Monoclonal antibodies and mechanistic studies on recA protein.

A protein has various epitopes, and a monoclonal antibody specifically binds to the protein by recognizing 1 of the epitopes. This characteristic of the monoclonal antibody has opened various new approaches in a wide variety of research works. In studies about recA protein and its promoted various reactions relating to genetic recombination, anti-recA protein-monoclonal antibodies are very useful to analyse reaction mechanisms and to detect transition in the higher order-structure of the protein, as well as to measure the amounts of recA protein in vitro or in vivo and to identify the related proteins. In this article, we will review studies on recA protein in which monoclonal antibodies were used as major tools. By using anti-recA protein-monoclonal IgGs as specific inhibitors, the partial reactions of the homologous pairing and strand exchange promoted by recA protein were separated, and by use of a set of anti-recA protein IgGs the stages of activation of recA protein in the above reactions were discriminated.     

Monoclonal antibodies against rat kidney gamma-glutamyl transpeptidase show species and tissue specificity.

The existence of a GTP-binding protein of the Ns type in Trypanosoma cruzi was explored. Epimastigote membranes were labelled by cholera toxin in the presence of [adenine-14C]NAD+. After SDS/polyacrylamide-gel electrophoresis of extracted membrane proteins, a single labelled polypeptide band of apparent Mr approx. 45,000 was detected. Epimastigote cells were treated with N-ethylmaleimide and electrofused to lymphoma S49 cells lacking the Ns protein. Evidence indicates that in such electrofusion-generated cell hybrids a heterologous adenylate cyclase system was reconstituted with the Ns protein provided by T. cruzi epimastigotes.     

Monoclonal antibodies to rhamnogalacturonan I backbone

Monoclonal antibodies were raised against rhamnogalacturonan I backbone, a pectin domain, using Arabidopsis thaliana seed mucilage-derived rhamnogalacturonan I oligosaccharides—BSA conjugates. Two monoclonal antibodies, designated INRA-RU1 and INRA-RU2, selected for further characterization, were specific for the backbone of rhamnogalacturonan I, displaying no binding activity against the other pectin domains i.e. homogalacturonans, galactans or arabinans. A range of oligosaccharides was prepared by enzymatic digestion of rhamnogalacturonan I isolated from Arabidopsis thaliana seed mucilage and from sugar beet pectin, purified by low-pressure chromatography and characterized by high-performance anion-exchange chromatography and mass spectrometry. These rhamnogalacturonan I oligomers were used to characterize the binding site of the two monoclonal antibodies by competitive inhibition. Both INRA-RU1 and INRA-RU2 showed maximal binding to the [→2)-α-l-rhamnosep-(1→4)-α-d-galacturonic acid p-(1→]₇ structural motif but differed in their minimum binding requirement. INRA-RU2 required at least two disaccharide (rhamnose–galacturonic acid) repeats for the antibody to bind, while INRA-RU1 required a minimum of six disaccharide repeats. Furthermore, the binding capacity of INRA-RU1 decreased steeply as the number of disaccharide repeats go beyond seven. Each of these antibodies reacted with hairy regions isolated from sugar beet pectin. Immunofluorescence microscopy indicated that both antibodies can be readily used to detect rhamnogalacturonan I epitopes in various cell wall samples.     

Monoclonal antibodies showing sequence specificity in their interaction with single-stranded DNAs.

Six hybridoma cell lines which secrete monoclonal antibodies binding to nucleic acids were produced from autoimmune NZB/NZW mice. Four of the antibodies were IgG's and the other two were IgM's. Using a solid phase radioimmunoassay (SPRIA) the binding of the antibodies to over thirty different nucleic acids was estimated. All the antibodies were extremely specific. There was no detectable interaction with various RNAs, and single-stranded DNAs bound more antibodies than duplex or multi-stranded DNAs. In every case the antibodies also showed considerable sequence preferences. For example one monoclonal antibody bound to d(TTC)n but not to d(TCC)n while another interacted strongly with D(TG)n and d(CA)n but not with d(TC)n, d(GA)n or homopolymers. In other cases the patterns of sequence specificity were extremely difficult to interpret although it seems clear that monoclonal antibodies have the potential to distinguish between any two nucleic acids however similar.     

