An Enzyme-Linked Immunosorbent Assay (Elisa) for Measurement of Human Igg Subclass Levels in Serum

Abstract

We established an enzyme-linked immunosorbent assay (ELISA) to quantify human IgG subclasses in serum. IgG subclasses captured with subclass-specific mouse MoAbs could be detected with enzyme-labeled goat anti-human IgG. We screened the MoAbs which were suitable for the method from the commercially available MoAbs raised against human IgG subclasses. The specificity of the present ELISA for each subclass was clarified by the experiments as follows: (a) identification of individual IgG subclasses purified from total IgG by protein A column chromatography and (b) identification of a certain specific subclass which increased relatively in monoclonal (M)-proteinemia-patient sera compared with normal. We could detect each subclass with the sensitivity of 4.0 ng/ml for IgG₁, 13 ng/ml for IgG₂, 0.5 ng/ml for IgG₃ and 0.4 ng/ml for IgG₄ using pooled normal serum calibrated against a reference serum 67/86 from the World Health Organization (WHO) as a standard.     

Use of Normal IgG and its Fragments to Lower the Non-Specific Binding of Fab'-Enzyme Conjugates in Sandwich Enzyme Immunoassay

Abstract

An antibody IgG-coated polystyrene ball was incubated with an antigen and then with affinity-purified Fab'-enzyme conjugate in the presence of normal IgG, F(ab')₂, Fab' or Fab'-bovine serum albumin conjugate. After washing by incubation at 30[ddot]C for 10 min with shaking, the enzyme activity bound to the polystyrene ball was assayed. The non-specific binding of the Fab'-enzyme conjugate to the polystyrene ball considerably decreased in the presence of normal IgG and the other related proteins, while the specific binding decreased only slightly. As a result, the detection limit of hCG, human IgE and human α-fetoprotein was improved 3 to 10-fold.     

IgG adsorption on a new protein A adsorbent based on macroporous hydrophilic polymers: II. Pressure–flow curves and optimization for capture

Pressure–flow curves are obtained for a new protein A adsorbent matrix based on macroporous hydrophilic polymer beads with average diameter of 57 μm and a narrow particle size distribution. Experimental data are obtained in a 1 cm diameter laboratory column and in preparative scale columns with diameters of 20, 30, and 45 cm. The results are consistent with a model that assumes a linear relationship between bed compression and relative flow velocity. Surprisingly, the packing compressibility is essentially independent of column diameter for the preparative columns. As a result, after accounting for the variation in extraparticle porosity caused by compression, the column pressure drop is accurately predictable using the Carman–Kozeny equation. A model is also developed to predict productivity for IgG capture as a function of operating conditions based on dynamic binding capacity data presented in Part I of this work. For typical conditions, the model predicts maximum productivity at low residence times, between 1 and 1.5 min, when the dynamic binding capacity is at about 70–80% of the maximum. Combining the two models for column pressure and for dynamic binding capacity allows the design of preparative scale columns that maximize productivity while meeting specified pressure constraints.     

Application of conjoint liquid chromatography with monolithic disks for the simultaneous determination of immunoglobulin G and other proteins present in a cell culture medium

The aim of this study was to develop a chromatographic method, as a substitute for enzyme-linked immunosorbent assays, for the rapid and simultaneous detection of IgG, insulin, and transferrin present in a cell culture medium. Conjoint liquid chromatography (conjoint LC) using monolithic disks was applied for this purpose. An anion-exchange disk was combined with a Protein G affinity disk in a preparative HPLC system. IgG bound to the Protein G disk, whereas transferrin and insulin were captured on the quaternary ammonium (QA) disk. Using this method, it was possible to simultaneously determine the concentrations of IgG, transferrin, and insulin in the cell culture medium. Thus, conjoint LC could be used for the rapid and simultaneous detection of different proteins present in a cell culture medium.     

The Core Fucose on an IgG Antibody is an Endogenous Ligand of Dectin‐1

The core fucose, a major modification of N‐glycans, is implicated in immune regulation, such as the attenuation of the antibody‐dependent cell‐mediated cytotoxicity of antibody drugs and the inhibition of anti‐tumor responses via the promotion of PD‐1 expression on T cells. Although the core fucose regulates many biological processes, no core fucose recognition molecule has been identified in mammals. Herein, we report that Dectin‐1, a known anti‐β‐glucan lectin, recognizes the core fucose on IgG antibodies. A combination of biophysical experiments further suggested that Dectin‐1 recognizes aromatic amino acids adjacent to the N‐terminal asparagine at the glycosylation site as well as the core fucose. Thus, Dectin‐1 appears to be the first lectin‐like molecule involved in the heterovalent and specific recognition of characteristic N‐glycans on antibodies.     

毛细管电泳免疫分析法研究鼠IgM、兔抗鼠IgG及其免疫复合物

根据鼠IgM能与兔抗鼠IgG发生特异性免疫反应,采用毛细管电泳免疫法分析鼠IgM及其免疫复合物.以Tris为电解质,探讨了缓冲溶液浓度和pH值、进样时间、进样电压等因素对鼠IgM、兔抗鼠IgG及其免疫复合物分离的影响.在缓冲体系浓度为40 mmo1·L-1 TAE、1.0 mmo1.L-1EDTA(pH为8.5),进样时间为12 s,进样电压为18 kV的条件下,鼠IgM、兔抗鼠IgG及其反应形成的免疫复合物获得了很好的分离.     

