A convenient enzyme-linked immunosorbent assay for testing whether monoclonal antibodies recognize the same antigenic site. Application to hybridomas specific for the beta 2-subunit of Escherichia coli tryptophan synthase

Seven hybridoma clones, producing antibodies directed against the β₂-subunit of Escherichia coli tryptophan synthase, have been obtained from mouse cells. To test whether the corresponding monoclonal antibodies recognize different epitopes on β₂, an ELISA double antibody binding system has been developed and is reported here. The antigen is first coated onto a microtitration plate. Two monoclonal antibodies are then added either separately or simultaneously, and the amount of bound antibody is quantitatively measured by use of immunoglobulin (rabbit anti-mouse IgG) linked to β-galactosidase. Additivity of the bound enzymatic activity is observed when the monoclonal antibodies bind to distinct epitopes.

Using this test, it is shown that, of the 7 anti-β₂ monoclonal antibodies obtained, 5 recognize the same antigenic site and the 2 others recognize a second site.     

Production of monoclonal antibodies against glucose oxidase, alkaline phosphatase and peroxidase. Their application in a highly sensitive antigen spot microassay

The production of monoclonal antibodies against peroxidase, alkaline phosphatase and glucose oxidase and the use of the respective enzyme monoclonal anti-enzyme complexes in immunoassays are described. A micromethod using nitrocellulose membranes as solid phase was developed. This microassay has the advantage of being a rapid and simple test procedure, using only 0.2 microliter of antigen solution and 25 microliter of test reagents. A high sensitivity was achieved by repeated incubation of monoclonal enzyme anti-enzyme complexes bridged by anti-mouse immunoglobulin. As little as 200 pg human IgG and 400 pg human IgM could be detected. The glucose oxidase assay together with the alkaline phosphatase assay displays the highest sensitivity and has significant advantages including easy evaluation due to high color contrast of the enzymatic reaction, availability of non-toxic substrate reactions, and no interference with endogenous enzyme activity in mammalian antigen preparations.     

Highly sensitive enzyme immunoassay for human lymphotoxin (tumor necrosis factor β) in serum

We have developed a rapid, simple and highly sensitive ‘sandwich’ enzyme immunoassay (ELISA) for the detection and quantification of human lymphotoxin (= tumor necrosis factor β) in serum. The assay performed in microtiter plates, employs two monoclonal murine antibodies able to neutralize the cytotoxic activity of lymphotoxin. In a one-step procedure, antibody LTX-21 (IgG2b) coated on to the solid phase captures antigen present in the sample; subsequently antibody LTX-22 (IgG1), covalently coupled to horseradish peroxidase, labels the bound antigen. The assay is able to detect lymphotoxin spiked into human serum in concentrations as low as 7 pg/ml, whereas human tumor necrosis factor does not cross-react even at 10⁷-fold higher concentrations. Only biologically active protein is recognized by the antibodies, since inactivation of lymphotoxin measured by bioassay results in a parallel decrease in immunoreactivity. Natural, glycosylated lymphotoxin shows the same reactivity as recombinant, unglycosylated protein. The assay will be useful for the qunatification of endogenous human lymphotoxin in serum, other body fluids, and culture supernatants of human cells, and can also be used to monitor levels of recombinant human lymphotoxin in animal studies and clinical trials.     

A biotin-streptavidin enzyme immunoassay for detection of antibodies to porcine granulosa cell antigens.

A colorimetric solid-phase enzyme immunoassay has been developed which quantifies antibodies to porcine granulosa cell membrane antigens in rabbits immunized with porcine granulosa cells. A cell-free, particulate membrane preparation of porcine granulosa cells was used as coating antigen. A biotinylated second antibody in conjunction with a streptavidin-beta-galactosidase conjugate was utilized to amplify reactivity. The enzyme beta-galactosidase was used due to high background obtained using peroxidase, presumably due to endogenous peroxidase activity of the tissue. Sigmoidal serum dilution curves were obtained with immune rabbit sera indicating that absorbance was related to the concentration of antibodies. Assay activity was reduced by preincubation of immune serum with granulosa cell membranes. Sera from ovariectomized or pre-immune rabbits did not yield any specific binding in the assay. This assay has potential applicability for quantifying antiovarian and antigranulosa cell antibodies in women suspected of having autoimmune premature ovarian failure.     

