Preparation monoclonal β-type anti-idiotype antibody of zearalenone and development of green ELISA quantitative detecting technique

Abstract

Immunoassay has been widely used in the screening of mycotoxins, which may be hazardous to the operator or the environment. This study was to develop a green way to measure zearalenone (ZEN) with a monoclonal β-type anti-idiotype antibody (Ab2β) against ZEN in place of ZEN standard. Six monoclonal β-type anti-idiotype antibodies were prepared. The 50% inhibitory concentration (IC₅₀) value to ZEN of the six antibodies was between 34.45?±?1.12–182.12?±?15.40?nM. A green ELISA was then developed and validated. The quantitative conversion formula between ZEN and the monoclonal Ab2β against ZEN was y?=?0.092x^0.722, R² = 0.990. The working range was 2.63–100.64?ng ml^?1. The recovery rate in spiked feed samples was from 82.15% to 102.79%, and the within-assay and between-assay coefficient variation (CV) level were less than 10.00%. A good correlation was obtained by high-performance liquid chromatography method (HPLC) to validate the developed method.     

Comparison of the properties of concanavalin A and anti-α-d-glucose antibodies

Concanavalin A and anti-α-d-glucose antibodies form precipitin complexes with antigens havingα-d-glucose as terminal units. The sedimentation rates, molecular weights, gel electrophoretic mobilities, isoelectric points, and immunoglobulin type of Con A andα-Ab have been determined. The interactions of the compounds with antigens in the presence of potential inhibitors have been compared. The data show that the interaction of Con A with glucose units occurs with hydrogen bonding at hydroxyl groups at C1, 3,4, and 6 and van der Waals bonding at the pyranose ring oxygen. In theα-Ab complex with glucose units, in addition to the above bond types, a hydrogen bond at the hydroxyl at C2 occurs and this bond is essential for interaction.     

Production and characterization of monoclonal antibodies to estrogen-related receptor alpha (ERRα) and use in immunoaffinity chromatography

Estrogen-related receptor alpha (ERRα) is an orphan nuclear receptor whose elevated expression is thought to contribute to breast, colon, and ovarian cancers. In order to investigate the role of ERRα in human disease, there is a need for immunological reagents suitable for detection and purification of ERRα. We expressed recombinant human ERRα in Escherichia coli, purified the protein, and used it to generate monoclonal antibodies (mAbs) to ERRα. Nine high-affinity mAbs were chosen for their abilities to detect overexpressed ERRα in enzyme-linked immunosorbent assays (ELISAs) and Western blots, after which isotyping and preliminary epitope mapping was performed. The mAbs were all IgG subtypes and reacted with several different regions of full-length ERRα. A majority of the mAbs were found to be useful for immunoprecipitation of ERRα, and several could detect DNA-bound ERRα in electrophoretic mobility supershift assays (EMSAs) and chromatin immunoprecipitation (ChIP). The suitability of mAbs to detect ERRα in immunofluorescence assays was assessed. One mAb in particular, 2ERR10, could specifically detect endogenous ERRα in mammary carcinoma cells. Finally, we performed assays to screen for mAbs that gently release ERRα in the presence of a low-molecular-weight polyhydroxylated compound (polyol) and nonchaotropic salt. Using gentle immunoaffinity chromatography, we were able to isolate ERRα from mammalian cells by eluting with a polyol-salt solution. Our characterization studies show that these monoclonal antibodies perform well in a variety of biochemical assays. We anticipate that these novel reagents will prove useful for the detection and purification of ERRα in research and clinical applications.     

Use of monoclonal antibodies to pregnanediol-3α-glucuronide for the development of a solid phase chemiluminescence immunoassay

Monoclonal antibodies to pregnanediol-3α-glucuronide were produced by hybridomas between P3-X63-Ag8 variants and spleen cell of mice immunized with a bovine serum albumin conjugate of the homologous hapten. The ascites fluid collected from mice inoculated with the cloned hybridoma cells contained antibodies with high specifity and affinity to pregnanediol-3α-glucuronide. A sensitive solid-phase chemiluminescence immunoassay for urinary pregnanediol-3α-glucuronide was established utilizing these antibodies. The assay was validated in terms of specificity, accuracy, sensitivity and precision. When urine samples were assayed for pregnanediol-3α-glucuronide, the results obtained by the solid-phase chemiluminescence immunoassay method and the conventional gas liquid chromatographic method agreed well (n = 30, r=0.96). The method may be of value for monitoring luteal function since it is fast, sensitive and does not require the use of radioisotopes or purification of the biological sample. Monoclonal antibody preparations facilitate rigorous standardization of the assay.     

