Production of monoclonal antibodies to Shigella. Enzyme-linked immunosorbent assay for screening hybridoma antibodies with intact bacteria

A simple and rapid enzyme-linked immunosorbent assay (ELISA)-type assay has been developed to screen hybridoma supernatant fluids with whole viable or killed bacteria as the antigen. The optimum concentration of acetone-killed and dried cell antigen for coating was 25–100 μg/ml. Screening of hybridoma supernatant fluids against whole cells, both with and without fixation, was assessed and both were equally sensitive. The data indicate that bacteria] fixation is detrimental in ELISA probably because of loss of antigenic structure. A highly specific monoclonal antibody (laM3) was produced against Shigella flexneri la and was employed to optimize the assay procedure.     

Molecular mimicry: frequency of reactivity of monoclonal antiviral antibodies with normal tissues.

More than 600 monoclonal antiviral antibodies made against 11 different viruses were screened against 14 different organs from normal uninfected mice. Of these antiviral antibodies, 21, or approximately 3.5%, reacted with specific cells in these organs. Several of these antibodies were of the multiple-organ-reactive type and recognized antigens in more than one organ. It was concluded that the reactivity of monoclonal antiviral antibodies with normal tissues is a common phenomenon.     

Analysis of herpesvirus saimiri structural proteins with monoclonal antibodies.

Herpesvirus saimiri is a lymphotrophic virus isolated from squirrel monkeys that causes highly malignant lymphomas in owl monkeys, marmosets, and rabbits, but not in its natural host. We have been interested in exploring immunological and biological aspects of this phenomenon and describe in this report the isolation of 27 monoclonal antibodies (MCAs) to herpesvirus saimiri which were grouped into 11 distinct sets based on the proteins they immunoprecipitated. In total, these 11 groups of MCAs identify the majority of the proteins present in the virion. Immunofluorescence was used to study how the viral antigens are compartmentalized in infected cells. One antibody produced intense nuclear staining and immunoprecipitated both the largest protein seen in gels (150,000 daltons) and one of the smallest (13,000 daltons). All of the other groups of MCAs principally stained the cytoplasm of infected cells. One of the unexpected results of this study was the observation that a majority of the MCAs precipitated more than one protein (15 of 27 antibodies, 7 of 11 groups). Whereas one group of MCAs, which normally precipitated four proteins, identified only single polypeptides after dithiothreitol pretreatment, the other sets of antibodies continued to recognize two or more viral antigens under reducing conditions. Immunoprecipitation of viral proteins with polyvalent sera obtained from virus-infected rabbits and monkeys was carried out to verify and extend previously published reports on the proteins of herpesvirus saimiri.     

100-kD coated vesicle proteins: molecular heterogeneity and intracellular distribution studied with monoclonal antibodies

Proteins with molecular weights of around 100,000 (designated 100K) are found in all coated vesicles. Five monoclonal antibodies have been raised against the major 100K proteins of bovine brain coated vesicles, which migrate on SDS gels as three closely spaced bands. One antibody stains the middle band (band B), two stain both upper and lower bands (bands A and C), and two stain the lower band (band C) only. Thus, the polypeptides in bands A and C are related (but not identical), a result confirmed by NH2-terminal sequencing. Other tissues were found to express proteins corresponding to, and co-migrating with, bands B and C but not band A. Only the two antibodies that recognize both A and C stained fixed and permeabilized tissue culture cells; they both showed a punctate pattern in the plane of the plasma membrane. Double labeling with anti-clathrin antibodies confirmed that the dots correspond to coated pits and vesicles. However, perinuclear staining seen with anti-clathrin, corresponding to Golgi-derived coated vesicles, was conspicuously absent with the two monoclonal antibodies. Affinity-purified polyclonal antisera against the 100K proteins, reported earlier, gave perinuclear as well as punctate staining; these included one antiserum which gave mainly perinuclear staining (Robinson, M. S., and B. M. F. Pearse, 1986, J. Cell Biol., 102:48-54). Thus, different 100K proteins appear to be found in different membrane compartments. Since the 100K proteins are thought to lie between clathrin and the membrane proteins of the vesicle, these results may help to explain how different membrane proteins can be sorted into coated vesicles in different parts of the cell.     

