Preparation and characterization of a polyclonal antibody from rabbit for detection of trinitrotoluene by a surface plasmon resonance biosensor

A polyclonal antibody against trinitrophenyl (TNP) derivatives was raised in rabbit, and the antibody was applied to detection of trinitrotoluene (TNT) using a surface plasmon resonance (SPR) biosensor. TNP-keyhole limpet hemocyanine (TNP-KLH) conjugate was injected into a rabbit, and a polyclonal anti-TNP antibody was realized after purification of the sera using protein G. Aspects of the anti-TNP antibody against various nitroaromatic compounds, such as cross-reactivities and affinities, were characterized. The temperature dependence of the affinity between the anti-TNP antibody and TNT was also evaluated. The quantification of TNT was based on the principle of indirect competitive immunoassay, in which the immunoreaction between the TNP-β-alanine-ovalbumin (TNP-β-ala-OVA) and anti-TNP antibody was inhibited in the presence of free TNT in solution. TNP-β-ala-OVA was immobilized to the dextran matrix on the Au surface by amine coupling. The addition of a mixture of free TNT to the anti-TNP antibody was found to decrease the incidence angle shift due to the inhibitory effect of TNT. The immunoassay exhibited excellent sensitivity for the detection of TNT in the concentration range of 3 × 10⁻¹¹ to 3 × 10⁻⁷ g/ml. To increase the sensitivity of the sensor, anti-rabbit IgG antibody was used. After flowing the mixture of free TNT and anti-TNP antibody, anti-rabbit IgG antibody was injected, and the incidence angle shift was measured. Amplification of the signal was observed and the detection limit was improved to 1 × 10⁻¹¹ g/ml.     

Production of a monoclonal antibody and development of enzyme-linked immunosorbent assay for alkyl ethoxylates

A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) has been developed for the determination of alkyl ethoxylates (AEs) that are the most widely used nonionic surfactants in the world. Three types of hapten, hemi-succinated AEs (C12EO7suc, C16EO23suc, and C18EO10suc), were synthesized and conjugated to bovine serum albumin (BSA) for mouse immunization. The mice immunized with the C12EO7suc-BSA that showed the high immune responses were used for cell fusion. The obtained monoclonal antibody (TFG2-76) was specific to AEs, which had alkyl (C) and ethoxy (EO) chain lengths of C10-12 and EO5-15, respectively. Two types of solid support, namely, a polystyrene tube and a 96-well microplate, were used for antibody immobilization. The working ranges of the tube-type and plate-type ELISAs were 2-100 and 20-1000 μg/L with IC₅₀ values of 12 and 71 μg/L AE (C12EO7), respectively. Moreover, the lowest quantification limit of plate-type ELISA could be lowered to 5 μg/L by decreasing the coated antibody concentration. Cross-reactivities with non-AE surfactants were determined, and the assay proved highly selective for AEs. The application of plate-type ELISA to determine spiked AEs in distilled water, tap water and river water provided good recoveries without matrix effects.     

Characterization of a monoclonal antibody that specifically inhibits pullulanase activity ofbacillus circulans amylase-pullulanase enzyme

A monoclonal antibody (MAb) against amylase-pullulanase enzyme fromBacillus circulons, which hydrolyzes not only theα-1,6-glycosidic linkage but also theα-1,4-glycosidic linkage to the same extent, has been produced by the fusion of BALB/c mouse spleen cells immunized with the native enzyme and P3x63Ag8U1 myeloma cells, and examined for inhibition of pullulanase activity in order to characterize the catalytic site of the pullulanase. The MAb recognizes active enzyme, but not the SDS-denatured or heat-inactivated protein, indicating that the antibody is highly conformational-dependent, specific for active enzyme. The antibody inhibited the pullulanase activity, but not amylase activity. The monoclonal antibody immunoblotted the enzyme and immunoprecipitated the enzyme. The immunoprecipitation was inhibited in the presence of substrate, pullulan, and the MAb competitively inhibited the binding of pullulan to the enzyme. The MAb, therefore, recognizes the pullulanbinding site of the enzyme. Kinetic analysis showed that the MAb inhibited pullulanase activity with inhibition constant (K _i ,) of 0.77Μg/mL, providing evidence that the antibody decreases the catalytic rate of enzyme activity and has an effect on substrate binding. These results strongly confirm the previous observations that APE may have two different active sites responsible for the expression of amylase and pullulanase activities (Kim, C. H. and Kim, Y. S.Eur. J. Biochem. 1995,227, 687–693).     

利用pⅧ展示系统改进噬菌体抗体芯片

将展示单链抗体的重组噬菌体与羧基终止的硅片偶联, 制成噬菌体抗体芯片, 可用于检测多类蛋白质和蛋白质组. 通常抗体被展示于噬菌体外壳蛋白pⅢ上, 由此制备的芯片灵敏度和信噪比较低. 我们选用凝血酶特异的单链抗体为代表, 比较了pⅢ展示系统和pⅧ展示系统制成芯片的检测效果. 由于pⅧ展示系统的融合蛋白拷贝数多, 所受空间位阻小, 大幅度提高了噬菌体抗体芯片的灵敏度和信噪比, 有望用于制备新型蛋白质芯片.     

Fabrication of a novel immunosensor using functionalized self-assembled monolayer for trace level detection of TNT by surface plasmon resonance

We have developed a new immunosensor based on self-assembly chemistry for highly sensitive and label-free detection of 2,4,6-trinitrotoluene (TNT) using surface plasmon resonance (SPR). A monolayer of amine terminated poly(ethylene glycol) hydrazinehydrochloride (PEG-NH₂) thiolate was constructed on an activated gold surface and immobilized with trinitrophenyl-β-alanine (TNPh-β-alanine) by amide coupling method. The binding interaction of a monoclonal anti-TNT Ab (M-TNT Ab) with TNPh-β-alanine immobilized thiolate monolayer surface was monitored and evaluated for detection of TNT based on the principle of indirect competitive immunoreaction. Here, the competition between the self-assembled TNT derivative and the TNT in solution for binding with antibody yields in the response signal that is inversely proportional to the concentration of TNT in the linear detection range. With the present immunoassay format, TNT could be detected in the concentration range from 0.008 ng/ml (8 ppt) to 30 ng/ml (30 ppb). The response time for an immunoreaction was 2 min and one immunocycle could be done with in 4 min including surface regeneration. Bound antibodies could be easily eluted from the self-assembled immunosurface at high recoveries (more than 100 cycles) using pepsin solution without any damage to the TNT derivatives immobilized on the surface. The compact self-assembled monolayer was highly stable and prevented the non-specific adsorption of proteins on the surface favoring error free measurement.     

A microscale approach for predicting the performance of chromatography columns used to recover therapeutic polyclonal antibodies

A microscale approach is described which screens conditions for recovering polyclonal antibodies from ovine sera by mixed-mode cation-exchange chromatography. The impact of pH and loading buffer salt concentration were assessed using robotically operated 20 μL packed pipette tips. Low salt concentrations delivered capacities up to 41 mg/mL, while only half this level was obtained at high salt concentrations. Two of the screened conditions were then tested in a 10 mL packed bed and overall trends in capacity, yield and purity were found to be retained. Microscale pipette tips thus provided a useful basis for the rapid, approximate definition of a chromatography design space.     

Docking of B-cell epitope antigen to specific hepatitis B antibody

The interaction of pres1 region of hepatitis B virus B-cell epitope antigen with specific hepatitis B neutralizing monoclonal antibody was examined by docking study. We modelled the 3D complex structure of B-cell epitope antigen residues CTTPAQGNSMFPSCCCTKPTDGNCY by homology modelling and docked it with the crystal structure of monoclonal antibody specific for the pres1 region of the hepatitis B virus. At the optimized docked conformation, the interactions between the amino acids of antigen and antibody were examined. It is found that the docked complex is stabilized by 59.3 kcal/mol. The stability of the docked antigen-antibody complex is due to hydrogen bonding and van der Waals interactions. The amino acids of the antigen and antibody responsible for the interaction were identified.     

单克隆抗体药物糖基化修饰分析研究进展

单克隆抗体药物是一类以免疫球蛋白G的结构为基础的大分子糖蛋白药物,为癌症、自身免疫疾病以及病毒感染等多种疾病的治疗提供了全新的途径。单抗药物的糖基化修饰类型及水平对其稳定性、清除率、免疫原性、抗体依赖细胞毒性及补体依赖细胞毒性等都有一定的影响。单抗药物的迅速发展及其在多种疾病治疗中日益凸显的重要性都对单抗药物的研发及用药安全等方面提出了更高的要求。因此,建立规范可靠的单抗药物糖基化修饰分析方法有着十分重要的意义。该综述将简要介绍单克隆抗体药物糖基化修饰及相关的定性、定量分析方法。     

Safe and Objective Assay of Enterovirus 71 Neutralizing Antibodies via Pseudovirus

Current serum neutralization assays based on the inhibition of the cytopathic effect(Nt-CPE) need to manipulate live viruses, which are time-consuming, labor-intensive, and have the potential exposure to infectious agents, so a safe and objective assay via pseudovirus for the fast and efficient detection of enterovirus 71(EV71) neutralizing antibodies was developed. First, we generated EV71 pseudovirus containing firefly luciferase gene in place of the capsid gene P1 in EV71 genome. Vero cells infected with 200 CCID₅₀(50% cell culture infective dose) of EV71 pseudovirus for 24 h were found to have the best performance. Seval sera were measured by EV71 pseudoparticle neutralization assay(Nt-PPN) and the conventional serological method Nt-CPE. Neutralizing antibody titers measured by Nt-PPN and those obtained by Nt-CPE demonstrate a high correlation between the two methods. Overall, the PPN assay represents a valid alternative to conventional serological methods for the evaluation of EV71 neutralizing antibodies. This method can be used for detecting neutralizing antibodies of other picornaviruses, such as hepatitis A virus( HAV) and coxsackievirus 16(CVA16), and make it possible to determine whether there is cross-reactivity between EV71 and CVA16.     

Anti-Metatype Antibody Screening,Sandwich Immunoassay Development,and Structural Insights for β-Lactams Based on Penicillin Binding Protein

Theoretically, sandwich immunoassay is more sensitive and has a wider working range than that of competitive format. However, it has been thought that small molecules cannot be detected by the sandwich format due to their limited size. In the present study, we proposed a novel strategy for achieving sandwich immunoassay of β-lactams with low molecular weights. Firstly, five β-lactam antibiotics were selected to bind with penicillin binding protein (PBP)2x* to form complexes. Then, monoclonal and polyclonal antibodies against PBP2x*-β-lactams complexes were produced by animal immunization. Subsequently, the optimal pairing antibodies were utilized to establish sandwich immunoassay for detection of 18 PBP2x*-β-lactam complexes. Among them, ceftriaxone could be detected at as low as 1.65 ng/mL with working range of 1–1000 ng/mL in milk. To reveal the detection mechanism, computational chemistry and molecular recognition study were carried out. The results showed that β-lactams with a large size and complex structures maybe conducive to induce conformational changes of PBP2x*, and then exhibit greater possibility of being detected by sandwich immunoassay after combination with PBP2x*. This study provides insights for subsequent investigations of anti-metatype antibody screening and sandwich immunoassay establishment for small-molecule detection.