, is mostly carried out by using suitable cross-linking reagents. The use of homobifunctional reagents such as glutaraldehyde or bisimidate tends to result in a heterogeneous mixture containing molecular species of different sizes and specific activities. Ishikawa [7] has developed a procedure that uses heterobifunctional cross-linking reagents to prepare enzyme-labeled reagents of high specific activity and with a narrow molecular size distribution. The use of heterobifunctional cross-linking reagents allows a reaction sequence to be more easily controlled. The most useful of these reagents are succinimidyt-4(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and its more water-soluble sulfo-derivative (sulfo-SMCC), N-succinimidyl (4-iodoacetyi) aminobenzoate, succinimidyl-m-maleimidobenzoate and N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). In addition to chemical methods for preparing labeled immunochemical reagents, certain enzyme-labeled antigens can be prepared by using gene fusion methods. For example, Peterhans et al. [8] have carried out the in-frame fusion of the gene encoding the amino-terminal 461 bp of human interferon-0t2 with the gene for Escherichia coli J3-galactosidase. The expression of this fused protein in E. coli produced a conjugate that was enzymatically active as well as immunochemically reactive towards a monoclonal antibody directed against the amino-terminal region of human interferon
Introduction Immunoassay is a quantitative analytical technique that is characterized by a very high degree of sensitivity and specificity. It is based on the interaction between an antibody and its complementary antigen or hapten. Immunoassays can be classified into six groups [1o]. Group one includes the classic 'competitive' immunoassays of antigens or haptens, using limiting amounts of both antibody and labeled analyte. Group two is similar to Group one in that all reagents are present in limiting amounts. The difference is that the labeled reagent is the antibody rather than the analyte, and the immobilized reagent is a derivative of the analyte instead of the antibody. Nephelometric, turibidimetric and gravimetric precipitation assays comprise Group three. These assays involve a quantification of the sizes of molecular complexes and aggregates formed through the antibody-antigen interactions. Group four includes assays in which all reagents are used in excess. One example is a two-site sandwich immunoassay in which both the immobilized antibody and labeled antibody are used in excess. Similarly, in the one-site immunoenzymometric assay for hapten, a calculated excess of labeled antibody is first incubated with the analyte. Any labeled antibody that is not occupied by analyte is removed by the addition of an excess of immobilized analyte to which it binds. The amount of labeled antibody with bound analyte is then quantified. Group five includes assays for specific antibodies using immobiliT.ed antigens and labeled secondary antibodies. Finally, group six consists of labeled reagent immunoassays that do not require separation of bound from unbound reagents before the intensity of the signal is measured. These assays are generally known as separation-free or homogeneous assays, and involve the signal from the labeled reagent being modulated by the immunochemical binding reaction [2,3].
All immunoassays that require a separation of either antibody or antigen-bound labeled reagents from the unbound labeled reagents will have, at some points during the assay, a heterogeneous phase to facilitate the separation process. Accordingly, such assays are termed heterogeneous assays. With the possible exception of assays in Group three, all immunoassays require, in addition to antibody, labeled reagents. Additionally, heterogeneous assays require a solid phase. This review will focus on recent developments in novel detection systems and new assay principles as well as methods for preparing labeled reagents and solid phases.
Label conjugation A direct covalent attachment of monomeric labels to the amino groups of either antibody or antigen can be achieved for labels carrying any of the following groups: aldehyde, carboxyiate, isocyanate, imidate, epoxide, aziridine, activated halogen, sulfonyi chloride or 2-alkoxy N-methylpyridiniu [4]. For example, labels which carry maleimide groups can be reacted with the thiol groups of a protein. Similarly, the histidine or tyrosine groups of a protein can be linked to labels carrying/>aminophenyl groups. The advent of hybridoma technology has enabled the production of large quantities of monoclonal antibodies with defined specificities. This has created an opportunity for using monoclonal antibodies in vivo for either radio-immunotherapeutic or tumor imaging applications [5]. For instance, monodonal antibodies reactive with human glioma were successfully labeled with the 0t-emitting nuclide astatine 211At [6]. Although At is a halogen, proteins that are labeled with 211At using the procedure commonly used for protein iodination do not give a stable product. A new procedure of labeling employs N-succinimidyt, 31211At] astatobenzoate to acyiate the protein to give a stable product of high specific activ-
Abbreviations AMPPD--3•(2••spir•adamantane)•4-meth•xy•4-(3•-ph•sph•ry••xy)pheny•-1•2-di•xetane; CEA-carcinoembryonic antigen; DAB---p-dimethylaminobenzene; DNP~dinitrophenyl; EA~nzyme-acceptor; ED enzyme-donor; ELISA--enzyme-linked immunosorbent assay; HRP--horseradish peroxidase;Ig--immunoglobulin; Q ~ u a r t z crystal microbalance; SERRS---surface-enhanced resonance Raman scattering; SMCC---succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate; TSH--thyroid-stimulating hormone. 102
~) Current Biology Ltd ISSN 0958-1669
ImmunoassayNgo 103 ity (4 mCi/mg protein), within the time-frame consistent with the half-life of the radionuclide. The label, N-succinimidyl 31211At]astatobenzoate, was synthesized by reacting N-succ'mimidyl 3-(trimethylstannyl)benzoate with 211At in the presence of t-butyl hydroperoxide [6]. The radio-labeling of antibody with metal ion can be carried out with the aid of a bifunctional reagent which reacts with the antibody at one end whilst allowing a metal ion to chelate to the other end. A series of heterobifunctional reagents have been prepared. The new cross-linking reagents are capable of reacting at one end of the reagent with thiol groups of a protein, whilst the thiol groups at the other end of the reagent are protected. Upon deprotection, metal ions such as technetium-99m can chelate to these thiol groups [plo]. The labeling of antibody or antigen with polymeric and poly-functional molecules, such as enzymes or phycobiliproteins, is mostly carried out by using suitable cross-linking reagents. The use of homobifunctional reagents such as glutaraldehyde or bisimidate tends to result in a heterogeneous mixture containing molecular species of different sizes and specific activities. Ishikawa [7] has developed a procedure that uses heterobifunctional cross-linking reagents to prepare enzyme-labeled reagents of high specific activity and with a narrow molecular size distribution. The use of heterobifunctional cross-linking reagents allows a reaction sequence to be more easily controlled. The most useful of these reagents are succinimidyt-4(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and its more water-soluble sulfo-derivative (sulfo-SMCC), N-succinimidyl (4-iodoacetyi) aminobenzoate, succinimidyl-m-maleimidobenzoate and N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). In addition to chemical methods for preparing labeled immunochemical reagents, certain enzyme-labeled antigens can be prepared by using gene fusion methods. For example, Peterhans et al. [8] have carried out the in-frame fusion of the gene encoding the amino-terminal 461 bp of human interferon-0t2 with the gene for Escherichia coli J3-galactosidase. The expression of this fused protein in E. coli produced a conjugate that was enzymatically active as well as immunochemically reactive towards a monoclonal antibody directed against the amino-terminal region of human interferon
