BACKGROUND: Detection methods for ABO antibody (Ab) titers vary across laboratories, and the results are different depending on the method used. We aimed to compare titer values using different detection methods for the measurement of ABO Ab titers. METHODS: For ABO Ab detection, pooled group A or B red blood cells (RBCs) were reacted with each of 20 sera from blood groups A, B, or O without dithiothreitol treatment. The room-temperature (RT) incubation technique and the indirect antiglobulin test (IAT) were used in the tube test and gel card test. Flow cytometry (FCM) was performed by using anti-IgM and anti-IgG Abs. RESULTS: Regardless of the blood groups tested, the FCM assay with anti-IgM showed the highest titer compared to the tube test and gel card test with RT incubation in both. The tube test with IAT showed a higher titer than the gel card test with IAT (Gel-IAT) or FCM with anti-IgG in blood group A and B, while Gel-IAT showed the highest titer relative to the other tests, only for the anti-A Ab in blood group O. CONCLUSIONS: There were significant differences in the titers depending on the detection method used, and each method showed a different detection capacity for each ABO Ab depending on the ABO blood group tested. Therefore, caution should be exercised in interpreting ABO Ab titer results, taking into consideration the detection method used and the blood group.
ABO Blood-Group System/*immunology ; Adult ; Aged ; Agglutination Tests/instrumentation/*standards ; Antibodies/*analysis ; Antibodies, Anti-Idiotypic/analysis ; Erythrocytes/chemistry/metabolism ; Female ; *Flow Cytometry ; Humans ; Male ; Middle Aged ; Temperature

Swine hepatitis E virus (HEV) is widespread throughout pigs in both developing and industrialized countries. This virus is an important zoonotic agent and a public concern worldwide. Infected pigs are asymptomatic, so diagnosing swine HEV relies on detection of the virus or antibodies against the virus. However, several obstacles need to be overcome for effective and practical serological diagnosis. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) that used a purified recombinant capsid protein of swine HEV. The potential clinical use of this assay was evaluated by comparing it with a commercial kit (Genelabs Technologies, Diagnostics, Singapore). Results of the ELISA were highly correlated with those of the commercial kit with a sensitivity of 97% and specificity of 95%. ROC (receiving operator characteristic) analysis of the ELISA data produced a value of 0.987 (95% CI, 0.977~0.998, p < 0.01). The cut-off value for the ELISA was also determined using negative pig sera. In summary, the HEV-specific ELISA developed in the present study appears to be both practical and economical.
Animals ; Antibodies, Anti-Idiotypic/*analysis/blood/genetics ; Capsid Proteins/*genetics/metabolism ; Enzyme-Linked Immunosorbent Assay/*methods/veterinary ; Hepatitis E/diagnosis/immunology/*veterinary/virology ; Hepatitis E virus/genetics/*isolation & purification/metabolism ; Immunoglobulin G/blood/genetics ; ROC Curve ; Recombinant Proteins/genetics/metabolism ; Swine ; Swine Diseases/*diagnosis/immunology/virology

Total mRNA was extracted from lymphocytes separated from the peripheral blood of allergic patients, and then variable region of heavy chain (VH) and variable region of light chain (VL) cDNA library were constructed by RT-PCR. Human scFv templates for rabbit reticulocyte lysate ribosome display were assembled by primers and linker peptide (Gly4Ser)3. mRNA bound in antibody-ribosome-mRNA complexes was recovered using in-situ single primer RT-PCR, and three rounds of anti-IgE scFv DNA were enriched. The target DNA fragments were double enzyme digested and ligated into plasmid pET22b (+), followed by transformation in E. coli Rosseta (DE3). Positive clones were screened using clone PCR, Dot blotting and antigen ELISA. The correct lengths of VH (400 bp) and VL (710 bp) PCR products were obtained. The expected 1,000 bp ribosome display templates were also observed in agarose gel electrophoresis. After three rounds of ribosome display target sequences were effectively enriched, leading to a library of 10(13) members. Antibodies with the highest ELISA value for IgE were generated in the strain pET-IgE-6. A human anti-IgE scFv library was successfully constructed as described herein. Ribosome display using single primer in-situ RT-PCR as the recovery procedure effectively enriched target sequences. Anti-IgE scFv with high affinity and specificity were identified. The prepared human anti-IgE scFv fragment might be self-developed to a lead drug for treating asthma. Our study provides an alternative method for rapid discovery of human antibodies of therapeutic importance.
Amino Acid Sequence ; Antibodies, Anti-Idiotypic ; genetics ; isolation & purification ; Antibody Affinity ; Asthma ; blood ; Base Sequence ; DNA, Complementary ; metabolism ; Escherichia coli ; metabolism ; Humans ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Lymphocytes ; chemistry ; Peptide Library ; RNA, Messenger ; isolation & purification ; Recombination, Genetic ; genetics ; Ribosomes ; chemistry ; genetics ; immunology ; Single-Chain Antibodies ; genetics ; isolation & purification ; Transformation, Genetic

Aptamers are synthetic, relatively short (e.g., 20-80 bases) RNA or ssDNA oligonucleotides that can bind targets with high affinity and specificity, similar to antibodies, because they can fold into unique, three-dimensional shapes. For use in various assays and experiments, aptamers have been conjugated with biotin or digoxigenin to form complexes with avidin or anti-digoxigenin antibodies, respectively. In this study, we developed a method to label the 5' ends of aptamers with cotinine, which allows formation of a stable complex with anti-cotinine antibodies for the purpose of providing another affinity unit for the application in biological assays using aptamers. To demonstrate the functionality of this affinity unit in biological assays, we utilized two well-known aptamers: AS1411, which binds nucleolin, and pegaptanib, which binds vascular endothelial growth factor. Cotinine-conjugated AS1411/anti-cotinine antibody complexes were successfully applied to immunoblot, immunoprecipitation, and flow cytometric analyses, and cotinine-conjugated pegaptanib/anti-cotinine antibody complexes were used successfully in enzyme immunoassays. Our results show that cotinine-conjugated aptamer/anti-cotinine antibody complexes are an effective alternative and complementary technique for aptamer use in multiple assays and experiments.
Animals ; Antibodies, Anti-Idiotypic/immunology/metabolism ; *Aptamers, Nucleotide/chemistry/immunology ; Biological Assay ; *Cotinine/administration & dosage/chemistry ; Flow Cytometry ; Hep G2 Cells ; Humans ; Mice ; NIH 3T3 Cells ; Phosphoproteins/*chemistry/immunology ; Protein Binding ; RNA-Binding Proteins/*chemistry/immunology ; *Vascular Endothelial Growth Factor A/chemistry/immunology

The elastin metabolism in systemic sclerosis (SSc) has been known to be abnormal. The authors investigated relationship between the clinical manifestations of systemic sclerosis (SSc) and serum levels of soluble elastin-derived peptide (S-EDP) and anti-elastin antibodies. Serum samples were obtained from 79 patients with SSc and 79 age- and sex-matched healthy controls. Concentrations of serum S-EDP and anti-elastin antibodies were measured by ELISA. The serum concentrations of S-EDP in SSc patients were significantly higher than in healthy controls (median, 144.44 ng/mL vs 79.59 ng/mL, P < 0.001). Serum EDP concentrations were found to be correlated with disease duration in SSc (P = 0.002) and particularly in diffuse cutaneous SSc (P = 0.005). Levels of anti-elastin antibodies were found to be more elevated in SSc patients than in healthy controls (median, 0.222 U vs 0.191 U, P = 0.049), more increased in diffuse cutaneous SSc than limited cutaneous SSc (median, 0.368 U vs 0.204 U, P = 0.031). In addition, levels of anti-elastin antibodies were also found to be negatively associated with presence of anti-centromere antibody (P = 0.023). The S-EDP levels were not found to be correlated with levels of anti-elastin antibodies. The increased S-EDP and anti-elastin antibody levels and association with clinical and laboratory characteristics may reflect the abnormal metabolism in SSc.
Adult ; Antibodies, Anti-Idiotypic/*blood/immunology ; Centromere/immunology ; Elastin/*blood/immunology ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Male ; Middle Aged ; Peptides/*blood/immunology ; Scleroderma, Systemic/*metabolism/pathology

Allergic diseases have become global social health problems. The binding of IgE with its high affinity receptor FcepsilonRI plays a key step in I-type allergy. Recently, more and more key molecules on the IgE/FcepsilonRI signaling transduction pathway were to be the drug candidates against allergic diseases, with in-depth study of FcepsilonRI signal pathway gradually. The main drugs include molecule antibodies, peptides, vaccines, fusion proteins, small molecules, and other drugs related to IgE/FcepsilonRI. The recent progress in the study of mechanisms of representative drugs targeting on IgE/FcepsilonRI signaling pathway was reviewed in this article.
Aminophenols ; pharmacology ; therapeutic use ; Animals ; Anti-Allergic Agents ; pharmacology ; therapeutic use ; Antibodies, Anti-Idiotypic ; pharmacology ; therapeutic use ; Antibodies, Monoclonal, Humanized ; pharmacology ; therapeutic use ; Humans ; Hypersensitivity ; drug therapy ; immunology ; Immunoglobulin E ; metabolism ; Intracellular Signaling Peptides and Proteins ; antagonists & inhibitors ; Molecular Targeted Therapy ; Omalizumab ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; Pyrimidines ; pharmacology ; therapeutic use ; Receptors, IgE ; metabolism ; Signal Transduction ; Syk Kinase

OBJECTIVE: To construct a eukaryotic expression vector encoding human nasopharyngeal carcinoma anti-idiotype antibody single chain fragment (ScFv) gene G22, and to identify its expression in rectal cancer cells (CMT-93). METHODS: The G22 gene was ligated into the sites of EcoRI and NotI of eukaryotic expression vector pcDNA3.1(+). After the identification and DNA sequencing, the recombinant plasmid pc DNA3.1(+)-G22 was stably transfected into CMT-93 cells, and the expression of G22 was detected by Western blot, flow cytometry and immunofluorescence staining. RESULTS: Restriction enzyme digestion and DNA sequencing showed that the recombinant plasmid contained the human nasopharyngeal carcinoma anti-idiotype antibody ScFv gene G22. Transfection experiment verified that G22 gene could be expressed in CMT-93 cells in the right way. CONCLUSION The eukaryotic expression vector containing the human nasopharyngeal carcinoma anti-idiotype antibody ScFv gene G22 is successfully constructed and expressed, which is the basis for further study of its DNA vaccine.
Antibodies, Anti-Idiotypic ; genetics ; Antibodies, Neoplasm ; genetics ; immunology ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; Humans ; Immunoglobulin Variable Region ; genetics ; Nasopharyngeal Neoplasms ; immunology ; Recombinant Proteins ; biosynthesis ; genetics

The V(H) and V(L) gene fragments of anti-CD28 mAb were combined to form anti-CD28 ScFv gene by using TP-PCR method. Sequence analysis showed that 6 x His tag was added to it for the ease of purification and the V(H), and V(L) gene fragments were connected by a linker containing 15 amino acids which are biased by the baculovirus promoter, ph. Then ScFv gene fragment was inserted into baculovirus transfer vector pBacPAK8. The recombinant transfer vector, pBacPAK8/CD28-ScFv was constructed successfully. The pBacPAK8/CD28-ScFv and the linear Bm-BacPAK6 were co-transfected into the cell line of Bombyx mori (BmN) with the help of Lipofectin,then the product was purified by plaque assay and identified by PCR method. The recombinant virus, Bm-BacPAK6 CD28-ScFv, was obtained successfully. The BmN cells and the larvae of Bombyx mori were infected by the recombinant baculovirus and harvested every 24h postinfection. SDS-PAGE and Western Blotting analysis confirmed the expression of ScFv with the molecular weight of about 28 kD. The expression in BmN cells was detected 24h post infection and it peaked at 72 h, while in the larvae of Bombyx mori, the expression was detected 48 h post infection and it peaked at 120 h.
Animals ; Antibodies, Anti-Idiotypic ; biosynthesis ; genetics ; Antibodies, Monoclonal ; immunology ; Baculoviridae ; genetics ; metabolism ; Bombyx ; cytology ; genetics ; metabolism ; CD28 Antigens ; genetics ; immunology ; Cell Line ; Humans ; Immunoglobulin Fab Fragments ; genetics ; immunology ; Immunoglobulin Variable Region ; genetics ; immunology ; Larva ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection

Human transferrin receptor (TfR) was isolated from homogenates of placental tissues by affinity chromatography on transferrin-Sepharose, and then used to screen human scFv against it from a fully-synthesized phage scFv library. After verifying the specificity, gene fragment of one of the selected scFv was inserted into the plasmid pET22b(+) and transformed into E. coli BL21(DE3) . Expression of scFv in transformant was induced with 0.5mmol/L IPTG. ELISA assay on HeLa cells showed that scFv protein could recognize and bind to TfR on the surface of HeLa cells. The scFv was purified by one-step affinity chromatography with Ni+ -NTA agarose, and injected into Kunming mouse via tail veins. This scFv was detected in brain tissues 1h later by capillary depletion method, which indicates that scFv protein can permeate through the blood brain barrier by mediation of the TfR receptor. Our works lay the foundation for the treatment of tumors and central nervous system diseases.
Animals ; Antibodies, Anti-Idiotypic ; genetics ; isolation & purification ; metabolism ; Escherichia coli ; genetics ; metabolism ; HeLa Cells ; Humans ; Immunoglobulin Fragments ; biosynthesis ; genetics ; immunology ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; immunology ; Mice ; Receptors, Transferrin ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; Transferrin ; metabolism

OBJECTIVETo study the regulation of Fc receptor expression by immune complexes (ICs) on neutrophils and U937 cells.

METHODSIgA ICs, IgG1 ICs, IgG2 ICs, IgG3 ICs, IgG4 ICs, and IgM ICs were incubated with neutrophils or U937 cells for 1 h. Then their surface Fc receptors were stained by anti-Fc gammaR I, anti-Fc gammaR II , anti-Fc gammaR III, and anti-Fc alphaR I monoclonal antibodies and analyzed by fluorescent activated cell sorting (FACS).

RESULTSIgG1 ICs and IgG3 ICs up-regulated Fc gammaR II and Fc gammaR III on U937 cells, Fc gammaR I and Fc alphaR I on neutrophils. Almost all ICs down-regulated Fc gammaR II on neutrophils.

CONCLUSIONSICs can regulate Fc receptor expression on neutrophils and U937 cells, among which IgG1 ICs and IgG3 ICs are most effective.

Antibodies, Anti-Idiotypic ; immunology ; Antibodies, Monoclonal ; pharmacology ; Antigen-Antibody Complex ; immunology ; metabolism ; Antigens, CD ; immunology ; Humans ; Immunoglobulin A ; classification ; immunology ; Immunoglobulin G ; chemistry ; classification ; immunology ; metabolism ; Neutrophils ; metabolism ; Receptors, Fc ; biosynthesis ; genetics ; Receptors, IgG ; immunology ; U937 Cells ; immunology