OBJECTIVESTo obtain a single-chain antibody with high affinity against hepatocellular carcinoma (HCC).

METHODSA second single-chain antibody mutant library was established using an error-prone PCR and a phage display. Single-chain antibodies with high affinity for hepatocellular carcinoma were selected using ELISA.

RESULTSThe content of the second single-chain antibody mutant library was about 4.5 x 10(7). Two selected mutants, M90 and M116, were obtained after 3 rounds of panning and ELISA. Immunoassay showed that both M90 and M116 could bind to human HCC cells. The relative affinity of M90 was 1.7-fold higher than that of the original antibody, and M116 was 2-fold higher than that of the original antibody.

CONCLUSIONError-prone PCR is an effective and simple method for affinity maturation of antibodies isolated from a phage antibody library.

Antibodies, Neoplasm ; immunology ; Antibody Affinity ; Antibody Specificity ; Carcinoma, Hepatocellular ; immunology ; pathology ; Humans ; Immunoglobulin Fragments ; immunology ; Immunoglobulin Variable Region ; genetics ; immunology ; Liver Neoplasms ; immunology ; pathology ; Mutation ; Peptide Library

Monoclonal antibodies (mAbs) contribute a lot to the development of numerous fields in life science as a pivotal tool in modern biological research. Development of the PCR methods and maturation of antibody production have made it possible to generate mAbs from single human B cells by single cell RT-PCR with successional cloning and expression in vitro. Compared to traditional monoclonal antibody technologies, single B cell technologies require relatively fewer cells, which are highly efficient in obtaining specific mAbs in a rapid way with preservation of the natural heavy and light chain pairing. With so many advantages, single B cell technologies have been proved to be an attractive approach for retrieval of naive and antigen-experienced antibody repertoires generated in vivo, design of rationale structure-based vaccine, evaluation and development of basic B cell biology concepts in health and autoimmunity, and prevention of infectious diseases by passive immunization and therapy for disorders. Accordingly, this review introduced recent progresses in the single B cell technologies for generating monoclonal antibodies and applications.
Antibodies, Monoclonal ; biosynthesis ; genetics ; immunology ; Antibody Specificity ; B-Lymphocytes ; cytology ; immunology ; metabolism ; Humans ; Immunologic Techniques

OBJECTIVETo explore whether the antibody subtypes against the extracellular 1-2 (EC1-2) epitopes of pemphigus vulgaris antigen (PVA) are related to the pathogenesis of PVA.

METHODSEC1-2 fusion protein, emulsified with complete Freund's adjuvant (CFA) or aluminum hydroxide hydrate [Al ( OH)3], was used to immunize C57BL/6 mouse. After immunization, the cytokine types, specific antibody titers, and antibody subtypes were detected. Also, a neonatal mice model was used to evaluate the pathogenesis of different antibodies.

RESULTSTh1 type cytokine interferon gamma (IFNgamma) was elevated in CFA group, while Th 2 type cytokine interleukin-4 (IL-4) was increased in Al (OH)3 group. The antibody subtypes were different in both groups. After the two groups were transferred with antisera separately, the neonatal mice developed erosion on the skin from Al(OH)3 group, with acantholysis histopathologically and bright immuno-fluorescence deposition, which was not seen in CFA group.

CONCLUSIONDifferent antibody subtypes may contribute to the pathogenesis of disease.

Animals ; Antibody Specificity ; Autoantibodies ; immunology ; Desmoglein 3 ; immunology ; Epitopes ; immunology ; Mice ; Mice, Inbred C57BL ; Pemphigus ; immunology ; pathology

OBJECTIVETo characterize the specific monoclonal antibodies to Aspergillus conidia.

METHODSFlow cytometry was used to examine the reactivity of the specific monoclonal antibodies to Aspergillus conidia.

RESULTSBoth the monoclonal antibodies MA3 and Con2 showed specific reactivity to Aspergillus conidia suspensions. MA3 was capable of binding to the conidia of A.fumigatus, A.flavus, A.niger and A.terreus, while Con2 was reactive only to the conidia of A.fumigatus.

CONCLUSIONTwo specific monoclonal antibodies (MA3 and Con2) to Aspergillus conidia have been obtained.

Antibodies, Fungal ; immunology ; isolation & purification ; Antibodies, Monoclonal ; immunology ; isolation & purification ; Antibody Specificity ; Aspergillus ; immunology ; Flow Cytometry ; Spores, Fungal ; immunology

