BACKGROUNDWe previously reported that iodine-131((131)I)-labeled anti-pro-gastrin-releasing peptide (ProGRP(31-98)) monoclonal antibody D-D3 could selectively accumulate in the tumor sites of nude mice bearing small cell lung cancer (SCLC) xenografts. However, (131)I-D-D3 was cleared slowly from the body, and the best radioimmunoimaging time for SCLC was 72 - 96 hours after injection. The aims of this study were to radiolabel anti-ProGRP(31-98) D-D3 monoclonal antibody with technetium-99m ((99m)Tc) and to investigate the biodistribution of this antibody in healthy ICR mice.

METHODSD-D3 was labeled with (99m)Tc via the 2-mercaptoethanol reduction method. (99m)Tc-D-D3 was purified by the gel column separation method. The labeling efficiency and radiochemical purity were measured by thin-layer chromatography. The immunological activity of (99m)Tc-D-D3 was determined with cell conjugation assays. (99m)Tc-D-D3 was injected into healthy ICR mice via a tail vein, and all the healthy ICR mice were sacrificed by cervical dislocation at a designated time. Then, the blood and major organs were removed and weighed, and counted in a gamma scintillation counter to determine the percentage of the injected dose per gram (%ID/g).

RESULTSThe labeling rate and the radiochemical purity of (99m)Tc-D-D3 were (73.87 ± 2.89)% and (94.13 ± 4.49)%, respectively. The immunobinding rates of (99m)Tc-D-D3 to the human small cell lung cancer NCI-H446 cell line and lung adenocarcinoma A549 cell line were (81.2 ± 2.37)% and (24.3 ± 1.46)%, respectively. The distribution data of normal ICR mice demonstrated that (99m)Tc-D-D3 was mainly distributed in the liver, kidney and lung, and less in the brain tissue and muscle.

CONCLUSIONS(99m)Tc-D-D3 antibody not only had high radiochemical purity, but also had good stability both in vitro and in vivo, and maintained good immunological activity. (99m)Tc-D-D3 was metabolized mainly in the kidney and liver, and the blood radioactivity decreased rapidly. Thus, (99m)Tc-D-D3 is conducive to the radioimmunoimaging of SCLC.

Animals ; Antibodies, Monoclonal ; chemistry ; immunology ; metabolism ; Female ; Male ; Mice ; Mice, Inbred ICR ; Peptide Fragments ; immunology ; Recombinant Proteins ; immunology ; Technetium ; chemistry

As the dominant antigens, early secreted antigenic target 6 (ESAT6, E6) and culture filtrate protein 10 (CFP10, C10) had once been the focus of tuberculosis (TB) vaccine due to their capability of inducing strong cell immune response in the host. They are also endowed with promising future of prevention against and diagnosis of TB. In this review, we systematically introduce recent research progress of E6 and C10, especially in structure-function, biological characteristics, protein expression and secretion, host immunity and vaccine development, and the prospects of their application are also discussed.
Antigens, Bacterial ; chemistry ; genetics ; immunology ; Bacterial Proteins ; chemistry ; genetics ; immunology ; Humans ; Immunodominant Epitopes ; immunology ; Molecular Biology ; Peptide Fragments ; chemistry ; genetics ; immunology ; Tuberculosis Vaccines ; genetics ; immunology ; Vaccines, DNA ; immunology

BACKGROUNDTo characterize the intertype epitopes on human adenovirus (HAdV) hexon.

METHODSBased on computerized analysis on adenoviruses sequence of genomic alignment, antigenicity prediction and 3-D structure characteristics of hexon subunit, several peptides of hexon of adenoviruses were chosen to be synthesized or recombinant proteins of the hexon were expressed in E. coli by use of PGEX-5X. To identify the existence of intertype epitopes, the antisera raised with synthetic peptides or purified recombinant proteins were analyzed with Western blot and immunofluorescent assay.

RESULTSThe results of Western blot indicated that both peptide and recombinant antibodies showed specific reactivities with hexons of HADv-3, 4, 7 individually. Meanwhile, typical stain of immunofluorescence was found on HeLa cells infected with these HAdV by incubation with peptide as well as recombinant antibodies. Also, antibodies raised against peptide recognized the recombinant hexon protein in which a corresponding region of peptides was covered.

CONCLUSIONSMost of the predicted intertype epitopes of HAdV hexon wer e exclusively found in synthetic peptides and recombinant proteins. These intertype epitopes showed to be continuous and sequential which could be employed for development of antibodies of diagnostic use.