孕妇产前抗A（B）IgG效价对高胆红素血症新生儿的影响研究

目的探讨孕妇产前抗A（B）IgG效价对高胆红素血症新生儿的黄疸情况及血红细胞参数等指标的影响。方法选取由O型血孕妇生产的且出生后1周内发生新生儿高胆红素血症的足月A（B）型血新生儿174例，按孕妇产前抗A（B）IgG效价分为低值效价组、中值效价组和高值效价组，比较3组新生儿溶血症（HDN）发生率、胆红素指标（TBil、DBil、IBil）、黄疸出现时间、入院治疗开始时间、入院治疗持续时间、血红细胞参数（RBC、Hb、HCT、RDW）和贫血发生率。结果3组新生儿胆红素指标（TBil、DBil、IBil）均无统计学差异（均P＞0.05），但孕妇产前抗A（B）IgG效价越高，HDN发生率越高，黄疸出现时间、入院治疗开始时间越早，入院治疗持续时间越长，RBC、Hb、HCT越低，RDW越高，贫血发生率也增高（均P＜0.05）。结论孕妇产前抗A（B）IgG效价可影响新生儿高胆红素血症的发生、发展进程，效价越高，新生儿血红细胞的破坏程度越大。     

Development of a Rapid Single‐Drop Analysis Biosensor for Screening of Phenanthrene in Water Samples

Detection techniques for biosensors often require bulky instruments or cells that are not feasible for in‐field analysis. Our single‐drop cell design, optimized in this work, comprised a screen‐printed three‐electrode (SPE), strip in horizontal position onto which a volume of 100 μL of sample or substrate solution was placed to ensure electrical contact (complete circuit). Together with optimized linear sweep voltammetry (LSV), parameters for the detection of the enzyme alkaline phosphatase (AP), the system was applied to a biosensor for the analysis of polycyclic aromatic hydrocarbons (PAHs), in environmental samples. A limit of detection (LOD), of 0.15 ppb was achieved for a model system with an IC₅₀ value of 0.885 ppb and a linear range (LR), of 0.2–10 ppb. Application of the single drop analysis (SDA), format to a PAH biosensor gave a LOD of 1.4 ppb for detection of phenanthrene with an IC₅₀ value of 29.3 ppb and linear range of 2–100 ppb. Proof of concept is shown with spiked sample analysis of phenanthrene in matrices such as sea, river and tap water.     

基于pH敏感相分离技术的新型荧光免疫测定体系

In this paper, it was discovered that a novel pH-sensitive copolymer of N-isopropylacrylamide (NIP) and N-(3-dimethylaminopropyl)methacrylamide (DMAPM) could be gotten by polymerization. The phase transition pH (pHtr) of P(NIP-DMAPM) polymer was found to be 7.4 at 37℃. The polymer was precipitated out of water above a critical pH=7.4 and re-dissolved below pH----7.4. The characteristic of this polymer made it possible to carry out the immunochemical steps of an immunoassay in a true solution and then to quickly separate the resulting product from the reaction mixture. In a competitive fluorescence immunoassay, the standard rabbit IgG and rabbit IgG immobilized on P(NIP-DMAPM) first competitively reacted with the fluorescein isothiocyanate (FITC) labeled antibody, then the pH of solution was adjusted above the pHtr of polymer to precipitate the polymer-immune complex,and the polymer-immune complex precipitate was separated and re-dissolved by the adjustment of pH, finally the FITC-labeled antibody in the immune complex was quantified by fluorescence measurement. The calibration graph for rabbit IgG was linear over the range of 100-1000 ng/mL with a detection limit of 11 ng/mL. The method is rapid, sensitive and simple. Owing to neutral pHtr of P(NIP-DMAPM), the damage to antigen-antibody immune complex was greatly decreased in the course of separation. In addition, a sandwich enzyme-linked fluorescence immunoassay method for the determination of human IgG was also developed, showing that the pH-sensitive phase separating immunoassay could be performed in the competitive method as well as the sandwich method.     

Development of a tetramethylrhodamine-labeled probe for a capillary electrophoresis-based competitive immunoassay of staphylococcal enterotoxin B

Staphylococcal enterotoxin B (SEB) was labeled with tetramethylrhodamine isothiocyanate (TRITC) and used as a probe for a competitive immunoassay. Labeling conditions such as solution pH and time were varied to observe the effect on the fluorescent product. It was found that solution pH of the labeling reaction had little effect on the fluorescence signal of the resulting products. However, labeling at pH 7.0 produced a probe that had a higher affinity for the antibody used in this study than the probes produced at pH 8.0 and 9.0. The fluorescent probes were used to perform a competitive assay for SEB in model skim milk samples. Detection limit was approximately 300 fg of SEB. Quantitation was achieved by curve fitting of fluorescent signals for bound/free probe versus log[SEB] with logarithmic functions. Accuracy in the model skim milk samples was acceptable for 3 and 5 nM SEB, but decreased considerably for a concentration of less than 1 nM SEB. The error was attributed to deviation in linearity in the standard curve at lower concentrations. Reproducibility for the analysis of both standard solutions used for the calibration curves and the model skim milk samples was excellent, with standard deviations of approximately 10% from data collected over a 3-week period. No cross-reactivity was found when the assay was tested with a 700 nM sample of staphylococcal enterotoxin A. Although competitive immunoassays are usually used for small molecules, such as therapeutic drugs, the results demonstrate that relatively large molecules (SEB, 27 kDa) can also be assayed with the technique.