Further characterization of cooperative interactions of monoclonal antibodies

We reported previously that mixtures of some monoclonal antibodies directed against the glycoprotein hormone human chorionic gonadotropin (hCG) had a higher affinity for the antigen than either monoclonal antibody separately. The synergistic interaction could no longer be detected when one of the antibodies was replaced with its F(ab) fragment. This cooperative interaction has now been further characterized. One-half of 10 possible pairs prepared from five IgG1 monoclonal antibodies against hCG result in a synergistic interaction. The addition of an IgG2b monoclonal antibody to one of the IgG1 monoclonal antibodies also induces a cooperative interaction, which shows that the effect is not subclass restricted. Cooperative interactions between antibodies are also not restricted to solution conditions; adsorption of one antibody to a solid support appears to increase the cooperative effect. Indeed, one pair of antibodies that failed to bind hCG synergistically in solution did so when one antibody was bound to a solid surface. The liquid phase antibody also has an effect on the specificity of the solid phase antibody. The sensitivity of the solid phase assay system has enabled us to develop a rapid method of determining if two monoclonal antibodies can bind to an antigen simultaneously. A quantitative theoretical model has been devised that successfully predicts the cooperative behavior observed between antibodies and should be useful in devising conditions that result in sensitive solid phase radioimmunoassays.     

Monoclonal antibodies for carcinoembryonic antigen and related antigens as a model system: determination of affinities and specificities of monoclonal antibodies by using biotin-labeled antibodies and avidin as precipitating agent in a solution phase immunoassay

A general method is described for the determination of affinity constants and antigen cross-reactivities of monoclonal antibodies. The method employs biotin-labeled antibody, radiolabeled antigen, and avidin as a precipitating agent in a homogeneous phase, competitive radioimmunoassay. This method eliminates incomplete or variable precipitation of antigen-antibody complexes often encountered in immunoassays in which monoclonal antibodies are employed. Using this assay system, we were able to rapidly determine the affinity constants for a number of monoclonal antibodies elicited to carcinoembryonic antigen (CEA). In the preceding paper it was shown that five of the monoclonal antibodies recognized distinct epitopes on CEA. In antigen-binding experiments with these five monoclonal antibodies, the percent of radiolabeled CEA bound in antibody excess ranged from 30 to 92%. The CEA cross-reacting antigens, normal cross-reacting antigen (NCA), and tumor-extracted, CEA-related antigen (TEX) were significantly bound by one, and to a lesser degree, by two of the five antibodies. Two antibodies did not bind significant amounts of NCA or TEX. In inhibition studies, the amount of unlabeled CEA leading to 50% inhibition of 125I-labeled CEA-binding was in the range of 3.7 to 760 ng per tube. The amount of TEX showing the same degree of inhibition was 23-fold greater than the amount of CEA for two antibodies and 351-fold greater than the amount of CEA for a third antibody. The affinity constants for CEA were in the range of 1.0 x 10(8) to 5.1 x 10(10) M-1. The affinity constants for NCA and TEX, determined for one of the antibodies, were three orders of magnitude lower in comparison to CEA. The heterogeneity of radiolabeled CEA as indicated by the low fraction bound by one of the monoclonal antibodies is shown to be most probably an artifact resulting from radioiodination damage. The application of the approach described in this report should eliminate the problems most commonly encountered in the determination of affinity constants for monoclonal antibodies or the use of monoclonal antibodies in competitive, homogeneous-phase immunoassays.     

Detection and characterization of monoclonal antibodies to platelet membrane proteins

We have devised a solid-phase radioimmunoassay for the detection and characterization of monoclonal antibodies directed against platelet surface antigens. Platelet membrane proteins, solubilized with 0.1% Triton X-100, were covalently coupled to cyanogen bromide (CNBr)-activated filter paper disks that were than used as the support in antibody binding assays. SDS PAGE of solubilized membrane proteins taken immediately before and after incubation with activated disks indicated that representative amounts of each membrane protein were bound to the disks. Either monoclonal or heterologous anti-platelet antibody could be detected on disks that had been prepared using as little as 50 micrograms of membrane protein per 100 disks. For the detection of antibody, disks were incubated with test sera for 2 h, washed, and incubated with 125I-labeled anti-immunoglobulin G, and the amount of bound radioactivity was determined. The sensitivity of the disk assay in detecting monoclonal antibodies was far greater than that of a corresponding radioimmunoassay that used whole platelets as the solid phase. By linking other proteins such as fibrinogen or anti-mouse subclass specific antisera to CNBr-activated disks, the method was adapted for antibody characterization. The sensitivity and ease with which the assay can be performed make this technique most suitable for screening and characterizing monoclonal antibodies.     

