A rapid and efficient method for the generation and screening of monoclonal antibodies specific for cell surface antigens

The generation of monoclonal antibodies (mAb) of desired specificity to cell surface antigens can serve as a valuable tool to study protein expression and function. However, traditional approaches to mAb generation usually involve large-scale protein purification and intensive screening, and may not result in mAb specificities to the native protein of interest. We describe a simple, inexpensive, high-throughput method for the generation and screening of hybridomas secreting mAb specific for cell surface receptors. Intact reporter cells expressing a CD3zeta-fusion receptor of the protein of interest are plated in 96-well arrays of captured, plate-bound hybridoma supernatants. A mAb to the protein of interest generates a signal leading to reporter-cell expression of beta-galactosidase, and enzyme activity can be screened in a single day using a non-radioactive substrate. Importantly, a single cell line can be used for immunization, screening, semi-quantitative affinity comparisons, and subsequent screening for physiological ligand expression, if the protein of interest is a receptor. We describe an application of this approach to generate mAb specific for a protein of previously unknown expression and undocumented function.     

Induction and rapid screening of monoclonal antibodies against cyclosporin A

Cyclosporin C-hemisuccinate was coupled to thyroglobulin by the mixed anhydride method. Cyclosporin C was used instead of cyclosporin A (CsA) because of lack of functional groups of CsA. The resulting protein-cyclosporin C conjugate allowed us to induce high antibody titers also against cyclosporin A in rabbit and mice. Polyclonal and monoclonal antibodies were prepared following standard procedure. Since no standard methods for screening and quantification of anti-CsA-antibodies were available, two methods were adapted: (a) liquid phase radio assay (RA) and (b) solid phase enzyme-linked immunoassay (ELI-SA). For the former procedure inactivated charcoal was applied to separate the antibody-bound and the unbound CsA. CsA-coated PVC microtiter plates were used for the latter.     

A rapid and efficient strategy to generate allele-specific anti-HLA monoclonal antibodies

That generation of allele-specific anti-human leukocyte antigen (HLA) monoclonal antibodies (ASHmAb) is very difficult is well known. This is thought to be due to the unique epitope structure, an assemblage of amino acid residues that lie separately in the amino acid sequence of human HLA, and to its low antigenicity compared with that of common epitopes recognized as xenogeneic determinants by mice. Here we report a rapid and efficient strategy to generate ASHmAb. Different from usual immunization methods is that we suppressed the production of non-allele-specific anti-HLA antibodies against xenogeneic determinants of HLA molecules by immunizing human HLA-B51 transgenic mice against non-HLA-B51 HLA tetramers. In addition, HLA-coated beads enabled rapid and efficient screening for ASHmAb. ASHmAb generated by this strategy will be useful for HLA typing and for clinical diagnosis, such as flow cytometry-based chimerism analysis for early detection of graft failure and relapse of leukemia after HLA-mismatched hematopoietic stem cell transplantation.     

A rapid method for generating and characterizing anti-variable region monoclonal antibodies

The generation of anti-variable region monoclonal antibodies (mAbs) against therapeutic antibodies is essential in the pharmacokinetic/pharmacodynamic (PK/PD) assessments of the drugs in clinical study samples. Sandwich EIA and other methods are typically employed to achieve sensitivity and selectivity for the PK/PD analyses. These assays usually require generation of mAb reagents that bind specifically to the therapeutic mAb candidate in non-competing pair combinations. Thus, large panels of anti-variable region mAbs must be generated in an expeditious manner to increase the probability of success. Previously, we described a novel immunization method using type 1 interferons (IFNs) coupled with an agonistic anti-CD40 mAb to drive immune responses (Staquet et al., Human Antibodies 15 (2006), 61-69). This protocol allows for rapid and robust generation of large panels of anti-variable region mAbs. In order to quickly characterize and efficiently identify optimal anti-variable region antibody pairs early in the hybridoma process using crude supernatants, an inexpensive, high-throughput ELISA method was developed. The ability to rapidly identify appropriate mAb pairs will save resources by eliminating the time-consuming and laborious process of subcloning irrelevant hybridomas.     

A rapid and efficient immunization protocol for production of monoclonal antibodies reactive with autoantigens

A simple, time-saving and efficient immunization method suitable for the production of mouse monoclonal antibody secreting hybridomas is described. Draining lymph nodes isolated 9 days after a primary immunization were used as the source of antibody producing cells. No systemic spread of antibody producing cells or specific antibodies could be detected. The present protocol was employed to generate a panel of collagen type II reactive monoclonal antibodies. Most of the monoclonals so generated were found to be of the IgG class.     

