Monoclonal IgG to the inner capsid of human rotavirus

Summary Monoclonal IgG to the inner capsid of human rotavirus was produced by mice immunized with hybrid cells derived from mouse myeloma and the spleens of mice immunized with rotavirus. This IgG reacted with an antigenic determinant of mammalian rotaviruses that was not present on that of avian rotaviruses.With 2 Figures     

人博卡病毒VP2蛋白单克隆抗体的制备

目的表达纯化人博卡病毒（human Bocavirus,HBoV）VP2蛋白,采用杂交瘤方法制备抗HBoVVP2蛋白的单克隆抗体。方法应用原核表达载体pET-30a和大肠埃希菌表达VP2蛋白,并用固定化金属亲和层析,纯化后的蛋白作为抗原免疫BALB／c小鼠,利用杂交瘤技术和间接ELASA方法筛选阳性的杂交瘤细胞,并对单克隆抗体进行分型检测和滴度测定。结果表达并纯化获得了重组HBoVVP2蛋白,利用杂交瘤技术得到单克隆IgG抗体,抗体效价达到1：4×10^5。结论利用HBoVVP2蛋白免疫制备了单克隆抗体,并具有较高的效价。本研究为快速诊断和研究HBoV打下基础。     

人博卡病毒VP2蛋白单克隆抗体的制备

目的 表达纯化人博卡病毒( human Bocavirus,HBoV) VP2蛋白,采用杂交瘤方法制备抗HBoV VP2蛋白的单克隆抗体.方法 应用原核表达载体pET-30a和大肠埃希菌表达VP2蛋白,并用固定化金属亲和层析,纯化后的蛋白作为抗原免疫BALB/c小鼠,利用杂交瘤技术和间接ELASA方法筛选阳性的杂交瘤细胞,并对单克隆抗体进行分型检测和滴度测定.结果 表达并纯化获得了重组HBoV VP2蛋白,利用杂交瘤技术得到单克隆IgG抗体,抗体效价达到1∶4×105.结论 利用HBoV VP2蛋白免疫制备了单克隆抗体,并具有较高的效价.本研究为快速诊断和研究HBoV打下基础.     

Serum antibody response to recombinant major inner capsid protein following human infection with group B rotavirus.

Recombinant major inner capsid protein (VP6) of the IDIR strain of group B rotavirus (GBR) was incorporated in a solid-phase immunoassay to access antibody response to infection in humans. Expression of VP6 in insect cells permitted design of a highly sensitive assay that avoided the contaminants present in GBR antigens obtained from fecal specimens. Among patients infected with the ADRV strain of GBR in China, increased reactivity with recombinant VP6 was observed in convalescent-phase sera in comparison with sera obtained shortly after infection (P = 0.0084). Anti-VP6 antibodies were detectable as soon as 7 days after onset of gastrointestinal symptoms, and serum reactivity persisted in specimens drawn more than 1 year after infection. Solid-phase immunoassay with recombinant VP6 was next employed in order to assess anti-GBR antibody in 513 serum specimens obtained from 423 Maryland residents (ages, 7 months to 96 years; median age, 42 years). Four individuals (< 1%) exhibited serum antibodies directed against the recombinant VP6 (ages, 54 to 95 years; mean age, 77 years). Examination of 129 additional serum specimens including some from other geographic regions of the United States failed to reveal the presence of anti-GBR antibody. Anti-GBR antibody was also not detected in any of 131 serum specimens from 60 staff and residents of a nursing home in Switzerland. While infection of humans with GBR has been uncommon in these locations outside of China, the detection of serum antibodies in older individuals in the United States either indicated an unknown, age-related risk factor or may have indicated infection in the more distant past. The availability of these reagents should allow surveys for GBR infection among additional populations that have not previously been investigated.     

Development of a monoclonal antibody specific for serotype 3 rotavirus strains

Monoclonal antibodies were prepared against serotype 3 simian rotavirus SA11. Antigenic analysis of 18 hybridoma cell lines secreting monoclonal antibodies by radioimmunoprecipitation and Western blot revealed that seven monoclonals were directed against the major inner capsid protein VP6, four against VP3, an outer capsid protein with hemagglutinating activity, and one against VP7, the main outer capsid protein of the virus. The specificity of six monoclonals could not be determined. One monoclonal (1P14E2) directed against VP3 showed serotype 3-specific neutralizing activity. This monoclonal, which recognized only serotype 3 viruses in an enzyme-linked immunosorbent assay, could be useful in assays for serotyping rotavirus directly in stool samples.     

Characterization of neutralization specificities of outer capsid spike protein VP4 of selected murine,lapine, and human rotavirus strains

Neutralization specificities of outer capsid spike protein VP4 of murine rotavirus strains EW (P?[16],G3) and EHP (P?[20],G3) and lapine rotavirus strains Ala (P?[14],G3), C11 (P?[14],G3), and R2 (P?[14],G3) as well as human rotavirus strains PA169 (P?[14],G6) and HAL1166 (P?[14],G8) were determined by two-way cross-neutralization. This was done by generating and characterizing (i) three murine x human, three lapine x human, and two human x human single gene substitution reassortant rotaviruses, each of which bore identical human rotavirus DS-1 strain VP7 (G2), and (ii) guinea pig hyperimmune antiserum raised against each reassortant. Reference rotavirus strains employed in the study represented 10 established VP4 (P) serotypes, including 1A[8], 1B[4], 2A[6], 3[9], 4[10], 5A[2], 5B[2], 5B[3], 6[1], 7[5], 8[11], 9[7], and 10[16] as well as a P serotype unknown P[18]. Murine rotavirus strains EW and EB were demonstrated to share the same P serotype (P10[16]) distinct from (i) 9 established P serotypes, (ii) lapine and human rotavirus strains bearing the P[14] genotype, and (iii) an equine rotavirus strain bearing the P[18] genotype. Both lapine (Ala, C11, and R2) and human (PA169 and HAL1166) rotaviruses were shown to belong to the same VP4 serotype, which represented a distinct new P serotype (P11[14]). P serotype 13[20] was assigned to murine rotavirus EHP strain VP4, which was shown to be distinct from all the P serotypes/genotypes examined in the present study.     

Characterization of monoclonal antibodies against human rotavirus hemagglutinin

Three monoclonal antibodies capable of specifically inhibiting hemagglutination of human rotavirus were produced. Their hemagglutination inhibition (HI) activity was specific to the homologous strain (KUN) used for immunization. The monoclonal antibodies with HI activity were highly effective in neutralizing the infectivity of the KUN strain. These antibodies reacted with Vp80, and 80,000 molecular weight (MW) protein present in the viral outer shell. It was confirmed by immunoblotting assay with the monoclonal antibodies that the antigenic site of human rotavirus hemagglutinin (HA) resides on Vp80 and on its smaller trypsin cleavage products Vp30 (MW 30,000) and Vp24 (MW 24,000). Immunofluorescence studies using the antibodies revealed that the HA antigen of the KUN strain developed at the final stage of virus maturation.     

Preparation of a monoclonal antibody against human lactase

A simple procedure for screening for anti-enzyme monoclonal antibodies is described. The properties of our first antibody identified this way, directed against human lactase, are reported.     

Two anti-parvovirus B 19 IgM capture assays incorporating a mouse monoclonal antibody specific for B 19 viral capsid proteins VP 1 and VP 2

Summary During an outbreak of parvovirus B 19 in 1989 in Northern Ireland, 7580 blood donors were screened for B 19 antigen. Two units screened positive, one of which was obtained for use as viral antigen. A monoclonal antibody (R92F6) made against this antigen was specific for B 19 capsid proteins VP 1 and VP 2. The monoclonal antibody was used in the development of 2 anti-B 19 IgM capture enzyme assays. These used a conventional substrate (O-phenylene diamine) and a chemiluminescent signal reagent. There was excellent concordance between the 2 assays. A total of 403 patients sera were tested and 65 sera were positive in each assay.     

抗肠道病毒EV71型外壳蛋白VP1单克隆抗体的制备和鉴定

目的 制备具有中和活性的抗肠道病毒EV71型外壳蛋白VP1的单克隆抗体.方法 人工合成SP55和SP70(分别包含VP1的第163-177,208-222位氨基酸)两段VP1的多肽,分别免疫BALB/c小鼠,常规杂交瘤技术进行细胞融合,用间接酶联免疫吸附试验(ELISA)筛选阳性杂交瘤细胞并测定效价.用分泌的单抗和EV71病毒在RD细胞上进行中和试验以检验其中和活性.结果 得到2株能稳定分泌抗肠道病毒EV71型VP1蛋白单克隆抗体的杂交瘤细胞株,2株单抗的中和效价分别为1:8和1:16.结论 成功制备出2株具有中和活性的抗肠道病毒EV71型VP1蛋白单克隆抗体,为其下一步应用打下基础.     

