This study is (1) to improve the stabilization of human scFv to rabies virus; (2) to prepare active human dsFv fragment; and (3) to evaluate the biological activities of dsFv. The dsFv V(H) and VL were separately expressed in PET22b(+)/BL21 (DE3), solublized and combined in appropriate molar ratio in refolding solution. The resultant dsFv fragments were evaluated for its protection against rabies virus, its affinity and stability, in reference to the cognate scFv. The dsFv was found to bind specifically to Vero vaccine of rabies virus. Compared to the scFv, the dsFv was more stable, had higher affinity, and was able to inhibit the infection of Rabies virus to Vero cell. This established a solid basis for the clinical application of dsFv to rabies virus.
Antibodies, Viral ; immunology ; Disulfides ; chemistry ; Humans ; Immunoglobulin Fragments ; immunology ; metabolism ; Immunoglobulin Variable Region ; immunology ; metabolism ; Rabies virus ; immunology

OBJECTIVETo screen HCV NS5 mimotopes by using monoclonal antibody and phage peptide library.

METHODSBy using HCV NS5 monoclonal antibody as selective molecule, a 7 peptide phage library was biopanned and positive clones were selected by ELISA, competition assay and DNA sequencing.

RESULTSTwelve positive clones were chosen for DNA sequencing. From the experiment and sequencing comparison results, one epitope was confirmed as the mimotope of HCV NS5.

CONCLUSIONSHCV mimotope is obtained by phage peptide library screening. The result provides a new approach for HCV therapy and vaccine development.

Amino Acid Sequence ; Antibodies, Monoclonal ; immunology ; Enzyme-Linked Immunosorbent Assay ; Epitope Mapping ; Hepatitis C ; therapy ; Peptide Library ; Viral Nonstructural Proteins ; chemistry ; genetics ; immunology ; Viral Vaccines ; immunology

Antibodies, Viral ; blood ; Betacoronavirus ; immunology ; Coronavirus Infections ; diagnosis ; Gold Colloid ; chemistry ; Humans ; Immunoassay ; methods ; Immunoglobulin G ; blood ; Immunoglobulin M ; blood ; Pandemics ; Pneumonia, Viral ; diagnosis ; Reagent Kits, Diagnostic

Adult ; Antibodies, Viral/isolation & purification* ; COVID-19/virology* ; Feces/chemistry* ; Female ; Humans ; Intestines/virology* ; Male ; RNA, Ribosomal, 16S/genetics* ; RNA, Viral/isolation & purification* ; SARS-CoV-2/pathogenicity*

OBJECTIVETo obtain monoclonal antibodies (McAbs) against severe acute respiratory syndrome (SARS) associated coronavirus (SARS-CoV) nucleocapsid (N) protein to develop diagnostic test for SARS and study the pathogenesis of the disease.

METHODSBALB/c mice were immunized with purified N protein of SARS-CoV. Hybridoma cell lines secreting monoclonal antibodies against SARS-associated coronavirus nucleocapsid were established after cell fusion with mouse splenic cells and SP2/0 cells. The specificity of the McAbs obtained was examined by Western blot and indirect fluorescence assay. Epitopes reacted with the McAbs were preliminarily located through Western blot by expressing truncated N proteins.

RESULTSAfter cell fusion and three rounds of cell cloning, six hybridoma cell lines secreting monoclonal antibodies specifically against SARS-CoV nucleocapsid were obtained. Western blot and indirect fluorescence assay showed that the McAbs reacted specifically with nucleocapsid protein and SARS-CoV. Among the six McAbs, three recognize the epitopes located in the N-terminus of the protein, whereas the others reacted with those located in the C-terminus.

CONCLUSIONThe anti-SARS-CoV nucleocapsid McAbs were developed and these McAbs may be useful in the development of diagnosis assays and basic research of SARS.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibodies, Viral ; biosynthesis ; immunology ; Antibody Specificity ; Female ; Hybridomas ; secretion ; Mice ; Mice, Inbred BALB C ; Nucleocapsid Proteins ; immunology ; isolation & purification ; SARS Virus ; chemistry ; immunology

The emerging CR6261 antibody could neutralize several subtype influenza virus with high affinity, whose VH domain binds to the HA protein conserved domain. Therefore, it has drawn much attention as a potential broad-spectrum therapeutic antibody against influenza virus. In this study, we constructed the eukaryotic expression vectors pCR6261V(H), pCR6261V(H)-GFP and pCR6261scFv and screened the monoclonal cell lines that could stably express CR6261V(H), CR6261V(H)-GFP and CR6261scFv on the cell membrane. After influenza virus infecting the stable cell lines, the titers of viruses were tested by hemagglutination inhibition test. The result shows that the titers of viruses in CR6261scFv and CR6261V(H)-GFP stable expression cell lines decreased and there was no obvious discrimination between the CR6261V(H) expression cell line and the negative control, suggesting that CR6261V(H) and CR6261scFv expressing on the cell membrane could partly inhibit the virus infection. Though the effective inhibition strategy is undergoing, our research will provide new clues for the breeding of anti influenza transgenic animals.
Antibodies, Monoclonal ; immunology ; Antibodies, Viral ; chemistry ; immunology ; Antibody Specificity ; Cell Line ; Genetic Vectors ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; immunology ; metabolism ; Humans ; Influenza, Human ; immunology ; Orthomyxoviridae ; drug effects ; immunology ; Single-Chain Antibodies ; immunology