A rapid and efficient method for generating anti-variable region monoclonal antibodies using type-1 interferons as immune modulators

The generation of anti-variable region monoclonal antibodies (mAbs) against therapeutic antibodies is essential in the pharmacokinetic/pharmacodynamic (PK/PD) assessments of the drugs in clinical study samples. Sandwich EIA and other methods are typically employed to achieve sensitivity and selectivity for the PK/PD analyses. These assays usually require generation of mAb reagents that bind specifically to the drug in non-competing pair combinations. Thus, large panels of anti-variable region mAbs must be generated in an expeditious manner to increase the probability of success. Herein we describe a novel immunization method that utilizes type 1 interferons (IFNs) as immunomodulators coupled with an agonistic anti-CD40 mAb as a B cell proliferative agent to drive immune responses. This novel protocol allows for rapid and robust mAb reagent generation without the use of conventional protein-denaturing adjuvants. The use of IFNs allowed for the generation of comparable and in some cases, increased numbers of anti-variable region mAbs in a dramatically shorter timeframe. This IFN based, immunostimulatory approach utilizing a non-denaturing adjuvant, likely presents conformational epitopes and may optimize the humoral response for the rapid generation of anti-variable region reagents to therapeutic mAb candidates.     

A rapid method for comparing monoclonal antibodies by limited proteolysis and electrophoresis

A rapid and sensitive method for comparing the primary structure of proteins has been adapted to the study of monoclonal antibodies. Samples were digested with alpha-chymotrypsin in the presence of sodium dodecyl sulfate after which peptide fragments were separated into distinctive banding patterns by polyacrylamide gel electrophoresis. This method could easily detect differences in the primary structure of antibodies with related as well as unrelated binding specificities. In addition, antibody molecules derived by somatic diversification from the same germ line gene segments could be distinguished from one another.     

Rat monoclonal antibodies. II. A rapid and efficient method of purification from ascitic fluid or serum

A simple and rapid procedure for the purification of C1q from human, guinea pig and mouse serum is described. This procedure allows the purification of C1q within one and a half days using euglobulin precipitation, chromatography on Superose 6B, followed by chromatography on Mono S by Fast Protein Liquid Chromatography (FPLC). The highly purified, hemolytically active C1q is free of any immunoglobulins. Since the purification of C1q of three different species was performed by the same purification procedure, a comparison of the subunit compositions was made under reducing and non-reducing conditions on SDS-PAGE. The yield was found to be more than 50%.     

Production and immunohistochemical application of monoclonal antibodies against delta sleep-inducing peptide

Monoclonal antibodies were produced following immunization of rats with delta sleep-including peptide (DSIP). The spleen cells of the rats were fused with the myeloma cell line SP2/0. The supernatants of hybridomas were screened on a solid-phase immunoassay using dot-immunobinding of DSIP and some DSIP fragments. The supernatants of six stable producer clones were found to react with DSIP. From this procedure it was also deduced that all these monoclonal antibodies recognized epitope(s) of the penta carboxy-terminal region of DSIP (DSIP5-9). Application of these monoclonal antibodies to rat median eminence sections gave a strong immunolabelling of a large population of fibres and terminal-like structures, mainly localized through the lateral areas. Elution-restaining experiments using a monoclonal antibody to DSIP and a polyclonal antiserum to luteinizing hormone-releasing hormone (LHRH) showed that the patterns of immunoreactivity respectively visualized overlap almost completely. Although numerous LHRH-immunoreactive neuronal elements were also easily demonstrated in the median eminence of the mouse, the hamster and the gerbil species, incubation of sections with monoclonal antibodies to DSIP failed to give any immunoreaction. Taken together these data argue for the independence of the DSIP/LHRH immunola belling systems Furthermore, it was demonstrated that DSIP5 9-related epitopes detected in the rat median eminence have no counterpart in the three other rodent species investigated. These species differences may reflect the fact that the carboxy-terminal sequence of the nonapeptide DSIP originally discovered in the rabbit is not conserved in all rodent species.     

A rapid and efficient method for purification of rat B cells for complement-dependent cytotoxicity testing

The development of an effective technique for the enrichment of rat B cells is described. This protocol is an adaptation of the ‘panning’ technique employing anti-Ig-coated plastic dishes. Rat B cells are bound directly to anti-rat Ig-coated wells in Terasaki test plates where they are typed by complement-dependent cytotoxicity. This technique enriches class II (Ia-like) antigen-positive B cells, facilitating detection with antisera specific for rat class II alloantigens. The utility of this technique in the cell surface phenotyping of minor lymphocyte subpopulations is discussed.     

Rapid screening method for monoclonal antibodies against cytoskeletal proteins

A horseradish peroxidase/anti-horseradish peroxidase monoclonal mouse antibody complex was prepared and used for the detection of mouse monoclonal antibodies against proteins of the whole cytoskeleton. A rapid qualitative assay of large number of antibody samples on the whole cytoskeleton and an early determination of their specificity by the immunoblot technique is described.     

A rapid method for processing very large numbers of tissue sections for immunohistochemical hybridoma screening

A hybridoma screening format which facilitates the processing of thousands of tissue sections for immunohistochemical analysis is described. The method utilizes sterile replica transfers of monoclonal antibody-containing test supernatants from 96-well culture plates to tissue sections mounted in appropriately sized 8 X 12 arrays, and is extremely rapid and inexpensive.     

A sensitive method for detection of polyclonal and monoclonal antibodies against the adenovirus hexon

Chromic chloride and tannic acid methods were elaborated to bind purified adenovirus hexons to sheep red blood cells (SRBC). Either method gave very sensitive haemagglutination (HA) with adenovirus antisera, but treatment with tannic acid was more sensitive. Using this method, specific antibodies present in polyclonal immune sera against adenoviruses and adenovirus hexons, and the reactivity of monoclonal antibodies directed against adenovirus hexon type 1 were tested to 11 adenovirus types. With certain types, the high haemagglutination titres exceeded those obtained with ELISA, while with other types they remained slightly below the ELISA titres. The elaborated passive HA method with tannic acid is easily accomplished and may serve as an effective and useful tool in the study of both polyclonal and monoclonal antihexon antibodies.     

A simple, rapid method for screening hybridomas producing monoclonal antibodies to platelet proteins

Several parameters influence the outcome of somatic cell fusions based on the K?hler and Milstein technology, and a number of steps are of critical importance, including the screening strategy. The procedure chosen, appropriate for the type of antibody required, should be rapid and sensitive, in order to clone the relevant hybrids as quickly as possible. A simple and quick dot blot-based method is reported, suitable for screening hybridoma culture supernatants in order to identify clones producing monoclonal antibodies to platelet constituents.     

Potential cross‐reactivity of monoclonal antibodies against clinically relevant mycobacteria

Tuberculosis is a disease caused by the Mycobacterium tuberculosis complex (MTb). In 2011, global mortality due to tuberculosis was 1·4 million individuals. The only available vaccine is the attenuated M. bovis [bacillus Calmette–Guérin (BCG)] strain, which confers variable protection against pulmonary tuberculosis. Some widely distributed non‐tuberculous mycobacteria (NTM), such as M. avium and M. arupense, are also potential pathogens for humans. This work aimed to produce and characterize monoclonal antibodies against the M. bovis BCG Mexico strain of the MTb, M. avium subs. hominissuis and the M. arupense strain from NTM. Hybridomas were produced from splenocytes of BALB/c female mice immunized with radiation‐inactivated mycobacteria, and the immunoglobulin (Ig)G2a antibody‐producing clones with the highest antigenic recognition were selected. The selected clones, Mbv 2A10 for M. bovis BCG Mexico, Mav 3H1 for M. avium and Mar 2D10 for M. arupense, were used in further studies. Enzyme‐linked immunosorbent assay (ELISA) and immune proteomics analyses characterized the clones as having the highest cross‐reactivity with mycobacteria. Using mass spectrometry, a number of proteins recognized by the monoclonal antibody (mAb) clones were identified. These proteins had roles in metabolic processes, hypoxia, cell cycle and dormancy. In addition, a Clustal W and Immune Epitope Database (IEDB) in‐silico analysis was performed in protein sequences that result in the conserved regions within probability epitopes that could be recognized for Mbv2A10 and Mav3H1 clones.     

A rapid and efficient method for DNA extraction from paraffin wax embedded tissue for PCR amplification

DNA from archival, formaldehyde fixed, paraffin wax embedded human tissue, suitable for amplification by the polymerase chain reaction (PCR), was obtained using a microwave method based on the capture of DNA by magnetic beads. Fragments of the α-1-antitrypsin gene (AAT) and the apolipoprotein E gene (APOE) were amplified successfully from human liver and brain tissue, respectively. This procedure provides a more rapid, simple and efficient method for reproducibly obtaining DNA from preserved tissue that has been kept in storage for up to 30 years.     

Characterization and formulation of multiple epitope-specific neutralizing monoclonal antibodies for passive immunization against cryptosporidiosis

The coccidian parasite Cryptosporidium parvum causes diarrhea in humans, calves, and other mammals. Neither immunization nor parasite-specific pharmaceuticals that are consistently effective against this organism are available. While polyclonal antibodies against whole C. parvum reduce infection, their efficacy and predictability are suboptimal. We hypothesized that passive immunization against cryptosporidiosis could be improved by using neutralizing monoclonal antibodies (MAbs) targeting functionally defined antigens on the infective stages. We previously reported that the apical complex and surface-exposed zoite antigens CSL, GP25-200, and P23 are critical in the infection process and are therefore rational targets. In the present study, a panel of 126 MAbs generated against affinity-purified CSL, GP25-200, and P23 was characterized to identify the most efficacious neutralizing MAb formulation targeting each antigen. To identify neutralizing MAbs, sporozoite infectivity following exposure to individual MAbs was assessed by enzyme-linked immunosorbent assay. Of 126 MAbs evaluated, 47 had neutralizing activity. These were then evaluated individually in oocyst-challenged neonatal mice, and 14 MAbs having highly significant efficacy were identified for further testing in formulations. Epitope specificity assays were performed to determine if candidate MAbs recognized the same or different epitopes. Formulations of two or three neutralizing MAbs, each recognizing distinct epitopes, were then evaluated. A formulation of MAbs 3E2 (anti-CSL [alphaCSL]), 3H2 (alphaGP25-200), and 1E10 (alphaP23) provided highly significant additive efficacy over that of either individual MAbs or combinations of two MAbs and reduced intestinal infection by 86 to 93%. These findings indicate that polyvalent neutralizing MAb formulations targeting epitopes on defined antigens may provide optimal passive immunization against cryptosporidiosis.