Preparation and characterization of a neutralizing monoclonal antibody directed to VP4 of rotavirus strain K8 which has unique VP4 neutralization epitopes

Summary For selecting the neutralizing monoclonal antibodies (N-MAbs) directed to VP4 of rotavirus strain K8, which has unique VP4 neutralization epitopes, we prepared several reassortant viruses by mixed infection of two different strains K8 (serotype 1) and P (serotype 3) in vitro: three reassortant clones having VP4 of K8 and VP7 of P and four clones having VP4 of P and VP7 of K8. By using these reassortants in screening hybridomas, a N-MAb (K8-2C12) directed to strain K8-specific VP4 was obtained. The MAb K8-2C12 neutralized only K8 when tested against numerous strains of different serotypes, while in enzyme-linked immunosorbent assay this MAb reacted also with simian rotavirus SA11 (serotype 3), bovine rotavirus NCDV (serotype 6), and human rotavirus (HRV) strain 69M (serotype 8). Neutralization-resistant mutants of K8 were selected by the K8-2C12 antibody and VP4 amino acid sequences of the mutants were determined. Single amino acid substitution was detected in the three mutant clones at position 394, which is included in the major cross-reactive neutralization region identified in other rotaviruses.     

Identification of a novel bluetongue virus 1-specific B-cell epitope using a monoclonal antibody against the VP2 protein

Bluetongue virus (BTV) VP2 is an important antigenic protein that can be used for the differential diagnosis of different BTV serotypes. Here, we generated a serotype-specific monoclonal antibody (mab) against BTV1. A series of peptides synthesized based on the amino acid sequence of BTV1 VP2 were screened to define ¹¹⁵AQPLKVGL¹²² as the minimal linear peptide epitope recognized by mab 4B6. Using an immunofluorescence assay (IFA), we found that mab 4B6 reacted strongly with BTV1, but did not react with other BTV serotypes (BTV2-24). The 4B6 will serve as a novel reagent in the development of diagnostic tests for BTV1 infection.     

Functional and molecular characterization of a monoclonal antibody against human interleukin 2

Human recombinant interleukin 2 (r-IL2) was used as an immunizing antigen to yield a murine monoclonal antibody (mAb) termed BO-7. Although the antibody binds to r-IL2 more avidly, it also reacted strongly with IL2 from natural sources in an enzyme-linked immunosorbent assay (ELISA), allowing the detection of the purified lymphokine at sensitivity levels closely approaching those found with the IL2 biological assay. Binding to the antigen is specific, as deduced from the close correlation of ELISA immunoreactivity with IL2 biological activity and from immunoblot analysis of electrophoretically separated IL2 from various sources. Binding studies with synthetic IL2-derived peptides revealed the location of the epitope, which is recognized by mAb BO-7: A peptide representing amino acid residues 59-72 (peptide 84) is strongly reactive with the antibody, while an overlapping peptide (residues 48-69) is not. Peptide 84, moreover, can be applied for immunopurification of mAb BO-7 and competes for binding to the antibody with the intact IL2 molecule. In turn, another monoclonal anti-IL2 antibody (35H10), showing the same reactivity pattern with peptides, competes with mAb BO-7 for binding to IL2. The application of mAb BO-7 as a specific reagent for the quantitation of IL2 in a sandwich-type ELISA is demonstrated.     

Preparation of a monoclonal antibody against human monocyte lineage

Preparation and characterization of a monoclonal antibody termed M206 directed to a human monocyte lineage are described. The antibody was produced by somatic cell hybridization between BALB/c spleen cells primed with human histiocytic lymphoma cell line U937 and murine myeloma cell line P₃U₁. The antibody reacted intensely with human peripheral blood monocytes as well as U937 but did not react with peripheral blood T and B cells. Granulocytes were weakly stained with the antibody by indirect immunofluorescence. M206 also reacted intensely with the immature leukemic cells from patients with acute monocytic leukemia but was unreactive with other types of leukemic cells except cells from chronic myelogenous leukemia which showed varied patterns of reactivities. M206 reacted with a single polypeptide chain with a molecular weight of 180,000 on the surface of U937 cells.     

Improved immunodetection of Taura syndrome virus using a monoclonal antibody specific for heterologously expressed VP1 capsid protein

vp1, a gene encoding one of the capsid proteins of Taura syndrome virus, was cloned into the pGEX-6P-1 expression vector, and the resulting construct was then used to transform E. coli strain BL21. After induction, an N-terminally glutathione-S-transferase-tagged VP1 (GST-VP1) protein with a molecular mass of 80 kDa was obtained. This protein was purified by SDS-PAGE and used for immunization of Swiss mice for monoclonal antibody (MAb) production. Three MAbs specific for the VP1 protein were selected that were suitable for detecting natural TSV infection in Penaeus vannamei by dot blotting, western blotting and immunohistochemistry. This detection occurs without cross-reaction to other shrimp tissues or other common shrimp viruses. As determined by dot blotting, the detection sensitivity of the MAbs was approximately 2 fmole/spot of the GST-VP1. These MAbs showed detection sensitivity comparable to that of MAbs specific for VP2, but they exhibited stronger immunoreactivity than previously studied MAbs specific for VP3. Although the sensitivity of the MAbs to VP1 was 1,000 times lower than one-step RT-PCR, they could be used in various types of antibody-based assays to confirm and enhance the detection sensitivity of TSV infection in shrimp.     

Production of a monoclonal antibody against a human osteosarcoma xenograft

Monoclonal antibodies (MAbs) against a human sarcoma xenograft carried in BALB/c nu/nu mice were produced by immunizing BALB/c mice with tumor cells and fusing their spleens with the SP2/O-Ag 14 mouse myeloma cell line. Hybridoma supernatants were screened using cryostat tissue sections and an immunoperoxidase reaction for ability to stain osteosarcoma xenograft tumor cells but not tonsil lymphocytes. Of 73 supernatants tested, 19 reacted with both osteosarcoma tumor cells and lymphocytes, while three reacted only with osteosarcoma. One of the latter hybridomas was cloned by limiting dilution to establish a line producing an IgG1 MAb (OS-1). By immunoperoxidase, this MAb stained tumor cells in surgical biopsies of primary (6 of 7) and metastatic (1) osteosarcomas and showed limited cross-reactivity with other tumors. It also cross-reacted with some basement membranes, endothelium and muscular media of blood vessels, and smooth muscle, but not with parenchymal cells of various normal tissues. This MAb may prove useful for the immunohistochemical confirmation of a diagnosis of osteosarcoma in surgical pathology.     

Reactivity of VP4-specific monoclonal antibodies to a serotype 4 porcine rotavirus with distinct serotypes of human (symptomatic and asymptomatic) and animal rotaviruses.

Thirteen hybridomas secreting VP4-specific monoclonal antibodies against the Gottfried strain of porcine rotavirus (serotype 4) were produced and characterized. Nine of the hybridomas secreted neutralizing monoclonal antibodies (N-MAbs) against Gottfried rotavirus. These N-MAbs were divided into five distinct groups (groups I to V) according to their patterns of reactivity with different serotypes of human and animal rotaviruses. Group I N-MAbs (n = 3) were cross-reactive with five different serotypes of human rotavirus examined by a plaque reduction virus neutralization test. Group II N-MAbs (n = 3) neutralized all symptomatic human rotavirus serotypes tested and asymptomatic human rotavirus serotype 4 to a low titer. The single group III N-MAb neutralized mainly symptomatic human rotavirus serotypes 2 and 9 and none of the asymptomatic human rotavirus serotypes. The one N-MAb in group IV reacted at low titers with only asymptomatic human rotavirus serotypes 1 through 4. A group V N-MAb recognized serotype 4 porcine rotaviruses (Gottfried and SB-2) but no other human or animal rotaviruses examined. None of the N-MAbs recognized any animal rotaviruses tested (SA-11, RRV, OSU, NCDV, and B223), except for the Gottfried and SB-2 rotaviruses. The failure of N-MAbs (groups I to IV) to react with any animal rotaviruses tested but their ability to react variably with all human rotaviruses tested suggest that neutralizing epitopes on the VP4 protein are highly conserved between the Gottfried porcine and human rotaviruses. The Gottfried rotavirus may possibly represent a naturally occurring reassortant between pig and human rotaviruses or a rotavirus which is human in origin but pathogenic for swine.     

Epitope mapping of 2E2-D3, a monoclonal antibody directed against human TDP-43

TDP-43 is now known to be a major component of ubiquitin-positive, tau-negative inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions and sporadic amyotrophic lateral sclerosis. In this study, we mapped the epitope for the monoclonal anti-TDP-43 antibody 2E2-D3. Our mapping and peptide competition experiments showed that the antibody reacted with human TDP-43, but not mouse or rat TDP-43, and recognized amino acids 205-222 of human TDP-43, corresponding to a part of the second RNA recognition motif. These findings suggest that 2E2-D3 is a useful antibody for the characterization of mouse lines transgenic for human TDP-43.     

A human monoclonal antibody against HLA-Cw1 and a human monoclonal antibody against an HLA-A locus determinant derived from a single uniparous female

Abstract: Two human monoclonal antibodies (HuMAbs) with widely different HLA specificities were raised from a uniparous HLA-seropositive female. Screening against a large panel of serologically HLA-typed lymphocytes in the complement-dependent cytotoxicity test showed that one of these HuMAbs, VP6G3, was specific for HLA-Cwl, thereby constituting the first HuMAb against an HLA-C locus product. The second HuMAb, VP5G3, was directed against an HLA-A-encoded determinant shared by HLA-A11, -A25, -A26 and -A66. The epitopes responsible for binding were determined by comparing the aminoacid sequences and were pinpointed to the 6K/9F combination for HuMAb VP6G3, and 163R with a critical contribution of aminoacids present at positions 166/167 for HuMAb VP5G3.