摘要:

We have previously demonstrated molecular mimicry between the S peplomer protein of mouse hepatitis virus (MHV) and Fc gamma R (FcγR). A monoclonal antibody (MAb) to mouse FcγR (2.4G2 anti-FcγR MAb), purified rabbit immunoglobulin, but not their F(ab′)2fragments, as well as mouse and rat IgG, immunoprecipitated (1) recombinant S peplomer protein expressed by a vaccinia virus recombinant in human, rabbit, and mouse cells, and (2) natural S peplomer protein from cells infected with several strains of MHV and MHV escape mutants. We report here results of studies documenting molecular mimicry between FcγR and S peplomer protein of viruses representing three distinct antigenic subgroups of the Coronaviridae. We have shown a molecular mimicry between the S peplomer protein of bovine corona virus (BCV) and FcγR. The 2.4G2 anti-FcγR MAb, rabbit IgG, but not its F(ab′)2fragments, as well as homologous bovine serum, free of anti-BCV antibodies, immunoprecipitated S peplomer protein of BCV (Mebus strain). In contrast, we did not find molecular mimicry between S peplomer protein of human corona virus (HCV-OC43) and FcγR. Although the OC43 virus belongs to the same antigenic group as MHV and BCV, MAb specific for human FcγR I or FcγR II and purified human IgG1, IgG2, and IgG3, myeloma proteins did not immunoprecipitate the S peplomer protein from HCV-OC43-infected RD cells. In addition, we did demonstrate molecular mimicry between the S peplomer protein of porcine transmissible gastroenteritis virus (TGEV) and FcγR. TGEV belongs to the second antigenic subgroup of coronaviridae. Homologous swine IgG, but not its F(ab′)2fragments, immunoprecipitated from TGEV-infected cells a 195-kDa polypeptide corresponding to the TGEV S peplomer protein. We have also examined whether there is a molecular mimicry between S peplomer protein of infectious bronchitis virus (IBV) and FcγR. Nonimmune chicken IgG did not immunoprecipitate the S peplomer protein from IBV-infected chicken embryo fibroblasts or Vero cells, suggesting that there is no molecular mimicry between the IBV-S and FcγR. In conclusion, we have demonstrated molecular mimicry between FcγR and S peplomer protein of three members of Coronaviridae, namely MHV, BCV, and TGEV. In contrast, the S peplomer protein of two other members of Coronaviridae, namely HCV-OC43 and IBV, did not exhibit any molecular

ISSN:0272-457X    

DOI:10.1089/hyb.1995.14.1

年代:1995

数据来源: MAL

摘要:

We describe here a two-phase approach for the development of high -affinity human anti-HIV immunoglobulin Fab domains in a bacterial expression system. The first phase of this technique involves the generation of human hybridoma cell lines producing high-affinity antibodies (MAbs). Anti-HIV-1 human MAbs from peripheral blood lymphocytes (PBLs) were prepared from an HIV-1-seropositive patient and from an HIV-1-seronegative volunteer immunized with HIV-1 rgpl60. One MAb (T15G1), derived from the blood of the seropositive donor, was specific for HIV-1 gp41, recognized gp41 on the surface of HIV-1-infected cells and bound this antigen with an apparent dissociation constant of 4x10-10M. A second MAb (M7B5), developed from the immunized volunteer, was specific for HIV-1 gpl20 with a dissociation constant on the order of 8 x 10-10M, but was unable to recognize cell surface antigen. In the second phase of this technique the Fab domains of these two MAbs were molecularly cloned into a bacterial expression vector. mRNA was isolated from the M7B5 and T15G1 hybridoma cell lines and used as a template for the production of cDNA. The cDNA was amplified using the polymerase chain reaction (PCR) technique, and then fused, in frame, into a bacterial expression vector. The recombinant Fabs (rFabM7B5 and rFabT15Gl) were expressed as dicistronic messages in bacteria using the IPTG-inducible lactose promoter (LacZ). DNA sequencing was used to define the γ chain isotypes and the VHand VLchain gene usage. The binding specificities of rFabM7B5 and rFabT15Gl were indistinguishable from their respective intact MAbs. The dissociation constants for the rFabs were approximately 1.4 x 10-9M for rFabM7B5 and 2 x 10-9M for rFabT15Gl. Our studies show that human Fab domains, which maintain their binding specificities and have high binding affinities for HIV, can be rescued from hybridoma cell lines and expressed in bacteria

ISSN:0272-457X    

DOI:10.1089/hyb.1995.14.9

年代:1995

数据来源: MAL

摘要:

The epitopes of 42 well-characterized monoclonal antibodies (MAbs) against carcinoembryonic antigen (CEA) from 10 different research groups were mapped in terms of domain structure (domains N, A1-151. A2-B2, and A3-B3) of the CEA molecule on the basis of the reactivities with recombinant CEA proteins expressed in Chinese hamster ovary cells. Thirty-six of the 42 MAbs tested have previously been classified into 5 essentially nonoverlapping epitope groups (GOLD 1-5) by cross-competition assays among MAbs for CEA binding (Hammarström S,et al.: Cancer Res. 1989; 49:4852-4858). The epitopes recognized by GOLD 2 MAbs were all present on domain A2-B2, those for GOLD 5 MAbs were all on domain N, and those for GOLD 4 were mapped around domains Al-Bl and A2-B2. On the other hand, the epitopes for GOLD 1 MAbs were distributed into domains N, A2-B2, and A3-B3, and those for GOLD 3 MAbs were separated into domains N and A3-B3. Although the exact reasons for the dispersed patterns of GOLD 1 and 3 MAbs on the domain structure of the CEA molecule are unclear at present, several factors, such as a spatial relation or a close proximity of epitopes, conformation dependency, and repetivity of epitopes, may be considered as possible explanations. The epitope mapping reported here helps form the basis for understanding the relation between the chemical structure and antigenic activities of the CEA molecule and may be useful to study the functions of the CEA molecule, especially those of the respective domains

ISSN:0272-457X    

DOI:10.1089/hyb.1995.14.19

年代:1995

数据来源: MAL

摘要:

A monoclonal antibody (MAb), KuN241, recognized an antigen present on all monocytes, polymorphonuclear leukocytes (PMNs), B cells, and a subpopulation of T cells. Cell surface expression pattern and immunoprecipitation studies indicated the molecule recognized by KuN241 to be complement receptor 1 (CR1). This was confirmed by sequential immunoprecipitation using Ell, a CR1-specific monoclonal, and by immunoprecipitation analysis of a truncated transfection product of CR1.In vitroactivation of PBLs for 7 days with pokeweed mitogen (PWM) abrogated surface expression of CR1 on B cells. Overnight culture with phorbol 12-myristate 13-acetate (PMA) downregulated the expression of CR1 detected by KuN241 both on PMNs and monocytes. Addition of MAb KuN241 to purified PMN and monocytes resulted in a transient increase in intracellular calcium ([Ca2+]i). This was blocked by the Fab fragment of MAb KuFc79 directed against the Fcγ receptor. Further, Fab fragment of KuN241 cross-linked with F(ab')2goat anti-mouse Ig failed to increase [Ca2+]ilevels. Taken together, these results suggested a novel mechanism for transmembrane signaling events by dual binding of KuN241, the Fab region to CR1 and the Fc region to Fc-γRI

ISSN:0272-457X    

DOI:10.1089/hyb.1995.14.29

年代:1995

数据来源: MAL

摘要:

A new monoclonal antibody to the Thomsen—Friedenreich (TF) antigen (or, more precisely, epitope; Galβl-3GalNAc-) has been developed that is specific for both anomeric forms of this disaccharide (TFα and TFβ, including related structures on glycolipids), and not assay restricted. We demonstrate that this avid antibody (A78-G/A7) is well suited for immunohistochemistry on paraffin-embedded and cryosectioned tissues, immunoblotting, ELISA techniques, and hemagglutination. Immunohistochemistry on paraffin sections does not require proteolytic or microwave pretreatment. The binding characteristics of this antibody are largely independent of variations in pH (6.0-8.2) and temperature (4-37°C). Immunoblotting with KG-1 (human acute myelogenous leukemia) cells revealed a series of TF-active glycoproteins with a main band at about 155 kDa. Immunoprecipitation was performed using a new technique applicable to IgM-type antib

ISSN:0272-457X    

DOI:10.1089/hyb.1995.14.37

年代:1995

数据来源: MAL

摘要:

A human B lymphoblastoid cell line JWCI-L94 secretes an IgM human monoclonal antibody (HuMAb) that reacts with human melanoma cell lines, M14 and M12. To identify the antigenic epitope of this antibody, we screened λgt11 expression libraries constructed from M14 and M12. A total of 12 immunoreactive clones were isolated, and their DNA sequences were determined. The only sequence shared by all these clones was alanine—proline (A—P) at the carboxyl (C) terminal. HuMAb L94 reacted not only with C-terminal A—P-containing fusion proteins, but also with the synthetic dipeptide A—P. None of the peptides containing A-P internally or amino terminally reacted to HuMAb L94. Proline or alanine alone had no ability to bind to HuMAb L94. When alanine was replaced by glycine (G—P) or proline (P—P), the binding activity of these peptides was similar to that of A—P. On the other hand, when alanine was replaced by serine, valine, leucine, glutamine, lysine, methionine, phenylalanine, or hydroxyl proline, the resulting peptide completely lost the antigenic activity of HuMAb L94. These results demonstrate that HuMAb L94 recognizes C-terminal A—P, G—P, or P—P, and that a human antibody can recognize peptides as small as a tw

ISSN:0272-457X    

DOI:10.1089/hyb.1995.14.45

年代:1995

数据来源: MAL

摘要:

A monoclonal antibody, EIV-E12, was produced against splenic ellipsoid-associated cells. In postnatal chicks, the antigen defined by EIV-E12 associated with about 90% of bursal cells and accumulated in B cell-rich areas of lymphoid organs. The antigen was expressed by many splenic cells on day 9 of embryogenesis and about 1 day later in the bursal anlage, where the cells positive for the antigen localized in close proximity to the plical epithelium. In the bursa, these early EIV-E12 cells appeared before Bu-1-positive cells and may represent a dendritic cell precursor. B cells within the developing bud acquired the EIV-E12 antigen. Immunoprecipitation studies revealed a molecular weight of 233 (nonreduced) and 205 (reduced) for the EIV-E12 antigen. These values were markedly different from the molecular weight of the antigen identified by Bu-1 but similar to that of a CB10 antigen. The CB10 and EIV-E12 MAbs exhibited some differences in cellular staining. Taken together these data suggest that EIV-E12 MAb recognizes a unique cellular population during embryogenesis.

ISSN:0272-457X    

DOI:10.1089/hyb.1995.14.51

年代:1995

数据来源: MAL

摘要:

An Indian isolate ofP. falciparumwas propagatedin vitroand monoclonal antibodies were raised against this parasite. Of 11 positive hybrids obtained, 3 were cloned and characterized. When tested in IFA with synchronized cultures dominated by mature schizonts, all three monoclonal antibodies showed distinct fluorescent patterns. These three monoclonals reacted with seven of the eightP. falciparumisolates tested. One of these monoclonal antibodies, which showed a fluorescence pattern similar to the merozoite dot fluorescence reported by previous investigators, also inhibited parasite growthin vitro. The antigens that reacted with the monoclonal antibodies have been isolated by using MAb-affinity columns.

ISSN:0272-457X    

DOI:10.1089/hyb.1995.14.59

年代:1995

数据来源: MAL

摘要:

Four murine monoclonal antibodies (MAbs) of the IgM class were raised against human acetylcholinesterase (AChE; Ec 3.1.1.7). The MAbs BMS-3E4, BMS-7G10, and BMS-9F4 all recognized human erythrocyte AChE, while BMS-6D6 bound specifically to human soluble brain AChE, on the basis of immunobinding assays. Dose-response studies gave an ELISA ED50titer of 4.5 x 10-4M for BMS-6D6, while BMS-3E4 gave the best titer at 8.8 x 10-4M. Sucrose density gradients demonstrated sedimentation of antigen-antibody complexes, consistent with earlier findings (i.e., BMS-6D6 bound with brain AChE while BMS-3E4 preferred erythrocyte (AChE). No cross-reactivity between the two MAbs against the two antigens was noted.

ISSN:0272-457X    

DOI:10.1089/hyb.1995.14.67

年代:1995

数据来源: MAL

摘要:

The early identification of antibody-secreting hybridomas significantly reduces the time and resources devoted to unproductive colonies. To quantify the immunoglobulin concentration in hybridoma supernatants within 2 weeks of fusion, we used immunomagnetic microspheres to capture immunoglobulin in the hybridoma culture supernatant. The captured immunoglobulin was detected using a goat anti-mouse second antibody liked to β-galactosidase. With data transformation to correct for the nonlinear accumulation of the fluorescent reaction product, the enzymatic hydrolysis of fluorescein digalactoside permitted the reliable detection of less than 10 pg of immunoglobulin per milliliter. To determine the value of quantifying immunoglobulin concentration within 2 weeks of fusion, the amplified fluorescence microassay was applied to the evaluation of 3 consecutive fusions and more than 1200 growing hybridoma colonies. Using antibody concentrations greater than 10 ng/ml as a threshold for routine subculture, the selection of hybridoma colonies based on antibody secretion was threefold more efficient than selection based on colony growth alone. These results suggest the utility of the early determination of immunoglobulin concentration in the selection of hybridoma colonies

ISSN:0272-457X    

DOI:10.1089/hyb.1995.14.75

年代:1995

数据来源: MAL