Monoclonal antibodies demonstrating GABA-like immunoreactivity

Summary Mouse monoclonal antibodies (mAb) to GABA were developed following immunization with GABA coupled to bovine serum albumin (GABA-BSA). The selection of hybridoma cell lines producing antibodies which reacted with GABA-BSA but not with glutamate-BSA conjugates as well as the characterization of chosen mAb was performed by enzyme linked immunosorbent assays (ELISA). The five mAb selected were all of the IgG class and displayed different patterns of crossreactivities with the amino acid- and dipeptide-BAS conjugates tested. MAb 3A12 reacted approximately 4,000 times better with GABA-BSA than with -alanine-BSA conjugates according to serial dilution experiments of the antibody in ELISA. Immunoreactivity was even lower for other conjugates tested including glycine-, taurine-, glutamate-, and glutamine-BSA. Immunohistochemical results in rat and chicken brain indicated that the patterns of GABA-like immunoreactivity observed with these mAb were consistent with the available information on the distribution of GABA-containing neurons.     

Specificity of human anti-NOR antibodies,a distinct species of "natural" anti-alpha-galactosyl antibodies

Natural anti-NOR antibodies are common in human sera and agglutinate human erythrocytes of a rare NOR phenotype. The NOR phenotype-related antigens are unique neutral glycosphingolipids recognized by these antibodies and Griffonia simplicifolia IB4 isolectin (GSL-IB4). The oligosaccharide chains of NOR glycolipids are terminated by Galalpha1-4GalNAcbeta1-3Galalpha units. To characterize the specificity of anti-NOR antibodies and compare it with specificities of GSL-IB4 and known anti-Galalpha1,3Gal antibodies, alpha-galactosylated saccharides and saccharide-polyacrylamide conjugates were used. New synthetic oligosaccharides, corresponding to the terminal di- and trisaccharide sequence of NOR glycolipids and the conjugate of the NOR-tri with HSA were included. These compounds were tested by microtiter plate ELISA and hemagglutination inhibition. Anti-NOR antibodies reacted most strongly with Galalpha1-4GalNAcbeta1-3Gal (NOR-tri), and over 100 times less strongly with Galalpha1-4GalNAc (NOR-di). The antibodies reacted also with Galalpha1-4Gal and Galalpha1-4Galbeta1-4GlcNAc, similarly as with NOR-di but not with other tested compounds. In turn, anti-Galalpha1,3Gal antibodies reacted most strongly with Galalpha1-3Gal and were very weakly inhibited by the NOR-related oligosaccharides (weaker than by galactose), and NOR-tri was less active than NOR-di. GSL-IB4 reacted with all tested alpha-galactosylated saccharides and conjugates, including the similarly active NOR-tri and NOR-di. These results showed that anti-NOR represent a new species of anti-alpha-galactosyl antibodies with high affinity for the Galalpha1-4GalNAcbeta1-3Gal sequence present in rare NOR erythrocytes.     

Development and characterization of monoclonal antibodies specific for the genus Listeria

Abstract Monoclonal antibodies were obtained by the classic hybridoma technique with lymphocytes of BALB/c mice immunized with formalin killed Listeria monocytogenes cells. Among 1000 hybridomas issued from the fusion, four monoclonal antibodies (mAbs A6 A E4, C10 A F7, G4 A D6, G7 A D5) gave interesting results. By Western-blot analysis with various soluble extracts of different Listeria species, the four mAbs reacted with two major antigens of 38 and 41 kDa, with all Listeria species tested. The mAb A6 A E4 is an IgG2b with κ light chains and reacted only with Listeria antigens without any cross reaction with other organisms tested by ELISA, dot-blotting and Western-blotting. With the same conditions, the three other mAbs reacted with Listeria and with other genus extracts, particularly with Streptococcus and Enterococcus . mAb A6 A E4-reactive antigens are proteins, and glycoprotein immunoassay indicated that the epitope is devoid of carbohydrate moiety. This mAb A6 A E4-reactive protein was neither expressed on cell surface nor released outside the bacteria; immunogold electron microscopy showed that these antigens were localized in the cytoplasma area.     

