Recombinant human-mouse chimeric monoclonal antibody specific for human adenocarcinoma associated antigen

We have recently described a class-switched (IgM to IgG1) human-mouse chimeric antibody. In the present study, a human-mouse chimeric antibody specific for human adenocarcinoma-associated antigen YH206 antigen was constructed by fusing murine variable region genes (V kappa and VH) to human constant region genes (gamma 1, kappa). The murine variable domain genes were isolated from a functional murine hybridoma cell line, YH206, which secreted IgM monoclonal antibody specific for YH206 antigen. The fusion genes of heavy and light chains were introduced into the immunoglobulin non-producing mouse myeloma cell line X63-Ag8.653 by electroporation. We obtained transformants which secreted class-switched human-mouse chimeric antibodies specific for YH206 antigen. A dot immunobinding assay demonstrated that the class-switched chimeric antibody retained the ability to bind to the YH206 antigen.     

The strain-specific diagnosis of influenza by using lanthanide immunofluorescence analysis based on monoclonal antibodies to the hemagglutinin of the influenza A virus]

Nine monoclonal antibodies (MCA) to hemagglutinin of influenza A/Taiwan/1/86 (H1N1) virus and 5 MCA to influenza A/Mississippi/1/85 (H3N2) virus were generated and characterized. The MCA were used for the development of diagnostic test systems on the basis of time-resolved fluoroimmunoassay. The same MCA were used as primary and detecting antibodies in the test system specific for HA of the H1 serosubtype, whereas in the test system specific for influenza A serosubtype H3 virus MCA of different epitope appurtenance were used as primary and secondary antibodies. The sensitivity of the test system for HA of serosubtype H1 was found to be 10 ng/ml and that for serosubtype H3 5 nh/ml. The developed test systems were tried on the clinical material collected during the epidemic periods of 1983-1989.     

Characterization of H2 influenza virus hemagglutinin with monoclonal antibodies: influence of receptor specificity

Antigenic analysis of human and avian H2 influenza viruses were done with monoclonal antibodies to the HA molecules in hemagglutination inhibition (HI) assays. These studies revealed that the receptor-binding specificity of the hemagglutinin can markedly influence the antigenic analysis obtained with monoclonal antibodies in HI tests. Influenza viruses that are sensitive or resistant to inhibition by horse serum inhibitors showed marked differences in their reactivity with monoclonal antibodies to the hemagglutinin. This was apparent with the A/RI/5+/57 and A/RI/5-/57 strains of H2N2 viruses isolated by Choppin and Tamm (1960a), half of the panel of different monoclonal antibodies failed to inhibit hemagglutination of the RI/5- variant, whereas all of the 18 monoclonal antibodies inhibited RI/5+. These findings have important implications in the antigenic analysis of influenza viruses where HI assays are conventionally used to determine the extent of antigenic drift in nature. Antigenic differences were detectable between different human H2 influenza virus isolates from 1957 that were sensitive to inhibition by horse serum, indicating that minor antigenic variation occurs within the first year of appearance of the new subtype. Minor antigenic variation continued in the H2 viruses until 1961, but by 1962 antigenically distinguishable variants that could be discriminated with both monoclonal antibodies and postinfection ferret antisera predominated. Analysis of avian H2 influenza viruses with a panel of monoclonal antibodies indicated that antigenic variation occurs and that multiple different variants cocirculate in the population. There was no progressive antigenic change in the avian H2 influenza viruses with time, as was found with the human H2N2 strains. Topographical mapping of the H2 hemagglutinin by selection of antigenic variants with monoclonal antibodies and analysis of their reactivity patterns by HI showed overlap between the epitopes examined. These results may reflect restriction in the antibody repertoire of the mice used in preparation of the monoclonal antibodies or that the H2 hemagglutinin does not have such discrete nonoverlapping antigenic regions found in the early H3 influenza virus.     

Anti-idiotypic antibodies of a predefined specificity generated against CDR3VH synthetic peptides define a private anti-CD4 idiotype

A synthetic peptide corresponding to the third complementarity determining region (CDR) of the heavy chain (CDR3VH) of anti-Leu3a, a monoclonal anti-CD4 antibody which inhibits HIV gp120 binding to CD4, was used to elicit specific anti-peptide antibodies in rabbits. The anti-peptide antisera showed anti-idiotypic antibody (anti-Id) activity and recognized both the immunizing peptide and the intact cognate protein by ELISA. In addition, the antisera reacted with isolated heavy chains of anti-Leu3a by Western blot analysis. The lack of reactivity with a panel of monoclonal anti-CD4 antibodies suggested that the anti-peptide antisera recognize a private idiotype (Id) associated with the anti-Leu3a CDR3VH region. Further studies demonstrated the inability of the rabbit antisera to inhibit the binding of anti-Leu3a to the CD4 molecule. In addition, soluble recombinant CD4 was unable to inhibit the binding of the rabbit anti-peptide antisera to anti-Leu3a indicating that the CDR3VH region may not be involved in CD4 recognition. Anti-Id containing sera from mice, rabbits and nonhuman primates immunized with the intact anti-Leu3a molecule did not bind the CDR3VH synthetic peptide, suggesting that the corresponding region of anti-Leu3a may not represent an immunodominant idiotypic determinant in thes e species. These results suggest the potential use of synthetic peptides corresponding to immunoglobulin variable (V) region amino acid sequences in generating anti-Id reagents of a predefined specificity. In addition, V-region synthetic peptides may be useful in mapping the idiotopes recognized by an anti-Id response to the cognate molecule.     

Structural basis of stimulatory anti-idiotypic antibodies

In order to design and produce effective vaccines based upon the idiotype network hypothesis of Jerne, a thorough understanding of the biological and structural aspects underlying the stimulating activities of anti-idiotypic antibodies is needed. Here we determined the nucleotide sequence of the variable heavy and light chain regions of two monoclonal anti-idiotypic antibodies which induce different anti-phosphorylcholine responses. The nucleotide sequences of the variable domains of two monoclonal anti-TEPC 15 (T15) antibodies (F6-3 and 4C11) were determined by the primer extension and Maxam-Gilbert techniques. The nucleotide sequence data show that 4C11 and F6-3 have homologous VH segments and JH segments, but different D regions. The VH segments of both clones belongs to the J558 VH family. Most of the differences among the VH segments are located in CDR2. The VK segments of 4C11 and F6-3 are homologous to the VK gene group 4 and group 8, respectively. Comparison of the sequences of 4C11 and F6-3 with other published anti-idiotype antibodies shows that there is no preferential utilization of immunoglobulin genes. An analysis of the distribution of charged residues and hydropathic comparison studies were used to interpret the sequence of 4C11 in terms of the biological mimicry of antigenic stimulation.     

Characterization and evaluation of monoclonal antibodies developed for typing influenza A and influenza B viruses.

Monoclonal antibodies that are broadly reactive with influenza A or influenza B viruses were produced as stable reagents for typing influenza viruses. Monoclonal antibodies to influenza A were specific for either matrix protein or nucleoprotein. The antibodies to influenza B were specific for nucleoprotein or hemagglutinin protein. In an enzyme immunoassay procedure, influenza A antibodies detected H1N1, H2N2, and H3N2 influenza A virus strains collected between 1934 and 1984. Each of the influenza B antibodies detected influenza B reference viruses collected between 1940 and 1984. Pools of either influenza A or influenza B monoclonal antibodies were used to detect influenza viruses reisolated from clinical specimens in tissue culture. At 48 h after inoculation, the influenza A monoclonal antibodies detected 64% of H1N1 and 94% of H3N2 influenza A specimens, and the influenza B monoclonal antibodies detected 79% of the influenza B specimens. The results of this study suggest that the monoclonal antibodies described should provide useful diagnostic reagents for workers in virology laboratories who wish to isolate and identify influenza virus but have been unable to obtain consistent supplies of animal sera specific for influenza A or B viruses.     