Bioimmunoassay (BIA): A Sandwich Immunoassay Scheme Employing Monoclonal Antibodies and Hormone Receptors to Quantify Analytes

Abstract

When some antigens bind to receptors, a portion of the antigen remains exposed and can be recognized by labeled monoclonal antibodies. By measuring the amount of antibody bound to the antigen-receptor complex, one can quantify the amount of antigen that is present. Since this assay procedure depends on simultaneous receptor recognition of a biologically active site and antibody recognition of a distal epitope on the analyte, we call it a bioimmunoassay. Bioimmunoassays have many of the advantages of radioligand receptor assays (RRA) used to quantify biological activity and, depending on the choice of antibodies employed, may be more specific than RRA. In addition, since they are sandwich assays, they are usually more sensitive than RRA. Bioimmunoassays can be performed in several different modes and in the case described here we used a radiolabeled antibody to detect hormone-receptor complexes. Hence we term this example a bio-immunoradiometric assay or BIO-IRMA. We illustrate the properties of various assay procedures using a monoclonal antibody to the beta subunit of hCG which recognizes an epitope common to all other mammalian LH/hCG-like gonadotropins and which is capable of detecting 10 pg of hCG standard. In principle, this assay can be applied to any material capable of binding to a receptor, enzyme, etc. which can also be recognized by an antibody. Since it is a sandwich type of assay, it is subject to the same advantages and limitations of other sandwich assays except that it can be used to discriminate some biologically active and inactive analytes. Monoclonal antibodies which are prepared from spleen cells of animals immunized with antigen-receptor complexes and selected for their ability to bind antigen-receptor complexes should prove most useful for bioimmunoassay procedures.     

A radiometric immunosorbent assay for the detection of anti-hormone-binding protein antibodies

A radiometric immunosorbent assay (RISA) for the detection of monoclonal antibodies to hormone-binding proteins has been developed. The assay involves incubating hybridoma supernatants in microtiter wells that have been coated with goat anti-mouse IgG antibodies. Any mouse IgG in the test supernatant is thus specifically retained in the wells. Radioactive ligand-binding protein complexes are then incubated in the wells. The presence of anti-binding protein antibodies in the supernatant is indicated by specific retention of radioactive ligand-binding protein complexes in the wells. Crude antigen preparations, such as tissue homogenates, can be used to detect antibodies. The assay is capable of detecting antibody at concentrations 20 ng/ml (approximately 100 pM IgG). The RISA has been used successfully to screen for monoclonal antibodies to the intracellular receptor for 1,25-dihydroxyvitamin D3 and should be useful for the detection of antibodies to ligand-binding proteins in general.     

Near real-time biosensor-based detection of 2,4-dinitrophenol

A fluorescent biosensor assay has been developed for near real-time detection of 2,4-dinitrophenol (DNP). The assay was based on fluorescent detection principles that allow for the analysis of antibody/antigen interactions in solution using the KinExA immunoassay instrument. Our KinExA consisted of a capillary flow observation cell containing a microporous screen that maintains a compact capture antigen-coated bead bed. The bead bed was comprised of polymethylmethacrylate (PMMA) beads coated with dinitrophenol-human serum albumin (DNP-HSA) conjugate. Phosphate buffered saline (PBS) solutions, containing various concentrations of free DNP, were incubated for 30 min with mouse anti-DNP monoclonal antibody to equilibrium. Solutions containing the DNP-monoclonal antibody complex and possible excess free antibodies were then passed over DNP-HSA labeled beads. The free monoclonal anti-DNP antibody, if available, was then bound to the DNP-HSA fixed on the beads. The system was then flushed with excess PBS to remove unbound reactants in the bead bed. The beads were then subjected to brief contact with PBS solutions containing goat anti-mouse fluorescein isothiocyanate (FITC)-labeled secondary antibody, once again, followed by a short PBS flush. The fluorescence was recorded during the addition of the FITC labeled secondary antibody to the bead bed through the final PBS flushing with the KinExA. The amount of DNP detected could then be determined from the fluorescent slopes that were generated or by the remaining fluorescence that was retained on the beads after final PBS flushing of the system. This assay has been able to detect a minimum of 5 ng/ml of DNP in solution and can be adapted for other analytes of interest simply by changing the capture antigen and antibody pairs.     

长叶车前花叶病毒上海分离株单克隆抗体的制备及其免疫学特性 Ⅲ 病毒抗原决定簇的单克隆抗体识别

前文分别报道了长叶车前花叶病毒上海分离侏(RMVsh)单克隆抗体的制备及根据它们在不同免疫反应中的特性,将它们分为两组,分别识别性质不同的抗原决定簇。本文采用修改的Friguet方法测定了各组内各单克隆抗体之间的增值反应(Additivity Reaction)特性,并分析了它们识别抗原决定簇的特性。村料与方法一、病毒及单克隆抗体长叶车前花叶病毒上海分离株及其单克隆抗体1H2、7H1、10H1、11H2、12H3、17H6、29H1来源、制备和特性见前文报道。二、抗原饱和曲线的测定抗原饱和曲线测定采用间接ELISA办法,抗原浓度为2μg/ml。     

Monoclonal antibodies to antigens in the peribacteroid membrane from Rhizobium-induced root nodules of pea cross-react with plasma membranes and Golgi bodies

Three rat hybridoma lines that produced monoclonal antibodies reacting with the peribacteroid membrane from Pisum sativum were isolated, and these all appeared to recognize the same antigenic structure. Using one of these monoclonal antibodies, AFRC MAC 64, electron microscopy of immunogold-stained thin sections of nodule tissue revealed that the antigen, present in the peribacteroid membrane, was also found in the plant plasma membranes and in the Golgi bodies, but not in the endoplasmic reticulum. When peribacteroid membrane proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose by electro-blotting, it was found that MAC 64 bound to a series of protease-sensitive bands that migrated in the mol. wt. range 50-85 K. The epitope was sensitive to periodate oxidation and its structure may therefore involve the carbohydrate component of a membrane glycoprotein. We suggest that this structure originates in the Golgi apparatus and is subsequently transferred to the peribacteroid membranes and plasma membranes. The monoclonal antibody also reacted with peribacteroid membranes from nodules of Vicia and lupin, and with plasma membranes and Golgi membranes from uninfected plant cells, including root tip cells from onion (Allium cepa), indicating that the antigen is highly conserved in the plasma membranes of plant cells.     

The molecular chaperone calnexin associates with the vacuolar H(+)-ATPase from oat seedlings.

Acidification of endomembrane compartments by the vacuolar-type H(+)-ATPase (V-ATPase) is central to many cellular processes in eukaryotes, including osmoregulation and protein sorting. The V-ATPase complex consists of a peripheral sector (V1) and a membrane integral sector (V0); however, it is unclear how the multimeric enzyme is assembled. A 64-kD polypeptide that had copurified with oat V-ATPase subunits has been identified as calnexin, an integral protein on the endoplasmic reticulum. To determine whether calnexin interacted physically with the V-ATPase, microsomal membranes were Triton X-100 solubilized, and the protein-protein interaction was analyzed by coimmunoprecipitation. Monoclonal antibodies against calnexin precipitated both calnexin and V-ATPase subunits, including A and B and those of 44, 42, 36, 16, and 13 kD. A monoclonal antibody against subunit A precipitated the entire V-ATPase complex as well as calnexin and BiP, an endoplasmic reticulum lumen chaperone. The results support our hypothesis that both calnexin and BiP act as molecular chaperones in the folding and assembly of newly synthesized V1V0-ATPases at the endoplasmic reticulum.     

An immunoradiometric assay for 1,25-dihydroxyvitamin D3 receptor

A ligand-independent, quantitative assay has been developed for the measurement of 1,25-dihydroxyvitamin D receptor utilizing purified receptor from pig intestine as a standard and two high affinity monoclonal antibodies directed to two different epitopes on the receptor. In this assay a fixed amount of 125I-labeled antibody is incubated with a fixed amount of a second antireceptor antibody linked to biotin and increasing amounts of purified receptor protein or sample. Antibody-receptor complexes can then be immunoprecipitated with avidin-Sepharose beads and counted. This method is highly reproducible and can detect 150 pg of 1,25-dihydroxyvitamin D3 receptor in crude extracts with intra- and interassay coefficients of variation of 8.6 and 18.2%. The monoclonal antibodies used recognize both native and denatured receptors from several different species, including human. This immunoradiometric assay should prove useful for studies of receptor regulation, occupancy, distribution, and turnover.     

Solid-phase assay for the detection of low-abundance enzymes, and antibodies to enzymes in immune reactions, using acid sphingomyelinase as a model

The development of a solid-phase immunosorbent assay, suitable for use with enzyme antigens, is described. Acid sphingomyelinase and a mouse monoclonal anti-sphingomyelinase antibody have been used to determine optimal conditions for the assay. The assay involves immobilization of a second antibody (anti-mouse IgG) in the wells of a polyvinyl microtiter plate. Soluble immune complexes of first antibody (monoclonal anti-sphingomyelinase) and antigen (sphingomyelinase), incubated in separate vials, are then reacted in the anti-mouse IgG-coated assay wells, and the extent of the cross-reaction between antibody and antigen is measured by direct assay of enzyme retained in the well. A necessary condition of the assay is that antibody must not inhibit enzyme activity, which makes it especially suitable for monoclonal antibodies. The assay finds useful application in hybridoma fluid screening, equivalence point determination, and demonstration of cross-reacting enzyme from various tissue sources.     

Functional cholesteryl binding agents: evaluation of viability and specificity of antibodies bound to modified phospholipid vesicles

A cholesterol derivative was incorporated into small unilamellar phospholipid vesicles, and antibodies were bound covalently to the vesicles. More than one antibody was bound to each vesicle. The antigen binding viability and specificity were determined using a modified radioimmunoassay and an in vitro cell assay. Both of these tests showed good antibody activity and specificity. The antigen affinity of the bound antibodies was higher than for the unbound antibody due to more than one viable antibody being bound to each vesicle. The modified vesicles can be used as immunodirected drug delivery systems for both diagnosis and therapy.     

Use of Radioactive Antibodies for Characterizing Antigens and Application to the Study of Flagella Synthesis

A simple rapid immunochemical procedure has been developed which provides information about the qualitative and quantitative nature of antigens. It involves the use of purified radioactive ((125)I-labeled) antibodies. The amount of antibody bound to the antigen is determined by filtering the mixture through diethylaminoethyl (DEAE)-cellulose paper. All of the antigen, as well as the antibody complexed with it, is trapped on the paper, whereas free antibody is removed by repeated washing. This technique has been applied to the study of three immune systems, bovine serum albumin, Escherichia coli tryptophan synthetase B protein, and Bacillus subtilis flagella. The results obtained by the DEAE-antibody binding technique were comparable, in terms of sensitivity, specificity, and accuracy, to data obtained by microcomplement fixation and precipitin methods. The assay was used to measure the kinetics of flagella regeneration in B. subtilis.     

Quality control test for immunoreactivity of radiolabeled antibody

A rapid method for the measurement of the immunoreactive fraction of a radiolabeled monoclonal antibody or antibody fragment has been developed. This may be used as a quality control test prior to patient administration of the radiolabeled antibody preparation. The test employs solid phase antigens and the assay is conducted under conditions of antigen excess. Assay parameters have been evaluated and a standardized procedure has been developed. The assay has been compared to a standard extrapolation method and found to give approximately the same result. The test has been used on four different radiolabeled antibodies currently in clinical trials in patients with colorectal cancer. Mean immunoreactive fractions for these radiolabeled antibodies ranged from 35 to 65% and the variability of the immunoreactive fraction ranged from 140 to 240% for different antibodies. We conclude that the quality, defined as the immunoreactive fraction, of radiolabeled antibodies is both low and highly variable, indicating the need for a quality control test of these radiopharmaceuticals in the clinic prior to patient administration.     

Internalizing Cancer Antibodies from Phage Libraries Selected on Tumor Cells and Yeast-Displayed Tumor Antigens

A number of approaches have been utilized to generate antibodies to cancer cell surface receptors that can be used as potential therapeutics. A number of these therapeutic approaches, including antibody-drug conjugates, immunotoxins, and targeted nucleic acid delivery, require antibodies that not only bind receptor but also undergo internalization into the cell upon binding. We previously reported on the ability to generate cancer cell binding and internalizing antibodies directly from human phage antibody libraries selected for internalization into cancer cell lines. While a number of useful antibodies have been generated using this approach, limitations include the inability to direct the selections to specific antigens and to identify the antigen bound by the antibodies. Here we show that these limitations can be overcome by using yeast-displayed antigens known to be associated with a cell type to select the phage antibody output after several rounds of selection on a mammalian cell line. We used this approach to generate several human phage antibodies to yeast-displayed EphA2 and CD44. The antibodies bound both yeast-displayed and mammalian cell surface antigens, and were endocytosed upon binding to mammalian cells. This approach is generalizable to many mammalian cell surface proteins, results in the generation of functional internalizing antibodies, and does not require antigen expression and purification for antibody generation.     

Microimmunoassay permits determination of concentrations in immunohistochemistry controls

A competition enzyme-linked immunosorbant assay (CELIA) can be used to determine the amount of antigen needed to immunoabsorb an antiserum. This is demonstrated using both polyclonal and monoclonal antibodies against human amylase. This method is of especial value when the tissue is particularly difficult to obtain, and especially valuable when CELIA has already been completed.     

A simple hybridoma screening method for high-affinity monoclonal antibodies using the signal ratio obtained from time-resolved fluorescence assay

In hybridoma screening, quantitative kinetic evaluation is difficult since the concentration of each antibody in the hybridoma supernatant is unknown. From modeling calculations, we hypothesized that the ratio of two different antigen-antibody concentrations might allow discrimination of high-affinity monoclonal antibodies irrespective of the antibody concentration. Using anti-alpha-fetoprotein monoclonal antibodies of known affinity, we set the signal ratio of a time-resolved assay at >0.1, in which the antigen concentrations were 10 and 100 ng/mL. From anti-alpha-fetoprotein hybridoma screening with this assay, it was possible to effectively select high-affinity monoclonal antibodies with KD values below 1x10(-8) M. High-sensitivity sandwich enzyme-linked immunosorbent assay which detects domain III of alpha-fetoprotein has been established using selected high-affinity monoclonal antibodies. This screening method is useful for selection of high-affinity monoclonal antibodies of potential diagnostic value.     

Regional distribution of prostaglandin endoperoxide synthase studied by enzyme-linked immunoassay using monoclonal antibodies

Prostaglandin endoperoxide synthase transforms arachidonic acid to prostaglandin H2 via prostaglandin G2. The enzyme purified from bovine vesicular gland was given to mice as antigen, and monoclonal antibodies were raised by the hybridoma technique. Two species of the monoclonal antibody recognizing different sites of the enzyme were utilized to establish a peroxidase-linked immunoassay of prostaglandin endoperoxide synthase. Fab' fragment of one of the antibodies was prepared and conjugated to horseradish peroxidase. The conjugate was then bound to prostaglandin endoperoxide synthase, and the labeled enzyme was precipitated by the addition of the other antibody. The peroxidase activity of the immunoprecipitate correlated linearly with the amount of prostaglandin endoperoxide synthase. This sensitive and convenient method to determine the enzyme amount rather than the enzyme activity was utilized to extensively screen the amount of prostaglandin endoperoxide synthase in various bovine tissues. In addition to vesicular gland, platelets and kidney medulla previously known as rich enzyme sources, the immunoenzymometric assay demonstrated a high content of the enzyme in various parts of alimentary tract and a low but significant amount of enzyme in some parts of brain.     

A monoclonal antibody to an oocyte-specific poly(A) RNA-binding protein

Xenopus oocyte-specific poly(A) RNA-binding proteins were isolated and used to prepare monoclonal antibodies. One antibody was used to characterize one particular antigen by immunoblot analysis. The antigen had a molecular weight of 56,000 was oocyte-specific, and decreased in amount during oogenesis. The antigen was localized in the cytoplasm throughout oogenesis and sedimented mainly at 40-60 S. The antigen also was shown to bind poly(A) RNA following chromatography of ribonucleoprotein particles on oligo(dT)-cellulose. The antibody was used to immunoadsorb nontranslating ribonucleoprotein particles. Fifty-five per cent of the poly(A) RNA sedimenting between 40-60 S was shown to be bound by the antigen. The further use of this antibody in attempting to examine other components of the ribonucleoprotein particle is discussed.