A combined phage display ScFv library against Myxobolus rotundus infecting crucian carp, Carassius auratus auratus (L.), in China

Immunological methods have been developed for the diagnosis of Myxobolus rotundus but their use has been limited for the prevention and therapy of this serious parasitic pathogen. Phage display antibody libraries are a powerful technique for the development of antibodies to molecules of interest and have advantages over traditional hybridroma approaches. In the present study, four antigen fractions related to M. rotundus were prepared and a combined phage display single-chain antibody fragments (ScFv) library was constructed against this parasite. Preliminary analysis indicated that a combined antibody library of about 2.08 X 10(5) individual clones and high diversity was generated. After four rounds of screening (bio-panning) against soluble spore protein prepared from lysed, intact, mature M rotundus spores, a strain monoclonal phage display ScFv, termed pCAN-6H9, with better affinity, was isolated. The pCAN-6H9 gene fragment was sequenced and analysed. The specificity of pCAN-6H9 was further demonstrated by dot-blot. In competition enzyme-linked immunosorbent assay, both the original and enriched phage-displayed ScFv repertoire showed significant inhibition of mouse anti-M rotundus serum binding to coated antigen, while the inhibition rate of monoclonal pCAN-6H9 phage particles was only 11.83%.

Use of nitrocellulose films for affinity-directed mass spectrometry for the analysis of antibody/antigen interactions

Combination of affinity extraction procedures with mass spectrometric analyses is termed affinity-directed mass spectrometry, a technique that has gained broad interest in immunology and is extended here with several improvements from methods used in previous studies. A monoclonal antibody was immobilized on a nitrocellulose (NC) membrane, allowing the corresponding antigen to be selectively captured from a complex solution for analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). This method was also used to rapidly determine the approximate binding region responsible for the antibody/antigen interaction. The tryptic fragments of antigen protein in buffer were applied to the antibody immobilized on NC film and allowed to interact. The NC film was then washed to remove salts and other unbound components, and subjected to analysis by MALDI-TOFMS. Using interferon-alpha (2a) and anti-interferon-alpha (2a) monoclonal antibody IgG as a model system, we successfully extracted the antigen protein and determined the approximate binding region for the antigen/antibody interaction (i.e., the tryptic fragment responsible). Copyright (C) 2001 John Wiley & Sons, Ltd.

Study of Interaction Force between Antigen and Antibody Using Flow Chamber Method

A parallel plate flow chamber was used to study the interaction force between human IgG (immobilized on a chip surface as ligand) and goat anti-human IgG (immobilized on microspheres surface as receptor). First, it was demonstrated that the binding force between the microspheres and the chip surface came from the bio-specific interaction between the antigen and the antibody. Secondly, it was obtained that the critical shear rate to detach microspheres from the chip surface increases with the ligand surface concentration. Finally, two models to estimate the antigen-antibody bond strength considering bonds' positions were proposed and analyzed.

Visualization of molecule interaction between antigen and antibody - One of ellipsometric imaging applications

It is to investigate molecule interactions between antigen and antibody with ellipsometric imaging technique and demonstrate some features and possibilities offered by applications of the technique. Molecule interaction is an important interest for molecule biologist and immunologist. They have used some established methods such as immufluorcence, radioimmunoassay and surface plasma resonance, etc, to study the molecule interaction. At the same time, experimentalists hope to use some updated technique with more direct visual results. Ellipsometric imaging is non-destructive and exhibits a high sensitivity to phase transitions with thin layers. It is capable of imaging local variations in the optical properties such as thickness due to the presence of different surface concentration of molecule or different deposited molecules. If a molecular mono-layer (such as antigen) with bio-activity were deposited on a surface to form a sensing surface and then incubated in a solution with other molecules (such as antibody), a variation of the layer thickness when the molecules on the sensing surface reacted with the others in the solution could be observed with ellipsometric imaging. Every point on the surface was measured at the same time with a high sensitivity to distinguish the variation between mono-layer and molecular complexes. Ellipsometric imaging is based on conventional ellipsometry with charge coupled device (CCD) as detector and images are caught with computer with image processing technique. It has advantages of high sensitivity to thickness variation (resolution in the order of angstrom), big field of view (in square centimeter), high sampling speed (a picture taken within one second), and high lateral resolution (in the order of micrometer). Here it has just shown one application in study of antigen-antibody interaction, and it is possible to observe molecule interaction process with an in-situ technique.

