Development of monoclonal antibodies against bovine mucin core 2 β6 N-acetylglucosaminyltransferase

Molecular cloning techniques have been used to produce abundant amounts of recombinant glycosyltransferases for biochemical studies. We recently cloned a cDNA which encoded bovine mucin core 2 6N-acetylglucosaminyl transferase (C2TF). Poly-histidine-C2TF fusion protein was generated from the cloned cDNA in the E. coli Xpress system and used to produce monoclonal antibodies (MAbs). We obtained seven hybridomas which secreted MAbs against bovine C2TF in mouse ascites with titers ranging from 1:1280 to 1:40960 as assessed by immunofluorescence assay (IF). Isotyping revealed that all seven MAbs were IgG (4 IgG1, 2 IgG2b and 1 IgG2a). The affinity constants (M^–2) for these MAbs range from 5.4 × 10⁷ to 1.2 × 10⁹. These MAbs recognized bovine C2TF in tissue sections and on Western blottings. Six of these MAbs reacted with human core 2-M enzyme and one with both core 2-L and core 2-M enzymes on Western blottings. Therefore, These antibodies should be useful for further study of bovine and human core 2 enzymes.     

T-cell receptor-specific monoclonal antibodies against a V β 11-positive mouse T-cell clone

Two monoclonal antibodies specific for the mouse T-cell receptor (Tcr) have been established by immunization with a V 11⁺ T-cell clone, clone C6. One is a rat antibody, KT11 (IgG2b, k), specific for the V chain of C6, V 11. This was demonstrated by the fact that the strain distribution pattern of KT11⁺ cells was similar to that of V 5, 8, 9, 11, 12, and 13 and that the gene that encodes the molecule detected by KT11 was closely linked to V 8 in (B10 × SJL)F₁ × SJL backcross mice. Furthermore, V of C6 has been cloned from a gt10 cDNA library and was demonstrated to be identical to the V 11 published sequences. All strains of mice that do not express major histocompatibility complex class II E molecules had higher numbers of KT11^– cells than E⁺ strains. The KT11⁺ population in A strain mice and its H-2 congenic strains, however, was not affected by the presence or absence of E molecules. The other is a mouse antibody, KTL2 (IgM), specific for the idiotope of the Tcr expressed on the clone C6. Both antibodies were mitogenic and induced cytotoxicity. Expression of epitopes detected by KT11 or KTL2 was down-modulated by a T3-specific antibody 145-2C11.     

Monoclonal antibodies against plasma protease inhibitors: II. Production and characterization of 25 monoclonal antibodies against human α1-antitrypsin. Correlation between antigenic structure and functional sites

Twenty-five hybridomas secreting monoclonal antibodies against human ₁-antitrypsim have been produced by the cell-fusion techmque (Köhler and Milstein, 1976). All antibodies are specific for ₁-antitrypsim and carry ₁-antitrypsim heavy chains and light chains. Inhibition experiments showed that these monoclonal antibodies define three independent antigenic regions on the ₁-antitrypsim molecule; one of these domains appears to be involved in the interaction between ₁-antitrypsim and trypsin. In addition, one monoclonal antibody, AATY39, was used to develop an enzyme-linked immunosorbent assay capable of detecting low levels of ₁-antitrypsim in the range of 1 to 2 ng/ml.     

High prevalence of antibodies against hepatitis A virus among captive nonhuman primates

Hepatitis A virus (HAV) can infect not only humans but also several other nonhuman primates. This study has been conducted to evaluate the comprehensive anti-HAV seroprevalence in captive nonhuman primate populations in Thailand. The prevalence of antibodies against HAV in 96 captive nonhuman primates of 11 species was evaluated by competitive enzyme immunoassay (EIA). HAV antibodies were found in 64.7% (11/17) of macaques, 85.7% (6/7) of langurs, 28.4% (10/35) of gibbons, and 94.6% (35/37) of orangutans. However, anti-HAV IgM was not found in any sera. These results indicate that the majority of captive nonhuman primates in Thailand were exposed to HAV. It is possible that some of the animals were infected prior to capture.     

Generation of mouse–human hybridomas secreting antibodies against peanut allergen Ara h1

Two clones of mouse–human hybridomas, secreting human monoclonal antibodies to a peanut allergen Ara h1, were generated from human peripheral blood lymphocytes transformed with Epstein--Barr virus, followed by cell fusion with mouse myeloma cells. Epitope analysis with overlapping peptides synthesized on a multi-pin apparatus revealed antibody-binding sequences of Ara h1 protein.     