Adenoviruses, Human ; immunology ; Amino Acid Sequence ; Animals ; Capsid ; chemistry ; immunology ; Capsid Proteins ; immunology ; Epitopes ; immunology ; HeLa Cells ; Humans ; Mice ; Peptide Fragments ; immunology

CD99 plays an critical role in the diapedesis of monocytes, T cell differentiation, and the transport of MHC molecules. Engagement of CD99 by agonistic monoclonal antibodies has been reported to trigger multifactorial events including T cell activation as well as cell-cell adhesion during hematopoietic cell differentiation. In this study, to identify the functional domains participating in the cellular events, we mapped the epitopes of CD99, which are recognized by two agonistic CD99 monoclonal antibodies, DN16 and YG32. Using recombinant fusion proteins of GST with whole or parts of CD99, we found that both antibodies interact with CD99 molecules independently of sugar moieties. DN16 mAb detected a linear epitope located in the amino terminal region of CD99 while YG32 mAb bound another linear epitope in the center of the extracellular domain. To confirm that the identified epitopes of CD99 are actually recognized by the two mAbs, we showed the presence of physical interaction between the mAbs and the fusion proteins or synthetic peptides containing the corresponding epitopes using surface plasmon resonance analyses. The dissociation constants of DN16 and YG32 mAbs for the antigen were calculated as 1.27 X 10(-7) and 7.08 X 10(-9) M, respectively. These studies will help understand the functional domains and the subsequent signaling mechanism of CD99.
Amino Acid Sequence ; Antibodies, Monoclonal/*immunology ; Antigens, CD/*chemistry/*immunology ; Blotting, Western ; Cell Adhesion Molecules/*chemistry/*immunology ; *Epitope Mapping ; Epitopes/*chemistry/*immunology ; Glutathione Transferase ; Human ; Molecular Sequence Data ; Peptide Fragments/chemistry/immunology ; Recombinant Fusion Proteins/chemistry/immunology

The nucleocapsid protein (N protein) has been found to be an antigenic protein in a number of coronaviruses. Whether the N protein in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is antigenic remains to be elucidated. Using Western blot and Enzyme-linked Immunosorbent Assay (ELISA), the recombinant N proteins and the synthesized peptides derived from the N protein were screened in sera from SARS patients. All patient sera in this study displayed strong positive immunoreactivities against the recombinant N proteins, whereas normal sera gave negative immunoresponses to these proteins, indicating that the N protein of SARS-CoV is an antigenic protein. Furthermore, the epitope sites in the N protein were determined by competition experiments, in which the recombinant proteins or the synthesized peptides competed against the SARS-CoV proteins to bind to the antibodies raised in SARS sera. One epitope site located at the C-terminus was confirmed as the most antigenic region in this protein. A detailed screening of peptide with ELISA demonstrated that the amino sequence from Codons 371 to 407 was the epitope site at the C-terminus of the N protein. Understanding of the epitope sites could be very significant for developing an effective diagnostic approach to SARS.
Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Epitopes ; chemistry ; immunology ; Humans ; Nucleocapsid Proteins ; chemistry ; immunology ; Peptide Fragments ; chemical synthesis ; Plasmids ; Recombinant Proteins ; immunology ; isolation & purification ; metabolism ; SARS Virus ; genetics ; immunology ; metabolism

The N-terminal fragment of prohormone brain natriuretic peptide (NT-proBNP) is a commonly used biomarker for the diagnosis of congestive heart failure, although its biological function is not well known. NT-proBNP exhibits heavy O-linked glycosylation, and it is quite difficult to develop an antibody that exhibits glycosylation-independent binding. We developed an antibody that binds to the recombinant NT-proBNP protein and its deglycosylated form with similar affinities in an enzyme immunoassay. The epitope was defined as Gly63-Lys68 based on mimetic peptide screening, site-directed mutagenesis and a competition assay with a peptide mimotope. The nearest O-glycosylation residues are Thr58 and Thr71; therefore, four amino acid residues intervene between the epitope and those residues in both directions. In conclusion, we report that an antibody reactive to Gly63-Lys68 of NT-proBNP exhibits O-glycosylation-independent binding.
Amino Acid Sequence ; Animals ; Antibodies/*immunology ; Antigen-Antibody Reactions ; Epitope Mapping ; Epitopes/chemistry/genetics/*immunology ; Glycosylation ; HEK293 Cells ; Heart Failure/immunology ; Humans ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Natriuretic Peptide, Brain/chemistry/genetics/*immunology ; Peptide Fragments/chemistry/genetics/*immunology ; Rabbits ; Recombinant Fusion Proteins/chemistry/genetics/immunology

OBJECTIVETo investigate the pathogenesis of cytotoxic T cell (CTL) dysfunction in patients with HCV infection.

METHODSBALB/c mice were immunized by subcutaneous injection of polypeptides from HCV core region, and the CTL activity of mouse spleen cells was detected by the LDH release test. Two polypeptides which can enhance CTL function and two polypeptides which can inhibit CTL function were selected and cross-combined. BALB/c mice were immunized using the combined polypeptides and the CTL activities were detected afterwards.

RESULTSCTL activity was inhibited by CPA9 (39-74 amino acids), CPB7 (67-76 amino acids) and CPB8 (71-80 amino acids), and promoted by CPA10 (5-23 amino acids), CPB6 (63-72 amino acids) and CPB2 (131-140 amino acids). Using single factor analysis of variance, the CTL activity in the mice could be enhanced by polypeptides from the HCV core region, CPB2+CPB8, CPB6+CPB8, respectively. There was no obvious difference between CPB2+CPB7, CPB6+CPB7 and negative control.

CONCLUSIONSCPA9, CPB7, and CPB8, the 3 polypeptides from HCV core region play an inhibition role and CPA10, CPB6, and CPB2 play an enhancement role in CTL activity in mice. The inhibition and enhancement functions of the polypeptides from HCV core region interact each other.

Animals ; Cytotoxicity Tests, Immunologic ; Cytotoxicity, Immunologic ; drug effects ; Mice ; Mice, Inbred BALB C ; Peptide Fragments ; administration & dosage ; immunology ; Spleen ; cytology ; drug effects ; immunology ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; Viral Core Proteins ; administration & dosage ; chemistry ; immunology

AIMTo establish methods and requirements for the quality control of recombinant human neu epitope peptide 12.

METHODSBiological activity of recombinant human neu epitope peptide 12 was evaluated in FVB/N transgenic mice (TgN MMTV neu 202 Mul, Jackson Lab., USA) administered with samples. The percentage of antibody-positive mice detected by ELISA was used in the biological activity evaluation. The peptide map was performed by peptic digestion. The antigen content was determined by SEC-HPLC.

RESULTS AND CONCLUSIONThe quality control methods, such as biological activity, peptide map, antigen content, and the requirements for the quality control of recombinant human neu epitope peptide 12 were established. The established methods and requirements were already used for the quality control of recombinant human neu epitope peptide 12 products.

Animals ; Antibodies, Monoclonal ; analysis ; Biotechnology ; methods ; Epitopes ; analysis ; chemistry ; immunology ; Female ; Genes, erbB-2 ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Transgenic ; Peptide Fragments ; chemistry ; immunology ; Peptide Mapping ; Quality Control ; Receptor, ErbB-2 ; analysis ; chemistry ; immunology ; Recombinant Fusion Proteins ; analysis ; chemistry ; immunology ; Technology, Pharmaceutical ; standards

Calreticulin (CRT) is a multifunctional molecule in both intracellular and extracellular environment. We have previously found that a recombinant CRT fragment (rCRT/39-272) could modulate T cell-mediated immunity in mice via activation and expansion of CD1d(hi)CD5⁺ B cells as well as induction of CRT-specific regulatory antibodies. Antibody secreting cells (ASCs) are terminally differentiated B cells responsible for producing antibodies to participate in positive immune response as well as immune regulation. In this study, we demonstrate that rCRT/39-272 differentiates murine CD1d(hi)CD5⁺ B cells into ASCs marked by increased expression of plasma cell-associated transcription factors and production of polyreactive antibodies against DNA and CRT in vitro. Intraperitoneal administration of rCRT/39-272 augmented differentiation of CD1d(hi)CD5⁺ B cells into ASCs in naïve mice or mice with experimental autoimmune encephalomyelitis. Thus, we propose that ASC differentiation and subsequent antibody production of CD1d(hi)CD5⁺ B cells are key steps in CRT-mediated immunoregulation on inflammatory T cell responses.
Animals ; Antigens, CD1d ; metabolism ; Autoantibodies ; biosynthesis ; B-Lymphocytes ; cytology ; drug effects ; immunology ; metabolism ; CD5 Antigens ; metabolism ; Calreticulin ; chemistry ; Cell Differentiation ; drug effects ; Encephalomyelitis, Autoimmune, Experimental ; immunology ; Humans ; Mice ; Peptide Fragments ; chemistry ; pharmacology ; Solubility

Prokaryotic expression plasmids carrying N-terminal(1-163aa) and C-terminal(141-306aa) gene of HCoV-NL63 nucleocapsid protein were constructed with pET-30a(+) vector. Consequently, we prepared two purified proteins, Np and Cp, respectively, and established a Western blotting-based line assay (WBLA) for detection of antibodies against HCoV-NL63 using three purified proteins: Np , Cp and Nf, a full-length HCoV-NL63 nucleocapsid protein as previously reported. We detected anti-HCoV-NL63 antibodies among 50 sera samples collected from adult for health-examination by WBLA. The results showed that: 25 (50%), 27 (54%), 36 (72%) of 50 sera were indentified as anti-HCoV-NL63 antibody positive when the antigen was from Nf, Np and Cp, respectively. Among these sera with positive anti-HCoV-NL63 antibody,Cp showed highest antibody positive rate in WBLA,and consistent rates of detection were 64% between Nf and Np, 54% between Nf and Cp, 54% between Np and Cp. Our study provides the foundation for development of HCoV-NL63 serological detection reagents and an experimental tool for immunological research of HCoV-NL63 infection.
Adult ; Antibodies, Viral ; blood ; Blotting, Western ; Coronavirus ; chemistry ; immunology ; Humans ; Nucleocapsid Proteins ; genetics ; immunology ; isolation & purification ; Peptide Fragments ; genetics ; isolation & purification ; Recombinant Proteins ; biosynthesis ; immunology ; isolation & purification ; Serologic Tests