Antigens of selected Acanthamoeba species detected with monoclonal antibodies

Acanthamoeba species are ubiquitous soil and freshwater protozoa that have been associated with infections of the human brain, skin, lungs and eyes. Our aim was to develop specific antibodies to aid in rapid and specific diagnosis of clinically important isolates. Mice were variously immunised with live mixtures of Acanthamoeba castellanii strain 112 (AC112) trophozoites and cysts, or with sonicated, formalin-fixed or heat-treated trophozoites, or with a trophozoite membrane preparation. Eight hybridoma cell lines secreting monoclonal antibodies reactive with A. castellanii epitopes were generated. Seven of the new antibodies (designated AMEC1-3 and MTAC1-4) were isotyped as IgMkappa and one (MTAC5) as IgG1kappa. All of the novel antibodies bound to AC112 cysts, and MTAC4 and MTAC5 also bound to trophozoites as measured by flow cytometry on unfixed cells. Single chain antibody fragments that retained parental antibody binding characteristics were engineered from three of the hybridomas (AMEC1, MTAC3 and MTAC4). Four monoclonal antibodies (AMEC1, AMEC3, MTAC1, MTAC3) bound reliably to unfixed cysts of clinical isolates of A. castellanii (two strains) and Acanthamoeba polyphaga (two strains), belonging to Pussard-Pons morphological group II, and to Acanthamoeba lenticulata and Acanthamoeba culbertsoni, belonging to Pussard-Pons morphological group III. None of the antibodies bound to cysts or trophozoites of the environmental group I species, Acanthamoeba tubiashi. Antibodies AMEC1, MTAC3, MTAC4 and MTAC5 reacted with buffered formalin-fixed AC112 by immunohistochemistry, and also stained Acanthamoeba in sections of infected rat cornea and buffered formalin-fixed, paraffin-embedded infected human cornea. These antibodies may be useful in diagnosing pathogenic Acanthamoeba species in clinical specimens, provided that cysts are present.     

Variation in the biological function of envelope protein epitopes of yellow fever vaccine viruses detected with monoclonal antibodies.

Three different virus strains (17D-204, 17DD and the French neurotropic vaccine) have been used as live attenuated yellow fever (YF) vaccines and are manufactured in different centres around the world. The envelope proteins of these vaccine viruses were examined and compared using mouse monoclonal antibodies (MAbs) in haemagglutination inhibition (HAI) and neutralization (N) tests. The epitopes eliciting HAI and/or N were found to vary depending on the virus examined. Such variation was also found between vaccine viruses of the same strain manufactured in different centres. These data were confirmed by the use of mouse polyclonal antisera. On the basis of the MAb results in HAI tests a dendrogram of the similarity coefficients between the viruses was constructed and showed that the viruses could be placed into three major groups. Thus, it is concluded that YF vaccines manufactured in different centres are antigenically distinct as recognized by the mouse immune system.     

A simple in vitro method for raising monoclonal antibodies to cochlear proteins.

Monoclonal antibodies have been produced to mammalian hair cell antigens using a simple in vitro kit. Antigen was crudely prepared from dissected cochlear tissue by detergent extraction. There was no need to purify hair cells. Hybridoma supernatents were screened most efficiently on dissociated cells fixed with acetone. The immunisation method is sensitive to nanograms of antigen and can generate responses to conserved or weak antigens. The kit requires very little previous experience with cell culture and generates monoclonal antibodies within 3-4 weeks. It has overcome a number of problems with production of antibodies to hair cells and it should prove to be a very valuable tool in many laboratories.     

Lysosomal membrane proteins of Amoeba proteus, as studied with monoclonal antibodies.

Monoclonal antibodies were prepared against lysosomal membrane proteins of amoebae and used to follow lysosome-phagosome fusion after induced phagocytosis. The specificity of antibodies was checked by indirect immunofluorescence microscopy, immunoelectron microscopy, and localization of the antigen in subcellular fractions. The antibody-recognized proteins started to appear on the membranes of phagolysosomes about 5 min after phagocytosis as detected by indirect immunofluorescence, and the intensity of fluorescence increased for up to 1 h. Results of injection experiments in which purified antibodies had been injected into living cells and probed by indirect fluorescence indicated that the antigens were located on the cytoplasmic side of the lysosomal membranes. Lysosomes fuse with phagosomes on the one hand but not with non-fusible vesicles such as symbiosomes on the other. The results support the view that a membrane component(s) of non-fusible vesicles somehow prevents lysosomes from fusing with them.     

Common epitopes of serum retinol-binding proteins: a study with monoclonal antibodies.

Monoclonal antibodies were raised against purified chicken retinol-binding protein. These were characterised extensively with respect to their ability to recognize retinol-binding proteins from different species. The monoclonal antibodies exhibited differential recognition characteristics. Though the majority presented restricted reactivities, one out of the four monoclonal antibodies studied cross-reacted with retinol-binding proteins from all species tested so far.     

Cytoplasmic proteins of porcine adipocytes: identification with monoclonal antibodies

In the present study, monoclonal antibodies were produced using porcine adipocyte extracts as the immunogen. Two of the monoclonal antibodies, designated CB6 and IB4, exhibited reactivity toward only cells containing lipid in stromal-vascular cell cultures. The antigens recognized by the CB6 and IB4 monoclonal antibodies were 50 kD and 55 kD proteins, respectively. In vivo, IB4 immunoreactivity was detected only in lipid-containing cells, whereas immunofluorescence using CB6 was also detectable around muscle fiber bundles underlying the subcutaneous mesenchyme. In fetal subcutaneous mesenchyme, CB6 and IB4 immunoreactivities toward lipid-containing cells increased with developmental age, but each was not detectable in cells containing the smallest lipid droplets. In stromal-vascular cultures containing adipocytes, 48 hour treatment with the anti-lipogenic agent, growth hormone, only slightly altered CB6 immunoreactivity, whereas IB4 immunoreactivity was reduced by more than sixfold. The exact identity of the CB6 and IB4 antigens was not determined, but each may be useful as markers for studying regulation of adipocyte metabolism.     

Engineering antibodies and proteins for molecular in vivo imaging

The rapid and ongoing discovery of new disease related biomarkers leads to a dramatic paradigm change in human healthcare and constitutes the basis for a truly personalized medicine. Molecular imaging enables early detection and classification of human diseases and provides valuable data for optimized, target-oriented therapies. By now, the biochemical and physiological properties of antibody derivatives or alternative protein scaffolds can be engineered for the detection of a wide range of target structures. The successful application of these reagents in animals, xenograft models and cells in preclinical research clearly demonstrate their utility for molecular imaging. Despite these promising perspectives, only a few antibodies and recombinant proteins are used yet for molecular imaging in human medicine. Especially the high safety demands and the need to eliminate off target effects in humans require extensive research and development efforts.     

A platform for high-throughput molecular characterization of recombinant monoclonal antibodies

We describe quantitative characterization of a sample preparation platform for rapid and high-throughput analysis of recombinant monoclonal antibodies (MAbs) and their post-translational modifications. MAb capture, desalting and in situ reduction/alkylation were accomplished by sequential adsorption of analyte to solid phase beads (protein A, reverse-phase) suspended in microtiter plate wells. Following elution and rapid tryptic digestion in the presence of acid-labile surfactant (RapiGest), peptides were fractionated by stepwise elution from reverse-phase pipet tips and the fraction containing Fc N-glycopeptides isolated. Direct quantitative analysis of the relative abundance of peptide glycoforms by MALDI-TOF MS in linear mode closely correlated with normal phase HPLC analysis of fluorophore labeled N-glycans released by PNGaseF.     

Characterization of monoclonal antibodies to Acanthamoeba myosin-I that cross-react with both myosin-II and low molecular mass nuclear proteins

We characterized nine monoclonal antibodies that bind to the heavy chain of Acanthamoeba myosin-IA. Eight of these antibodies bind to myosin-IB and eight cross-react with Acanthamoeba myosin-II. All but one of the antibodies bind to a 30-kD chymotryptic peptide of myosin-IA that derives from the COOH terminus of the molecule, and to tryptic peptides as small as 17 kD, hence these epitopes are clustered closely together on the heavy chain. None of the antibodies prevent heavy chain phosphorylation by myosin-I heavy chain kinase. One antibody inhibits the K+-EDTA ATPase activity and three antibodies inhibit the actin- activated Mg++-ATPase activity of myosin-I under the set of conditions that we tested. When fluorescent antibody staining of both whole cells and isolated nuclei is done, several of these monoclonal antibodies react strongly with nuclei. These antibodies also stain the cytoplasmic matrix, especially the cortex near the plasma membrane. All nine of the monoclonal antibodies bind to polypeptides of 30-34 kD that are highly enriched in nuclei isolated from Acanthamoeba. There is no myosin-I in the isolated nuclei, so the 30-34-kD polypeptides, not myosin-I, are responsible for the nuclear staining.     

Novel recombinant monoclonal antibodies for vitellogenin assays in cyprinid fish species

Various polyclonal and monoclonal antibodies have been developed for vitellogenin (Vtg) bioassays in different aquatic species. Preparation of these reagents is time-consuming and expensive. In the present study, a phage-displayed, recombinant, single-chain variable fragment (scFv) format antibody library was constructed using splenic mRNA from non-immunized mice. After 3 rounds of panning, 3 scFv antibodies with specificity for the highly conserved N-terminal region of cyprinid fish Vtg were isolated. One of these, antibody H4, bound purified Vtg from common carp Cyprinus carpio, zebrafish Danio rerio and Chinese rare minnow Gobiocypris rarus with similar affinities and detected Vtg in zebrafish plasma samples. This study provides a simple, low cost Vtg bioassay for plasma samples from a variety of cyprinid fish.     

Comparison of the cell envelope proteins of Micrococcus cryophilus with those of Neisseria dnd Branhamella species.

In an attempt to elucidate the relation between Micrococcus cryophilus, Neisseria caviae, Neisseria ovis, and Branhamella catarrhalis, fractions derived from outer membranes of a strain of each organism were examined for protein composition by SDS - polyacrylamide gel electrophoresis. Micrococcus cryophilus outer membrane protein showed extensive similarities to that of N. ovis and contained a heat-modifiable protein which behaved almost identically with the corresponding bands previously shown to exist in N. caviae and N. ovis. Branhamella catarrhalis protein was distinctly different from those of M. cryophilus and the two 'false neisserias' N. caviae and N. ovis.     

Mammalian cell surface display for monoclonal antibody-based FACS selection of viral envelope proteins

The elicitation of broadly and efficiently neutralizing antibodies in humans by active immunization is still a major obstacle in the development of vaccines against pathogens such as the human immunodeficiency virus (HIV), influenza virus, hepatitis C virus or cytomegalovirus. Here, we describe a mammalian cell surface display and monoclonal antibody (mAb)-mediated panning technology that allows affinity-based selection of envelope (Env) variants from libraries. To this end, we established an experimental setup featuring: 1) single and site specific integration of Env to link genotype and phenotype, 2) inducible Env expression to avoid cytotoxicity effects, 3) translational coupling of Env and enhanced green fluorescent protein expression to normalize for Env protein levels, and 4) display on HEK cells to ensure native folding and mammalian glycosylation. For proof of concept, we applied our method to a chimeric HIV-1 Env model library comprising variants with differential binding affinities to the V3-loop-directed mAbs 447–52D and HGN194. Fluorescence-activated cell sorting selectively enriched a high affinity variant up to 56- and 55-fold for 447–52D and HGN194, respectively, after only a single round of panning. Similarly, the low affinity variants for each antibody could be selectively enriched up to 237-fold. The binding profiles of membrane-bound gp145 and soluble gp140 chimeras showed identical affinity ranking, suggesting that the technology can guide the identification of Env variants with optimized antigenic properties for subsequent use as vaccine candidates. Finally, our mAb-based cellular display and selection strategy may also prove useful for the development of prophylactic vaccines against pathogens other than HIV.     

Microwaving for double indirect immunofluorescence with primary antibodies from the same species and for staining of mouse tissues with mouse monoclonal antibodies

Many methods have been devised for double immunocytochemical staining. We now describe that moderate microwaving does not elute antibodies, but prevents their reactions with subsequently applied reagents. Thus, microwaving performed in between the first and second staining cycles permits double indirect immunofluorescence staining with antibodies raised in the same species. Moreover, microwaving also inhibits reactions with endogenous immunoglobulins present in extracellular compartments. This substantially reduces background in indirect immunostaining of mouse tissues with mouse monoclonal antibodies. Accepted: 19 October 1999     

The molecular and biochemical characterization of mutant monoclonal antibodies with increased antigen binding.

Mutant mAb with increased Ag binding were generated from a hybridoma cell line, 36-65, that secretes an IgG1,kappa anti-p-azophenylarsonate-(Ars) specific antibody. The mutant antibodies were identified using an Ars-specific ELISA and sib selection so that approximately 10(6) cells could be analyzed. The ELISA used as Ag a low ratio of Ars coupled to BSA and was set up so that only those antibodies that had higher binding than the parent would be detected. Seven mutant producing cell lines were isolated from five independent clones of 36-65. The mutant antibodies bind Ag 20 to more than 200-fold better than the parent and have wild type V region sequences. All have C region mutations that result in an increased avidity. At least five different genetic events are responsible for the C region mutations.