Effects of monoclonal antibodies on α-staphylotoxin action against erythrocytes and model phospholipid membranes

It was found that one of twenty tested monoclonal antibodies (MABs) existed which drastically enhanced ability of Staphylococcus aureus α-tosin (ST) to both lysis of human erythrocytes and increase of planar phospholipid bilayer conductance more than 10 and 1000 times respectively. Other 19 MABs possessed only neutralized effect. The activation could only be observed if the activating MAB (AMAB) interacted with ST in solution but not in membrane. The one molecule of AMAB was able to activate approximately 2–4 molecules of ST. It was assumed that this activation was a result of the AMAB-induced transition of ST from a hydrophilic to an amphiphilic form. The activation could not be observed when the activity of AMAB/ST mixtures was tested on highly sensitive rabbit erythrocytes. All the tested MABs (including AMAB) were able to inhibit the ST-induced lysis of rabbit erythrocytes. The activating effects of AMAB on ST action in BLM and in human erythrocytes as well as their inhibiting influence on the ability of toxin to cause a lysis of rabbit erythrocytes indicate the presence of an ST-specific receptor on the membrane of rabbit erythrocytes.     

Phage antibodies: will new 'coliclonal' antibodies replace monoclonal antibodies?

Antibodies can now be rapidly isolated from large and diverse recombinant libraries by displaying functional antibody fragments on the surface of bacteriophage particles and directly selecting with antigen. This method has been used to isolate antibodies, including human antibodies, with and without immunization, and to improve the affinity and specificity of antigen binding.     

Circulating antibodies against neonatal cardiac muscarinic acetylcholine receptor in patients with Sjögren's syndrome

Isolated congenital heart block may be associated with Primary Sjogren's Syndrome. In this work we demonstrated that IgG present in the sera ofpatients with Primary Sjogren's Syndrome (PSS) could bind and activate muscarinic acetylcholine receptors of rat neonatal atria. These antibodies were able to inhibit in a irreversible manner the binding of ³H-QNB to muscarinic cholinergic receptors of purified rat atria membranes. Moreover, IgG from PSS individuals could modify biological effects mediated by muscarinic cholinoceptors activation, i.e. decrease contractility and cAMP and increase phosphoinositide turnover and cGMP. Atropine blocked all of these effects and carbachol mimicked them; confirming muscarinic cholinergic receptors-mediated PSS IgG action. Neither binding nor biological effect were obtained using adult instead of neonatal rat atria. IgG from sera of normal women were not effective in the studied system. The prevalence of cholinergic antibody was 100% in PSS and was independent of Ro/SS-A and La/SS-B antibodies. It could be concluded that antibody against muscarinic cholinergic receptors may be another serum factor to be considered in the pathophysiology of the development of congenital heart block.     

Purification and characterization of monoclonal antibodies against the free α-subunit of human chorionic gonadotrophin

Monoclonal antibodies (Mabs) against human chorionic gonadotropin hormone (hCG) were raised by hybridoma technology using Sp2/0 myeloma cells as fusion partner. Sixty-five percent of the total culture wells exhibited hybrid growth and 8% of the total wells (13 culture wells) contained anti-hCG secreting hybrids. A positive hybrid cell line secreting antibodies against the free alpha-subunit of hCG was cloned twice by limiting dilution method and eighty four clones were obtained that secreted monoclonal antibodies anti-alpha hCG. One of these hybridoma clones (1C4) secreting monoclonal antibodies against the free alpha-subunit of hCG was selected for purification and characterization purposes. This hybridoma cell line secreted monoclonal antibodies of IgG1 subclass, which were purified by affinity chromatography on Protein A Sepharose CL-4B column with a final relative recovery of antibody activity of 75% and a purification factor of about 12. The purified preparation was analyzed by SDS-PAGE, native PAGE, and IEF. Specificity studies of this Mab revealed that it recognized specifically an epitope on the free alpha-subunits of hCG, FSH, LH, and TSH as determined by enzyme immunoassays. On the other hand, this Mab exhibited crossreactivity with other pituitary hormones either as free subunits or intact molecules as follows: alpha hCG 100%; intact hCG 1.8%; beta hCG 0.14%; alpha FSH 24.5%; intact FSH 0.8%; beta FSH 0.09%; alpha LH 20.5%; intact LH 0.9%; beta LH 0.08%; alpha TSH 50.5%; intact TSH 3.7%; beta TSH 0.07%; The affinity constant (K) of this Mab with respect to free alpha-subunit of hCG was found to be 1.5 x 10(7) I/mol as determined by the simple antibody dilution analysis method.     