Endogenous peroxidase activity in primary culture of human thyroid cells. Localization and measurement

Intracellular localization of and an assay method for endogenous peroxidase (PO) activity were studied using primary culture of thyroid cells obtained from patients with hyperthyroidism. PO activity was visualized by cytochemical reaction and was located mainly in perinuclear cisternae and rough endoplasmic reticulum. With increased culture time, the number of cells showing positive PO activity and amount of the enzyme reaction product in individual cells showed a parallel decrease. For measurement of PO activity, cultured thyroid cells were frozen and thawed and then incubated with citric acid buffer solution containing o-phenylenediamine (opd) and hydrogen peroxide. After incubation, the optical density (OD) of the solution colorized by endogenous peroxidase was measured at 405 nm using a microplate reader. About 1 X 10(4) cells were sufficient for assay of PO activity. Using the above method to assay PO activity and sandwich enzyme immunoassay for thyroglobulin (TG), chronological changes in the PO activity and TG concentration in the culture medium were examined. Although the cells showed no decrease in number, PO activity and TG concentration decreased chronologically. When the ratio of PO activity to TG concentration was calculated, in 3 cases the ratio was almost constant, and in the remaining two, it decreased chronologically. The present biochemical method thus seems useful for determining peroxidase activity of cultured thyroid en masse.     

CEDIA in vitro diagnostics with a novel homogeneous immunoassay technique Current status and future prospects

CEDIA assays represent a state of the art technique utilizing two genetically engineered, enzymatically inactive fragments of β-galactosidase as the basis for a homogeneous enzyme immunoassay. The smaller, amino-terminal polypeptide, designated the enzyme donor (ED), can recombine spontaneously with the large residual fragment, called the enzyme acceptor (EA), to form active β-galactosidase, in a process called complementation. ED have been designed in such a way that a ligand, such as a hormone or drug, can be chemically attached to a specific amino acid residue without affecting the enzyme complementation. However, the binding of a ligand-specific antibody to the ED-ligand conjugate will inhibit complementation. If a sample containing ligand is added to the reaction mixture, the ligand will compete with the ED-ligand conjugate for the limited number of antibody binding sites. Thus, the ligand concentration in the sample will modulate enzymatic activity by influencing the amount of free ED-ligand conjugate available for complementation.

The basic technology of CEDIA assays has a number of inherent advantages, the most important of these being a linear calibration curve with high precision over the whole assay range, lack of endogeneous enzyme activity and minimal serum interference, chemically defined conjugates and flexibility in assay design. These provide significant advantages in comparison to other homogeneous immunoassay techniques. As a result, CEDIA assays have been successfully developed for high concentration drugs such as theophylline, phenobarbital and phenytoin as well as for very low concentration analytes such as digoxin, B12 and folate. In a modified assay format, even the determination of binding proteins has been accomplished, an example being thyroxine binding proteins in the CEDIA T-uptake assay. More recently, the methodology has been extended to the measurement of high molecular weight analytes like ferritin.     

ENDOGENOUS PEROXIDASE ACTIVITY IN PRIMARY CULTURE OF HUMAN THYROID CELLS Localization and Measurement

Intracellular localization of and an assay method for endogenous peroxidase (PO) activity were studied using primary culture of thyroid cells obtained from patients with hyperthyroidism. PO activity was visualized by cytochemical reaction and was located mainly in perinuclear cisternae and rough endoplasmic reticulum. With increased culture time, the number of cells showing positive PO activity and amount of the enzyme reaction product in individual cells showed a parallel decrease. For measurement of PO activity, cultured thyroid cells were frozen and thawed and then incubated with citric acid buffer solution containing o-phenylenediamine (opd) and hydrogen peroxide. After incubation, the optical density (OD) of the solution colorized by endogenous peroxidase was measured at 405 nm using a microplate reader. About 1 × 10⁴ cells were sufficient for assay of PO activity. Using the above method to assay PO activity and sandwich enzyme immunoassay for thyroglobulin (TG), chronological changes in the PO activity and TG concentration in the culture medium were examined. Although the cells showed no decrease in number, PO activity and TG concentration decreased chronologically. When the ratio of PO activity to TG concentration was calculated, in 3 cases the ratio was almost constant, and in the remaining two, it decreased chronologically. The present biochemical method thus seems useful for determining peroxidase activity of cultured thyroid en masse.     

