抗玉米赤霉烯酮单克隆杂交瘤细胞系的建立及单抗生产

为快速检测食品中的玉米赤霉烯酮 (Zearalenone以下简称ZEN) ,建立了抗ZEN的单克隆杂交瘤细胞株。用ZEN BSA偶联物免疫 8～ 10周龄雌性BalB c小鼠后 ,取脾细胞与小鼠骨髓瘤细胞Sp2 0融合 ,经过 4～ 5次亚克隆建立了稳定分泌抗ZEN抗体的杂交瘤细胞株 ,分别命名为Z2A4、Z8D6。将上述 2种杂交瘤细胞分别注入BalB c小鼠腹腔 ,获得含抗ZEN单克隆抗体的腹水。将腹水用饱和硫酸铵盐析法纯化 ,得到Z2A4、Z8D6单克隆抗体。经鉴定 ,其亚类为IgG1,抗体腹水效价Z2A4和Z8D6分别为 1∶1 6× 10 6和 1∶3 2× 10 6;分子量均为 15 0 0 4 0 (15 0kD) ;参考工作浓度分别为1∶4 0 0 0 0和 1∶10 0 0 0 0 ;纯化后抗体IgG含量分别为 34和 39mg ml ;抗体与其它霉菌毒素无交叉反应(交叉反应率 <1% ) ,具有较高特异性 ;抗体亲和力常数Z2A4和Z8D6分别为 5 5 2× 10 8和 4 6 8×10 9mol L。     

Partial purification and preparation of bovine lactoperoxidase and characterization of kinetic properties of its immobilized form incorporated into cross-linked alginate films

Lactoperoxidase (LPS), purified directly from bovine rennet whey by Toyopearl-SP cation-exchange chromatography and lyophilized by using dextran as supporting material, maintained almost 70 and 60% of its activity after almost 2 and 5 months storage at −18 °C, respectively. Incorporation of the prepared LPS into alginate films between 0.08 and 0.69 mg/cm² (516–4325 U/cm²) caused the immobilization of most of the enzyme and gave films with LPS activity between 0.05 and 2.8 U/cm², determined in the presence of 8 μM H₂O₂. Between 2 and 24 μM H₂O₂ concentrations, a two-fold increase in H₂O₂ concentration caused 1.5–2.5-fold increase in LPS activity of films incorporated with 0.24–0.28 mg/cm² (1200 U/cm²) LPS. The Q₁₀ and E_a of immobilized enzyme activity between 4 and 16 °C were 1.69 and 34.6 kJ/mol, respectively. However, in the 16–30 °C range, the temperature change had almost no effect on LPS activity of films. The optimal activity of immobilized LPS was observed at pH 6.0, but the enzyme maintained 30–85% of its activity between pH 3.0 and 7.0. The immobilized LPS also had a high stability between pH 4.0 and 6.0. The results of this study showed the good potential of LPS-incorporated alginate films in forming a natural antimicrobial mechanism in different foods.     

Detection of the sour-rot pathogen Geotrichum candidum in tomato fruit and juice by using a highly specific monoclonal antibody-based ELISA

Geotrichum candidum is a common soil-borne fungus that causes sour-rot of tomatoes, citrus fruits and vegetables, and is a major contaminant on tomato processing equipment. The aim of this work was to produce a monoclonal antibody and diagnostic assay for its detection in tomato fruit and juice. Using hybridoma technology, a cell line (FE10) was generated that produced a monoclonal antibody belonging to the immunoglobulin class M (IgM) that was specific to G. candidum and the closely related teleomorphic species Galactomyces geotrichum and anamorphic species Geotrichum europaeum and Geotrichum pseudocandidum in the G. geotrichum/G. candidum complex. The MAb did not cross-react with a wide range of unrelated fungi, including some likely to be encountered during crop production and processing. The MAb binds to an immunodominant high molecular mass (> 200 kDa) extracellular polysaccharide antigen that is present on the surface of arthroconidia and hyphae of G. candidum. The MAb was used in a highly specific enzyme-linked immunosorbent assay (ELISA) to accurately detect the fungus in infected tomato fruit and juice. Specificity of the ELISA was confirmed by sequencing of the internally transcribed spacer (ITS) 1-5.8S-ITS2 rRNA-encoding regions of fungi isolated from naturally-infected tomatoes.     