This study was purposed to prepare and primarily identify the specific monoclonal antibodies (McAbs) against the apoptosis related protein PNAS-2 so as to provide the essential tool for study of PNAS-2 function. The McAbs against PNAS-2 were prepared via the immunization of mice, cell fusion and cloning using synthetic peptide of PNAS-2 as immunogen; the specificity, titer and subtype of McAb were detected by Western blot, ELISA and immunofluorescence. The results showed that the stable hybridoma cell line S-31-7 producing McAbs against PNAS-2 protein was successfully obtained. The immunoglobulin of the McAb was identified to be IGg1lambda. The titer of ascetic fluid fled McAb were 1:8,000. A single specific band with 28 kD was shown in Western blot test, and the antigen recognized was present in cell cytoplasm by immunofluorescence. In conclusion, the obtained McAb against PNAS-2 displays strong specificity and high titer, which may be applied to the advanced research on PNAS-2 protein.
Animals ; Antibodies, Monoclonal ; immunology ; isolation & purification ; Antibody Specificity ; immunology ; Apoptosis Regulatory Proteins ; immunology ; Female ; Mice ; Mice, Inbred BALB C

7 strains of stable cell lines secreting monoclonal antibodies against AAV2 capsids were obtained by immunizing BALB/C mice with highly purified recombinant adeno-associated virus. Among them, the monoclonal antibodies B10 and G4 had neutralizing activity, and their subtypes were IgG1 and IgG2a, respectively. The binding characterizations of the two neutralizing antibodies were studied. Both B10 and G4 showed serotype specific binding activities to rAAV2 virus particles other than AAV1, AAV5, and AAV8, and the binding could not be blocked by heparin. After incubating with the two antibodies separately, rAAV2 viruses could still bind to sensitive cell line BHK-21, suggesting that the binding sites of the two antibodies to rAAV2 located at different positions on viral particle surface from the primary receptor binding sites of AAV2. Western blotting assay showed that B10 could bind to VP1, VP2 and VP3 of rAAV2. However, G4 bound none of them. The results suggested that B10 recognized a linear epitope of AAV2 capsid, whereas G4 probably recognized a conformational epitope on the surface of AAV2 virus particle. The two antibodies with different characteristics provided valuable tools for AAV2 virus particles detection and infection processes.
Animals ; Antibodies, Monoclonal ; immunology ; Antibodies, Viral ; immunology ; Antibody Specificity ; Capsid ; immunology ; Cell Line ; Dependovirus ; genetics ; immunology ; Epitopes ; immunology ; Female ; Humans ; Immunoglobulin G ; immunology ; Mice ; Mice, Inbred BALB C

To prepare monoclonal antibodies (mAbs) against structural proteins of severe fever with thrombocytopenia syndrome bunyavirus (SFTSV), BALB/c mice were immunized using purified inactivated SFTSV virions as the antigens. Subsequently, hybridoma cell lines that secreted monoclonal antibodies against nucleoprotein (NP) and glycoproteins (GP) were obtained using a hybridoma technique. The antigen specificities of prepared mAbs were examined by indirect immunofluorescence and immunoprecipitation assays. Functional analyses were then performed,including the detection of IFA antibody titers,the levels of neutralizing activity and antibody affinities. After cell fusion and cloning,13 hybridoma cell lines secreted mAbs specifically against SFTSV-GP and 7 hybridoma cell lines secreted mAbs specifically against SFTSV-NP. Immunofluorescence and immunoprecipitation assays showed that the mAbs had high levels of antigen specificity. Among the 13 anti-SFTSV-GP mAbs,6 recognized Gn,whereas the others reacted with Gc. IFA titers of most anti-SFTSV-GP mAbs were between 1,280 and 20,480, and four anti-SFTSV-Gn mAbs showed neutralizing activity. Seven of the obtained anti-SFTSV-NP mAbs reacted specifically with NP,of which the IFA titers ranged from 5,120 to 20,480 with no observed neutralizing activity. Furthermore, two anti-SFTSV-GP mAbs, 1C8 and 1G8, showed high levels of affinity via a non-competitive ELISA. Our study lays the foundation for the development of further diagnostic assays and basic research into SFTSV.
Animals ; Antibodies, Monoclonal ; immunology ; Antibodies, Viral ; immunology ; Antibody Specificity ; Bunyaviridae Infections ; immunology ; virology ; Female ; Humans ; Hybridomas ; immunology ; Mice ; Mice, Inbred BALB C ; Phlebovirus ; immunology ; Viral Structural Proteins ; immunology

Oxidized low density lipoprotein (LDL) seems to take a part in atherogenesis through direct interactions with macrophages, endothelial cells, and smooth muscle cells, and is thought to participate in renal glomerular injury. For the purpose of illustrating the role of oxidized LDL in the human diseases, monoclonal antibodies were developed and characterized, recognizing oxidized LDL-specific epitopes that do not exist on native LDL. LDL was oxidized by the incubation with CuSO4, and used as immunogen. Splenocytes from the immunized mouse and mouse myeloma cells were fused to produce hybridomas, which were screened for the secretion of oxidized LDL-specific antibodies. Immunoblot analysis and binding affinity assay showed that these monoclonal antibodies recognize malondialdehyde-conjugated peptide epitopes.
Antibodies, Monoclonal ; Antibody Affinity ; Antibody Specificity ; Enzyme-Linked Immunosorbent Assay ; Epitopes ; Human ; Lipoproteins, LDL/immunology* ; Malondialdehyde/immunology ; Malondialdehyde/analysis ; Peptide Fragments/immunology ; Thiobarbituric Acid Reactive Substances/analysis

ZCH-2B8a (IgG2a) is a novel monoclonal antibody (McAb) generated in laboratory of Children Hospital of Medical College, Zhejiang University recently using human myeloblastic leukemia cell line KG1a as immunogen. This antibody has been submitted to the 8th International Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA8) and the results showed that the antibody recognized an unknown molecule on the surface of some blood cells. The aim of this study was to investigate the reactivity of this antibody on normal blood cells and malignant cell lines and to explore its possible application in clinical practice. The multi-parameter flow cytometry was used to analyze the expression pattern of 2B8a antigen in triplicate on normal blood components including T cells, B cells, natural killers (NK), neutrophils, monocytes, dendritic cells (DC), red blood cells (RBC), platelets (Plt), hematopoietic stem/progenitor cells derived from either bone marrow or G-CSF mobilized peripheral blood CD34(+) cells and malignant cell lines including 14 hematopoietic, 5 neuroblastoma, 1 colon cancer and 1 amniotic epithelium cell lines. The amount of positive cells > or = 20% was considered as positivity. The results showed that 2B8a antibody reacted to 3/3 specimens of blood B cells with a positive rate of 26.29% and 2/3 specimens of monocytes with an average positive rate of 59.84%. 2B8a was weakly reactive to neutrophils (23.72%) and negative for T cells, NK, DC, RBC and Plt. The antibody reacted to all 3 marrow CD34(+) cells with an average positive rate of 39.33% while it was negative for G-CSF-mobilized CD34(+) peripheral blood stem/progenitor cells (PBSC, 1.25%). Cell line analysis showed that the antibody notably reacted to three out of 4 cell lines (Raji, SMS-SB, Nalm-6 and Nall-1) with the positive rates of 98.78%, 98.61%, 94.93% respectively and weakly to one of them with 5.68% in B lineage cell lines and monoblastic cell line (U937, 67.78%) while it was only weakly positive or negative for other myeloid leukemia cell lines including Meg01 (33.40%), HL-60 (29.70%), K562 (28.19%), KG1a (16.23%) and HEL92.1.7 (8.02%). Among 4 T lineage leukemia, 5 neuroblastoma and 1 colon cancer cell lines tested, only Molt-3 was found weakly positive (31.40%) for 2B8a, while the remaining 3 T cell lines (Molt4, JM and CCRF-CEM), 5 neuroblastoma cell lines (LA-N1, KCNR, BE, SK-N-SH, SK-N-AS) and the colon cancer cell line (HR8348) tested were negative. An amniotic epithelium cell line (FL) was showed positive for the antibody (45.03%). It is concluded that 2B8a antibody primarily reacts to B lineage and monocytic lineage cells which may bear the diagnostic and therapeutic applications among different types of hematopoietic malignancies.
Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibody Specificity ; Antigen-Antibody Reactions ; Antigens, Neoplasm ; analysis ; immunology ; Antigens, Surface ; immunology ; B-Lymphocytes ; cytology ; immunology ; HLA Antigens ; immunology ; Hematopoietic Stem Cells ; cytology ; immunology ; Hematopoietic System ; cytology ; Humans ; Leukemia, Myeloid, Acute ; immunology ; pathology ; Tumor Cells, Cultured

Although the confirmative diagnosis of typhoid fever is by culture of the causative organism, usually from blood, a serological test is still necessary to provide a more rapid method of diagnosis. The indirect fluorescent antibody test, using a Salmonella typhi Vi antigen and a FITC-conjugated rabbit anti-human polyvalent immunoglobulin, was evaluated for the diagnosis of typhoid fever. Serum specimens were collected from patients with febrile diseases on admission. Of the 32 patients with titers of 1:64 or more, 22 were confirmed to have typhoid fever by blood culture and 7 had fever of undetermined origin that was considered to be typhoid fever clinically. Three patients were diagnosed to have salmonellosis other than typhoid fever. Of the 121 patients with titers of 1:32 or less, 105 patients had non-typhoidal febrile disease, 15 patients had fever of undetermined origin, and one patient was confirmed to have typhoid fever by blood culture. When a Vi antibody titer of 1:64 or more was taken as serological evidence for the diagnosis of typhoid fever, the sensitivity and specificity were 95.7% and 97.2%, respectively. The incidence of positive test results following fever onset was 70.0% within 1 week of fever onset, 88.9% from 1 to 2 weeks, and 100% after 2 weeks. In conclusion, the Vi-indirect fluorescent antibody test(Vi-IFAT) can be employed as a useful serologic test in the diagnosis of typhoid fever.
Antigens, Bacterial/*analysis ; Fluorescent Antibody Technique/*standards ; Human ; Salmonella typhi/immunology ; Sensitivity and Specificity ; Typhoid Fever/*diagnosis