A sensitive assay for the IFN-regulated 2–5A synthetase enzyme

2–5A synthetase is the central enzyme of the 2–5A system, an important mediator of interferon action. An assay capable of detecting low, yet biologically important levels of 2–5A synthetase enzyme activity is described. The purification of enzyme reaction products on SepPak C-18 cartridges resulted in a significant reduction in background, when a high specific activity substrate was used to label the 2–5A. Quantitation of labeled 2–5A by chromatography and scintillation counting provided a means of detecting femptomolar amounts of 2–5A. The combination of these procedures accounts for a 3–4 log increase in sensitivity over existing assays. This degree of sensitivity should permit a more accurate determination of the 2–5A synthetase activity in vivo leading to a better understanding of the role of the 2–5A system in virus infection and other cellular processes.     

Isoelectric focusing spectra of antibodies which activate mutant -galactosidases

Antibodies raised against wild type β-galactosidase of Escherichiu coli activate certain inactive mutant β-galactosidases to enzymic activity. We have developed a technique to detect 7 S activating antibody after isoelectric focusing in thin layers of polyacrylamide gels. Enzymic activity bound to antibodies is detected using the histochemical substrate 5-bromo-4-chloro-3-indoxyl-β-D-galacto-pyranoside. When split, this substrate produces insoluble, blue-green indigo stain at the sites of enzymic reaction. With this technique antisera raised in five rabbits against the wild type enzyme have been analyzed for their capacity to activate inactive mutant enzyme. Each rabbit produces a characteristic spectrum of activating antibodies when tested with a given mutant enzyme, which is different from that produced by another rabbit. A restricted population of antibodies, e.g. the product of one to five clones of antibody-forming cells activate a mutant enzyme. The spectrum of activating antibodies obtained with a mutant enzyme can be correlated to the position of the mutation in the gene for β-galactosidase which gives rise to this inactive enzyme. Three genetically different groups of mutant enzymes are activated by three distinct populations of antibodies within the serum of one rabbit. Mutation at a fourth site within the gene yields a mutant enzyme which is activated by one of the three populations of specific antibodies. This indicates that one antibody, in binding to one antigenic site, can restore the activity of two mutant enzymes altered at two different sites within the polypeptide chain of the enzyme.     

Removal of Alpha-Gal Epitopes from Porcine Aortic Valve and Pericardium using Recombinant Human Alpha Galactosidase A

It has been reported that the immune response due to α-Gal epitopes is an important factor in tissue valve failure. The elimination of the interaction between the natural anti-Gal antibodies and α-gal epitopes on the xenografts is a prerequisite to the success of xenografts in humans. Previously, we reported that the green coffee bean α-galactosidase could remove all α-Gal epitopes from cell surface of porcine aortic valve and pericardial tissue, but it has limitations on cost effectiveness. In this study we wanted to know whether the recently produced recombinant human α-galactosidase A has the same effective enzymatic activity as green coffee bean α-galactosidase in removing α-Gal epitopes from the same tissues. After treating fresh porcine aortic valve and pericardial tissue with recombinant α-galactosidase A, each sample was stained with Griffonia simplicifolia type I isolectin B4 indirect immunoperoxidase avidin-biotin technique. We then examined whether the α-Gal epitopes were reduced or abolished in each consecutive concentration of recombinant α-galactosidase A by comparing the degree of the Griffonia simplicifolia isolectin B4 staining. As a result, the recombinant α-galactosidase A could remove cell surface α-Gals on porcine aortic valve and pericardial tissue as effectively as green coffee bean α-galactosidase.     