Multifaceted antibodies development against synthetic α-dystroglycan mucin glycopeptide as promising tools for dystroglycanopathies diagnostic

Glycoconjugate Journal - Dystroglycanopathies are diseases characterized by progressive muscular degeneration and impairment of patient’s quality of life. They are associated with altered...     

Is the identification of antibodies against the nervous tissue an indicator of brain injury?

Using a modification of the ELISA method, auto-antibodies against the own nervous tissue have been identified in the serum of laboratory rats. The prevalence of the IgM class of antibodies suggests their physiological significance. Antibody levels are higher in females than in males. Brain hypoxic injury brings about a shift in the spectrum of antibodies towards the IgG class. It may thus serve as an indicator of brain impairment caused by a lack of oxygen.     

Characterization of a panel of mouse single-chain antibodies against human recombinant interferon β1b

A panel of single-chain Fv-antibodies (ScFv’s) against recombinant human interferon beta 1b (rhIFN-β1b) has been obtained from immune and naïve combinatorial cDNA libraries of the mouse variable immunoglobulin genes. ScFv’s were expressed in Escherichia coli cells. For producers isolated from the immune library a difference in production yield of ScFv’s in periplasm and incubation medium as well as their expression and storage stability have been demonstrated. After sequencing of target DNA the multiple alignment and structural analysis of ScFv’s sequences with different primary structures were carried out and significant difference in both complementarity-determining (CDR) and framework (FR) regions of theirs variable domains has been shown. For the ScFv’s isolated from the immune library, specificity of their binding with native and denatured rhIFN-β1b in ELISA and Western-blotting as well as their high storage stability have been shown. The affinity constants for each representatives of the ScFv’s panel were in the range from 1.96 × 10^?8 to 1.69 × 10^?9 M.     

Dissolution of pre-existing platelet thrombus by synergistic administration of low concentrations of bifunctional antibodies against β3 integrin

Most antithrombotic approaches target prevention rather than the more clinically relevant issue of resolution of an existing thrombus. In this study, we describe a novel and effective therapeutic strategy for ex vivo clearance of pre-existing platelet thrombus by the combination of two bifunctional platelet GPIIIa49-66 ligands that target different parts of the arterial thrombus. We produced an additional GPIIIa49-66 agent (named APAC), which homes to activated platelets. Like our previously described SLK (which targets newly deposited fibrin strands surrounding the platelet thrombus), APAC destroys platelet aggregates ex vivo in an identical fashion with 85% destruction of platelet aggregates at 2 hours. The combined application of APAC and SLK demonstrated a ~2 fold greater platelet thrombus dissolution than either agent alone at a low concentration (0.025 μM). Platelet-rich clot lysis experiments demonstrated the time required for 50% platelet-rich fibrin clot lysis (T(50%)) by APAC (95 ± 6.1 min) or SLK (145 ± 7.1 min) was much longer than that by combined APAC + SLK (65 ± 7.6 min) at the final concentration of 0.025 μM (APAC + SLK vs APAC, p<0.05; APAC + SLK vs SLK, p<0.01). Thus these low concentrations of a combination of both agents are likely to be more effective and less toxic when used therapeutically in vivo.     

Immune-protective antibodies against capsular polysaccharides do not affect natural competence of Streptococcus pneumoniae: implications for current conjugate vaccination strategies?

We studied the effect of opsonization of Streptococcus pneumoniae with capsular antibodies on horizontal transfer of DNA. Opsonization did not inhibit DNA uptake. This suggests that horizontal transfer of capsular genes, which is an important escape mechanism of the pathogen, remains a potential threat for the efficacy of conjugate vaccination.     