Analysis of antibody response to synthetic peptides derived from the fusion protein of measles virus

A computer program combining of hydrophilicity, flexibility, surface probability, secondary structure and antigenic index parameters of the amino acid sequence of measles virus (MV) fusion protein was used to select four possible epitopes. Rabbits were immunized with the synthesized peptides conjugated to purified protein derivative using the homobifunctional cross-linker bis-sulfosuccinimidyl suberate. Immune stimulating complexes were prepared with the peptides conjugated to the purified protein derivative carrier using a dialysis method. All antisera raised in rabbits against the peptide conjugates had a high titer to the homologous peptides and reacted well with denatured MV as tested by plate ELISA. None of the sera had neutralizing antibody. Human sera positive for MV antibody reacted strongly with the synthesized peptides indicating that the selected locations function as partial antigenic sites. Antisera against peptide conjugates reacted weakly in immunofluorescence and none of these antisera reacted with purified MV proteins in Western blot. The results obtained in this study indicated that although the computer program could not predict epitopes important for the neutralization of the MV, the predicted epitopes are useful for detecting antibodies against MV.     

Tropomyosin shares immunologic epitopes with group A streptococcal M proteins

Tropomyosin is an alpha-helical coiled-coil protein with structural similarities to the streptococcal M protein. In order to show serologic cross-reactivity between streptococcal M proteins and tropomyosin, we selected from a panel of murine mAb those which reacted with M proteins and tropomyosins in the ELISA. Western blots were used to study the reactions of each mAb with human and rabbit cardiac and rabbit skeletal tropomyosins. The antibodies were further characterized for their reactions with the additional autoantigens myosin, actin, keratin, and DNA. Five mAb were found which reacted with either PepM5 or ColiM6 protein and tropomyosin in Western blots or ELISA. Two of the tropomyosin positive mAb were also antinuclear antibodies and were inhibited with DNA. In Western blots of cardiac tropomyosins, the mAb reacted with either the 70-kDa dimer of tropomyosin, the 35-kDa monomer, or both. Some differences were observed in the reactions of the mAb with the different tropomyosins in Western blots. The heart cross-reactive epitopes shared between M proteins and tropomyosin were in most instances shared with cardiac myosin. Differences were observed among the reactions of the mAb with the different tropomyosins. This report constitutes the first evidence of serologic cross-reactivity between streptococcal M proteins and tropomyosins.     

Heart cross-reactive antigens of mutans streptococci share epitopes with group A streptococci and myosin

Monoclonal antibodies (mAb) generated by immunization of mice with membranes of Streptococcus pyogenes Manfredo (M type 5) were tested for their reactivity with sodium dodecyl sulfate extracts of Streptococcus mutans GS5, Streptococcus rattus BHT (formerly S. mutans, serotype b), and S. mutans Ingbritt 175 in the Western blot. The reaction of the sodium dodecyl sulfate-extracted proteins of whole S. mutans, serotype b), and S. rattus with the mAb revealed that the shared cross-reactive antigens were near m.w. 62,000 to 67,000. Several higher m.w. proteins in S. mutans Ingbritt 175 reacted with the mAb but were not observed in the other two strains of streptococci. Cytoplasmic membranes of S. rattus BHT reacted with these mAb in the enzyme-linked immunosorbent assay and Western blot. The most reactive BHT membrane component detected by these mAb was a 62-kDa polypeptide that was similar in m.w. to the membrane component recognized by these antibodies in purified S. pyogenes membranes. The reactivity of the mAb with BHT membranes in the enzyme-linked immunosorbent assay was absorbed by rabbit skeletal muscle myosin. The patterns of reactivity observed in the BHT membrane immunoblots in this study closely resemble those previously obtained using polyclonal rabbit anti-human heart sera.     