Biphasic addition strategy of hypoxanthine and thymidine for improving monoclonal antibody production

In our previous study, we demonstrated that combinatorial addition of hypoxanthine (10 mg/L) and thymidine (2 mg/L) was able to stimulate initial cell growth and elevate volumetric concentration of antibody by 22% (Chen et al., Appl. Microbiol. Biotechnol., 93, 169-178, 2012). In this study, a systematic study was carried out to investigate the effects of hypoxanthine and thymidine (H&T) on cell growth and antibody production in a much wider range of concentration. In addition, we pursued to establish a highly productive fed-batch culture via rationally designing H&T addition regime. It was found that both cell growth and antibody production in batch cultures were H&T concentration-dependent. Specifically, a low concentration stimulated cell growth while exerting no influence on specific productivity (q(mAb)), and a high concentration inhibited cell growth, however, significantly enhancing q(mAb). Subsequent experiments with fed-batch shaking flasks demonstrated the feasibility of improving antibody production using a biphasic addition strategy for H&T: supplementing a low concentration of H&T during initial cell growth phase and a high concentration of H&T at the production phase. By applying the optimized feeding regime, a maximum viable cell density (VCD) of 6.45 × 10(6)cells/mL and volumetric antibody production of 632 mg/L were achieved in a 2 L-B.Braun bioreactor. Taken together, in this study, a biphasic H&T addition strategy for cell culture was developed, which hold great promise to improve antibody production.     

Immunological characteristics of monoclonal antibodies against shellfish major allergen tropomyosin

Two types of murine monoclonal antibodies (MAbs) against American lobster (Homarus americanus) were generated and characterized. Three purified MAbs were characterized to be specific to the shellfish major allergen tropomyosin. MAbs 5G5E1 and 1A3A7 were reactive to tropomyosin from crustacean species only, whereas MAb 2A7H6 was reactive to both crustacean and mollusk tropomyosins. None of the antibodies reacted to vertebrate tropomyosins. Competitive ELISA indicated that the antigenic epitopes recognized by the two types of MAbs were different from each other. In addition, competitive immunoblot results showed that the binding of shellfish-allergic patient IgEs to lobster tropomyosin was inhibited by the MAb 2A7H6 only. This finding suggests that the antigenic epitope for the 2A7H6 antibody might be similar or close to the allergenic epitope shared by crustaceans and mollusks. Consequently, the MAbs recognizing the different common antigenic epiotopes obtained in the present study would not only facilitate the allergen characterization of shellfish, but may also be useful for the development of specific and sensitive immunoassays for allergen quantification or epitope mapping.     

Comparison of alginate and pectin based beads for production of poultry probiotic cells

A comparative study on the stability and potential of alginate and pectin based beads for production of poultry probiotic cells using MRS medium in repeated batch fermentation was conducted. The bead cores, made of three types of materials, i.e., ca-alginate, ca-pectinate and ca-alginate/pectinate, were compared. The effect of single and double layer coatings using chitosan and core material, respectively, on the bead stability and cell production were also studied. The pectin based beads were found to be more stable than that of the alginate beads and their stability was further improved by coating with chitosan. The cell concentration in pectin based beads was comparable to that in the alginate beads. On the other hand, pectin based beads gave significantly lower cell concentration in the growth medium for the initial fermentation cycles when compared to the alginate beads. In conclusion, pectin was found to be potential encapsulation material for probiotic cell production owing to its stability and favourable microenvironment for cell growth.     

Development of a new monoclonal antibody based direct competitive enzyme-linked immunosorbent assay for detection of brevetoxins in food samples

Brevetoxin B (PbTx-2) was covalently linked to carrier protein bovine serum albumin and human gamma globulin. A monoclonal antibody against PbTx-2, which showed high cross-reactivity values with PbTx-1, PbTx-3 and PbTx-9 (more than 89%) was obtained from ascites and some characteristics of monoclonal antibody were studied. An direct competitive enzyme-linked immunosorbent assay (ELISA) for detection of PbTxs was developed, which showed an IC50 value of 5.3 ng mL⁻¹ with a detection limit of 0.6 ng well⁻¹. The recoveries of PbTxs from cockle (88.4%–102.3%) and oyster (89.4%–104.3%) demonstrated that the matrices of cockle and oyster where PbTxs are found do not interfere with the assay. The newly developed competitive ELISA appears to be a reliable and useful method for mass monitoring of PbTxs in mollusk.     