Human antibodies generated by phage antibody technology have been widely used in the immunotherapy of various diseases. Among the characteristics of these therapeutic antibodies, affinity is one of the most important determinants of their biological efficacy. The binding of an antibody and its corresponding antigen could be disrupted by thiocyanate solution of different concentrations, depend upon the affinity of the antibody. This mechanism has been adopted to determine the relative affinity of monoclonal or polyclonal antibodies in routine immunological practice. Correlation between the elution method and other techniques that measure the affinity such as equilibrium dialysis and biospecific interaction analysis (BIA) has been established. Here we describe the applications of the thiocyanate elution method in the determination of the relative affinity index (RAI) of phage antibodies (Phabs). Five clone antibodies, including 3 clones of anti-keratin antibodies (AK1, AK2 and AK3) and 2 clones of anti-HBsAg antibodies (HB1 and HB2) were selected to express Phabs and Fabs, and the RAI were determined by ELISA after thiocyanate elution. A HRP-conjugated anti-M13 was used as secondary antibody for Phabs and HRP-goat-anti-human Fab was used for Fabs. The affinity ranks of the Phabs were compared with that of the Fab fragments. The results showed that all the Phabs tested were tolerant to thiocyanate treatment. The relative affinity rank of 5 Phabs coincided well with that of their corresponding Fabs. We conclude that the thiocyanate elution can be used as an easy and rapid method to measure and compare the relative affinity of Phabs.
Antibodies ; immunology ; Antibodies, Monoclonal ; immunology ; Antibodies, Viral ; immunology ; Antibody Affinity ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; methods ; Hepatitis B Antibodies ; immunology ; Hepatitis B Surface Antigens ; immunology ; Keratins ; immunology ; Peptide Library ; Proteomics ; methods ; Thiocyanates ; chemistry ; Transfection

The N protein of the rinderpest virus (RPV) was analyzed topologically and antigenically by using anti-N monoclonal antibodies (Mabs). Ten Mabs were raised against the N protein of the RPV. At least six non-overlapping antigenic sites (sites A-F) were delineated by competitive binding assays using biotinylated Mabs. Of them 5 sites (A, C, D, E and F) on the N protein were recognized by RPV-specific Mabs in ELISA and IFA while site B was recognized by Mabs reacting with both RPV and PPRV. Non- reciprocal competition was found among sites C, D and E. Recombinant RPV N protein after exposure to 0.2% SDS exhibited higher ELISA titers in all Mabs recognizing 6 sites. Four sites (A, B, E and F) on 2% SDS-treated N protein lost completely reactivity with Mabs while the remaining sites (C and D) on the protein retained their antigenicity to some degree. It indicates that two sites (C and D) were sequential. Six representative Mabs bound to each site exhibited competition with rinderpest antibodies in a blocking ELISA, indicating that the sites were actively involved in antigenicity in cattle.
Antibodies, Monoclonal/*immunology ; Antigens, Viral/chemistry/*immunology ; Binding, Competitive ; Enzyme-Linked Immunosorbent Assay ; Epitopes/immunology ; Nucleocapsid Proteins/chemistry/*immunology ; Rinderpest virus/*immunology

This study evaluated the effect of Matricaria chamomilla and vaccination frequency on cattle immunization against rabies. Four groups (n = 15 /group) were treated with or without Matricaria chamomilla CH(12) and vaccinated with one or two doses of rabies vaccine (30 day interval). No effect of chamomile was found on cattle immunization against rabies; however, antibody titers were protective in cattle vaccinated twice, while 93.3% of cattle vaccinated only once had titers under 0.5 UI/ml after 60 days. In conclusion, the use of chamomile did not alter the humoral immune response in cattle, and two vaccine doses are suggested for achieving protective antibody titers.
Animals ; Antibodies, Viral/blood ; Cattle ; Dose-Response Relationship, Immunologic ; Drug Administration Schedule ; Drug Interactions ; Matricaria/*chemistry ; Plant Extracts/chemistry/*pharmacology ; Rabies Vaccines/administration & dosage/*immunology

BACKGROUNDTo study the antigenic and genetic characteristics of influenza (H3N2) virus circulated in China in 2004.

METHODSSingle-way and cross-way hemagglutination inhibition (HI) tests were firstly used to determine the reactivity with the reference serum of virus isolates. Based on the serological results, virus isolates were selected according to the different time and location in China in 2004. The HA1 domain of HA gene of those virus isolates were then sequenced in order to analyze the gene characterization.

RESULTSSingle-way HI test results showed that 52.3% of isolates showed 4 folds or more HI titer difference compared to A/Fujian/411/2002 (H3N2) itself (international reference strain in 2004). Cross-way HI test results showed that the antigenic ratio was 4. The nucleic acid and amino acid sequence data of HA1 domain showed that the mutated virus appeared in early February of 2004, and became the dominant circulating strain gradually. There were four important mutant positions, they were 159 Y>F, 189 S>N, 145 K>N, 226 V>I, respectively. The results also indicated that the mutated viruses originated from southern China, then transmitted to northern China, according to the analysis of time and location distribution.

CONCLUSIONThe HA1 domain of HA gene of influenza virus (H3N2) isolated from 2004 in China showed mutation and antigenic drift, and the mutated viruses were becoming the dominant circulating strain in China, and showed amino acid sequence difference compared to A/Fujian/411/2002 (H3N2) A/Wellington/1/2004 (H3N2), the vaccine components pronounced by WHO for 2004-2005 northern hemisphere and 2005 southern hemisphere respectively, which suggested that further surveillance should be conducted to monitor the virus mutation in circulation.

Animals ; Antibodies, Viral ; blood ; Antigens, Viral ; immunology ; Cell Line ; China ; DNA, Complementary ; chemistry ; genetics ; Humans ; Influenza A Virus, H3N2 Subtype ; classification ; genetics ; immunology ; Phylogeny ; RNA, Viral ; genetics ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA