Monoclonal antibody (mAb) is an important biological macromolecule and widely used in immune detection, in vitro diagnostics, and drug discovery. However, the inherent properties of mAb restrict its further development, such as high molecular weight and complex structure. Therefore, there is an urgent need to develop alternatives for mAb. Various types of miniaturized antibodies have been developed, among which the variable domain of immunoglobulin new antigen receptor (VNAR) is very attractive. The shark single-domain antibody, also known as shark VNAR, is an antigen-binding domain obtained by genetic engineering technology based on the immunoglobulin new antigen receptor (IgNAR) that naturally exists in selachimorpha. It has a molecular weight of 12 kDa, which is the smallest antigen-binding domain found in the known vertebrates at present. Compared with mAb, the shark VNAR exhibits various superiorities, such as low molecular weight, high affinity, tolerance to the harsh environment, good water solubility, strong tissue penetration, and recognition of the hidden epitopes. It has attracted wide attention in the fields of immunochemical reagents and drug discovery. In this review, various aspects of shark VNAR are elaborated, including the structural and functional characteristics, generating and humanization techniques, affinity maturation strategies, application fields, advantages and disadvantages, and prospects.
Animals ; Antibodies, Monoclonal ; immunology ; Antibodies, Monoclonal, Humanized ; immunology ; Antigens ; Epitopes ; metabolism ; Protein Domains ; immunology ; Receptors, Antigen ; chemistry ; immunology ; Sharks

BACKGROUND: The interaction between killer immunoglobulin-like receptors (KIRs) and HLA class I regulates natural killer (NK) cell cytotoxicity and function. The impact of NK cell alloreactivity through KIR in liver transplantation remains unelucidated. Since the frequency of HLA-C and KIR genotypes show ethnic differences, we assessed the impact of HLA-C, KIR genotype, or KIR-ligand mismatch on the allograft outcome of Korean liver allografts. METHODS: One hundred eighty-two living donor liver transplant patients were studied. Thirty-five patients (19.2%) had biopsy-confirmed acute rejection (AR), and eighteen (9.9%) had graft failure. The HLA-C compatibility, KIR genotypes, ligand-ligand, and KIR-ligand matching was retrospectively investigated for association with allograft outcomes. RESULTS: Homozygous C1 ligands were predominant in both patients and donors, and frequency of the HLA-C2 allele in Koreans was lower than that in other ethnic groups. Despite the significantly lower frequency of the HLA-C2 genotype in Koreans, donors with at least one HLA-C2 allele showed higher rates of AR than donors with no HLA-C2 alleles (29.2% vs 15.7%, P=0.0423). Although KIR genotypes also showed ethnic differences, KIR genotypes and the number of activating KIR/inhibitory KIR were not associated with the allograft outcome. KIR-ligand mismatch was expected in 31.6% of Korean liver transplants and had no impact on AR or graft survival. CONCLUSIONS: This study could not confirm the clinical impact of KIR genotypes and KIR-ligand mismatch. However, we demonstrated that the presence of HLA-C2 allele in the donor influenced AR of Korean liver allografts.
Adult ; Alleles ; Asian Continental Ancestry Group/*genetics ; Female ; Genotype ; Graft Rejection ; Graft Survival ; HLA-C Antigens/*genetics ; Homozygote ; Humans ; Killer Cells, Natural/cytology/immunology ; Ligands ; *Liver Transplantation ; Male ; Middle Aged ; Proportional Hazards Models ; Receptors, KIR/chemistry/*genetics/metabolism ; Republic of Korea ; Tissue Donors ; Transplantation, Homologous

The levels of Streptococcus (S.) mutans infections in saliva were evaluated and a comparison for specific antibody levels among children with different levels of S. mutans infection was made. The promising epitopic regions of antigen AgI/II (PAc) and glucosyltransferase (GTF) for potential vaccine targets related to S. mutans adherence were screened. A total of 94 children aged 3-4 years were randomly selected, including 53 caries-negative and 41 caries-positive children. The values of S. mutans and those of salivary total secretory immunoglobulin A (sIgA), anti-PAc and anti-Glucan binding domain (anti-GLU) were compared to determine the correlation among them. It was found the level of s-IgA against specific antigens did not increase with increasing severity of S. mutans infection, and the complete amino acid sequence of PAc and GTFB was analyzed using the DNAStar Protean system for developing specific anti-caries vaccines related to S. mutans adherence. A significantly positive correlation between the amount of S. mutans and children decayed, missing, and filled teeth index was observed. No significant difference was detected in specific sIgA against PAc or GLU between any two groups. No significant correlation was found between such specific sIgA and caries index. A total of 16 peptides from PAc as well as 13 peptides from GTFB were chosen for further investigation. S. mutans colonization contributed to early children caries as an important etiological factor. The level of sIgA against specific antigens did not increase with increasing severity of S. mutans infection in children. The epitopes of PAc and GTF have been screened to develop the peptide-based or protein-based anti-caries vaccines.
Antibodies, Bacterial ; biosynthesis ; Antigens, Bacterial ; chemistry ; immunology ; Bacterial Proteins ; chemistry ; immunology ; Case-Control Studies ; Child, Preschool ; Dental Caries ; immunology ; pathology ; prevention & control ; Epitopes ; chemistry ; immunology ; Female ; Glucosyltransferases ; chemistry ; immunology ; Humans ; Immunoglobulin A, Secretory ; biosynthesis ; Male ; Peptides ; chemistry ; immunology ; Saliva ; chemistry ; microbiology ; Severity of Illness Index ; Streptococcal Vaccines ; biosynthesis ; chemistry ; immunology ; Streptococcus mutans ; chemistry ; immunology ; pathogenicity ; Vaccines, Subunit ; Virulence Factors ; chemistry ; immunology

Infection of schistosomiasis japonica may eventually lead to liver fibrosis, and no effective antifibrotic therapies are available but liver transplantation. Hedgehog (HH) signaling pathway has been involved in the process and is a promising target for treating liver fibrosis. This study aimed to explore the effects of pentoxifylline (PTX) on liver fibrosis induced by schistosoma japonicum infection by inhibiting the HH signaling pathway. Phorbol12-myristate13-acetate (PMA) was used to induce human acute mononuclear leukemia cells THP-1 to differentiate into macrophages. The THP-1-derived macrophages were stimulated by soluble egg antigen (SEA), and the culture supernatants were collected for detection of activation of macrophages. Cell Counting Kit-8 (CCK-8) was used to detect the cytotoxicity of the culture supernatant and PTX on the LX-2 cells. The LX-2 cells were administered with activated culture supernatant from macrophages and(or) PTX to detect the transforming growth factor-β gene expression. The mRNA expression of shh and gli-1, key parts in HH signaling pathway, was detected. The mRNA expression of shh and gli-1 was increased in LX-2 cells treated with activated macrophages-derived culture supernatant, suggesting HH signaling pathway may play a key role in the activation process of hepatic stellate cells (HSCs). The expression of these genes decreased in LX-2 cells co-cultured with both activated macrophages-derived culture supernatant and PTX, indicating PTX could suppress the activation process of HSCs. In conclusion, these data provide evidence that PTX prevents liver fibrogenesis in vitro by the suppression of HH signaling pathway.
Animals ; Antigens, Helminth ; isolation & purification ; pharmacology ; Cell Culture Techniques ; Cell Differentiation ; drug effects ; Cell Line ; Culture Media, Conditioned ; chemistry ; pharmacology ; Gene Expression Regulation ; Hedgehog Proteins ; agonists ; antagonists & inhibitors ; genetics ; immunology ; Hepatic Stellate Cells ; cytology ; drug effects ; metabolism ; Humans ; Liver Cirrhosis ; metabolism ; parasitology ; prevention & control ; Macrophage Activation ; drug effects ; Macrophages ; cytology ; drug effects ; immunology ; Models, Biological ; Monocytes ; cytology ; drug effects ; metabolism ; Pentoxifylline ; pharmacology ; Phosphodiesterase Inhibitors ; pharmacology ; RNA, Messenger ; genetics ; immunology ; Schistosoma japonicum ; chemistry ; Signal Transduction ; Tetradecanoylphorbol Acetate ; pharmacology ; Zinc Finger Protein GLI1 ; genetics ; immunology ; Zygote ; chemistry

Lipooligosacharide (LOS) of Neisseria gonorrhoeae (gonococci, GC) is involved in the interaction of GC with host cells. Deletion of the alpha-oligosaccharide (alpha-OS) moiety of LOS (lgtF mutant) significantly impairs invasion of GC into epithelial cell lines. GC opacity (Opa) proteins, such as OpaI, mediate phagocytosis and stimulate chemiluminescence responses in neutrophils in part through interaction with members of the carcinoembryonic antigen (CEA) family, which includes CEACAM3 (CD66d), a human neutrophil specific receptor for phagocytosis of bacteria. In the present work, we examined the effects of OpaI-expressing lgtF mutant on phagocytosis by HeLa-CEACAM3 cells and chemiluminescence responses in neutrophils. The results showed that lgtF mutant even expressing OpaI completely lost the ability to promote either phagocytosis mediated by CEACAM3 interaction in HeLa cells or chemiluminescence responses in neutrophils. These data indicated that Opa proteins in the lgtF mutant, which might result from the conformational change, cannot be functional.
Antigens, Bacterial ; chemistry ; genetics ; immunology ; metabolism ; Carbohydrate Sequence ; Carcinoembryonic Antigen ; genetics ; immunology ; Gene Expression Regulation ; HeLa Cells ; Host-Pathogen Interactions ; Humans ; Lipopolysaccharides ; chemistry ; immunology ; Luminescent Measurements ; Mutation ; Neisseria gonorrhoeae ; genetics ; metabolism ; pathogenicity ; Neutrophils ; immunology ; microbiology ; Phagocytosis

Alleles ; Crystallography, X-Ray ; HIV Infections ; HIV-1 ; HLA-B Antigens ; chemistry ; immunology ; Humans

The Rhei Radix et Rhizoma was one of the most widely used traditional Chinese medicine for its special biological activities. The content of rhein, one of its major compounds, was an important standard for the quantity control of Rhei Radix et Rhizoma. The major method used for the detection of rhein was instrumental analysis like HPLC, but it was complex, time-consuming and cannot detect large samples at the same time. The enzyme-linked imunmosorbent assay (ELISA) was accurate, reliable, simple, low costs, and of a high-throughout. Recently, it was widely used for the determination of those small molecule compounds in some traditional Chinese medicinal plants. In this study, an artificial antigen were synthesized by the carbodiimide (CDI) method. Rhein-bovine (rhein-BSA) conju gate and rhein-ovalbumin (rhein-OVA) conjugate, were produced as the immunogen and coating antigen, respectively. The conjugate and the hapten number in the conjugate were determined by UV-Vis spectrophotometry (UV). The conjugation ratio of Rhein and BSA was about 4.0:1, rhein acid and OVA was 2.6 : 1, respectively. Rhein-BSA conjugate was used to immunize Bal b/c mice to produce antiserum. The antiserum titer of the Rhein were higher than 8000 detected by ELISA. The successfully synthesized conjugate antigen rhein-BSA implies its feasibility in the establishment of fast immunoassay for the rhein content determination.
Animals ; Anthraquinones ; analysis ; immunology ; Antibodies ; analysis ; immunology ; Antigens, Plant ; analysis ; immunology ; Drugs, Chinese Herbal ; analysis ; Enzyme-Linked Immunosorbent Assay ; instrumentation ; methods ; Mice ; Mice, Inbred BALB C ; Rheum ; chemistry ; immunology ; Rhizome ; chemistry ; immunology

The method of monoclonal antibody-based immunoassay has a great importance in the study of quality control of traditional Chinese medicine (TCM) and detection of trace components in vivo animals. Synthesis of small molecule artificial antigen is the prerequisite for the establishment of this method. In present study, catalpol-BSA was synthesized by sodium periodate oxidation method. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry ( MALDI-TOF-MS) and molecular exclusion chromatography showed that catalpol was successfully conjugated with BSA. The mice could specifically produce anti-catalpol antibodies with titer up to 1:8000. The artificial antigen of catalpol was successfully synthesized.
Animals ; Antibodies ; immunology ; Antigens ; chemistry ; immunology ; Immunoassay ; Iridoid Glucosides ; chemistry ; immunology ; Male ; Medicine, Chinese Traditional ; Mice ; Mice, Inbred BALB C ; Serum Albumin, Bovine ; chemistry ; immunology

Proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. For detection of antigens from Haemaphysalis longicornis, 1-dimensional electrophoresis (1-DE) quantitative immunoblotting technique combined with 2-dimensional electrophoresis (2-DE) immunoblotting was used for whole body proteins from unfed and partially fed female ticks. Reactivity bands and 2-DE immunoblotting were performed following 2-DE electrophoresis to identify protein spots. The proteome of the partially fed female had a larger number of lower molecular weight proteins than that of the unfed female tick. The total number of detected spots was 818 for unfed and 670 for partially fed female ticks. The 2-DE immunoblotting identified 10 antigenic spots from unfed females and 8 antigenic spots from partially fed females. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF) of relevant spots identified calreticulin, putative secreted WC salivary protein, and a conserved hypothetical protein from the National Center for Biotechnology Information and Swiss Prot protein sequence databases. These findings indicate that most of the whole body components of these ticks are non-immunogenic. The data reported here will provide guidance in the identification of antigenic proteins to prevent infestation and diseases transmitted by H. longicornis.
Animals ; Antigens/*analysis/*immunology ; Arthropod Proteins/*analysis/*immunology ; Electrophoresis ; Immunoblotting ; Ixodidae/*chemistry ; Mass Screening ; *Proteomics ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. For detection of antigens from Haemaphysalis longicornis, 1-dimensional electrophoresis (1-DE) quantitative immunoblotting technique combined with 2-dimensional electrophoresis (2-DE) immunoblotting was used for whole body proteins from unfed and partially fed female ticks. Reactivity bands and 2-DE immunoblotting were performed following 2-DE electrophoresis to identify protein spots. The proteome of the partially fed female had a larger number of lower molecular weight proteins than that of the unfed female tick. The total number of detected spots was 818 for unfed and 670 for partially fed female ticks. The 2-DE immunoblotting identified 10 antigenic spots from unfed females and 8 antigenic spots from partially fed females. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF) of relevant spots identified calreticulin, putative secreted WC salivary protein, and a conserved hypothetical protein from the National Center for Biotechnology Information and Swiss Prot protein sequence databases. These findings indicate that most of the whole body components of these ticks are non-immunogenic. The data reported here will provide guidance in the identification of antigenic proteins to prevent infestation and diseases transmitted by H. longicornis.
Animals ; Antigens/*analysis/*immunology ; Arthropod Proteins/*analysis/*immunology ; Electrophoresis ; Immunoblotting ; Ixodidae/*chemistry ; Mass Screening ; *Proteomics ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization