Specificity of monoclonal antibodies reactive with Fusobacterium nucleatum: effect of formalin fixation

Monoclonal antibody specific for Fusobacterium nucleatum was reacted with untreated and formalin fixed F. nucleatum cells by an enzyme-linked immunosorbent assay (ELISA) and by indirect immunofluorescence. Treatment of bacterial cells with formalin destroyed the antigenic determinant responsible for reactivity with this monoclonal antibody in both assays. Formalin fixation had no effect on hemagglutination activity (HA) of F. nucleatum cells or reactivity with polyvalent rabbit antiserum in double diffusion in agar. Scanning electron microscopy demonstrated that formalin fixation did not affect binding of F. nucleatum cells to microtiter plates. When developing monoclonal antibodies to be used as diagnostic reagents, the antigenic form utilized for immunization should be identical to the antigenic form which will eventually be used in the diagnostic assay.     

A method of membrane preparation for immunoassay

Membranes prepared from a variety of solid tissues were used as solid-phase antigens for ELISA or RIA after fixation onto polylysine-primed 96-well plates. The preservation of antigens in these membrane preparations was tested by reactivity in ELISA using 2 monoclonal antibodies: W6/32, which recognizes an HLA framework antigen (a protein antigen) and anti-SSEA-1, directed to a carbohydrate antigen carried on glycoproteins. Levels of antigen deposition and usefulness as solid-phase antigens were assessed for ELISA as compared to RIA. Coated plates may be frozen for many months with preservation of antigenic activity. This method is relatively simple, rapid, and is useful for preparation of tissue antigens for immunoassay, especially for screening monoclonal antibodies.     

Enzyme-linked immunosorbent assay for screening monoclonal antibody production: Use of intact cells as antigen

An enzyme-linked immunosorbent assay (ELISA) was developed for screening production of monoclonal antibodies with specificity for surface membrane components on human mononuclear cells. Whole cells used as antigen were dessicated under vaccum in flexible polyvinyl chloride plates or in rigid plates coated with protein-detergent solution. Rabbit or goat anti-mouse IgG conjugated with peroxidase was used as indicator after affinity column purification. Under these conditions, the sensitivity of ELISA proved comparable to other binding techniques or microcytoxicity and allowed rapid, reproducible, and efficient detection of antibody producing hybridomas.     

Detection of monoclonal antibodies against cell surface antigens: the use of antiglobulins coupled to red blood cells

An antiglobulin-coupled red cell assay is described for screening monoclonal antibodies against cell surface antigens. A monoclonal antibody specific for rat immunoglobulin kappa chains was coupled to red blood cells and used to detect binding of rat monoclonal antibodies to cells attached to the wells of microtitre plates. The method was found to be simpler and more rapid than the alternative enzyme-linked binding assay and useful for rapid screening and selection of antibodies for use as differentiation markers of human and mouse haemopoietic cells.     

A solid-phase immunoassay to screen anti-HLA monoclonal antibodies

A solid-phase assay to detect anti-HLA monoclonal antibodies was developed. In this assay microtiter plates are coated with antigens solubilized from cultured lymphoid cells by sonication and then incubated with anti-HLA monoclonal antibodies. The antigen-antibody interaction is indicated by the development of color following the addition of peroxidase-conjugated anti-mouse Ig xenoantibodies and its substrate. The assay is rapid since it does not require centrifugations during the washing steps. Furthermore the assay is simple, reproducible and suitable to screen large numbers of samples and to detect antibodies recognizing determinants not exposed on the membrane of viable cells. The sensitivity of the assay is influenced by the pH of the buffer used to coat plates with antigens, by the number of cells used to prepare soluble antigens, by the incubation time of antigen preparations with plates and by the incubation time of antibody preparations with antigen-coated plates. Titration of anti-HLA monoclonal antibodies with known specificity and screening of hybridomas generated with splenocytes from mice immunized with cultured human lymphoid cells indicate that the sensitivity of the solid-phase assay is similar to that of the ELISA with lymphoid cells.     

An enzyme-linked immunosorbent assay for measuring anti-sheep erythrocyte antibodies

A simple ELISA assay for detecting murine anti-SRBC antibodies of IgG class was developed and the variation of the results according to different experimental conditions was investigated. Erythrocytes were left to settle in flexible plastic microtiter plates, after which they were fixed with glutaraldehyde and the remaining binding sites in the plates saturated with ovalbumin. Serum or monoclonal IgG antibodies were then allowed to react with the erythrocytes. Protein A coupled to alkaline phosphatase caused a color change in the subsequently added enzyme substrate. The results proved to be of good reproducibility, specificity and sensitivity. The assay can be used for measuring IgG concentration, estimating antibody avidity and number of antigenic determinants on the SRBC, as well as screening IgG anti-SRBC hybridomas. The precision of concentration estimates was very good when standard curves were used.     

A rapid method for the detection of antibodies to cell surface antigens: a solid phase radioimmunoassay using cell membranes

Cell membranes isolated from murine lymphocytes or ascites tumors bind tightly to the surface of flexible plastic microtiter plates in the absence of additional proteins. This allows the detection of membrane associated molecules by specific antibodies and thus forms the basis for a rapid and sensitive radioimmunoassay for antibodies to membrane-bound components. The assay compares favorably with a variety of methods currently used to detect antibodies to cell surface antigens. The assay detects a variety of well characterized murine cell surface antigens (H-2, I-A, T-200, Thy-1.2, Ig). The level of antibody binding to membranes on plates correlates well with antigen density on intact cells. A modification of the assay involving competition between cross-reacting antibodies allows detection and resolution of closely spaced antigenic determinants.     

A monoclonal antibody directed against an organ-specific liver cell membrane antigen in rabbits

We generated monoclonal antibodies after immunization of mice with rabbit liver-specific protein (LSP) preparations. One of these antibodies (2D3) showed an organ-specific and species-specific binding pattern as determined by immunohistological and ELISA techniques. Immunoelectron microscopy studies demonstrated that this antibody is bound exclusively to the liver cell membrane except in the region of the bile canaliculi. We further describe a simple ELISA technique for the detection of anti-LSP antibodies. Our study clearly demonstrates the presence of at least 1 organ-specific liver cell membrane antigen in rabbit LSP and shows antigenic differences between areas of the plasma membrane of hepatocytes.     

Antigenic mapping and functional analysis of the F protein of Newcastle disease virus using monoclonal antibodies

Summary Twelve monoclonal antibodies to the F protein of a velogenic strain of Newcastle disease virus (NDV) were established. Each of these antibodies inhibited virus-induced plaque formation in BHK-21 cells. Seven antibodies neutralized viral infectivity in eggs; thus, antigenic variants could be selected with these antibodies and used for antigenic mapping. Based on the reactivity of the antigenic variants with the antibodies used in selection, 4 distinct antigenic sites (I–IV) were defined on the F protein molecule. In competitive binding assay, sites II and III were found to be spatially close to each other. Each antibody to sites I, II and III inhibited both virus-induced hemolysis of chicken erythrocytes and syncytium formation of BHK-21 cells. On the other hand, some of the antibodies to site IV selectively inhibited either hemolysis or cell fusion. This finding may indicate that the fusion of the viral envelope with erythrocytes and host cell membrane is modulated through different ways. Comparative analysis of different NDV strains using monoclonal antibodies to each of the different antigenic sites showed that the antigenicity of the F protein is highly conserved.With 1 Figure     

A simple ELISA for the classification of monoclonal antibodies according to their recognition of native epitopes

A sandwich ELISA for testing whether pairs of monoclonal antibodies recognize the same native antigenic site was developed. The assay was performed with murine anti-lysozyme monoclonal antibodies. A monoclonal antibody, adsorbed onto a microtiter plate, was used as a capture antibody for native lysozyme. After the reaction with the antigen a second monoclonal antibody, the test antibody, was added. The amount of bound antibody was quantitatively measured using rabbit anti-mouse immunoglobulin serum conjugated to alkaline phosphatase. The simultaneous binding of pairs of monoclonal antibodies to lysozyme was further substantiated by structural considerations.     

Identification of monoclonal antibodies to pancreatic islet cells by immunoperoxidase staining

Immunoperoxidase staining and enzyme-linked immunosorbent assay (ELISA) were used to identify monoclonal antibodies that reacted with pancreatic islet cells. All monoclonal antibodies produced against isolated human or rat pancreatic islets including one mouse autoantibody reacted with pancreatic islets in formalin-fixed pancreas sections, but not with rat kidney or thyroid. Reactivity was also found with suspensions of normal rat islet cells and rat insulinoma cells using a 3-stage immunoperoxidase procedure and an ELISA technique. Differences were observed in staining intensity between the various antigenic substrates tested suggesting variable cross-reactivity and/or number of epitopes. The sensitivity of the immunoperoxidase technique proved to be favourable for identification of monoclonal antibodies that recognize cellular constituents such as islet cell antigens present in low concentrations.     

A simple and sensitive ELISA to detect immune (gamma) interferon induced I-A on a macrophage line

A convenient and sensitive cell surface ELISA is described to detect immune (gamma) interferon induced I-A determinants on a murine macrophage line, P388D1. The ELISA is performed on monolayers of P388D1 cells grown in 96-well culture plates for 2 days in the presence of recombinant gamma IFN or supernatants from cultures of antigen-activated T cells. After glutaraldehyde fixation, the cultured cells are overlayed with fetal calf serum to block nonspecific antibody binding during the assay. A double sandwich technique employing a monoclonal anti-I-Ad (MKD6) antibody followed by peroxidase-conjugated goat anti-mouse gamma chain antibody is then used to detect the presence of I-A on the cell monolayers. Detection of I-A induced by both r gamma IFN and the supernatant from an antigen-stimulated T cell line was highly reproducible and sensitive using this method. Furthermore, the assay is easily performed and many sample supernatants can be rapidly screened for this activity. The assay is used by this laboratory to detect the antigen-stimulated production of gamma IFN by T cell clones and hybridomas.     

A microELISA assay for detection of anti-HLA activity of mouse monoclonal antibodies using an Astroscan 2100 automated plate reader.

A microenzyme-linked immunosorbent assay (microELISA) method has been developed using an Astroscan 2100 system automated plate reader which was initially designed for tissue typing by a two colour fluorescent microcytotoxicity assay. A 96-well plate ELISA used for screening mouse monoclonal antibodies raised against surface HLA antigens has been modified for use with the Astroscan plate reader and 72-well typing trays. The existing substrate 4-methylumbelliferyl-beta-D-galactoside (4MUG) has been replaced with fluorescein-di-beta-D-galactopyranoside (FDG), to provide a wavelength (530 nm) detectable by the Astroscan or other automated plate readers designed for reading microcytotoxicity assay plates. The assay volumes have also been reduced tenfold for use with Terasaki microtest plates. The assay now has the major advantage of requiring only 5 microliters of test supernatant allowing hybridomas to be screened earlier during a fusion and on a wider cell panel. The use of the large panel which includes B lymphoblastoid cell lines (B-LCL) and mouse L cell transfectants expressing HLA genes, reduces the length of time the hybridomas need to be kept in tissue culture before selection. Other advantages include the reduction in the number of target cells required, smaller volumes of reagents throughout the assay and the ability to screen cytotoxic as well as non-cytotoxic monoclonal antibodies. The sensitivity of this microELISA proved to be comparable with the original assay and so provides an efficient screening method for monoclonal antibodies.     

A rapid solid-phase enzyme-linked binding assay for screening monoclonal antibodies to cell surface antigens

A solid-phase enzyme-linked binding assay is described for screening monoclonal antibodies to cell surface antigens. E. coli β-galactosidase was coupled to rabbit anti-rat Ig and used to detect the binding of rat monoclonal antibodies to cells which had been fixed to the wells of microtitre plates using a combination of poly-l-lysine and glutaraldehyde. This method was found to be advantageous for the large scale screening of monoclonal antibodies with a panel of cell types, and has been useful in the selection of antibodies which would be candidates for differentiation markers within the human and mouse haemopoietic systems.     

A live cell enzyme-linked immunosorbent assay for detecting human hepatoma membrane antigens

Live cell enzyme-linked immunosorbent (ELISA) and fixed cell indirect immunofluorescence (IF) assays were compared to screen mouse hybridomas producing immunoreactive monoclonal antibodies against cell membrane antigens expressed on Ha22T, a human hepatoma cell line. While performing live cell ELISA, two parameters were tested to improve the viability of the target cells. The first parameter was the inclusion of growth medium in the assay buffers, and the second was performing the assay incubations at 37 degrees C in an incubator containing 5% CO2 in the air. Fixed cell IF detected and classified 46% of the hybridomas secreting monoclonal antibodies reactive with membrane, cytoplasm, cytoskeleton, and nuclear antigens of Ha22T cells. Fixed cell IF was able to reveal mixtures of two or more hybridomas growing in the same well secreting antibodies to different cell organelles. The live cell ELISA, on the other hand, identified 12 additional membrane reactive monoclonal antibodies from the hybridoma supernatants that were not reactive by IF. These results disclose that cell fixation procedures used for IF either completely or partially inactivated some of the cell membrane antigens. We, therefore, propose the use of a combination of immunoassays to select the maximum number of hybridomas secreting useful monoclonal antibodies from somatic cell fusions.     

Antigenic analysis of H4 influenza virus isolates using monoclonal antibodies to defined antigenic sites on the hemagglutinin of A/budgerigar/Hokkaido/1/77 strain

Summary Three non-overlapping antigenic sites were defined on the hemagglutinin of avian influenza virus A/budgerigar/Hokkaido/1/77 (H4N6) by competitive binding assay of monoclonal antibodies to the virus and comparative antigenic analysis of variants selected with monoclonal antibodies. Antigenic relationship among 25 H4 influenza viruses of different bird origin was examined by ELISA with the monoclonal antibodies to each of defined antigenic sites. Two of the three antigenic sites contained epitopes specific to the H4 influenza viruses of budgerigar and mynah origin, and the remaining site contained an epitope which was cross-reactive with almost all of the H4 influenza viruses.     

Induction and detection of antibodies to squalene. II. Optimization of the assay for murine antibodies

An improved high throughput assay for measuring murine antibodies to squalene (SQE) is described. The assay is highly reproducible and sensitive and can detect 80 ng/ml of antibody to SQE. The assay, an ELISA, is similar to our previously described assay in which plates containing PVDF membranes were used [J. Immunol. Methods 245 (2000) 1]. The PVDF plates worked well for detection of murine monoclonal antibodies (mAbs) to SQE, but substantial PVDF plate variation was observed, resulting in significant loss of signal and reproducibility between different lots of plates. In the new assay, the PVDF plates were replaced with Costar round bottom 96-well sterile tissue culture plates. These latter plates, which are not normally used for ELISA assay, gave high absorbances for monoclonal antibodies and anti-SQE serum binding to SQE and low absorbances for solvent-treated wells. Other commercially available polystyrene ELISA plates were unsuitable, in that either the background was high or the absorbance for antibodies binding to SQE was low, or both. This change in plate from PVDF to polystyrene allowed the use of an ELISA plate washer, which dramatically increased the throughput rate over the hand-washed PVDF plates. The improved assay also replaced fetal bovine serum (FBS), which contained SQE in lipoproteins, with fatty acid-free bovine serum albumin (BSA) as the blocker/diluent. Fifteen nanomoles of SQE were selected as the optimal amount of SQE to add to the wells. The binding of monoclonal antibodies and anti-SQE serum was dependent upon both the amount of antibody added to the wells and the amount of SQE added to the wells. Antibody concentration curves were hyperbolic in shape, as seen with most other antibodies. Antibody binding first increased with SQE amount and then reached a plateau around 10 nmol of SQE/well. At high SQE amounts (>75 nmol/well), antibody binding decreased with the amount of SQE added. Using 3H-SQE, the amount of SQE bound to the wells increased linearly, up to 50 nmol of SQE added. Approximately 90% of the added SQE bound to the well. When amounts greater than 100 nmol of SQE were added, the amount of SQE bound to the wells was greatly reduced to approximately 5-10% of the added SQE. The assay was highly reproducible both from lot to lot of plates and from experiment to experiment.     

The antigenic structure of the influenza B virus hemagglutinin: operational and topological mapping with monoclonal antibodies

We have probed the antigenic structure of the influenza B virus hemagglutinin (HA) with monoclonal antibodies specific for the HA of influenza B virus, B/Oregon/5/80. Seventeen laboratory-selected antigenic variants of this virus were analyzed by hemagglutination-inhibition (HI) assays or ELISA and an operational antigenic map was constructed. In addition, the monoclonal antibodies were tested in a competitive binding assay to construct a topological map of the antigenic sites. In contrast to the influenza A virus HA, only a single immunodominant antigenic site composed of several overlapping clusters of epitopes was defined by the HI-positive antibodies. Three variants could be distinguished from the parental virus with polyclonal antisera by HI and infectivity reduction assays suggesting that changes in this antigenic site may be sufficient to provide an epidemiological advantage to influenza B viruses in nature. In addition, two nonoverlapping epitopes of unknown biological significance were identified in the competitive binding analysis by two monoclonal antibodies with no HI activity and little or no neutralizing activity. We previously identified single amino acid substitutions in the HAs of the antigenic variants used in this study (M. T. Berton, C. W. Naeve, and R. G. Webster (1984), J. Virol. 52, 919-927). These changes occurred in regions of the molecule which, by amino acid sequence alignment, appeared to correspond to proposed antigenic sites A and B on the H3 HA of influenza A virus. Correlation with the antigenic map established in this report, however, demonstrates that the amino acid residues actually contribute to a single antigenic site on the influenza B virus HA and suggests significant differences in the antigenic structures of the influenza A and B virus HAs.     

Evaluation of monoclonal antibodies to human plasma low density lipoproteins. A requirement for lipids to maintain antigenic structure

Human plasma low density lipoproteins (LDL) are composed of approximately 25% apoproteins and 75% lipids (w/w). Immunochemical properties of LDL were studied using monoclonal antibodies. BALB/c mice were immunized with LDL and the spleen cells from these mice were then fused with a non-immunoglobulin secreting myeloma cell line (F0). The clones producing desirable antibodies were selected to study the antigenic properties of LDL by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay. First, it was found that the maximal binding of 125I-labeled LDL to polyvinyl chloride microtiter dishes was not temperature dependent. The binding affinity was high with a Ka value of approximately 1.9 X 10(10) M-1 while the monoclonal antibodies possessed an affinity to LDL of 5 X 10(8) M-1 which was 2 orders less than the affinity of LDL to the dishes. The former binding, once established, was irreversible as judged by a subsequent incubation with an excess of unlabeled LDL. The latter binding could be displaced by unlabeled LDL. Therefore, the ELISA technique offered a satisfactory approach to study the interaction between LDL and monoclonal antibodies. Removal of lipids from bound LDL by organic extraction resulted in a 50% loss of immunoreactivity, suggesting that the lipids of LDL are important in maintaining the antigenic structure of LDL. Since the apoprotein of LDL also constitutes approximately 40% of the mass (w/w) of very low density lipoproteins (VLDL), the immunoreactivity of VLDL assessed by LDL-monoclonal antibodies was also carried out. Removal of triglycerides from VLDL by lipoprotein lipase resulted in a substantial loss of immunoreactivity as determined by radioimmunoassay. These findings are consistent with the concept that lipids play a role in maintaining the integrity of the antigenic structure of LDL.     

The Rose Bengal Assay for Monoclonal Antibodies to Cell Surface Antigens: Comparisons with Common Hybridoma Screening Methods

An automated colorimetric procedure for detection of antibodies specific for cell surface antigens (1) has been compared for specificity and sensitivity to other methods of hybridoma supernatant screening. The Rose Bengal colorimetric assay (RBA) compared favourably in these respects with whole cell radioimmunoassay and indirect immunofluorescence with manual or flow cytometric analysis (FACS). A major advantage of the method is that it allows a large number of samples to be screened in a comparatively short time. Unlike other semi-automated colorimetic assays, such as ELISA, the procedure does not require cell fixation, which can destroy some antigenic determinants. The original assay of O'Neill and Parish (1) has been modified to give increased sensitivity and also to enable the detection of erythrocyte specific antibodies by elimination of the dye staining step and direct measurement of haemoglobin by spectrophotometry. The RBA allows detection of monoclonal antibodies (MoAb's) binding to only a proportion of the cells in a sample, which is an important feature when hybridoma supernatants are screened for reactivity to a minor cell population, for example against leukaemic cell samples with low percentages of blast cells.