Enzyme immunoassay techniques an overview

In spite of the great variety of enzyme immunoassays (EIA) they can be classified into two groups ‘analyte-observed’ and ‘ reagent-observed’ assays, depending on their reaction principle. The latter are favored by use of monoclonal antibodies and are characterized by a greater sensitivity, a larger measuring range, a lower susceptibility to disturbing influences. They can be used only for detection of macromolecules. For heterogeneous EIAs to be used on laboratory scale, simple adsorption of antigens and antibodies is still recommendable though affinity constants decrease by at least one order of magnitude and antibody density at the solid phase and analyte binding capacity are not parallel due to increasing steric hindrance. For this reason, the antibody with the higher affinity constant should therefore always be used as solid-phase antibody. Microparticles used as solid phase for heterogeneous assays, due to their very high binding capacity for the analyte and extremely short diffusion distances, guarantee ‘one step’ assays of only a few minutes.

Of the limited number of enzymes suitable as markers in immunoassays, horseradish peroxidase is the enzyme of choice followed by alkaline phosphatase. Although enzyme and enzyme-labelled reagents are detectable by fluorogenic product measuring with a sensitivity, which is 10–1000 times higher than using chromogenic substrates, the sensitivity of the assays can be increased only by factor 2–10.

Labelling enzymes cannot only be covalently bound to the antibody, but also via anti-enzyme antibodies. Pros and cons of the different methods of coupling the enzyme/anti-enzymes complex to analyte-containing immune complexes are discussed.

Different EIA variants to detect specific antibodies are reviewed. Among them only capture EIAs permit precise isotype analysis of antibodies of a distinct idiotype. Homogeneous EIAs are widely spread for hapten determination but even variants based on proximal linkage are no alternatives to heterogeneous EIAs for determination of macromolecules.

Different parameters are defined which permit to assess the quality of an immunoassay and which should be used in routine assays as internal controls in the laboratory.     

A rapid and simple immunoperoxidase staining procedure for blood and bone marrow samples

A fast and simple indirect immunoperoxidase staining technique is described, which can also be used for the characterization of cells with high endogenous peroxidase activity such as many haemopoietic cells. It is based on the combination of a newly developed glucose-oxidase plus glucose procedure for the inhibition of endogenous peroxidase activity with a standard 2 or 3 layer immunoperoxidase staining protocol. Glucose-oxidase plus glucose mixture completely inhibits endogenous peroxidase activity without having a detectable deleterious effect on any of the cellular antigens so far studied. In many instances this permits the use of only 1 layer of horseradish peroxidase (HRP)-labelled antibodies. The glucose-oxidase plus glucose mixture can also be added to cells together with the HRP-labelled antibody solution without losing its inhibitory effect for endogenous peroxidase activity and without leading to a visually detectable loss of the activity of the HRP conjugate. Consequently, the separate incubation step previously necessary for the inhibition of endogenous peroxidase activity becomes superfluous.     

Improved accuracy in diagnostic immunohistochemistry, lectin histochemistry and in situ hybridization using a gold-labeled horseradish peroxidase antibody and silver intensification.

BACKGROUND: Improvements in the use of the avidin-biotin peroxidase complex technique and direct as well as indirect labeled avidin-biotin methods for application in diagnostic immunohistochemistry, lectin histochemistry and in situ hybridization are reported. The new technology combines the advantages of immunoenzyme and immunogold silver staining techniques and can be performed on routinely fixed and paraffin-embedded tissues. EXPERIMENTAL DESIGN: The basic modification of the labeling procedures was introduced at the final revealing step. The histochemical visualization of catalytic activity of horseradish peroxidase by the diaminobenzidine reaction was replaced by the detection of horseradish peroxidase immunoreactivity using anti-horseradish peroxidase-gold complexes and their intensification with silver acetate which is relatively light insensitive. RESULTS: The use of gold-labeled anti-horseradish peroxidase antibodies eliminates the need for quenching of endogenous peroxidase activity. Furthermore, the immunogold silver staining provides improved lateral resolution, higher contrast, and lower background staining as compared with the diaminobenzidine reaction. The new technology has been applied for the localization of different polypeptides in endocrine cells, cytoskeletal elements, cell surface receptors, basal lamina type IV collagen, endothelial cell marker, lectin binding sites, and DNA of various viruses. CONCLUSIONS: We concluded that the anti-horseradish peroxidase-gold complex is of general use in a variety of techniques applying horseradish peroxidase as a marker and should be a valuable alternative to existing enzyme substrate techniques.     