天然产物莪术醇单克隆抗体的制备

为制备小分子化合物莪术醇的单克隆抗体,先将莪术醇(curcumol)与载体蛋白牛血清蛋白(BSA)偶联形成完全抗原,用基质辅助激光解吸飞行时间质谱法(MALDI-TOF-MS)鉴定莪术醇人工抗原的偶联率,然后采用杂交瘤技术获得杂交瘤株,并对其进行小鼠腹水的制备与纯化.结果表明:莪术醇半抗原与载体的偶联比为19.6,单克...     

Identification of a major hepatitis B core antigen (HBcAg) determinant by using synthetic peptides and monoclonal antibodies

To map the location of hepatitis B core and e Ag (HBcAg and HBeAg) on the hepatitis B virus core particle, we produced and analyzed four synthetic peptides which correspond to the most hydrophilic regions of the core P22 protein. Each peptide was tested in an ELISA for the ability to inhibit the binding between rHBcAg or rHBeAg and either polyclonal or monoclonal anti-HBc or anti-HBe antibodies. The former comprised 20 antisera positive for anti-HBc (anti-HBs and anti-HBe negative) and five antisera positive for anti-HBe and anti-HBc; the latter included three anti-HBc mAb developed in independent laboratories: G6F5, C51B10, and F8, as well as two anti-HBe mAb, E2 and E6. These experiments revealed the presence of a major HBcAg epitope expressed on C3, a peptide which covers amino acids 107-118 and reacted with all polyclonal and monoclonal antibodies tested. Another peptide, C2, sequence 73-85, reacted with 26% of human antisera but none of the anti-HBc mAb. None of the peptides seemed to express HBeAg activity because they do not cause any significant inhibition of the HBeAg/anti-HBe reaction. These data indicate the expression of an immunodominant HBcAg determinant on a linear dodecapeptide and argue against a strict conformation dependency of this Ag.     

Characterization of antisera to glutamate and aspartate

Antisera were raised in rabbits against glutamate (Glu) and aspartate (Asp) conjugated to the invertebrate carrier protein hemocyanin (HC) with glutaraldehyde (GA). The antisera were characterized by testing their immunocytochemical staining properties on sections cut at the level of the ventral cochlear nucleus (VCN) from fixed brains of normal rats after absorption with conjugates of compounds structurally similar and biologically relevant to Glu and Asp. Optimal staining with Glu antiserum was obtained at a dilution of 1:10,000 and was completely blocked by 303 micrograms/ml of the Glu-HC conjugate. No crossreactivity with any of 11 compounds tested was observed. Optimal staining with the Asp antiserum was obtained at 1:8000 dilution and was completely blocked by 225 micrograms/ml of the Asp-HC conjugate. Of 10 compounds tested for crossreactivity, only L-asparagine demonstrated a measurable (about 10%) crossreactivity with the Asp antiserum. The specificity of the two antisera was also tested by immunoblot analysis against 11 compounds conjugated to HC with GA. Listed in order of staining intensity, from greatest to least, conjugates that reacted with the Glu antiserum were Glu greater than Gly-Glu greater than Asp-Glu = Asp greater than N-carbamyl (NC)-Glu greater than Asn = Gln = GABA. Conjugates that reacted with the Asp antiserum, in order of decreasing staining intensity, were Asp greater than Glu-Asp = Asn greater than Gly-Asp greater than Glu. No other compounds tested for crossreactivity reacted with the two antisera in the immunoblot analysis. Glu-like immunoreactivity in rat dorsal root ganglia and somatosensory cortex, and the comparative distribution of Glu- and Asp-like immunoreactivities in the latter tissue, are presented as examples of staining patterns obtained with the two antisera.     

