Production and characterization of a murine monoclonal IgM antibody to human C1q receptor (C1qR)

A hybridoma cell line that produces a monoclonal antibody (MAb) to cell surface C1q receptor (C1qR) has been produced by fusion of the P3 X 63-Ag8.653 mouse myeloma cell line with the spleen cells of a CD-1 mouse that had been hyperimmunized with viable Raji cell suspensions (5 X 10(7) cells/inoculum). This MAb, designated II1/D1, is an IgM antibody with lambda-light chain specificity. Radiolabeled or unlabeled, highly purified II1/D1 was used to determine that: this antibody competes for C1q binding sites on C1qR-bearing cells; the molecule recognized by this MAb is the C1qR; and cells that are known to bind C1q also bind II1/D1 in a specific manner. Western blot analysis of solubilized Raji, or U937 cell membranes, showed that the 125I-MAb detected a major protein band of approximately 85,000 m.w. in its unreduced state, indicating that the C1qR is similar, if not identical, in both types of cells. Analyses of 125I-II1/D1 binding experiments revealed that the antibody bound to Raji cells or U937 cells in a specific manner. Uptake of the antibody was saturable, with equilibrium virtually attained within 35 min. Scatchard analysis of the binding data using the intact MAb suggests that the affinity constant KD is 2.9 X 10(-10) M, and at apparent saturation, 24.6 ng of the antibody were bound per 2 X 10(6) cells, giving an estimated 7.8 X 10(3) antibody molecules bound per cell. That the II1/D1 antibody is specifically directed to the C1q was further evidenced by an ELISA in which the ability of C1qR-bearing cells to bind the MAb was abrogated by c-C1q in a specific and dose-dependent manner. These results indicate that the II1/D1 is a specific antibody directed against the C1q and can be a useful tool in studying the biologic interaction of human C1q with its receptors on a variety of cells.     

Preparation of a monoclonal antibody to common amino acid sequence of LHRH and its application

In order to prepare an antibody directed at the common amino acid sequence of mammalian, avian, and fish luteinizing hormone-releasing hormones (LHRHs), C-terminal free LHRH was conjugated with bovine thyroglobulin, and was used as the antigen. A monoclonal antibody (LRH13) was obtained as an ascitic fluid by fusing the spleen cells of a BALB/c donor mouse immunized with the antigen to X63.Ag8.653 mouse myeloma cells followed by limiting dilution cloning and transplanting a positive clone to BALB/c mice. This monoclonal antibody seems to belong to IgG2b as it was eluted from protein A-Sepharose CL-4B with citrate buffer pH 3.5. competitive binding experiment using fragment peptides of LHRH indicated the binding site of LRH13 was a region around serine and tyrosine, and modification of mammalian LHRH by radioiodination caused a marked decrease in the binding activity. LRH13 has an affinity constant of 0.134 X 10(9) M-1 to native mammalian LHRH, and binds C-terminal free LHRH with a similar affinity (1.6X), however, it binds with higher affinities to N- and C-terminal free LHRH (12.9X), N-terminal free LHRH (10.4X), salmon LHRH (8.3X) and chicken LHRH-I (6.0X). Chicken LHRH-II, where tyrosine is replaced for histidine, has a lower affinity (0.3X) than that of mammalian LHRH. From its high affinity to N-, C-terminal free LHRH, LRH13 is also expected to bind possible precursor peptides of LHRH. Immunohistochemical staining of the brain sections obtained from rats, mice, chickens, Japanese quail, and rainbow trout successfully visualized cell bodies and fibers distributed from the olfactory bulb to the median eminence, indicating high LHRH specificity and wide crossreactivity in animal classes of this monoclonal antibody. With this antibody, LHRH-like immunoreactive substance in the pineal gland was also stained with fixation at neutral pH.     