Complement Fixing and Macrophage Opsonizing Activities Associated with β2 Microglobulin

Human β² microglobulin has been tested for 3 of the known effector functions of human immunoglobulin G, namely complement activation, opsonization and sensitization of mast cells. The ability of β microglobulin to fix complement was demonstrated by a complement fixation assay following aggregation of the protein by adsorption on latex or chemical cross linking with bisdiazobenzidine (BDB) and also by a CI inhibition assay using the native protein. In the latter assay β microglobulin was as effective as human Fc in binding CI and showed a similar dose response curve. When coated on sheep erythrocytes β microglobulin was able to promote rosette formation by guinea pig peritoneal macrophages. These properties have previously been shown to be associated with different homology regions of the Fc fragment. β microglobulin was not able to participate in PCA or RPCA reactions in the guinea pig.     

Identification of Haemophilus influenzae type b by a monoclonal antibody coagglutination assay.

A coagglutination assay using monoclonal antibody is described for the identification of Haemophilus influenzae type b. An immunoglobulin G2a monoclonal antibody, Hb-2, directed against a serotype-specific outer membrane protein of H. influenzae type b was adsorbed to Staphylococcus aureus Cowan 1 cells. In a dot enzyme immunoassay, Hb-2 reacted with 453 of 455 H. influenzae type b isolates and did not react with H. influenzae of other serotypes, untypeable H. influenzae strains, or other bacterial species. The Hb-2 coagglutination assay was evaluated by testing 136 H. influenzae type b strains selected on the basis of multilocus enzyme genotypes, 5 strains of another serotype, and 94 untypeable H. influenzae strains. The specificity of the coagglutination assay was demonstrated by the inhibition of the reaction by free Hb-2 monoclonal antibodies. The coagglutination assay was as specific as the dot enzyme immunoassay and can be rapidly performed and easily interpreted.     

A novel enzyme-linked immunosorbent assay for cortisol using a long-chain biotinylated cortisol-3-CMO derivative

An enzyme-linked immunosorbent assay (ELISA) using streptavidin-biotin system as a bridge between antibodies bound antigen and reporter molecule (horseradish peroxidase enzyme) has been described. The cortisol antiserum was generated against cortisol-3-O-carboxylmethyl oxime-bovine serum albumin (F-3-CMO-BSA). We have prepared biotin-labelled cortisol as a primary probe and utilized streptavidin-labelled horseradish peroxidase (SA-HRP) as secondary probe to monitor the antigen-antibody interaction. To the cortisol antibody coated micro wells, 25 microL of standard or samples, along with 100 microL of biotinylated cortisol, were kept for 1 h at room temperature. Thereafter, wells were washed and 100 microL of SA-HRP was added to all wells and kept again for 20 min at room temperature. Bound enzyme activity was measured using tetramethyl benzidine/hydrogen peroxidase (TMB/H2O2) as substrate. The incorporation of streptavidin-biotin system as a bridge between antibody bound antigen and reporter molecule (horseradish peroxidase enzyme) increased sensitivity and specificity of the cortisol assay. The use of low molecular weight primary label (F-3-CMO-biotin) might have facilitated the easy and selective access of the analyte present in serum to compete with the antigen-binding pocket of antibody, thereby detecting as low as 3.42 ng/mL of analyte specifically.     