Poly(Glu60Ala30Tyr10) (GAT)-induced IgG monoclonal antibodies cross-react with various self and non-self antigens through the complementarity determining regions. Comparison with IgM monoclonal polyreactive natural antibodies

Previous studies have shown that the antibodies of the preimmune repertoire are able to bind to various auto- and xenoantigens including chemical haptens. Sequence analysis of two such murine monoclonal IgM natural autoantibodies showed that they are encoded by unmutated germ-line variable regions of the light and heavy chain (V alpha and VH) genes which were also found in various murine immune responses, like phenyl-oxazolone, dinitrophenyl, arsonate, phosphorylcholine and influenza virus hemagglutinin. These data raised the question as to whether induced antibodies possessing germ-line sequence are also able to react with autoantigens. To study this problem, anti-poly(Glu60Ala30Tyr10) (GAT) and anti-alprenolol (Alp) monoclonal antibodies, carrying similar VH and V alpha genes and the same IgG1 isotype, were examined for their capacity to react with several self and non-self antigens. The results showed that: (a) the anti-GAT antibodies tested reacted with different autoantigens, such as murine tubulin, actin and myosin as well as trinitrophenyl (TNP) and bovine serum albumin. Similarly, one of the anti-Alp showed weak reactivities for myosin, DNA, actin and TNP; (b) in contrast two other anti-Alp antibodies did not react with any of the tested antigens. Since the major differences between the oligoreactive anti-GAT and the monoreactive anti-Alp antibodies are in the complementarity determining regions (CDR) our results suggest that the observed cross-reactions are mediated by hypervariable loops. Sequence comparison of these antibodies indicate a possible correlation between cross-reactivity and the presence of aromatic and charged amino acids in the CDR.     

Monoclonal anti-VH141 antibodies that specifically recognize the heavy chain variable region of, and are closely related to, MOPC141 myeloma protein whose VH gene belongs to VHQ52 family.

To raise monoclonal antibodies that specifically recognize the heavy chain variable region of MOPC141 myeloma protein (VH141), which belongs to VHQ52 family, rats were immunized with Fd'-conjugated keyhole limpet haemocyanin (KLH) (Fd': Fd' fragments of MOPC141), and the spleen cells were fused with mouse myeloma cells. The resulting 900 hybridomas were screened for antibody activity against Fd'1 fragments having no constant H-chain sequences, which were prepared by cleavage of the Fd' fragments with cyanogen bromide, and two monoclonal antibodies, designated 3-2-7h and 3-5-6f, were obtained. Radioimmunoassay inhibition test showed that the two monoclonal antibodies specifically recognized the VH141, but each was directed to a different determinant on the VH141. When the functional VH gene of Abelson virus-transformed mu-producing pre-B cells, which could be strongly stained with 3-5-6f monoclonal antibody, was cloned and sequenced, the VH gene was closely relate to that of MOPC141 (88% and 94% homology at amino acid and DNA level, respectively). Taken together, the results indicated that 3-2-7h had high specificity only for the VH141, whereas 3-5-6f specifically reacted not only with the VH141 but also with the VH region closely related to that of MOPC141, and that both the monoclonal anti-VH141 antibodies were specific for a limited range of VH regions within the VHQ52 family rather than being VHQ52 family specific. These monoclonal anti-VH141 antibodies should be very useful to determine at a single cell level by immunofluorescence the usage of the VH gene(s) identical or closely related to that of MOPC141 during early B-cell development.     

Monoclonal antibodies with high virus-neutralizing activity against pandemic influenza virus A/llV-Moscow/01/2009 (H1N1)swl

The authors have obtained a panel of 7 monoclonal antibodies (MAbs) against pandemic influenza virus A/IIV-Moscow/01/2009 (HIN1)swl isolated in Russia. One MAb is directed to a NP protein linear epitope and interacts with all the influenza A viruses under study. Six other MAbs are directed to H1 hemagglutinin conformation-dependent determinants and detect homologous virus in the hemagglutination-inhibition test, enzyme immunoassay, immunofluorescence and virus neutralization tests. MAbs differentiate pandemic influenza viruses A(H1N1)swl from seasonal influenza A(H1N1), A(H3N2), and B viruses. The high neutralizing activity of MAbs permits their use to study the fine antigen structure of influenza virus hemagglutinin and to differentiate the A(H1N1) pandemic influenza viruses and offers promise for obtaining humanized antibodies in order to make specific prevention and treatment of influenza.     