A monoclonal antibody to factor IX that inhibits the factor VIII:Ca potentiation of factor X activation

A murine monoclonal antibody (IgG1k, Kd approximately 10(-8) M) specific for an epitope located on the heavy chain of human factor IXa was used to study structure-function relationships of factor IX. The antibody inhibited factor IX clotting activity but did not impair activation of factor IX either by factor XIa/calcium or by factor VIIa/tissue factor/calcium. The antibody also did not impair the binding of factor IXa to antithrombin III. Moreover, the antibody did not prevent calcium and phospholipid (PL) from inhibiting the binding of factor IXa to antithrombin III. The antibody also failed to impair activation of factor VII by factor IXa/calcium/PL. Furthermore, the antibody did not interfere with the very slow activation of factor X by factor IXa/calcium/PL. In contrast, the antibody did interfere with factor X activation when reaction mixtures also contained factor VIII:Ca/von Willebrand factor. The marked acceleration of factor X activation observed in control mixtures was not observed in mixtures containing the antibody. Similar results were obtained in reaction mixtures containing the Fab portion of the antibody and factor VIII:Ca free of von Willebrand factor. In additional experiments, factor VIII:Ca/von Willebrand factor was found to inhibit the binding of the antibody to 125I-factor IXa as determined using an immunosorbent assay. Moreover, the antibody displaced factor VIII:Ca from the factor X activator complex (IXa/calcium/PL/VIII:Ca) as evidenced by an altered elution pattern on gel filtration chromatography. From these observations, we conclude that the antibody impairs the clotting activity of factor IXa through interference with its binding of factor VIII:Ca. This suggests a significant role for the heavy chain (residues of 181-415) of factor IXa in binding factor VIII:Ca.     

Developmental acquisition of type X collagen in the embryonic chick tibiotarsus

The temporal and spatial distribution of short chain skeletal (Type X) collagen was immunohistochemically examined in the chick tibiotarsus from 6 days of embryonic development to 1 day posthatching. The monoclonal antibody employed (AC9) was recently produced and characterized as being specific for an epitope located within the helical domain of the type X collagen molecule (T. M. Schmid and T. F. Linsenmayer, J. Cell Biol., in press). The earliest detectable appearance of type X collagen was at 7.5 days, at which time it was restricted to a middiaphyseal location (i.e., in the primary center of ossification). This was in marked contrast to type II collagen, which appears earlier and is distributed throughout the cartilaginous anlagen. With increasing embryonic age, the reactivity with the type X antibody progressively extended toward the epiphyses, lagging somewhat behind the progression of chondrocyte hypertrophy. The anti-type X collagen antibody also reacted with the bony matrix itself, but the immunofluorescent signal produced by this source was considerably less than that produced by cartilage. At 19 days of development, a new small site of type X deposition was initiated in an epiphyseal location, which subsequently enlarged in circumference. These results are consistent with our previous biochemical studies suggesting that, in cartilage, type X collagen is specifically a product of that population of chondrocytes which have undergone hypertrophy.     

Monoclonal antibody to rat apoC: multiple binding to apoC-I on lipoproteins increases its affinity constant

Using solid phase systems, the kinetics of binding of monoclonal antibody (LRB 45, IgG2b,kappa) to apoC-I and apoC-I on lipoproteins were investigated. At 25 degrees C, the association constant of LRB 45 antibody to apoC-I (3.56 X 10(6) M-1 X sec-1) was almost three times slower than the association constant LRB 45 antibody to lipoproteins (10.4 X 10(6) M-1 X sec-1). However, the dissociation constant of apoC-I from LRB 45 antibody (0.865 X 10(-4) sec-1) was also slower than the dissociation constant of lipoprotein from antibody (1.5 X 10(-4) sec-1). Thus, the calculated affinity constant (association constant/dissociation constant) of LRB 45 antibody for apoC-I was approximately half of that for lipoproteins (4.12 X 10(10) M-1 vs. 6.92 X 10(10) M-1). The intrinsic affinity constants for antibody binding to apoC-I and apoC-I on lipoproteins were determined by Scatchard analysis. The intrinsic affinity constant of antibody bound to apoC-I was estimated to be 5.49 X 10(10) M-1 whereas that of antibody binding to lipoproteins was 30 to 200 times less. Furthermore, ascites fluid from LRB 45 cell lines could immunoprecipitate serum lipoproteins. Thus, it is concluded that there is multiple binding of antibody to apoC-I on lipoproteins. This binding appears to increase the calculated affinity constant (avidity) for antibody-antigen interaction.     

Evidence for male X chromosomal mosaicism in X-linked agammaglobulinemia

Summary X-Linked agammaglobulinemia (XLA) is a severe antibody deficiency disease in man, resulting from an arrest in differentiation of pre-B cells. XLA is recessive: female carriers do not exhibit antibody deficiency, but manifest an exclusive inactivation of the XLA-carrying X chromosome in all peripheral blood B lymphocytes. An exclusive inactivation of the paternal X chromosome in the B lymphocytes of all daugthers thers of a male who had no agammalobulineamia demonstrated that the XLA defect can originate from healthy males. These males are X chromosomal mosaics. X-Chromosomal RFLP segregation analyses in other XLA pedigrees suggest a frequent introduction of XLA by healthy males. This implies that XLA often originates from mitotic errors, either at postmeiotic or early postzygotic stages.     

Antibody Response in Mice Inoculated with DNA Expressing Foot-and-Mouth Disease Virus Capsid Proteins

Candidate foot-and-mouth disease (FMD) DNA vaccines designed to produce viral capsids lacking infectious viral nucleic acid were evaluated. Plasmid DNAs containing a portion of the FMDV genome coding for the capsid precursor protein (P1-2A) and wild-type or mutant viral proteinase 3C (plasmids P12X3C or P12X3C-mut, respectively) were constructed. Cell-free translation reactions programmed with pP12X3C (wild-type 3C) and pP12X3C-mut produced a capsid precursor, but only the reactions programmed with the plasmid encoding the functional proteinase resulted in P1-2A processing and capsid formation. Baby hamster kidney (BHK) cells also produced viral capsid proteins when transfected with these plasmids. Plasmid P12X3C was administered to mice by intramuscular, intradermal, and epithelial (gene gun) inoculations. Anti-FMD virus (FMDV) antibodies were detected by radioimmunoprecipitation (RIP) and plaque reduction neutralization assays only in sera of mice inoculated by using a gene gun. When pP12X3C and pP12X3C-mut were inoculated into mice by using a gene gun, both plasmids elicited an antibody response detectable by RIP but only pP12X3C elicited a neutralizing antibody response. These results suggest that capsid formation in situ is required for effective immunization. Expression and stimulation of an immune response was enhanced by addition of an intron sequence upstream of the coding region, while addition of the FMDV internal ribosome entry site or leader proteinase (L) coding region either had no effect or reduced the immune response.     

Production of a monoclonal antibody against C terminus of atrial natriuretic factor by in vitro immunization

Spleen cells, sensitized in vitro with ANF-KLH conjugate, were fused with a non-producing mouse myeloma cell line, X63-Ag8.653. One of the isolated hybridomas secreted a monoclonal antibody which exhibited an association constant of 7.25 X 10(-9) M for ANF and recognized an epitope at the C-terminal of the synthetic rat ANF(101-126). Cross reactivity experiments with Auriculin B, Atriopeptin I and Atriopeptin II suggests Phe124-Arg125-Tyr126 in the epitope recognized by this antibody. The epitope appears to be similar to the portion of the hormone molecule essential for ANF binding to the B-receptor (high m.wt. receptor). This antibody may be useful for the generation of anti-idiotypic antibodies for the B-receptors.     

Structural analysis of the epitope recognized by a monoclonal antibody directed against tricyclic antidepressants

After somatic cell fusion between splenocytes of immunized BALB/c mice and NS-1 myeloma cells, a clone was obtained that secreted an anti-nortriptyline antibody of the IgG1 kappa isotype. The association constant of this antibody for pharmacologically active tricyclic antidepressant drugs ranged from 0.6 X 10(7) to 3 X 10(7) M-1. From thermodynamic and binding studies as well as tridimensional structures of tested compounds, the epitope recognized by the monoclonal antibody appeared to include both a hydrophobic tricycle in which the two phenyl rings form an angle of 120 to 130 degrees, and a side chain in which the amino group is separated from the two lateral rings of the tricyclic structure by a distance of approximately 5.9 A and 7.5 A, respectively. This conformation seems to be the one interacting with muscarinic acetylcholine brain receptors.     

Purification of the protein X of Streptococcus agalactiae with a monoclonal antibody

The protein X of Steptococcus agalactiae is a surface antigen included in the typing scheme of group B streptococci (GBS). We have developed a monoclonal antibody to the protein X and used it to purify this antigen by affinity chromatography. Electrophoresis in polyacrylamide, and immunoblotting using the monoclonal antibody or a rabbit antiserum raised with the affinity purified protein X, revealed a major band in the region of 200 kDa and a smaller one at 100 kDa. The isolated protein X will make possible investigations of its potential role in virulence and protection.     

Enzyme-linked coagulation assay. III. Sensitive immunoassays for clotting factors II, VII, and X

The solid-phase clotting assay utilizing fibrinogen coated on the wells of a microtiter plate and peroxidase-fibrinogen in solution as a substrate for thrombin (enzyme-linked coagulation assay, ELCA) has been modified for use as an immunoassay. Direct inhibition of factors II, VII, and X by polyclonal (rabbit) antibodies and of factor X by monoclonal antibodies has been demonstrated at high dilution of these antibodies and detection of the specific factors using ELCA. Using plates coated with a second antibody (goat anti-mouse IgG) as well as fibrinogen, monoclonal antibodies to factors X and VII were measured by binding the active factor to the plate and detection of the bound factor using ELCA. The assay was very sensitive, permitting the detection of as little as 0.2 ng/ml (30 pg/assay) of monoclonal antibody, or less than 0.4 ng/ml (60 pg/assay) of factor Xa. When plates were coated with monoclonal antibody to factor X and fibrinogen, the assay permitted the identification of distinct epitope specificities for two monoclonal antibodies to factor X by distinct competition of the monoclonal antibodies added in the solution phase for binding of factor Xa to the plate. This assay could be applied generally for immunoassay of clotting factors, and could have application in general as an immunoassay amplification system.     

Biochemical analysis of an antigen produced by both human sex chromosomes.

It has been argued on both evolutionary and functional grounds that genes must be shared by the mammalian sex chromosomes. The only direct evidence for such genes is our previous finding that loci on the human X (MIC2X) and Y (MIC2Y) chromosomes encode a species-specific cell surface antigen recognised by the monoclonal antibody 12E7. These loci map to the regions of the sex chromosomes which pair at meiosis, and MIC2X has been shown to escape X-inactivation. We have used immunoprecipitation and Western blot analysis combined with one- and two-dimensional polyacrylamide gel electrophoresis to compare the products of MIC2X and MIC2Y. The human specific molecule recognised by the 12E7 antibody is a membrane-associated polypeptide of mol. wt. 32.5 kd and pI = 5.0. No difference in size or charge has been detected between X and Y encoded forms of this molecule confirming that MIC2Y is a functional homologue of MIC2X. An intracellular polypeptide of mol. wt. 29 kd and pI = 7.0 present in the cytoplasm of both human and mouse cells is also recognised by the 12E7 antibody.     

Monoclonal antibodies to type X collagen. Biosynthetic studies using an antibody to the amino-terminal domain

Monoclonal antibodies to chick type X collagen have been used to study the structure, biosynthesis, and location of type X in cartilage. The antibodies were produced by injecting purified type X collagen into female SJL/J mice and then fusing their spleen cells with Sp2/0 myeloma cells. Hybridoma culture supernatants were screened for antibodies to type X collagen by enzyme-linked immunosorbent assay and Western blots. Positive supernatants did not cross-react with other collagen types (I, II, IX, XI) or with fibronectin. Three monoclonal antibodies were chosen for further characterization. Two of them (1A6 and 6F6) recognize a pepsin-sensitive domain of type X collagen. Rotary shadowing showed that 1A6 and 6F6 both recognize the same end of type X, probably the aminoterminal non-triple helical domain. Amino acid sequencing of the intact protein and of the epitope-containing peptide confirmed that the antibody recognition sites for 1A6 and 6F6 are within the amino-terminal domain. Monoclonal antibody 2B3 reacts with the pepsinized (45 kDa) and weakly with the nonpepsinized (59 kDa) forms of type X collagen. The monoclonal antibodies were used for immunolocalization of type X in hypertrophic chondrocytes and reacted only with tissue samples from areas undergoing endochondral ossification, e.g. growth plate and fracture callus. Antibody 6F6, when coupled to Sepharose, selectively binds to type X collagen from cell and organ cultures. In a pulse-chase experiment, no processing of the 59-kDa form of type X could be detected. Two components with molecular masses of approximately 70 and 85 kDa, arising from a disulfide-bonded aggregate, were synthesized by both the permanent and calcifying cartilage organ cultures but did not react with the antibody, suggesting that these proteins are not related to type X. In summary, the pulse-chase results and the immune precipitation with monoclonal antibody 6F6 did not detect biosynthetic precursors larger than 59 kDa or proteolytically processed forms of type X.     

Monoclonal antibodies against human abnormal (des-gamma-carboxy)prothrombin specific for the calcium-free conformer of prothrombin

A monoclonal antibody JO1 X 1 was prepared against human abnormal prothrombin using the hybridoma technique. The clone secreting this antibody was selected on the basis of the ability of this antibody to bind to abnormal prothrombin, but not to prothrombin, in the presence of calcium ions. The antibodies were purified by affinity chromatography in EDTA on columns of prothrombin-Sepharose. Bound antibodies were eluted with 15 mM CaCl2. The kinetics of dissociation of antibody from the antibody-prothrombin complex with the addition of calcium ions fit a first-order kinetic model. Increasing CaCl2 concentration increased the rate of antibody-prothrombin dissociation. Ca(II) and Mn(II) inhibited antibody-prothrombin interaction; half-maximal binding was observed at 0.9 and 4 mM, respectively. Mg(II) had little effect on antibody-antigen interaction. The JO1 X 1 antibody bound fragment 1, fragment (1-39), abnormal prothrombin, and prothrombin equivalently in the presence of EDTA, but did not bind to des(1-44)prothrombin in the presence of EDTA or prothrombin in the presence of CaCl2. These results indicate that the monoclonal antibody JO1 X 1 is conformation specific for the calcium-free conformer of prothrombin and directed against an antigenic determinant near the NH2 terminus of prothrombin expressed in the 1-39 region of the protein. This analysis provides confirmation of the presence of a metal-free conformer of prothrombin.     

Crystallization and preliminary X-ray diffraction studies of a monoclonal antibody Fab fragment specific for an influenza virus haemagglutinin and of an escape mutant of that haemagglutinin

Preliminary crystallographic data are given for two molecules involved in the interaction between the humoral immune response and the influenza virus. These molecules are the Fab fragment of an antibody specific for the haemagglutinin of influenza virus strain X31 (Hong Kong 1/68 (H3N2)) and a mutant of X31 haemagglutinin that escapes recognition by that antibody. Crystals of the haemagglutinin are isomorphous to those of X31, whose structure is known; they diffract to 3.4 A resolution. Crystals of the Fab fragment are trigonal with space group P3(1)21 (or P3(2)21) and diffract to 2.6 A resolution. The unit cell dimensions are a = b = 98.9 A, c = 89.2 A. A native data set has been collected for both proteins.     

The interaction of human blood coagulation factor VII and tissue factor: the effect of anti factor VII, anti tissue factor and diisopropylfluorophosphate.

The effect of Factor VII antibody and an antibody to the apoprotein of tissue factor has been tested on the product formed between Factor VII, tissue factor and calcium ions. The antibody to the apoprotein of tissue factor neutralized tissue factor but had no effect on the extrinsic Factor X activator activity when Factor VII had been allowed to react with tissue factor before the addition of the antibody. The Factor VII antibody neutralized Factor VII and it also blocked the Factor X activator activity when Factor VII had been incubated with tissue factor and calcium ions prior to the addition of Factor VII antibody.Diisopropylfluorophosphate (DFP) was found to neutralize native purified Factor VII and Factor VII in human plasma. This inhibition of Factor VII was very slow and required high concentrations of DFP. However, when the Factor VII had been preincubated with tissue factor and calcium ions, the neutralization of Factor VII by DFP occurred rapidly, and at much lower concentration of DFP.     

Properties of natural 19S antibodies in normal pig serum against the ΦX 174 and T2 phages

The physicochemical properties of natural phage-neutralizing antibodies were studied. Natural neutralizing antibodies against phages ΦX 174 and T 2 were found in the 19 S fraction isolated from normal pig serum by gel filtration on a Bio-Gel P-300 column. The 7 S fraction of normal pig serum possessed no neutralizing activity. The neutralizing activity of the 19 S fraction against phage ΦX 174 was not modified either by inactivation or by the addition of neutralizing cofactor; its activity against phage T 2 was lost by inactivation, but was restored by the addition of cofactor. The neutralizing activity of the 19 S fraction of normal pig serum was completely destroyed by 2-mercaptoethanol and was not restored by the subsequent addition of antibody cofactor. The results of attempts to release phage ΦX 174 from the neutralization complex with normal porcine serum 19 S macroglobulin antibody by the dilution method were the same as those of attempts to dissociate phage from the complex with 7 S type hyperimmune antibody. The virus particle was firmly and irreversibly adsorbed to both types of antibody and was not released by dilution. It is concluded from the results that neutralization of phage ΦX 174 by 19 S macroglobulin molecule of antibody is a simple, irreversible process, for which the thermolabile nonspecific serum components are not required.     

Use of monoclonal antibodies in the assay of hepatitis B core antigen and antibody

Hybridoma cells secreting antibody against hepatitis B core antigen (HBc Ag) were prepared. BALB/c mice were immunized with 0.2 ml of purified HBc Ag, and their spleen cells were fused with mouse myeloma (P3U1) cells by means of polyethylene glycol 1000. Activities of antibodies against HBc Ag (anti-HBc) were tested by the immune adherence hemagglutination (IAHA) and reverse passive hemagglutination inhibition (RPHI) techniques. Hybridoma cells found to contain antibodies accounted for 26.5% by IAHA and 52.1% by RPHI, respectively. Among 32 monoclonal anti-HBc antibodies, 18 were found to be positive by both IAHA and RPHI, and the remaining 14 positive by RPHI only. After cloning, they were injected intraperitoneally into ascitic mice. The highest anti-HBc activity with an IAHA titer of 1:4 X 10(6) and with an RPHI titer of 1:1 X 10(5) was detected in this ascitic fluid. Enzyme immunoassay (EIA) and RPHI with monoclonal antibody containing the highest anti-HBc activity were developed. All the sera in which anti-HBc was detected by IAHA and RPHI with polyclonal antibody were positive in EIA. RPHI titers obtained with monoclonal antibody were in good agreement with usual IAHA and RPHI titers obtained with polyclonal antibody. These results indicate that monoclonal antibody can be used in the HBc Ag and anti-HBc assay system.     

Rat monoclonal antibody specific for prostaglandin E structure

Six different monoclonal antibodies directed against prostaglandin E2 were obtained from hybrid myelomas, following fusion of mouse NS-1 myeloma cells with spleen cells from a rat immunized with bovine serum albumin conjugates of prostaglandin E2. Four of them were of the IgG2a subclass and the other two were an IgG2b and an IgG2c. Affinities of antibodies for prostaglandin E2 were in the range 5.8 X 10(6)-6.7 X 10(8) M-1. Cross-reactivity experiments showed that one monoclonal antibody was directed almost exclusively against the prostaglandin E structure. The specific monoclonal antibody purified from ascites fluid was used for enzyme immunoassay, and as little as 30 pg of prostaglandin E1 and 100 pg of prostaglandin E2 were detected, which values are comparable to those obtained by radioimmunoassay. These results reveal that the hybridization technique is a reliable way to obtain prostaglandin E-specific antibody and that monoclonal antibodies can be valuable reagents for immunoassays.