A potential new radiopharmaceutical for parathyroid imaging: Radiolabeled parathyroid-specific monoclonal antibody—II. Comparison of 125I- and 111In-labeled antibodies

The biodistributions of ¹¹¹In-BB5-G1 and ¹¹¹In-F(ab′)₂ were compared with the biodistributions of the corresponding ¹²⁵I-labeled molecules. For BB5-G1 intact antibody, the relative uptake of the ¹¹¹In- and ¹²⁵I-labeled molecules in human parathyroid tissue implants was similar at 24 h, but by 96 h the uptake of the ¹¹¹In-BB5-G1 %ID/g was four times greater than that observed with the ¹²⁵I-labeled antibody. For the F(ab′)₂ fragments, the relative parathyroid uptake of the two preparations was similar at all times tested. The uptake by the clearance organs was significantly higher when the ¹¹¹In-labeled molecules were used. Imaging results suggest that ¹¹¹In-BB5-G1 or ¹¹¹In-F(ab′)₂ may be a useful radiopharmaceutical for parathyroid radioimmunodetection.     

Humanization and characterization of an anti-human TNF-α murine monoclonal antibody

A murine monoclonal antibody, m357, showing the highly neutralizing activities for human tumor necrosis factor (TNF-α) was chosen to be humanized by a variable domain resurfacing approach. The non-conserved surface residues in the framework regions of both the heavy and light chain variable regions were identified via a molecular modeling of m357 built by computer-assisted homology modeling. By replacing these critical surface residues with the human counterparts, a humanized version, h357, was generated. The humanized h357 IgG(1) was then stably expressed in a mammalian cell line and the purified antibody maintained the high antigen binding affinity as compared with the parental m357 based on a soluble TNF-α neutralization bioassay. Furthermore, h357 IgG(1) possesses the ability to mediate antibody-dependent cell-mediated cytotoxicity and complement dependent cytotoxicity upon binding to cells bearing the transmembrane form of TNF-α. In a mouse model of collagen antibody-induced arthritis, h357 IgG significantly inhibited disease progression by intra-peritoneal injection of 50 μg/mouse once-daily for 9 consecutive days. These results provided a basis for the development of h357 IgG as therapeutic use.     

Monoclonal antibodies against plasma protease inhibitors: I. Production and characterization of 23 monoclonal antibodies against human α2

23 hybridomas secreting monoclonal antibodies against human ₂ , the fast-acting inhibitor of plasmin present in plasma, have been produced by the cell-fusion technique. Isotyping of the monoclonal antibodies has revealed that 14 monoclonal antibodies belong to the class IgG1, 6 to the class IgG2a, and 3 to the class tgG2b. All light chains belong to the group. The specificity and relative avidity of these monoclonals have been determined Using an indirect enzyme-linked immunosorbent assay. 13 monoclonals exhibit a relatively high avidity for ₂ , 5 are of intermediate avidity, and 5 of low avidity. The epitope specificity of these 23 rnonoclonal antibodies, originating from a single mouse, have been examined in inhibition experiments. A group of 10 monoclonal antibodies exhibit a very similar inhibition pattern. Partial inhibition effects displayed by 10 other antibodies define partially overlapping antigenic regions. The binding of these antibodies seems to produce a conformational change in the ₂ molecule, reducing the binding of two other antibodies. The last antibody defines an independent epitope.     