Glutaraldehyde (GA)-hapten adducts, but without a carrier protein, for use in a specificity study on an antibody against a GA-conjugated hapten compound: histamine monoclonal antibody (AHA-2) as a model

In our recent study on monoclonal antibodies (mAbs AHA-1-5) against glutaraldehyde (GA)-conjugated histamine (HA), we identified one mAb (AHA-2) which can detect neuronal HA in the rat brain with an immunocytochemistry method (ICC) [Fujiwara et al. (1999) J. Biochem. 126, 503-509]. In the present study the specificity of AHA-2 mAb for use for ICC has been examined by means of competitive experiments involving HA and analogs, all of which had been allowed to react with GA followed by sodium borohydride, but not allowed to couple with the carrier protein. It was demonstrated that the antibody distinguished alterations in the chemical structure of the molecule, showing decreased immunoreactivity with all the GA-adducts of (R)-(-)-alpha-methylhistamine, 1- and 3-methylhistamine, L-histidine, and 1- and 3-methyl-L-histidine. On the other hand, AHA-1 mAb only reacted with GA-adducts of 3-MeHA (3-MeHA-GA) and HA (HA-GA), to almost the same degree, in relatively high concentration ranges. AHA-3, 4, and 5 mAbs reacted about 10-times more strongly with 1-MeHA-GA than with HA-GA, but reacted very little or not at all with the other analogs. These results may suggest that AHA-2 mAb recognized both the non-substituted imidazole and alpha-methine groups of a HA molecule in addition to the conjugation site of GA including the part(s) reduced with NaBH(4), and especially the imidazole group more strictly than the other mAbs. This may partly explain why AHA-2, among the five AHA mAbs, can detect neuronal HA with an ICC method. The present ELISA method for GA-hapten adducts should be applicable to other antibodies against GA-conjugated biologically active amines or amino acids, thus allowing the study of antibody specificity for ICC more easily and accurately than was previously possible with hapten-protein conjugates as antigens.     

Human monoclonal antibodies reactive with antigens of the group A Streptococcus and human heart

Human mAb were produced from tonsillar or PBL of normal individuals or patients infected with group A streptococci. Lymphocytes were purified on Ficoll-Hypaque gradients and stimulated in vitro with purified group A streptococcal membranes or M protein extracts. The mAb were selected for study based on their reaction with group A streptococci, pep M5 protein, and/or M6 Escherichia coli protein. Further analysis by Western immunoblot or competitive inhibition ELISA revealed that there were two types of antibodies: one type that reacted with myosin and DNA and the other type that reacted with myosin, keratin, and/or actin. The specificities of these human mAb are similar to specificities observed in our previous studies of murine mAb reactive with group A streptococci and heart Ag. For comparison, anti-myosin antibodies were affinity purified from the sera of infected or acute rheumatic fever patients and were shown to react with myosin and DNA as well as with group A streptococci and M protein. To affinity purify these antibodies from normal sera, five times the amount of sera was required to obtain detectable quantities. These data suggest that the human mAb reactive with group A streptococci and myosin reflect the antibodies seen in sera from infected patients or acute rheumatics and that the B lymphocyte clones capable of producing these cross-reactive antibodies are also present in normal individuals.     

Identification and partial characterization of multiple native and neoantigenic epitopes of human C-reactive protein by using monoclonal antibodies

Multiple mAb to human C-reactive protein (CRP) were prepared which reacted preferentially with either native CRP, modified CRP (expressing "neo-CRP" determinants) or both forms of the molecule. These mAb were divided into four groups according to their binding characteristics to various CRP preparations and CRP peptides by using a combination of ELISA, dot blot, and Western blot assays; they were further characterized based upon their reactivity with CRP in the presence of calcium and inhibition by phosphorylcholine. The first group consisted of mAb that reacted only with native CRP, and served to define four distinct native CRP epitopes. The second group consisted of mAb that reacted with native CRP and also with CRP modified by direct immobilization on polystyrene plates, urea-chelation or SDS treatment in the absence of calcium, thus identifying a fifth native CRP epitope; these mAb displayed significantly greater reactivity with native than with modified CRP. The third group included mAb that reacted only with modified CRP and with the larger amino-terminal fragment (residues 1-146) of pronase-cleaved CRP. The fourth group included mAb that reacted only with modified CRP and with the smaller carboxyl-terminal fragment (residues 147-206) of pronase-cleaved CRP; most of these antibodies also reacted with the carboxyl-terminal octapeptide (residues 199-206) of CRP. These experiments have identified mAb that react preferentially with distinct conformational and sequence-determined epitopes of native and modified forms of the CRP molecule, respectively; provide partial identification of the epitopes with which they interact; point to the presence of at least five epitopes on native CRP and at least three epitopes on modified CRP; and provide antibodies suitable for identification and quantitation of native and modified forms of CRP. The mAb directed against neo-CRP epitopes may help identify the presence of this pentraxin and antigenically-related proteins at previously unappreciated sites.     

Immunological characterization of rat kininogens with monoclonal antibodies to T-kininogen. Distinction between the different domains of T-kininogen and the multiple rat kininogens.

A panel of 16 monoclonal antibodies (mAb) were produced against rat T-kininogen to characterize this family of proteins. These mAbs bound 125I-T-kininogen by radioimmunoassay as well as reacting strongly with immobilized T-kininogen in an enzyme-linked immunosorbent assay (ELISA). The reactivity of these antibodies with proteolytic fragments of T-kininogen demonstrated the recognition of several different epitopes. One antibody was specific for the domain 1 of the heavy chain and/or the light chain, twelve antibodies were specific for domain 2 and three antibodies were specific for domain 3. All monoclonal antibodies recognized the two forms of T-kininogen encoded by the two different T-kininogen genes, TI and TII kininogen, except antibody TK 16-3.1 which uniquely reacted with TII kininogen. Two antibodies recognizing domain 2 cross-reacted with the high-molecular-mass kininogen (H-kininogen), whereas all the other monoclonal antibodies were specific to T-kininogen and did not recognize the heavy chain of H-kininogen. None of the antibodies tested altered the thiol protease inhibitory activity of T-kininogen, its partial proteolysis by rat mast cell chymase or the hydrolysis of H-kininogen by rat urinary kallikrein. The use of these antibodies in the development of sensitive ELISA to measure T-kininogen levels in plasma, urine, liver microsomes and hepatocytes is described. Two different forms of T-kininogen were distinguished by these monoclonal antibodies in Western blotting using rat plasma. The localization of T-kininogen was defined using these monoclonal antibodies by immunohistochemistry in rat liver hepatocytes and rat kidney.     

Production and specificity of poly- and monoclonal antibodies raised against Shewanella putrefaciens

B. FONNESBECH, H. FRØKIAER, L. GRAM AND C. MOSBY JESPERSEN. 1993. Polyclonal antibodies were raised in rabbits and mice against Shewanella putrefaciens. Murine monoclonal antibodies were produced against the type strain (ATCC 8071) as well as wild type strains isolated from fish products. The specificities of four polyclonal and 12 monoclonal antibodies were tested by dot-blotting, an indirect and a competitive ELISA against 16 Gram-negative strains; including six strains of S. putrefaciens and one strain of Pseudomonas rubescens (NC 10695). All polyclonal antibodies reacted strongly with S. putrefaciens and with Ps. rubescens and cross-reacted with the nine other bacteria ( Pseudomonas spp., Aeromonas spp. and Vibrio anguillarum ). The monoclonal antibodies could be divided into three groups with different patterns of specificity. The largest group (8 monoclonal antibodies) reacted strongly with S. putrefaciens and with Ps. rubescens and showed only weak reactions with the other strains. The results confirm that Ps. rubescens should be classified as S. putrefaciens.     

Flow cytometric analysis on reactivity of human T lymphocyte-specific and cytokine-receptor-specific antibodies with peripheral blood mononuclear cells of chimpanzee (Pan troglodytes), rhesus macaque (Macaca mulatta), and squirrel monkey (Saimiri sciureus)

Abstract: There are relatively few monoclonal antibodies (mAb) that have been characterized for their applicability in studies on the immune system of various nonhuman primates. In the present study, we identified a large number of mAb that can be used in future immunological studies in three different nonhuman primates, i.e., chimpanzees, rhesus macaques, and squirrel monkeys. The reactivity of 161 anti-human mAb to T-cell antigens and cytokine receptors were tested on peripheral blood mononuclear cells (PBMC) from the three primate species by flow cytometric analysis. A total of 105 (65%), 73 (45%), and 68 (42%) antibodies reacted with PBMC from chimpanzees, rhesus macaques, and squirrel monkeys, respectively. Out of the 161 mAb, 38 reacted with all three species and 112 reacted with one or two of the species. No specific reaction was observed with mAb to receptors to GM-CSF, 4–1BB, FLT3, FLX2, common β-chain, IL-1 (type I receptor), and IL-8.     