Characterisation of monoclonal antibody against aflatoxin B1 produced in hybridoma 2C12 and its single-chain variable fragment expressed in recombinant Escherichia coli

An anti-aflatoxin B₁ monoclonal antibody (anti-AFB₁ mAb) from the hybridoma 2C12 was established and its inhibition concentration fifty (IC₅₀) for AFB₁ and relative cross-reactivities (CRs) to other mycotoxins were estimated to be 8 ng/mL and less than 4% compared with AFB₁ by a competitive direct enzyme-linked immunosorbent assay. For production of anti-AFB₁ single-chain variable fragment (anti-AFB₁ scFv) in recombinant Escherichia coli, its scFv-coding genes were cloned from the hybridoma 2C12. The anti-AFB₁ scFv formed inclusion bodies in the cytoplasm of E. coli required in vitro refolding process and hence recovered to retain binding activity successfully. Surface plasmon resonance analysis resulted that anti-AFB₁ scFv possessed 1.16 × 10⁻⁷ M of equilibrium dissociation constant (K_D), which was about 17 times higher than the parental anti-AFB₁ mAb of 6.95 × 10⁻⁹ M.     

Changes in the quality of antibodies produced by Chinese hamster ovary cells during the death phase of cell culture

Although overproduction of recombinant proteins by mammalian cells is well established, little attention has been paid to analysis of the quality of the products. We focused on the quality of antibodies produced during the death phase of a recombinant Chinese hamster ovary (CHO) cell line. The quality of the monoclonal antibody against HM1.24 antigen and the post-translational characteristics of the subunits during CHO cell culture in a 160-L bioreactor were investigated. The culture supernatant of a stable cell line was collected and purified by affinity chromatography and then analyzed. There were no significant changes in gel-permeation chromatography variables, carbohydrate structure, or antibody-dependent cellular cytotoxicity activity during the death phase of cell culture. However, ion-exchange chromatography analysis revealed that antibody heterogeneity changed, as indicated by a decrease in cell viability. The results presented here provide useful information that will help in determining the time to end each batch culture.     

Development of monoclonal antibodies and a competitive ELISA detection method for glycinin,an allergen in soybean

Soybean glycinin is a major food allergen causing anaphylaxis. A sensitive detection method for glycinin is needed to evaluate soybean allergies in food and feed products. In the present study, monoclonal antibodies (Mabs) against glycinin were prepared using purified glycinin as the immunogen. The generated Mabs, named 3B2 and 4B2, were identified as being IgG_2b and IgG_2a iso-types respectively, and exhibited high specificity to glycinin. Then we developed a competitive ELISA based on Mab 4B2 to measure glycinin which showed an IC₅₀ value of 1.7 ng/mL with a detection limit of 0.3 ng/mL, and the linear portion of the curve was 0.3–11.2 ng/mL. Recovery tests indicated that the competitive ELISA based on Mab 4B2 gave reliable reproducibility. The produced Mab 4B2 and the developed ELISA could provide a valuable tool for sensitive determination of glycinin and for future studies conducted to reveal the mechanism of how glycinin functions in anaphylaxis.     

A monoclonal antibody-based sensitive enzyme-linked immunosorbent assay (ELISA) for the analysis of the organophosphorous pesticides chlorpyrifos-methyl in real samples

Chlorpyrifos-methyl hapten, O-methyl-O-(3,5,6-trichloro-2-pyridinyl)-N-(2-carboxyethyl)-phosphoramidothionte (H1), was synthesized and conjugated with bovine serum albumin (BSA) and ovalbumin (OVA) by the active ester method. Then H1–OVA conjugate was used as coating antigen, while H1–BSA conjugate was used as immunogen for producing monoclonal antibody. After optimisation, a monoclonal antibody-based effective competitive indirect enzyme-linked immunsorbent assay (ELISA) was developed and applied for determination of chlorpyrifos-methyl with a novel combination of antibody/antigen, I₅₀ of which was 75.22 ng/ml, limit detection (LD) was 0.32 ng/ml, and there was relative high cross-reactivity (CR) only with chlorpyrifos (1.4%), and CRs with other tested pesticides were all below 1% and regarded as negligible. The recoveries obtained by standard chlorpyrifos-methyl addition to real samples, including grape, Chinese cabbages, water and soil were all from 82.4% to 110.2%. Therefore, the optimised ELISA might become a convenient and satisfied analytical tool for monitoring chlorpyrifos-methyl residues in agriculture ecosystem.     

FTB单克隆抗体的研制与特性鉴定

为建立测定ＦＴＢ的ＩＣ－ＥＬＩＳＡ方法，用复合抗原ＦＴＢＳ－ＢＳＡ和ＦＴＢＳ－ＨＳＡ作为免疫原免疫ＢＡＬＢ／ｃ小鼠，取免疫小鼠脾细胞与鼠ＳＰ２／Ｏ－Ａｇ１４骨髓瘤细胞融合，经反复筛选，获得２株稳定分泌抗ＦＴＢ抗体的单克隆杂交瘤细胞株。免疫球蛋白亚型鉴定，１株为ＩｇＭ，１株为ＩｇＧ１。两株杂交瘤腹水的ＥＬＩＳＡ效价分别为１×１０－６和１×１０－８，用ＰＥＧ沉淀法和辛酸－饱和硫酸铵法对抗体腹水进行了纯化，并分别测定了２种单抗与ＦＴＢＳ－ＩｇＧ的亲和力常数以及与其它１１种真菌毒素的相对交叉反应，建立了测定ＦＴＢ的ＩＣ－ＥＬＩＳＡ法。     

三聚氰胺单克隆抗体制备与鉴定

目的制备高度特异的三聚氰胺单克隆抗体,经酶联免疫吸附试验鉴定并制备胶体金试剂盒。方法以牛血清白蛋白交联的三聚氰胺为抗原,免疫BALB/c小鼠,通过传统杂交瘤融合和筛选技术制备抗三聚氰胺单克隆抗体,经纯化制备腹水,采用酶联免疫吸附实验(enzyme linked immune sorbent assay, ELISA)对抗体进行效价测定、亚类测定、交叉鉴定和蛋白质免疫印迹试验(westernblot)特异性鉴定后制备胶体金试剂盒。结果筛选出1株抗三聚氰胺单抗,制备的三聚氰胺胶体金试剂盒敏感值能达到30μg/L。结论获得了高度特异的且能稳定分泌三聚氰胺抗体的细胞株。     

Development of monoclonal antibodies for the fusarin mycotoxins

The fusarins are a group of mycotoxins produced by fungi that commonly infest cereal crops, in particular by the fungus Fusarium verticillioides. This group of compounds is characterized by a substituted 2-pyrrolidone ring attached to a 12-carbon polyunsaturated backbone. Several of the fusarins contain an epoxide substitution on the pyrrolidone ring and are highly mutagenic. This paper describes the development of seven monoclonal antibodies and immunoassays for detecting fusarins C and A. Fusarin C was isolated and conjugated to ovalbumin to produce the immunogen. Competitive indirect enzyme-linked immunosorbent assays (CI-ELISAs) were developed based upon the isolated monoclonal antibodies. The concentrations of fusarin C able to inhibit colour development by 50% (IC₅₀) in CI-ELISAs were 1.0, 2.0, 3.6, 23.4, 28.9, 31.4, and 66.7 ng ml^-1 for clones 1-38, 1-30, 1-5, 1-7, 1-43, 1-25, and 1-21, respectively. Cross-reactivity with fusarin A was 44.8, 51.4, 41.1, 174.0, 62.6, 78.2, and 98.0% for clones 1-38, 1-30, 1-5, 1-7, 1-43, 1-25, and 1-21, respectively. Given the sensitivity of these antibodies for fusarins it is expected that, with further development, they may be useful for detecting fusarins at relevant levels in foods.     

Modifying the antigen-immunization schedule improves the variety of monoclonal antibodies obtained from immune-phage antibody libraries against HIV-1 Nef and Vif

Immune phage antibody libraries are an attractive technology for isolating antigen-specific monoclonal antibodies (mAbs). Here we show that the immunization schedule affects the immune phage antibody library properties. We subcutaneously (s.c.) administered HIV-1 Nef and Vif antigens with different schedules (25 μg × 2 s.c. and 10 μg × 3 s.c.). The variety of isolated mAbs in 25 μg × 2 s.c. groups (Nef: 11 clones, Vif: 9 clones) was superior to that in the 10 μg × 3 s.c. groups (Nef: 2 clones, Vif: 1 clone). This finding suggests that it is important to optimize the immunization schedule for isolating a wide variety of mAbs.     

Two novel analytical methods based on polyclonal and monoclonal antibodies for the rapid detection of Cronobacter spp.: Development and application in powdered infant formula

Cronobacter spp. are opportunistic pathogens, and infections are associated with a high mortality rate. In the current study, monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) were generated using heat-inactivated C. sakazakii strain ATCC29544 as the immunogen. Following assay optimization, an indirect enzyme-linked immunosorbent assay (ELISA) based on pAbs and a sandwich ELISA based on mAbs and pAbs were established for the detection of Cronobacter spp. The indirect ELISA detected all species of Cronobacter assayed, and the limit of detection (LOD) was established as 10⁵ cfu/mL. In contrast, the sandwich ELISA was specific for C. sakazakii and had greater sensitivity than the indirect ELISA (LOD of 2 × 10⁴ cfu/mL). Following 10 h of enrichment, Cronobacter spp. were detected using either of the two analytical methods in samples inoculated with 1 cfu/100 g powdered infant formula (PIF). The results from this study demonstrated that both of these novel ELISAs were specific, sensitive, and rapid assays for the screening of pathogenic Cronobacter spp. in PIF.