Preparation of monoclonal antibodies against a derivative of semicarbazide as a metabolic target of nitrofurazone

Monoclonal antibodies (McAb) were produced to detect semicarbazide (SEM), a metabolite as a marker residue of nitrofurazone in animal food production. A carboxyphenyl derivative (CPSEM) of SEM was synthesized following derivatisation with 4-carboxybenzaldehyde (CBA). CPSEM was purified by recrystallization and conjugated to bovine serum albumin (BSA) or ovalbumin (OVA) as immunogen or coating antigen, respectively. Hybridomas were obtained by fusing mouse myeloma cells SP2/0 with splenocytes from the mice immunized with CPSEM-BSA. Hybridomas 1F10 and 4F2 secreting antibodies against CPSEM were obtained and subcloned. Ascites of monoclonal antibodies were prepared by injecting 1 × 10⁶ cells of hybridoma 1F10 into mice abdomen. McAb obtained from hybridoma 1F10 was highly specific for CPSEM and had no cross-reaction with various nitrofuran metabolites and a range of veterinary drugs. The sensitivity of the McAb to SEM was 0.01 ng mL⁻¹ and the IC₅₀ value was 1.3 ng mL⁻¹ (SEM in the form of CPSEM). McAb 1F10 is suitable to develop an immunoassay for SEM with sufficient sensitivity for monitoring nitrofurazone residues.     

Recombinant antibodies for environmental analysis

Initial steps of antibody engineering in the late eighties revolutionized the technology of antibody production, particularly in the area of immunotherapy and diagnostics. Hallmarks that seemed to be out of reach for a long time are now the state of the art, e.g. tailoring of antibodies to match particular needs or by-passing immunization by use of antibody libraries. Despite the apparent benefits of recombinant antibody technologies, this field has been opened up hesitantly for other applications. This review addresses the development of recombinant antibody synthesis in environmental analysis. Examples are given of the molecular evolution of pesticide antibodies and their application for the analysis of real samples.     

Biological activity of functionalized SiO2 thin films prepared by sol-gel method

In this work we investigated the biological properties of sol-gel films in aqueous medium. Functionalized silica films were prepared by the sol-gel process, from organically modified silicon alkoxides with amino or thiol groups. Covalent binding of proteins with different orientations according to the hydrophilic or hydrophobic character of the surface was studied. This binding occurred via a covalent coupling agent providing a very stable linkage. No denaturation was detected and a good detection of the antigen was observed. Immunoassays have demonstrated the biological activity of grafted antibodies.     

Development of an enzyme-linked immunosorbent assay for metolcarb residue analysis and investigation of matrix effects from different agricultural products

The development of a direct competitive enzyme-linked immunosorbent assay based on polyclonal antibodies for N-methylcarbamate insecticide metolcarb is described. Two new haptens for the metolcarb were designed and synthesized. Both haptens were conjugated with keyhole limpet hemocyanin to form the immunogens. Four rabbits were immunized with the immunogens for production of polyclonal antibodies against metolcarb. Antisera titers were tested on the homologous coating antigens using a noncompetitive indirect enzyme-linked immunosorbent assay. The high titer antisera were used to develop the direct competitive enzyme-linked immunosorbent assay for the detection of metolcarb. The antibody–antigen combination with the highest selectivity for metolcarb was further optimized and its tolerance to changes in chemical conditions (ionic strength, pH value, and organic solvent) was studied. Under optimum conditions, the sensitivity and the limit of detection were determined to be 22 μg L⁻¹ and 1.2 μg L⁻¹ respectively. Determination of metolcarb in fruit juices and vegetables was accomplished by simple, rapid, and efficient extraction methods. Recoveries of metolcarb from spiked samples ranged from 80.5% to 109.5%. Validation of the developed immunosorbent assay was conducted by comparison of results from high-performance liquid chromatography. The correlation between the data obtained using developed immunosorbent assay and high-performance liquid chromatography was high (R ² = 0.9884). Therefore, the developed immunosorbent assay in this study was suitable for the rapid quantitative determination of metolcarb in agricultural products.     

Comparative surface plasmon resonance and enzyme-linked immunosorbent assay characterisation of a monoclonal antibody with N-acyl homoserine lactones

This study shows the detection of (N-acyl) homoserine lactones (AHLs or HSL) with monoclonal antibodies via a surface plasmon resonance (SPR)-based immunosensor in comparison to conventional microtiter plate-based enzyme-linked immunosorbent assay (ELISA). An HSL derivative, named HSL2 (Table 1), was attached to bovine serum albumin (BSA) and the conjugate (HSL2-BSA-r2) was either covalently immobilised on the SPR sensor chip surface via free amino groups or via adsorption on the ELISA polystyrene plate surface. With a newly developed rat monoclonal antibody (mAb HSL1/2 2C10), AHLs were detected sensitively in a competitive format with SPR and ELISA. Well comparable experiments between SPR and ELISA could be obtained in buffers. Moreover, the SPR sensor surface with the immobilised conjugate HSL2-BSA-r2 could be regenerated at least 340 times (regeneration cycles) without loss of activity. The measurement time per cycle was approximately 15 min. The competitive detection format for SPR and ELISA allowed the detection in the μg L⁻¹ range.     

Cell Differentiation and Proliferation in the Bone Marrow and Other Organs of 2D2 Mice during Spontaneous Development of EAE Leading to the Production of Abzymes

The exact cellular and molecular mechanisms of multiple sclerosis and other autoimmune diseases have not been established. Autoimmune pathologies are known to be associated with faults in the immune system and changes in the differentiation profiles of bone marrow stem cells. This study analyzed various characteristics of experimental autoimmune encephalomyelitis (EAE) in 2D2 mice. Differentiation profiles of six hematopoietic stem cells of bone marrow were found to significantly differ in 2D2 male and female mice during the spontaneous development of EAE. In addition, we found various properties of B and T cells, CD4+ and CD8+ lymphocytes in blood and several organs (bone marrow, spleen, thymus, and lymph nodes) of 2D2 male and female mice to be considerably different. These changes in hematopoietic stem cells differentiation profiles and level of lymphocyte proliferation in various organs of 2D2 mice were found to induce the production of IgGs against DNA, myelin basic protein, and myelin oligodendrocyte glycoprotein, increasing the number of autoantibodies hydrolyzing these substrates. We compared the changes of these immunological and biochemical parameters in 2D2 mice with those of mice of two other lines (Th and C57BL/6), also prone to spontaneous development of EAE. Some noticeable and even extreme variations were found in the time-related development of parameters between male and female mice of 2D2, Th, and C57BL/6 lines. Despite some differences, mice of all three lines demonstrated the changes in hematopoietic stem cells profiles, lymphocyte content, and production of catalytic autoantibodies. Given that these changes are harmful to mice, we believe them to cause the development of experimental autoimmune encephalomyelitis.     

Antibody-catalyzed benzoin oxidation as a mechanistic probe for nucleophilic catalysis by an active site lysine

Aldolase antibody 24H6, which was obtained by reactive immunization against a 1,3-diketone hapten, is shown to catalyze additional reactions, including H/D exchange and oxidation reactions. Comparison of the H/D exchange reaction at the alpha-position of a wide range of aldehydes and ketones by 24H6 and by other aldolase antibodies, such as 38C2, pointed at the significantly larger size of the 24H6 active site. This property allowed for the catalysis of the oxidation of substituted benzoins to benzils by potassium ferricyanide. This reaction was used as a mechanistic probe to learn about the initial steps of the 24H6-catalyzed aldol condensation reaction. The Hammett correlation (rho=4.7) of log(k(cat)) versus the substituent constant, sigma, revealed that the reaction involves rapid formation of a Schiff base intermediate from the ketone and an active site lysine residue. The rate-limiting step in this oxidation reaction is the conversion of the Schiff base to an enamine intermediate. In addition, linear correlation (rho=3.13) was found between log(K(M)) and sigma, indicating that electronic rather than steric factors are dominant in the antibody-substrate binding phenomenon and confirming that the reversible formation of a Schiff base intermediate comprises part of the substrate-binding mechanism.     

甲胺化衍生结合基于硅氢化物固定相的正相色谱用于单克隆抗体药物的N-糖基化表征

采用甲胺化衍生结合基于硅氢化物固定相的正相色谱（SiH-NPC）分析单抗的N-糖基化。样品经酶切、甲胺化衍生、纯化后由液相色谱-质谱进行分析。结果表明,相较于亲水相互作用色谱（HILIC）,SiH-NPC分离机制不同,使用常规的无盐流动相即可实现高分离度,避免污染质谱,色谱柱结构稳定,使用寿命长,更适合快速分析。结合唾液酸衍生方法,SiH-NPC在液相色谱-质谱联用鉴定酸性糖和糖异构体方面呈现显著优势,在生物制药行业中具有重要的应用潜力。