Mapping of B-cell epitopic sites and delineation of functional domains on the hemagglutinin-neuraminidase protein of peste des petits ruminants virus

A recombinant baculovirus expressing membrane bound form of hemagglutinin–neuraminidase (HN) protein of peste des petits ruminants virus (PPRV) was employed to generate monoclonal antibodies (mAbs) against PPRV-HN protein. Four different mAbs were employed for mapping of regions on HN carrying B-cell epitopes using deletion mutants of PPRV-HN and RPV-H proteins expressed in Escherichia coli as well as PPRV-HN deletion proteins expressed transiently in mammalian cells. The immuno-reactivity pattern indicated that all mAbs bind to two discontinuous regions of amino acid sequence 263–368 and 538–609 and hence the epitopes identified are conformation-dependent. The binding regions for three mAbs were shown to be immunodominant employing competitive ELISA with vaccinated sheep sera. Delineation of functional domains on PPRV-HN was carried out by assessing the ability of these mAbs to inhibit neuramindase activity and hemagglutination activity. Two mAbs inhibited NA activity by more than 63% with substrate N-acetyl neuraminolactose, while with Fetuin one mAb showed inhibition of NA activity (95%). Of the three antigenic sites identified based on competitive inhibition assay, site 2 could be antigenically separated into 2a and 2b based on inhibition properties. All the four mAbs are virus neutralizing and recognized PPRV-HN in immunofluorescence assay.     

Synthetic peptide conjugates with horseradish peroxidase and beta-galactosidase for use in epitope-specific immunocytochemistry and ELISA.

Synthetic peptide-alkaline phosphatase conjugates can be used to detect the epitope specificity of (i) antibody-forming cells in vivo by immunocytochemistry; (ii) of antibody secreting cells in vitro by spot-ELISA; and (iii) antibodies in solution by capture ELISA. The availability of synthetic peptide-enzyme conjugates using detector enzymes other than alkaline phosphatase would offer several important advantages, for example in double staining approaches. This paper reports the production of synthetic peptide-horseradish peroxidase conjugates and synthetic peptide-beta-galactosidase conjugates. A peptide of 21 amino acids (SP 29) was coupled to peroxidase in seven differing molar ratios of peptide over peroxidase, ranging from 1:3.4 to 1:575, using periodate oxidation of the enzyme. SP 29 was coupled to beta-galactosidase in four molar ratios ranging from 1.25 to 10, using glutaraldehyde pre-activation of the enzyme. The enzyme activity of the different conjugates was determined, the conjugates were tested in direct capture-ELISA with peptide-specific monoclonal antibodies, and the conjugates were tested in immunocytochemistry to detect peptide-specific B cells. The results show that the conjugates perform best if the peptide is coupled to the enzyme at relatively low molar ratios (1-30). The availability of these new peptide-enzyme conjugates broadens the applicability of synthetic peptides for detection purposes in several assay systems.     

Generation, characterization and ELISA of monospecific antibodies against the subunits of a Ca-dependent protein kinase and a Ca-transport ATPase from rabbit skeletal muscle

Monospecific precipitating sheep antibodies were generated for the first time against the purified, homogeneous -, β- and γ-subunits of the Ca²⁺-dependent protein kinase, phosphorylase kinase, from rabbit muscle. As reference, antibodies against the holoenzyme and the Ca²⁺-transport ATPase of sarcoplasmic reticulum were induced. In all cases antibody titers could be quantitated (standard error 5–10%) by enzyme-linked immunosorbent assay. Differentiation of antibody binding was achieved by quantitative precipitation and complement fixation assays. In general maximal antibody titers were reached 56 days after primary immunization and high titers ( 5000) were maintained for several weeks. Anti-, anti-β and anti-γ avidly precipitate the denatured subunits employed as immunogens as well as the native enzyme. No cross-reactivity between antibodies against a specific subunit and any of the other heterologous subunits was demonstrable in double immunodiffusion assays providing no evidence for immunologically identical sites on the -, β- and γ-subunits. Since anti-, anti-β and anti-γ strongly inhibit enzyme activity, it is likely that they do so primarily by sterically interfering with the binding of the large substrate phosphorylase b (M_r 2.0 × 10⁵) to phosphorylase kinase (M_r 1.3 × 10⁶). It cannot be excluded, however, that anti-β and anti-γ bind to the active sites on these 2 subunits.