Monoclonal antibodies to crayfish rhodopsin. I. Biochemical characterization and cross-reactivity

Five antibody secreting cell lines were selected on the basis of specific binding to photoreceptive structures from a fusion of myeloma cells with spleen cells from BALB/c mice immunized with photoreceptor membrane from crayfish compound eyes. On Western blots derived from one- and two-dimensional polyacrylamide gels of purified photoreceptor membrane the antibodies bound strongly to the major 35 kDa peptide and are therefore specific for the visual pigment, rhodopsin. Four antibodies also recognized a minor 24 kDa peptide probably representing a breakdown product generated in vivo by the action of lysosomal hydrolases. Epitope characterization of the antibodies using peptide maps of opsin after protease treatment revealed three grossly different specificities. Three antibodies recognize a major antigenic site located within the large proteolytic fragment of about 24 kDa, possibly derived from the aminoterminus of the molecule. Antibodies applied to lightly fixed frozen sections or semi-thin sections of crayfish retina embedded in Lowicryl or polyethyleneglycol specifically bound to the rhabdomeral structure formed by receptor cells R1-R7, but failed to show significant cross-reaction with R8, the blue receptor, proving significant differences in the primary structure of the apoproteins of visual pigments involved in crayfish colour vision. None of the antibodies revealed any cross-reactivity with Drosophila or squid rhodopsin, corroborating this finding. The antibodies also recognized granular material in the vicinity of the rhabdoms at sites occupied by secondary lysosomes containing degraded rhabdomeral membrane. No significant binding was observed to the outer plasma membrane of the retinula cells, or in any other part of the retina.     

Monoclonal antibodies against chordin. Use in structural and immunohistochemical studies

Eight MAbs have been developed against chordin and designated as At2-At9. It is shown that all antibodies are directed against identical, spatially overlapping or closely positioned epitopes of chordin. The chordin molecule has repetitive sites wherein epitopes for the eight MAbs are located. This site lies within a proteinase-resistant fragment of chordin, presumably a glycopeptide, of molecular mass between 2 and 10 kDa. Fluorescence staining of cryostat sections from stellate sturgeon with the use of At5 (indirect Coons' method) has revealed a positive reaction with notochord cells and sheath and with the spinal cord. No significant reaction with cartilage, muscle and kidney was detected.     

Murine polyspecific antibodies. I. Monoclonal and serum anti-DNA antibodies cross-reactive with 2,4,6-trinitrophenyl derivatives

Six anti-DNA hybridoma autoantibodies were prepared by fusing spleen cells from unimmunized MRL/MpJ/lpr/lpr female mice with BALB/c myeloma cells. The monoclonal antibodies were analyzed by solid-phase ELISA for antigen-binding specificities. Three antibodies (62A2, 85A5, and 43B2) bound ssDNA, TNP-KLH, and recognized an epitope(s) present on insolubilized proteins such as BSA, KLH, ferritin, and insulin. The antibodies bound, with a marked preference, TNP-KLH, either soluble or insoluble. The other three antibodies (35A1, 32C5, and 39D2) bound only ssDNA. However, this binding was inhibited by free flavinic acid. None of the six antibodies bound either cardiolipin or proteoglycans, indicating that they do not recognize the repeating negatively charge units common to cardiolipin, proteoglycans, and DNA. All six monoclonal antibodies were purified by affinity chromatography with TNP-Sepharose. Moreover, both anti-DNA and anti-TNP antibodies from sera of nonautoimmune and autoimmune mice were purified easily on TNP-Sepharose.     

Monoclonal antibodies against E- and F-type prostaglandins. High specificity and sensitivity in conventional radioimmunoassays

Polyclonal antisera against prostaglandins (PGs) are widely used for the assessment of the biological role of these mediators, but even the most specific contain antibodies against the major metabolites and degradation products of the haptens employed. To overcome this inherent problem we produced monoclonal antibodies (mAs) against PGE2, PGF2 alpha and 6-keto-PGF1 alpha using the somatic cell hybridization technique. The mAs against 6-keto-PGF1 alpha and PGF2 alpha proved to be highly specific, but allowed only for moderate detection limits (1-2 ng) in conventional fluid phase radioimmunoassays (RIAs). One of the mAs against PGE2 permitted a 100-fold improvement in the detection limit while being almost devoid of cross-reactivity with metabolites and other structurally related PGs. These results show that highly specific mAs against PGs can be produced to improve the available RIA technique for PG quantification.     

Monoclonal antibodies neutralizing mammalian DNA topoisomerase I activity

We have isolated three different monoclonal antibodies specific for mammalian type-I DNA topoisomerase. The antibodies react with three closely adjacent epitopes located in a central section of the enzyme (between amino acid residues 344 and 483). Two of the antibodies inhibit an early step of the nicking/closing pathway. We provide evidence showing that the antibodies do not block the association of the enzyme with DNA. The antibodies are useful for immunocytochemical investigation and for further exploration of the biochemical function of mammalian type-I DNA topoisomerase.     

Isotype specificity of antigen-specific helper clone in vivo

Inoculation of an immortalized clone of radiation leukemia virus (RadLV)-transformed antigen (ovalbumin, OVA)-specific T cells together with the relevant carrier (OVA) into unprimed syngeneic mice results in a preferential increase in the expression of anti-OVA antibodies of the immunoglobulin (Ig)G2b and IgG2a isotypes. Identical boosting of the clone-primed mice further augments the preferential production of anti-OVA antibodies of these two isotypes. The class-related helper activity is not due to nonspecific shift of class expression produced by the injected tumor cells, as a non-helper clone of RadLV-transformed T cells does not change the isotypic pattern of anti-OVA antibodies in the inoculated mice. A carrier-specific activation of the B cells is responsible for the class-restricted function of the helper clone. The isotypic profile of anti-hapten antibodies in mice injected with 2,4-dinitrophenyl (DNP)-bovine serum albumin and OVA-specific helper clone is not altered. On the other hand, mice inoculated with the OVA-specific helper clone and DNP-OVA respond with a preferential elevation of anti-DNP antibodies of the IgG2a and IgG2b isotypes. The preferential class augmentation may result from carrier-specific signals delivered by the helper clone which activate B cells in vivo toward certain CH expression. Alternatively, the observed class pattern may be induced by an isotype noncommited helper clone which triggers selected population of B lymphocytes of defined differentiation status toward secretion of a restricted array of isotypes. Regardless of the mechanism of the clone-dependent class expression, the isotypic profile in most of the experiments clearly demonstrates that an antigen-specific helper clone may be one of the elements which regulates the class of antibodies to be produced in vivo under normal physiologic conditions.     

Monoclonal antibodies against human pulmonary surfactant apoproteins: specificity and application in immunoassay

Monoclonal antibodies were prepared against pulmonary surfactant apoproteins which were isolated from lung lavages of patients with alveolar proteinosis with the following steps: solubilization of the surface-active fraction by Triton X-100, delipidation with butanol-ethanol extraction followed by column chromatographies on Blue-Sepharose and DEAE-Toyopearl in the presence of dithiothreitol. The fraction including 62 and 36 kDa proteins, i.e., pulmonary surfactant apoproteins, was used for the immunization. Monoclonal antibodies against the pulmonary surfactant apoproteins were prepared using hybridoma technology. The monoclonal antibodies prepared, PC6 and PE10, recognized the same proteins, i.e., 62 and 36 kDa proteins, in the patients' lavages. They also recognized 37 and 34 kDa proteins in human lung lavage and amniotic fluid. Quantitation of the apoproteins by enzyme-immunoassay using the monoclonal antibodies has been developed. A combination of PC6 and PE10 was found to be useful for a two-site sandwich enzyme-linked immunosorbent assay (ELISA), where it gave a good dose response and was capable of measuring 10-1280 ng of the apoprotein/ml. The specificity of the monoclonal antibodies in animal species was tested by this sandwich ELISA. The results indicated that the monoclonal antibodies obtained in this study are specific for the human lung.     