Molecular Modeling and Affinity Determination of scFv Antibody: Proper Linker Peptide Enhances Its Activity

One of existing strategies to engineer active antibody is to link VH and VL domains via a linker peptide. How the composition, length, and conformation of the linker affect antibody activity, however, remains poorly understood. In this study, a dual approach that coordinates molecule modeling, biological measurements, and affinity evaluation was developed to quantify the binding activity of a novel stable miniaturized anti-CD20 antibody or singlechain fragment variable (scFv) with a linker peptide. Upon computer-guided homology modeling, distance geometry analysis, and molecular superimposition and optimization, three new linker peptides PT1, PT2, and PT3 with respective 7, 10, and 15 residues were proposed and three engineered antibodies were then constructed by linking the cloned VH and VL domains and fusing to a derivative of human IgG1. The binding stability and activity of scFv-Fc chimera to CD20 antigen was quantified using a micropipette adhesion frequency assay and a Scatchard analysis. Our data indicated that the binding affinity was similar for the chimera with PT2 or PT3 and ~24-fold higher than that for the chimera with PT1, supporting theoretical predictions in molecular modeling. These results further the understanding in the impact of linker peptide on antibody structure and activity.

The role of anti-non-Gal antibodies in the development of acute humoral xenograft rejection of hDAF transgenic porcine kidneys in baboons receiving anti-Gal antibody neutralization therapy

Background. The present study was undertaken to determine the role of preformed and induced anti-non-Gal antibodies in the rejection of hDAF pig-to-baboon kidney xenotransplants after anti-Gal antibody neutralization therapy. Methods. Seven baboons receiv

SECRETORY MONOCLONAL IGA ANTIBODY TO HUMAN SPERM PRODUCED BY GASTROINTESTINAL IMMUNIZATION INHIBITS HUMAN SPERM ACTIVITY AND MOUSE INVITRO FERTILIZATION

BALB/c mice were immunized intragastrically with human sperm. Cells from the Peyer's patches and spleens of the immunized mice were for the preparation of hybridomas secreting antisperm monoclonal IgA (mcIgA). The specific ratio of IgA-secreting cells in Peyer's patches was much higher than that in spleen. The binding site on human sperm of 9 of 19 mcIgA was in the post-acrosomal region using an immunofluorescent assay. Two of eight selected mcIgA caused strong human sperm agglutination and three of them produced significant inhibition of mouse in vitro fertilization. No mcIgA tested caused obvious human sperm immobilization or inhibited mouse in vivo fertilization. In vitro assembly of selected mcIgA in ascites with mouse secretory component (SC) caused no significant changes in effects on sperm function and in vitro fertilization. By use of Western blotting, dimer or higher polymers were demonstrated in all selected mcIgAs and corresponding protein antigens in 6 of 8 selected mcIgAs. These results suggest that human sperm function may be inhibited and fertilization rate reduced by specific secretory IgA to human sperm and that secretory immunity to protein antigens of human sperm could be induced by intragastrointestinal immunization.