Molecular characterization of monoclonal anti-steroid antibodies: primary structures of the variable regions of seven antibodies specific for 17 alpha-hydroxyprogesterone or 11-deoxycortisol and their pH-reactivity profiles

The variable region nucleotide sequences of seven monoclonal anti-steroid antibodies that are specific for the closely related progesterone derivative, 11-deoxycortisol or 17 alpha-hydroxyprogesterone (17-OHP), have been determined by genomic cloning and DNA-sequencing or by direct mRNA-sequencing. As for their heavy chain variable regions, the nucleotide sequences of the SCET.M8.1 (SCET) and OHP 4B2.2.1 (4B2) antibodies were classified into the VH-9 family, while OHP 7D7.2.3 (7D7), 1E9.3.1 (1E9), 57.G6.1 (57) and 138.H8.1 (138) used VH-3 family genes. OHP 101.B11.1 (101) used a gene of the VH-1 family. For their light chain variable regions, SCET and 57 used VK-28 group genes, while 4B2, 7D7, 1E9, 101 and 138 antibodies used genes of the VK-21 subgroups (21A, 21B or 21C). All of the antibodies used different combinations of genes in the VH families and VK groups or subgroups. This indicates that the antibody response against the steroid hapten, 17-OHP, is fairly polyclonal, and several VH/VL combinations show high affinity for progesterone-related steroids. Although the primary structures of hypervariable loop regions of the mAbs were relatively diverse, generally, hydrophobic and aromatic amino acids were rich in these regions. Moreover, the length of heavy chain CDR3 was constant in all the antibodies investigated in this paper as well as the previously reported anti-progesterone monoclonal antibodies (mAbs). This suggests that the length of VH CDR3 in these mAbs has a considerable influence on the formation of antigen-combining pockets. The pH-reactivity profiles for the anti-17-OHP mAbs indicated that the the steroid-mAb binding was independent of pH between pH 4 and 11 in most of the mAbs. The results suggest that the steroid-mAb interactions are not largely affected by the electrostatic environments near the combining sites of these mAbs. Taken together, these data imply that the shape of hydrophobic depressions in the combining sites is important for the binding of relatively large, hydrophobic and rigid haptens like steroids.     

Heterogeneity of cross-reactive idiotypes. Serological and structural analysis of monoclonal anti-p-azobenzene-arsonate antibodies expressing major and minor idiotypes

Hybridoma-derived monoclonal anti-p-azobenzene-arsonate (ABA) antibodies were obtained from fusions of ABA-KLH primed A/J spleen cells with three different myeloma cell lines. Of the 156 antibody secreting hybridomas 24% carried the cross-reactive idiotype (CRI), which is known to be shared by 20-70% of anti-ABA serum antibodies in A/J mice. The isotypes, SDS-PAGE patterns and the partial amino acid sequences of the V-regions of one CRI negative and six CRI positive hybridoma proteins were determined. These antibodies were IgGl, kappa and IgG2b, kappa. Some idiotype carrying monoclonal antibodies appeared to be serologically identical. Although the partial VH amino acid sequences of these monoclonal antibodies showed great homology with each other and with serum antibody, several sequence variations in framework residues as well as in the first and second complementarily determining regions (CDRs) were found. The cross-reactive idiotype of the anti-ABA antibodies, therefore, exhibits structural microheterogeneity, i.e. it consists of a family of non-identical but closely related molecules as previously reported (Alkan et al., 1980; Estess et al., 1980; Marshak-Rothstein et al. 1980). Here the N-terminal sequence of the VH regions from 14 CRI+ and 8 CRI- antibodies as well as the VL regions from 11 CRI+ and 8 CRI- monoclonal antibodies are compared. Analysis of the available data demonstrated that there are pairs of hybridoma proteins (both CRI+ and CRI-) which have identical sequences for VH or VL. This suggests that there exist a minimum of 4 germ line genes coding for CRI+ VH, CRI+ VL, CRI- VH and CRI- VL respectively. In addition, CRI+ VL has always been found in association with a CRI+ VH.     

Molecular characterization of the VH region of murine autoantibodies from neonatal and adult BALB/c mice

We report on the molecular characterization of the heavy (H) chain variable (V) region of two murine autoantibodies reacting with a conventional self antigen, thyroglobulin (Tg), originated from unimmunized/unstimulated neonatal mice (clone B10H2) and from an adult hyperimmunized mouse (clone 62), respectively. Serologically, both hybridoma antibodies express the same idiotype (Id). By cloning and sequencing we demonstrated that their VH regions are encoded by identical VDJ gene elements including the VD and DJ joints. This VH belongs to the VH 7183 gene family, the most 3'-end proximal family in the murine genome. The D segment is unique and only 50% similar to any murine D segment. The J segment utilized is germ-line JH4. The cloned DNA rearrangement was transfected into the J558L myeloma cells and the secreted antibody was found to express the reference Id on the H chain, hence proving that the productive VDJ rearrangement had been identified. These results show that a spontaneously arising and an antigen-induced autoantibody use an identical VDJ gene rearrangement and that after hyperimmunization somatic mutation did not occur. The significance of this finding with respect to ontogeny of the B cell repertoire is discussed.     

Phage library panning against cytosolic fraction of cells using quantitative dot blotting assay: application of selected VH to histochemistry

Comprehensive preparations of antibodies against various kinds of proteins in cells would be useful in proteome research and antibody-based research. Here we report the panning of a human antibody heavy chain variable domain (VH) phage library against a cytosolic fraction of rat liver to obtain antibodies specific for certain cytoplasmic proteins. Rat liver specimens were homogenized and subjected to differential centrifugation. A 125000 x g supernatant (rat liver cytosol, RLC) was immobilized onto a nitrocellulose membrane and subjected to phage VH library panning. For efficient assessment of binding phages, we established a system that was a combination of monoclonal phage ELISA and quantitative dot blotting of phages. The VH genes of the binding phages were selected and expressed as VH--bacterial alkaline phosphatase (PhoA) conjugates (VH/RLC--PhoAs) in Escherichia coli. One of the VH/RLC--PhoAs stained one major band on Western blotting of RLC and also stained the cytoplasm of hepatocytes histochemically. This is the first report of phage library panning against the cytosolic fraction of cells to obtain human VH fragments, and the application of those human VH fragments to histochemical study.     

Antigenic analysis of H4 influenza virus isolates using monoclonal antibodies to defined antigenic sites on the hemagglutinin of A/budgerigar/Hokkaido/1/77 strain

Summary Three non-overlapping antigenic sites were defined on the hemagglutinin of avian influenza virus A/budgerigar/Hokkaido/1/77 (H4N6) by competitive binding assay of monoclonal antibodies to the virus and comparative antigenic analysis of variants selected with monoclonal antibodies. Antigenic relationship among 25 H4 influenza viruses of different bird origin was examined by ELISA with the monoclonal antibodies to each of defined antigenic sites. Two of the three antigenic sites contained epitopes specific to the H4 influenza viruses of budgerigar and mynah origin, and the remaining site contained an epitope which was cross-reactive with almost all of the H4 influenza viruses.     

Sequence changes at the V-D junction of the VH1 heavy chain of anti- phosphocholine antibodies alter binding to and protection against Streptococcus pneumoniae

X-linked immune deficient (Xid) mice fail to produce anti- phosphocholine (PC) antibodies even after immunization with Streptococcus pneumoniae. Consequently, Xid mice are extremely susceptible to infection with S. pneumoniae, PC-specific B cells appear to undergo clonal deletion in Xid mice; however, a new thymus-dependent form of PC, 6-(O-phosphocholine)hydroxyhexanoate (EPC), can rescue PC- specific B cells from the bone marrow presumably by providing T cell help before clonal deletion. Analysis of PC-specific IgG hybridomas from Xid mice revealed utilization of several V-D junctional variants of the VH1 gene segment rearranged to different D and JH gene segments. The majority of Xid anti-PC antibodies exhibit an Asp-->Gly95H replacement at the V-D junction. These Gly95H VH1 variants associate with kappa 1C L chains to produce anti-PC antibodies that: (1) have low relative affinity for PC, (ii) are heteroclitic for nitrophenylphosphocholine and (iii) fall to bind to or provide protection against S. pneumoniae. Single prototypic V-D variants of the T15 idiotype (Asp95H), M603 idiotype (Asn95H) and M167 idiotype (Asp95H- Ala96H) were also induced in Xid mice. The M603-like and M167-like antibodies bound to and protected against S. pneumoniae even though they exhibited Kas for PC which were lower than T15 idiotype+ antibodies. These data demonstrate that small changes in the V-D junctional sequence of the T15 (VH1) heavy chain alter L chain usage and the structure of the PC binding site so that the PC expressed on S. pneumoniae is no longer recognized.      

Molecular and idiotypic analysis of antibodies to Cryptococcus neoformans glucuronoxylomannan.

Antibodies to the Cryptococcus neoformans capsular glucuronoxylomannan (GXM) form the basis of two potential therapeutic intervention strategies, i.e., conjugate vaccines and passive antibody therapy. To better understand the molecular basis of the antibody response, the heavy- and light-chain immunoglobulin variable region (VH and VL, respectively) sequences of seven monoclonal antibodies (MAbs) to GXM were determined. Rabbit anti-idiotypic serum was made to the previously characterized murine MAb 2H1 and used to study MAb 2H1 idiotype expression in other GXM-binding MAbs and immune sera. MAb E1 originated from a C3H/HeJ mouse immunized with C. neoformans serotype A polysaccharide. MAbs 471, 1255, 339, 3C2, 386, and 302 originated from BALB/c mice immunized with polysaccharide of serotypes A, A, B, C, D, and D, respectively, conjugated to sheep erythrocytes. In the E1, VH uses V11 from the T15 gene family and JH3 and has a D segment of three amino acids, and the VL uses a VKSer-like gene family element and JK5. In MAbs 471 and 3C2, the VH uses VH7183-like gene family elements and JH2 and has D segments of seven amino acids, and the VL uses VK5.1 and JK1. In MAbs 1255 and 339, the VH uses VH10-like gene elements and JH4 and has six codon D segments, and the VL uses a VK21-like gene element and JK5. In MAbs 302 and 386, respectively, the VH uses VHGAM-like gene elements and JH2 and JH3 and has six and four codon D segments, and VL uses VK4/5-like gene elements and JK1.VH usage, MAb 2H1 idiotype expression, and fine specificity mapping define a minimum of three GXM epitopes which elicit protective antibodies. The results confirm that the antibody response is highly restricted, suggest a close relationship between molecular structure and serological properties, and provide insight into protein structural motifs important for GXM binding.     

Characterization of a progesterone-binding, three-domain antibody fragment (VH/K) expressed in Escherichia coli.

The heavy chain variable region (VH) and the kappa light chain of the anti-progesterone monoclonal antibody (mAb) DB3, have been expressed as a single-chain three-domain polypeptide, designated VH/K, and secreted into the periplasmic space of Escherichia coli (E. coli). The linker sequence was derived from the VH-CH1 elbow region. The C kappa domain provides a sensitive detection tail for Western blotting and enzyme-linked immunosorbent assay (ELISA). Periplasmic extracts of transformed E. coli contained material that bound progesterone and related steroids with similar specificity and affinity to DB3, and displayed the DB3 idiotype and kappa chain epitopes. Reference to the crystal structure of DB3 suggests that all the characteristics of the combining site interaction with steroids are retained in the bacterially expressed material. Western blotting demonstrated material with a molecular weight equivalent to three domains after reduction, but six domains in the unreduced state, suggesting that the VH/K polypeptide is assembled in the periplasm as a disulphide-bridged dimer. The VH/K construct provides a novel route to expression of antibody combining sites in E. coli for antibody engineering.     

Structural characterization of human monoclonal cold agglutinins: evidence for a distinct primary sequence-defined VH4 idiotype

Cold agglutinins that bind the developmentally regulated I red cell determinant occur naturally among human monoclonal IgM proteins. These autoantibodies are known to use light chains that derive mainly from the minor kappa III (kappa III) variable region subgroup. The kappa III subgroup is also highly expressed in monoclonal rheumatoid factors. However, while most monoclonal rheumatoid factors use structurally homologous heavy chains that derive from the VH1 family, information regarding the structure of the cold agglutinin heavy chains remains fragmentary. We demonstrate here that the kappa III cold agglutinin autoantibodies exclusively use heavy chains that derive from the VH4 family. Furthermore, these autoantibody heavy chains all express the same primary sequence-defined idiotype, corresponding to the second hypervariable region. These data indicate that cold agglutinins use a remarkably homogeneous subset of heavy chain variable regions. Moreover, unique patterns of preferential VH and VL pairing clearly distinguish the anti-I cold agglutinins from all other known monoreactive autoantibodies.     

抗H5N1禽流感病毒羊驼重链单域抗体的制备和功能鉴定

目的:获得具有中和活性、高特异性和稳定性的抗H5N1禽流感病毒血凝素蛋白(HA)的羊驼重链单域(VHH)抗体。方法:利用pET-22b表达载体诱导表达抗H5N1禽流感病毒HA VHH抗体蛋白,以包涵体形式表达的VHH抗体蛋白采用最优复性方法进行复性后,获得高纯度的VHH抗体,分别采用ELISA法鉴定VHH抗体的亲和力和热稳定性,采用血凝抑制实验鉴定抗体的特异性和体外中和活性。结果:经复性的抗H5N1禽流感HA VHH抗体对H5N1禽流感病毒HA具有良好的特异性。通过对三种不同复性方法比较,利用柱上复性的VHH23抗体具有较好的热稳定性,亲和力为9.1×10-7mol/L,同时对H5N1禽流感病毒HA具有良好的体外中和活性。结论:实验结果表明通过原核表达获得具有较好中和活性、特异性及稳定性的抗H5N1禽流感病毒VHH抗体,为进一步开展抗体的体内病毒中和试验奠定良好基础。     

Primary sequence and location of the idiotopes of V-88, a DNA-binding monoclonal autoantibody, determined by idiotope scanning with synthetic peptides on pins.

In this study, the primary sequence and location of the idiotopes of monoclonal antibody (mAb) V-88 have been examined. V-88 was derived from an adult (NZB x NZW)F1 mouse, has been partially defined previously with polyclonal anti-idiotype antisera, and is a member of the 16/6 idiotype (Id) family. From the inferred primary amino acid sequence of the antibody, sets of hexapeptides, overlapping by five residues, were synthesized on pins and used to scan the expression of epitopes (idiotopes) in the V regions of the light and heavy chains. A heterologous rabbit antiserum raised against the native antibody V-88, and absorbed to make it idiotype specific, was found to react with eight major epitopes distributed between the VH and VL regions. Half of these determinants mapped to the complementarity determining regions, with the others in framework sequences. Thus, the idiotype of antibody V-88 comprises, at least in part, continuous linear idiotopes in both hypervariable and framework areas. The process of absorbing the anti-idiotype antiserum on normal mouse immunoglobulin removed much of the background antibody activity against V region peptides, but left the activity against the dominant idiotopes. The sequence of a major idiotope, VATISG, in the FW2/CDR2 VH region is homologous to sequences of human antibodies that express the 16/6 idiotype, suggesting that Id.16/6 is at least in part defined by this region of the antibody. The same VH area is also homologous to sequences in bacterial and mammalian heat-shock proteins (hsp60-65). Thus there may be a functional link through idiotype connections, especially those involving Id.16/6, between anti-bacterial responses and production of autoantibodies, and some bacterial antigens may function indirectly as superantigens for B cells.