Histamine monoclonal antibody for brain immunocytochemistry.

Among five monoclonal antibodies (AHA-1 to 5 mAbs) prepared against glutaraldehyde (GA)-conjugated histamine (HA) in our previous study, only mAb AHA-2 was found to detect HA specifically in rat brain neurons by an immunocytochemistry method (ICC) using GA as a tissue fixative. All the other mAbs, except for AHA-5, reacted with HA in the enterochromaffin-like cells (ECL cells) of rat stomach [Fujiwara et al. (1997) Histochem. Cell Biol. 107, 39-45]. Enzyme-linked immunosorbent assay (ELISA) binding and inhibition tests demonstrated that AHA-2 is specific for HA, with almost no detectable cross-reaction with any other established or putative amino acid neurotransmitters, LH-RH, TRH, or peptides with N-terminal histidines. ELISA assays also suggested that the AHA-2 mAb recognizes a HA epitope structure different from the one recognized by the AHA-1 mAb. The immunostaining patterns with AHA-2 mAb, as seen in the five subgroups of the tuberomammillary nuclei in the rat posterior hypothalamus, were very similar to those described by Inagaki et al. [(1988) Brain Res. 439, 402-405; (1990) Exp. Brain Res. 80, 374-380] and Panula et al. [(1984) Proc. Natl. Acad. Sci. USA 81, 2572-2576; (1988) J. Histochem. Cytochem. 36, 259-269] using polyclonal anti-HA serum. However, it was also noted that moderate numbers of immunoreactive nerve fibers projected into the median eminence. The present HA ICC method using AHA-2 mAb allows highly sensitive HA detection in brain, and thus might permit detailed studies of HA localization hitherto impossible using previously available anti-HA polyclonal antibodies produced against carbodiimide-conjugated HA.     

Production and characterization of mouse monoclonal antibodies against human monocyte chemoattractant protein-1

We developed five different hybridoma cell lines that produced mAb against human monocyte chemoattractant protein-1 (MCP-1). The subclass of all five antibodies was IgG1. All five mAb formed complexes with metabolically labeled MCP-1 that could be demonstrated by immunoprecipitation. The antibodies were specific for MCP-1. They did not cross-react by immunoprecipitation with structurally related host defense cytokines present in metabolically labeled PHA- or LPS-stimulated mononuclear cell culture fluids, nor did they cross-react in a direct ELISA with neutrophil attractant/activation protein-1, with crude platelet lysate proteins, or with pure platelet proteins that have amino acids sequences similar to that of MCP-1. The mAb also reacted with rMCP-1 expressed in Escherichia coli, suggesting that they recognize protein structure rather than the glycosylated portion of human MCP-1. When the mAb were mixed with MCP-1, the monocyte chemotactic response to MCP-1 was inhibited. A sandwich ELISA was developed to detect MCP-1 in biologic fluids containing relatively high concentrations of other proteins. The sensitivity was 300 pg/ml, or 30 pg/ELISA well. An anti-MCP-1 mAb column was used in an improved method of MCP-1 purification. Approximately 240 micrograms of MCP-1 were purified from 5 liters of FCS-containing U-105MG cell culture supernatant. The yield was at least 60%. In addition to two forms of MCP-1 reported previously by us, two more forms of MCP-1 were found in a mixture of culture supernatants of PHA- and LPS-stimulated human PBMC.     

The C terminus of the anaphylatoxin C3a generated upon complement activation represents a neoantigenic determinant with diagnostic potential

Activation of the C component C3 results in generation of the anaphylatoxin C3a. The C3a polypeptide chain consists of 77 amino acids. The active site of this potent mediator, which also has immunoregulatory function resides in its C terminus. This report demonstrates that the C terminus of C3a (C3a-desArg) exposed by proteolytic cleavage from C3 represents a neoantigenic determinant. Two mAb specific for this epitope were obtained after immunization with the synthetic octapeptide (OP) Arg-Ala-Ser-His-Leu-Gly-Leu-Ala [C3a(69-76)] coupled to the carrier keyhole limpet hemocyanin (KLH). These anti-C3a(69-76) antibodies (H453 and H454) reacted in an ELISA system with C3a and KLH-OP but not with C3 or with KLH alone. Free OP efficiently blocked binding of the antibodies to C3a, whereas binding of another anti-C3a mAb (H13) remained unaffected. In immunoblotting analysis, the anti-C3a(69-76) mAb reacted with purified C3a but failed to react with the denatured, noncleaved C3. A novel quantitative C3a-ELISA was established with the anti-C3a(69-76) mAb. It had a sensitivity in the nanogram range (1 to 5 ng/ml). The C3a determination was not impaired by the presence of high concentrations of C3. Therefore, C3 removal was not required in contrast to the previously described C3a assays. This C3a ELISA might facilitate clinical C3a quantitation, e.g., in samples from patients with adult respiratory distress syndrome. In these patients, C3a determination in the early phase of the disease is of diagnostic relevance and has prognostic value.     

Antiidiotypic antibody related to the 84 kD human sperm membrane protein

Wistar rats were inoculated with purified YWK-I antibody.The anti-idiotypic antibodies were isolated from rat sera by successive passage over affinity chromatography columns of YWK-I mAb and normal mouse Igs.Specificity of anti-Id antibody was established by ELISA.The 84kD protein inhibited the binding of anti-Id to YWK-I mAb,but failed to repress antibody against normal mouse Ig binding to YWK-I mAb.In competitive inhibition assay,84kD protein had shown the ability to compete with anti-Id binding to YWK-I mAb in a dose-dependent manner.Crude sperm extract showed a lower competitive ability.No effect was found with the irrelevant 36kD sperm protein.The antisera from the Balb/C micr immunized with AId contained Ab3 that reacted with 84kD sperm protein.The binding of anti-Id to YWK-I mAb was inhibited by Ab3 in a dose-dependent fashion and Ab3 was shown to be able to induce human sperm agglutination.These results indicate that anti-Id which may mimic an epitope of the 84kD protein could be exploited as an antigen to raise antibodies against sperm protein.     

Production of Monoclonal Antibodies to Barley Mild Mosaic Virus and Their Use for Strain Differentiation

A panel of five stable hybridoma cell lines secreting mono- clonal antibodies (mAbs) were produced using a French mechanically transmitted isolate of barley mild mosaic virus (BaMMV-MF) as antigen. All mAbs reacted with BaMMV-MF in two enzyme-linked immunosorbent assay (ELISA) formats: triple antibody sandwich (TAS)-ELISA and antigen-coated plate (ACP)-ELISA. These mAbs recognized epitopes, present on both degraded virions and intact particles. Four mAbs (5C8, 1D5, 1B12, 1A12) belong to the immunoglobulin (Ig)G class and one mAb (3A9) represents an IgM. The five mAbs were compared in TAS- and ACP-ELISA for reactivity with numerous French isolates. These isolates were detected in TAS- and ACP-ELISA with four mAbs (5C8, 1D5, 1B12, 3A9). In both ELISA systems the mAb 1A12 recognized only an epitope specific for BaMMV-MF. All mAbs, except 1A12 recognized also the German (BaMMV-MG), Italian (BaMMV-I) and Japanese (BaMMV-Ka1) isolates in both TAS- and ACP-ELISA. The Japanese isolate (BaMMV-Na1) only reacted with two mAbs (1D5, 5C8) in TAS-ELISA. Only one mAb (3A9) reacted with BaMMV-MF, BaMMV-PF, BaMMV-I,BaMMV-MG and BaMMV-Ka1 in Western blot. These mAbs make it possible to distingish between the three BaMMV serotypes.