Migration of mouse antibody-secreting hybridoma cells from blood to genital tract and its regulation by sex hormones are associated with the differential expression patterns of adhesion molecules and chemokines in the tract rather than in the antibody-secreting cells

To understand better the molecular mechanisms of differential migration of antibody-secreting cells (ASCs) into mouse genital tracts, and regulation by sex hormones, surface markers, hormone receptors and adhesion molecules in mouse SG2 and PA4 hybridoma cells, respectively, secreting IgG2b and polymeric IgA antibody were detected by flow cytometry or RT-PCR. Semiquantitative RT-PCR was also used for measuring mRNA expression of adhesion molecules and chemokines (VCAM-1, ICAM-1, P-selectin, JAM-1 and CXCL12) in genital tracts of various adult mouse groups. The mRNAs of androgen receptor, estrogen receptor beta and CXCR4 were expressed in the ASCs. Sex hormones had no effect on expression of these molecules in ASCs. Except for VCAM-1, mRNA of all examined genes was expressed in normal mouse genital tracts. The mean of relative amounts of ICAM-1 and CXCL12 mRNA in all examined organs of females were higher (2.1- and 1.9-fold) than those in males. After orchiectomy or ovariectomy, the expression of ICAM-1, CXCL12 and P-selectin mRNA in the examined organs increased, except JAM-1 in male and CXCL12 in female. Sex hormone treatment recovered the changes to normal levels of mRNA expression in many examined genital tissues. In combination with our previous work, preferential migration of ASCs into female genital tract and regulation of migration by sex hormones are associated with expression patterns of adhesion molecules and chemokines in genital tract rather than in ASCs. (C) 2006 Elsevier Ireland Ltd. All rights reserved.

Detection of serum and mucosal antibody production and antibody secreting cells (ASCs) in large yellow croaker (Pseudosciaena crocea) following vaccination with Vibrio harveyi via different routes

The kinetics of mucosal and serum antibody response is well as antibody secreting cells (ASCs) production were studied in large yellow croaker following vaccination with inactivated Vibrio harveyi by different routes: oral administration. intraperitoneal (IP) injection and immersion. Indirect ELISA was used to measure the antibody level in serum and cutaneous mucus, and ELISPOT was used to monitor the ASCs derived from gill, blood and head kidney. The data demonstrated that IP injection resulted in the highest antibody levels in the systemic circulation, whereas immersion induced significant antibody levels in mucous. As for the ASCs response, IP injection induced high numbers of ASCs in the head kidney and blood; oral intubation only induced a slight ASCs response in the head kidney: immersion induced a much stronger ASCs response in the gill. These results indicate that mucosal antibodies following immersion immunization are independent of a systemic response and more sensitive, since it could be triggered earlier than serum antibodies. The mucosal antibodies following IP injection immunization may depend oil a systemic immune response. The protective effects of the three vaccination methods were compared by challenging with live V. harveyi. Survival of the three groups of vaccinated fish varied front 40 to 60%. while 100% mortality was found in control fish. Compared with IP and oral vaccination, immersion stimulated higher specific antibody titers in the mucosal system and achieved similar protection, so it is in effective and efficient method for immunizing a large number of fish against V harveyi (C) 2008 Elsevier B.V. All rights reserved.

Identification of a WSSV neutralizing scFv antibody by phage display technology and in vitro screening

White spot syndrome virus (WSSV) is one of the most significant viral pathogens causing high mortality and economic damage in shrimp aquaculture. Although intensive efforts were undertaken to detect and characterize WSSV infection in shrimp during the last decade, we still lack methods either to prevent or cure white spot disease. Most of the studies on neutralizing antibodies from sera have been performed using in vivo assays. For the first time, we report use of an in vitro screening method to obtain a neutralizing scFv antibody against WSSV from a previously constructed anti-WSSV single chain fragment variable region (scFv) antibody phage display library. From clones that were positive for WSSV by ELISA, 1 neutralizing scFv antibody was identified using an in vitro screening method based on shrimp primary lymphoid cell cultures. The availability of a neutralizing antibody against the virus should accelerate identification of infection-related genes and the host cell receptor, and may also enable new approaches to the prevention and cure of white spot disease.