Monoclonal antibody specific for α2→8-linked oligo deaminated neuraminic acid (KDN) sequences in glycoproteins. Preparation and characterization of a monoclonal antibody and its application in immunohistochemistry

Two particular types of sialoglycoproteins have been detected in fish: polysialoglycoproteins containing 28-linked polysialic acid (8Neu5Gc2) _n present in unfertilized Salmonidae fish eggs, and glycoproteins bearing oligo/polymers of deaminated neuraminic acids (KDN) found in the vitelline envelope of the eggs and ovarian fluid. We report the preparation and characterization of a monoclonal antibody specifically recognizing oligo/polymers of KDN sequences in glycoproteins and its application in immunohistochemistry. Fusion of spleen cells from a BALB/c mouse immunized with a KDN-rich glycoprotein (KDN-gp) containing (8KDN2) _n 6(KDN23Gal13GlNAc13) GalNAc1 residues, with mouse myeloma cells yielded a hybrid cell line producing a monoclonal antibody that bound to KDN-gp, but not to KDN-gp depleted of KDN residues. The specificity of the monoclonal antibody, designated mAb.kdn8kdn, was determined by an enzyme-linked immunosorbent assay using KDN-gp samples that varied in KDN content. These antigens were prepared by the selective removal of KDN residues from the native KDN-gp. The mAb.kdn8kdn reacted most strongly with the intact KDN-gp and less strongly with KDN-gp samples containing decreased numbers of KDN residues. The mAb.kdn8kdn was shown specifically to recognize the 28-linked oligo/polyKDN sequences, (8KDN2) _n , and to be able to distinguish specifically (8KDN2) _n chains from (8Neu5Ac2) _n and (8Neu5Gc2) _n chains. The antibody was used successfully for the immunohistochemical detection of reactive KDN epitopes in sections of paraffin embedded rat pancreas. Several controls verified the specificity of the immunohistochemical staining, thus providing the first demonstration of (8KDN2) _n sequences in a mammalian tissue. The mAb.kdn8kdn can now be used to search further for glycoconjugates containing (8KDN2) _n chains and will facilitate studies on their biosynthesis, intracellular localization and function.     

A monoclonal antibody for terminal β-galactose. Use in analysis of glycosphingolipids

A monoclonal antibody (mAb 8281) specific for the terminal -galactose (Gal) of glycosphingolipids (GSL) and glycoproteins was produced from mice immunized with lipid extract from fresh acute lymphocytic leukemia (ALL) cells. Immuno-thin layer chromatography (ITLC) and competition assays with purified neutral GSL standards, free sugars, and synthetic neoglycoproteins showed mAb 8281 to be strongly reactive with LacCer, GalCer and Gal--O-(CH₃)₂S(CH₃)₂-CONH-(Gal--O-CETE) linked to bovine serum albumin (BSA). The penultimate sugar also played a role in binding. The antibody was not reactive with carbohydrates with terminal Gal structures and unrelated terminal moieties. Indirect immunoperoxidase staining and flow cytometry with mAb 8281 demonstrated positive staining on numerous tissues, including smooth muscle, gastrointestinal mucosa, lymph node B cells and monocytes. ITLC analysis of the GSL composition of fresh B cell neoplasms using mAb 8281 confirmed the presence of lactosylceramide and galactosylceramide in neoplasms of varying stages of differentiation. Because of its specificity for terminal Gal carbohydrate residues, mAb 8281 may be useful in structural and functional analyses of GSL.     

α1 acute-phase globulins of rats. Isolation and some properties

1. A method is described for the isolation of certain of the alpha(1)-globulins of rat plasma that are known to increase in concentration after tissue damage (acute-phase globulins). 2. Although apparently homogeneous when examined by disc electrophoresis at pH9, these proteins could be subdivided further by isoelectric fractionation. 3. Treatment with neuraminidase removed approx. 60% of the sialic acid originally present in these proteins and gave almost completely homogeneous material of decreased mobility when examined by disc electrophoresis in polyacrylamide gel. When subjected to immunoelectrophoresis this material gave a single arc. 4. The homogeneity of the isolated materials was examined by ultracentrifugation. The single peak thus found is consistent with molecular weights of 45000-46000. 5. The isolated materials were shown to be glycoproteins containing approx. 15% of carbohydrate, and to have isoelectric points in the range pH4.4-4.8.     

Development and characterization of human monoclonal antibodies that neutralize multiple TGFβ isoforms

Transforming growth factor (TGF)β levels are elevated in, and drive the progression of, numerous disease states such as advanced metastatic cancer and systemic and ocular fibrosis. There are 3 main isoforms, TGFβ1, 2, and 3. As multiple TGFβ isoforms are involved in disease processes, maximal therapeutic efficacy may require neutralization of 2 or more of the TGFβ isoforms. Fully human antibody phage display libraries were used to discover a number of antibodies that bind and neutralize various combinations of TGFβ1, 2 or 3. The primary panning did not yield any uniformly potent pan-isoform neutralizing antibodies; therefore, an antibody that displayed potent TGFβ 1, 2 inhibition, but more modest affinity versus TGFβ3, was affinity matured by shuffling with a light chain sub-library and further screening. This process yielded a high affinity pan-isoform neutralizing clone. Antibodies were analyzed and compared by binding affinity, as well as receptor and epitope competition by surface plasmon resonance methods. The antibodies were also shown to neutralize TGFβ effects in vitro in 3 assays: 1) interleukin (IL)-4 induced HT-2 cell proliferation; 2) TGFβ-mediated IL-11 release by A549 cells; and 3) decreasing SMAD2 phosphorylation in Detroit 562 cells. The antibodies’ potency in these in vitro assays correlated well with their isoform-specific affinities. Furthermore, the ability of the affinity-matured clone to decrease tumor burden in a Detroit 562 xenograft study was superior to that of the parent clone. This affinity-matured antibody acts as a very potent inhibitor of all 3 main isoforms of TGFβ and may have utility for therapeutic intervention in human disease.     

Modulating carbohydrate–protein interactions through glycoengineering of monoclonal antibodies to impact cancer physiology

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Generation of potent mouse monoclonal antibodies to self-proteins using T-cell epitope “tags”

Immunization of mice or rats with a "non-self" protein is a commonly used method to obtain monoclonal antibodies, and relies on the immune system''s ability to recognize the immunogen as foreign. Immunization of an antigen with 100% identity to the endogenous protein, however, will not elicit a robust immune response. To develop antibodies to mouse proteins, we focused on the potential for breaking such immune tolerance by genetically fusing two independent T-cell epitope-containing sequences (from tetanus toxin (TT) and diphtheria toxin fragment A (DTA)) to a mouse protein, mouse ST2 (mST2). Wild-type CD1 mice were immunized with three mST2 tagged proteins (Fc, TT and DTA) and the specific serum response was determined. Only in mice immunized with the T-cell epitope-containing antigens were specific mST2 serum responses detected; hybridomas generated from these mice secreted highly sequence-diverse IgGs that were capable of binding mST2 and inhibiting the interaction of mST2 with its ligand, mouse interleukin (IL)-33 (mIL-33). Of the hundreds of antibodies profiled, we identified five potent antibodies that were able to inhibit IL-33 induced IL-6 release in a mast cell assay; notably one such antibody was sufficiently potent to suppress IL-5 release and eosinophilia infiltration in an Alternaria alternata challenge mouse model of asthma. This study demonstrated, for the first time, that T-cell epitope-containing tags have the ability to break tolerance in wild-type mice to 100% conserved proteins, and it provides a compelling argument for the broader use of this approach to generate antibodies against any mouse protein or conserved ortholog.     

Humoral and cellular responses of colorectal cancer patients treated with monoclonal antibodies and interferon γ

Summary Fifteen patients with metastatic gastrointestinal adenocarcinomas were treated with low doses of recombinant human interferon (rh-IFN) and a mixture of monoclonal antibodies (mAb) that bind to tumor cells. All antibodies were of the IgG2a isotype and interact with human effector cells to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Natural killer lysis against K562 cells by peripheral blood mononuclear cells purified from patients' blood was enhanced in all patients at day 3 during IFN treatment. Monocytes from two patients had increased ADCC levels. Increase in the percentage of monocytes able to bind mouse IgG2a was detected by Fc receptor flow cytometry analysis 24 h after the first IFN infusion. However, 3 days later, the percentage of fluorescent cells had fallen below baseline levels. The analysis of patients' sera showed that at day 2 after mAb infusion, only 50% of the circulating mouse IgG was immunoreactive, and after 1 week, only traces of immunoreactive mouse IgG were detected. All patients developed a human anti-(mouse Ig) response of IgG, IgM and IgA isotypes, although only low levels of anti-idiotypic antibodies were detected at the time of testing (up to 9 weeks) after mAb infusion. No difference in the IgG subclasses of anti-(mouse Ig) antibody was observed between patients treated with mAb and IFN and patients treated with mAb alone.This work was supported by a fellowship from the Minister of Foreign Affairs, France, and National Institutes of Health Grants CA10815, CA25874 and CA21124.     

Characterization of prostate-tissue-directed monoclonal antibody, α-Pro 13

Summary The -Pro 13-secreting hybridoma was produced by immunizing mice with an equal mixture of PC-3, DU145, and LNCaP established prostatic carcinoma cell lines. The specificity of -Pro 13 monoclonal antibody was evaluated by the criteria of differential binding to cultured cells; differential binding to extracts of malignant prostate, nonmalignant prostate, and malignant and nonmalignant tissues of various histiotypes in solid phase radioimmunoassay; and by immunoperoxidase staining of primary surgical tissues of varied histiotypes. The data generated by multiple assay investigation indicate that -Pro 13 exhibits preferential binding to the ductal epithelium of prostate tissue; immunoperoxidase evaluation indicates a considerable heterogeneity of staining of ductal epithelial cells. The most prevalent cross-reactivity of -Pro 13 monoclonal antibody with non-prostate tissue occurs with blood vessel endothelium of restricted tissues. Electrophoretic analysis of immunoprecipitates from radioiodinated prostatic tumor extracts indicates that the molecule recognized by -Pro 13 is of 120,000 dalton apparent nonreduced molecular weight. Under reducing conditions, the antigen (p40) consists of a major component of 40,000 dalton apparent MW and a minor component of 17,000 dalton MW. p40 has an isoelectric point of 3.5–4.5. The antigen is intrinsically stable on the PC-3 cell surface; its release into spent culture medium is negligible. p40 is also stable upon complexation with -Pro 13 antibody in that it is not shed from the cell surface as an immune complex nor is it endocytosed to any extent as an immune complex.Abbreviations MIg anti-mouse immunoglobulin - IEF isoelectric focusing - kD kilodalton - MW molecular weight - PA Staphylococcus protein A - RIA radioimmunoassay - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - SCM spent culture medium     

Immunocytochemical study of seminal γ-glutamyltransferase using monoclonal antibodies

We produced three monoclonal antibodies, SG1, SG2 and SG3, specific for human seminal -glutamyltransferase when characterized by enzyme-linked immunosorbent assay and immunoblotting. Seminal -glutamyltransferase was localized, by immunostaining, to the epithelial cells of the ductus epididymidis, seminal vesicle and prostate gland with SG1, those of the prostate gland with SG2, and those of the seminal vesicle with SG3. Rabbit polyclonal anti-seminal -glutamyltransferase serum reacted with the proximal convolution of the kidney and the bile capillaries of the liver, and with the epithelial cells of the reproductive organs. However, immunoreactivity was not observed in the kidney or liver with the monoclonal antibodies. Thus, these monoclonal antibodies are probably all specific to seminal -glutamyltransferase but recognize different epitopes.     

Generation of potent mouse monoclonal antibodies to self-proteins using T-cell epitope “tags”

Immunization of mice or rats with a "non-self" protein is a commonly used method to obtain monoclonal antibodies, and relies on the immune system's ability to recognize the immunogen as foreign. Immunization of an antigen with 100% identity to the endogenous protein, however, will not elicit a robust immune response. To develop antibodies to mouse proteins, we focused on the potential for breaking such immune tolerance by genetically fusing two independent T-cell epitope-containing sequences (from tetanus toxin (TT) and diphtheria toxin fragment A (DTA)) to a mouse protein, mouse ST2 (mST2). Wild-type CD1 mice were immunized with three mST2 tagged proteins (Fc, TT and DTA) and the specific serum response was determined. Only in mice immunized with the T-cell epitope-containing antigens were specific mST2 serum responses detected; hybridomas generated from these mice secreted highly sequence-diverse IgGs that were capable of binding mST2 and inhibiting the interaction of mST2 with its ligand, mouse interleukin (IL)-33 (mIL-33). Of the hundreds of antibodies profiled, we identified five potent antibodies that were able to inhibit IL-33 induced IL-6 release in a mast cell assay; notably one such antibody was sufficiently potent to suppress IL-5 release and eosinophilia infiltration in an Alternaria alternata challenge mouse model of asthma. This study demonstrated, for the first time, that T-cell epitope-containing tags have the ability to break tolerance in wild-type mice to 100% conserved proteins, and it provides a compelling argument for the broader use of this approach to generate antibodies against any mouse protein or conserved ortholog.     

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.