Monoclonal antibodies with a high degree of specificity for Listeria monocytogenes serotype 4b.

Strains of Listeria monocytogenes serotype 4b account for a large fraction of sporadic listeriosis cases, as well as all major food-borne epidemics attributed to this pathogen. We have identified a set of three monoclonal antibodies which showed a high degree of specificity for strains of L. monocytogenes serotype 4b. Two of these antibodies (c74.33 and c74.180, isotypes immunoglobulin M [IgM] and IgG3, respectively) recognized all serotype 4b strains, whereas antibody c74.22 (isotype IgG1) failed to recognize certain epidemic-associated strains. The corresponding antigens were located on the surface of the bacteria and were expressed following bacterial growth in different media and over a wide range of temperatures (4, 22, and 37 degrees C). Heating L. monocytogenes cells at 80,90, or 100 degrees C abolished reactivity for c74.22 but not for c74.33 MAb. These MAbs were negative for all of the non-Listeria strains tested, including representatives of several gram-negative and gram-positive species. The surface antigen recognized by c74.22 appeared to be associated with the ability of the bacteria to enter (invade) mammalian cells in culture.     

Monoclonal antibodies to the multienzyme enniatin synthetase. Production and use in structural studies

Monoclonal antibodies have been prepared against the multifunctional enzyme enniatin synthetase, which catalyses the biosynthesis of the cyclodepsipeptide antibiotic enniatin. Five different antibodies (designated 1.56, 21.1, 25.91, 28.7 and 28.34) were characterized. 1.56, 21.1 and 25.91 were of IgG1 and 28.7 and 28.34 of IgM subclass. Binding studies showed that 21.1 and 25.91 are obviously directed against determinants based on the primary structure of the enzyme, whereas 28.7, 28.34 and 1.56 bind to the native enzyme. All antibodies inhibited enniatin formation. Based on their ability to inhibit different partial reactions of the multienzyme the antibodies could be divided into three groups: 21.1 and 25.91 inhibit valyl thioester formation, 1.56 additionally inhibits D-2-hydroxyisovaleric acid thioesterification, and 28.7 and 28.34 block both thioester sites as well as the N-methylation step. None of the antibodies affected the formation of L-valyl or D-hydroxyisovaleryl adenylate by the enzyme. The results indicate that there must be distinct thioester activation sites for valine and D-hydroxyisovalerate close to each other and in the neighbourhood of the methyltransferase site. The adenylation sites for D-hydroxy-isovalerate and L-valine are obviously located at some distance.     

Monoclonal antibodies

The hybridoma technology developed by K?hler and Milstein has initiated a new era in biological sciences. In the last decade the possibility of generating limited amounts of monoclonal antibodies of predefined specificity has become a routine method in many laboratories throughout the world. The constant quality of various antibody preparations from 1 hybridoma cell line represents another important advantage of this method. Apart from the use for several purposes, e.g. HLA-typing, differentiation of lymphocyte subpopulations and blood group antigens, monoclonal antibodies play an important role in the determination of various tumor markers. In most modern immunoassays monoclonal antibodies are used. Furthermore, there is nowadays a limited experience concerning the in vivo use of monoclonal antibodies in malignant disease. Radiolabelled antibody immunodetection has been applied e.g. in colorectal and testicular tumors for the detection of tumor metastases. The therapeutic use of monoclonal antibodies has been reported in some patients with tumors of the hemopoietic system. The production of new murine and human monoclonal antibodies against various tumor types is subject of current investigations. The aim of these efforts is the development of monoclonal antibodies suitable for in vitro tumor diagnosis and application in vivo.     

Monoclonal antibodies against core and cellulose-binding domains of Trichoderma reesei cellobiohydrolases I and II and endoglucanase I.

Cellulases from Trichoderma reesei form an enzyme group with a common structural organization. Each cellulase enzyme is composed of two functional domains, the core region containing the active site and the cellulose-binding domain (CBD). To facilitate the specific detection of each domain, monoclonal antibodies (mAb) against cellobiohydrolase I (CBHI), cellobiohydrolase II (CBHII) and endoglucanase I (EGI) were produced. Five mAb were obtained against CBHI, ten against CBHII and eight against EGI. The location of the antigenic epitope for each antibody was mapped by allowing the antibodies to react with truncated cellulases, synthesized from deleted cDNA in Saccharomyces cerevisiae. Proteolytic fragments of Trichoderma cellulases, obtained by papain digestion, were used to confirm the results. Specific antibodies were detected against the core and the CBD epitopes for all three cellulases. Using the truncated enzymes, it was possible to locate the epitopes to a reasonably short region within the protein. To obtain a quantitative assay for each enzyme, a specific mAb against each antigen was chosen, based on the affinity to the corresponding antigen on Western-blot staining and on filter blots of the cellulolytic yeasts. The mAb were used to quantitative the corresponding enzymes in T. reesei culture medium. Specific quantitation of each cellulase enzyme has not been possible by biochemical assays or using polyclonal antibodies, due to their cross-reactions. Now, these mAb can be specifically used to recognize and quantitate different domains of these three important cellulolytic enzymes.     

Further studies of specificity of antibodies contained in antiserum against poliovirus.

Antigenic components of Mahoney strain (poliovirus type 1) involved in virus neutralization reaction were analyzed with mutant Mahoney strains resistant to inhibitors in equine serum (inhibitor-resistant mutants) by means of the kinetic neutralization test. It was shown that absorption of anti-Mahoney serum with five inhibitor-resistant mutants yielded sera with different antibodies, of which three had distinct specificities and two specificities possibly partly related to one of those three sera. Further, it was found that step wise selection of Mahoney variants resistant to one, two, three and four different inhibitors resulted in gradual deviation of its antigenic composition from that of the original strain. From these results, the possible presence of three or more distinct antigenic determinant sites on the surface of Mahoney strain was indicated.     

Monoclonal antibodies against phloem P-protein from plant tissue cultures. I. Microscopy and biochemical analysis

Most research involving phloem proteins is done with phloem exudates, which are not easily obtained from many plants. We report here on the use of tissue cultures to study phloem proteins. Monoclonal antibodies against the filamentous phloem protein, P-protein, were made by injecting mice with a phloem-enriched fraction isolated from Streptanthus tortuosus callus grown on a medium that stimulates the differentiation of xylem and phloem (phloem[+] cultures). Monoclonal antibodies specific for P-protein were identified by incubating free-hand stem sections of S. tortuosus in hybridoma supernatants, then in a goat anti-mouse antibody conjugated to fluorescein isothiocyanate (FITC), and observing the FITC under an epifluorescence microscope. Antibodies specific for P-protein in stem sections were used to probe nitrocellulose blots of polyacrylamide gels separating proteins isolated from both phloem(+) and phloem(-) tissue cultures. Immunoblots were incubated overnight in hybridoma supernatants followed by a secondary antibody conjugated to alkaline phosphatase. Three monoclonal antibodies—RS21, RS22, and RS23—bound to an 89-kD band in the phloem(+) lanes but failed to bind to any proteins in the phloem(—) lanes. In leaf sections of Arabidopsis thaliana processed by freeze-substitution, a mixture of RS21 and RS22 bound to the P-protein filaments in sieve elements, but not to any proteins in adjacent cells. A control antibody specific for tubulin did not bind to the P-protein filaments.