Isolation and identification of a novel WSSV nucleocapsid protein by cDNA phage display using an scFv antibody

In a previous study, a scFv phage display library against white spot syndrome virus (WSSV) was constructed and yielded a clone designated A I with conformational specificity against native but not denatured viral antigen. Although the clone A1 has been used successfully as a diagnostic antibody, its precise target antigen has not been elucidated. A different strategy was adopted involving the construction of a second T7 phage display library utilizing mRNA isolated from shrimp infected with WSSV. Following RT-PCR and T7 phage library construction, phages displaying the candidate epitope were selected with A I scFv. Since successive enrichment steps were not associated with an increased titer of the phages, enrichment after successive tests was confirmed by PCR resulting in the prefer-red selection of a specific DNA sequence encoding a novel nucleocapsid protein WSSV388. Immune electron microscopy revealed that WSSV388 is located on the nucleocapsid. This result demonstrated that unknown antigen could be identified by phage display using the epitope conformation dependent scFv. (c) 2006 Elsevier B.V. All rights reserved.

Effects of anti-bursin monoclonal antibody on immunosuppression in the duck (Cherry Valley duck)

To study the immunologic function of bursin, we analyzed the effects of anti-bursin monoclonal antibody (mAb) on the immunosuppression in ducks (Cherry Valley duck) by injecting various doses of the anti-bursin mAb into 13-d duck embryos. After hatch, cell-mediated immune activity and humoral responses were studied using lymphocyte proliferation test, tube agglutination test, and indirect enzyme-linked immuno-sorbent assay to detect anti-Escherichia coli antibodies and antibodies to Riemerella anatipester, respectively. Simultaneously, relative weights (BW-adjusted) of bursa of Fabricius (BF), spleen, and thymus were determined. Additionally, the morphology of BF, spleen, and thymus was examined at various ages using conventional histology. Follicle morphology of BF was analyzed by image analysis. The results indicated that anti-bursin mAb markedly decreased duck lymphocyte proliferation, the antibody-producing ability to bacteria, as well as the relative BF weight. Moreover, the anti-bursin mAb hindered the development of BF follicles.

Molecular cytogenetic detection of paternal chromosome fragments in allogynogenetic gibel carp, Carassius auratus gibelio Bloch

In gynogenesis, sperm from related species activates egg and embryonic development, but normally does not contribute genetically to the offspring. In gibel carp, Carassius auratus gibelio Bloch, however, gynogenetic offspring often show some phenotypes apparently derived from the heterologous sperm donor. This paternal effect of allogynogenesis is outstanding in an artificial clone F produced by cold treatment of clone E eggs after insemination with blunt-nose black bream (Megaloabrama amblycephala Yin) sperm. Karyotype analysis revealed 5-15 supernumerary microchromosomes in different individuals of clone F in addition to 156 normal chromosomes inherited from the maternal clone E. A painting probe was prepared from the microdissected microchromosomes, and used to investigate the origin of these microchromosomes. Strong positive signals were detected on each microchromosomes of clone F and on 4 pairs of chromosomes in blunt-nose black bream, whereas no signals were detected on the chromosomes of clone E. This result indicates that some paternal chromosome fragments of blunt-nose black bream have been incorporated into the artificial clone F. Therefore, the manipulation of allogynogenesis may provide a unique method to transfer DNA between diverse species for fish breeding.

Construction and characterization of a novel recombinant single-chain variable fragment antibody against White Spot Syndrome Virus from shrimp

An antibody phage display library against White Spot Syndrome Virus (WSSV) was constructed. After four rounds of panning against WSSV, 192 out of 480 clones displayed WSSV binding activity. One of the positive clones, designated A1, had relatively higher activity specifically binding to WSSV A1-soluble, single-chain fragment variable (scFv) antibody has an affinity constant (K-aff) of 2.02 +/- 0.42 x 10(9) M-1. Dot blot assays showed that A1-soluble scFv could detect WSSV directly from shrimp hemolymph after 24-h feeding infection by WSSV. A1 scFv has potential for the development of a cheap, simple and sensitive diagnostic kit for WSSV in the field. (C) 2003 Elsevier Science B.V. All rights reserved.