A novel synthetic na?ve human antibody library allows the isolation of antibodies against a new epitope of oncofetal fibronectin

Human monoclonal antibodies (mAbs) can routinely be isolated from phage display libraries against virtually any protein available in sufficient purity and quantity, but library design can influence epitope coverage on the target antigen. Here we describe the construction of a novel synthetic human antibody phage display library that incorporates hydrophilic or charged residues at position 52 of the CDR2 loop of the variable heavy chain domain, instead of the serine residue found in the corresponding germline gene. The novel library was used to isolate human mAbs to various antigens, including the alternatively-spliced EDA domain of fibronectin, a marker of tumor angiogenesis. In particular, the mAb 2H7 was proven to bind to a novel epitope on EDA, which does not overlap with the one recognized by the clinical-stage F8 antibody. F8 and 2H7 were used for the construction of chelating recombinant antibodies (CRAbs), whose tumor-targeting properties were assessed in vivo in biodistribution studies in mice bearing F9 teratocarcinoma, revealing a preferential accumulation at the tumor site.Key words: human antibody library, phage display, oncofetal fibronectin, vascular tumor targeting, scFv antibody fragments, chelating recombinant antibody (CRAb)     

A novel synthetic naïve human antibody library allows the isolation of antibodies against a new epitope of oncofetal fibronectin

Human monoclonal antibodies (mAbs) can routinely be isolated from phage display libraries against virtually any protein available in sufficient purity and quantity, but library design can influence epitope coverage on the target antigen. Here we describe the construction of a novel synthetic human antibody phage display library that incorporates hydrophilic or charged residues at position 52 of the CDR2 loop of the variable heavy chain domain, instead of the serine residue found in the corresponding germline gene. The novel library was used to isolate human mAbs to various antigens, including the alternatively-spliced EDA domain of fibronectin, a marker of tumor angiogenesis. In particular, the mAb 2H7 was proven to bind to a novel epitope on EDA, which does not overlap with the one recognized by the clinical-stage F8 antibody. F8 and 2H7 were used for the construction of chelating recombinant antibodies (CRAbs), whose tumor-targeting properties were assessed in vivo in biodistribution studies in mice bearing F9 teratocarcinoma, revealing a preferential accumulation at the tumor site.     

Specificity of human antibodies against galα1-3gal carbohydrate epitope and distinction from natural antibodies reacting with galα1-2gal or galα1-4 gal

Antibodies against galactosyl-1-3-galactose epitopes were characterized in normal and patient sera by radioimmunoassay binding to mouse laminin and oligosaccharide inhibition. Binding was strictly dependent on -linked galactose in a terminal position. Reduced affinities were observed for digalactoses with (1-2)-, (1-6)- and (1-4)-linkages and for the blood group B epitope, Gal1-3(Fuc1-2)Gal. Conformational models of various active and inactive oligosaccharides provided a clearer picture of the epitope requirements for the observed antibody specificity. Some antibody heterogeneity was detected by comparing individual sera and by hapten elution from a laminin adsorbent. New assays were developed with synthetic Gal1-3Gal-albumin conjugates and these were shown to be more sensitive than assays with mouse laminin. Two more ubiquitous human antibodies could be detected with Gal1-2Gal and Gal1-4Gal conjugates. They were distinct from Gal1-3Gal-specific antibodies as shown by carbohydrate inhibition. This demonstrates a considerable diversity in the recognition of -linked galactose epitopes by natural antibodies.     

A cyclic undecamer peptide mimics a turn in folded Alzheimer amyloid β and elicits antibodies against oligomeric and fibrillar amyloid and plaques

The 39- to 42-residue amyloid β (Aβ) peptide is deposited in extracellular fibrillar plaques in the brain of patients suffering from Alzheimer's Disease (AD). Vaccination with these peptides seems to be a promising approach to reduce the plaque load but results in a dominant antibody response directed against the N-terminus. Antibodies against the N-terminus will capture Aβ immediately after normal physiological processing of the amyloid precursor protein and therefore will also reduce the levels of non-misfolded Aβ, which might have a physiologically relevant function. Therefore, we have targeted an immune response on a conformational neo-epitope in misfolded amyloid that is formed in advance of Aβ-aggregation. A tetanus toxoid-conjugate of the 11-meric cyclic peptide Aβ(22-28)-YNGK' elicited specific antibodies in Balb/c mice. These antibodies bound strongly to the homologous cyclic peptide-bovine serum albumin conjugate, but not to the homologous linear peptide-conjugate, as detected in vitro by enzyme-linked immunosorbent assay. The antibodies also bound--although more weakly--to Aβ(1-42) oligomers as well as fibrils in this assay. Finally, the antibodies recognized Aβ deposits in AD mouse and human brain tissue as established by immunohistological staining. We propose that the cyclic peptide conjugate might provide a lead towards a vaccine that could be administered before the onset of AD symptoms. Further investigation of this hypothesis requires immunization of transgenic AD model mice.