乙型肝炎病毒表面抗原a、d、r亚型单克隆抗体的制备与应用

本文成功地建立了分泌抗乙型肝炎病毒表面抗原(抗-HBsAg)a,d、r3种亚型决定簇抗体的4株杂交瘤细胞。经一系列生化、免疫学鉴定,证明4株细胞所分泌的单克隆抗体(McAb)均具有各自的亚型特异性。反复克隆培养16周,并液氮冻存8个月后复苏,抗体的效价仍稳定不变。用纯化的McAb制备RPHA诊断试剂,检测了80例有乙型肝炎自觉症     

An antigen-capture enzyme-linked immunosorbent assay (ELISA) to detect isometamidium chloride in Oncorhynchus spp

An antigen-capture enzyme-linked immunosorbent assay (ELISA) was developed to detect and measure isometamidium chloride in the plasma of Oncorhynchus tshawytscha and O. mykiss. Isometamidium-ovalbumin conjugate and anti-isometamidium antibodies were used to coat polystyrene plates. The peroxidase saturation technique was used to optimize the coating antigen concentration; it demonstrated low affinity of the isometamidium-ovalbumin conjugate but high affinity of the anti-isometamidium antibodies for polystyrene surface sites. The optimal conditions of antiisometamidium antibodies to coat plates was at pH 7.3 and a 1:1000 dilution (0.0012 mg ml(-1) protein). The ELISA was sensitive as it detected 0.0006 mg ml(-1) of isometamidium in fish plasma. Isometamidium diluted with saline could not be detected at concentrations less than 0.05 mg ml(-1). The results indicate that this ELISA is much more sensitive when isometamidium is bound to plasma than unbound isometamidium in saline.     

R-ELISA: repeated use of antigen-coated plates for ELISA and its application for testing of antibodies to HIV and other pathogens.

In this paper we report on the evaluation of several procedures that allow for the repeated use of an antigen-coated, enzyme-linked immunosorbent assay (ELISA) plate for enzyme immunoassay (EIA). We have shown that antigen-coated ELISA plates that were incubated once with an aqueous solution containing 8 M urea, 2% sodium dodecyl sulfate and 2% mercaptoethanol, after an EIA, can be reused again for EIA without loss of antigenic capacity. Thus, in this procedure, after an EIA, the ELISA plates were washed once with the above solution and then in a buffer containing 20 mM Tris-HCl, pH 7.5, 0.1% Tween 20 and 500 mM NaCl. This washing protocol was shown to remove the primary antibody, enzyme-conjugated secondary antibody and substrate without removing the antigen from the ELISA plate microwells. Thus, an antigen-coated ELISA plate previously used for an assay could be reused. We tested this repeat ELISA (R-ELISA) procedure on high antigen-binding ELISA plates coated with two different plant virus proteins, a synthetic peptide, the p25/24 gag and the gp120 proteins of the human immuno-deficiency virus, or the staphylococcus enterotoxin protein. In each case tested, the procedure allowed for the repeated use of the same antigen-coated plates for EIA of the respective antibodies. This procedure should prove to be particularly valuable for mass screening of samples tested for HIV and other disease-causing agents.     

Sensitive detection of glucagon aggregation using amyloid fibril-specific antibodies

Sensitive detection of protein aggregates is important for evaluating the quality of biopharmaceuticals and detecting misfolded proteins in several neurodegenerative diseases. However, it is challenging to detect extremely low concentrations (<10 ppm) of aggregated protein in the presence of high concentrations of soluble protein. Glucagon, a peptide hormone used in the treatment of extreme hypoglycemia, is aggregation-prone and forms amyloid fibrils. Detection of glucagon fibrils using conformation-specific antibodies is an attractive approach for identifying such aggregates during process and formulation development. Therefore, we have used yeast surface display and magnetic-activated cell sorting to sort single-chain antibody libraries to identify antibody variants with high conformational specificity for glucagon fibrils. Notably, we find several high-affinity antibodies that display excellent selectivity for glucagon fibrils, and we have integrated these antibodies into a sensitive immunoassay. Surprisingly, the sensitivity of our assay—which involves direct (nonantibody mediated) glucagon immobilization in microtiter plates—can be significantly enhanced by pretreating the microtiter plates with various types of globular proteins before glucagon immobilization. Moreover, increased total concentrations of glucagon peptide also significantly improve the sensitivity of our assay, which appears to be due to the strong seeding activity of immobilized fibrils at high glucagon concentrations. Our final assay is highly sensitive (fibril detection limit of ~0.5–1 ppm) and is >20 times more sensitive than detection using a conventional, amyloid-specific fluorescent dye (Thioflavin T). We expect that this type of sensitive immunoassay can be readily integrated into the drug development process to improve the generation of safe and potent peptide therapeutics.     

Comparative evaluation of the methods of determining perfringens A antitoxin

The methods for determining the level of type A Perfringens antitoxin in human blood sera were examined and compared. The ratios for correlating the data obtained in the toxin neutralization test (NT) in vivo, in the passive hemagglutination test (PHT), and as a result of the enzyme-labeled immunosorbent assay (ELISA) with regard to the antitoxin level measured in the NT in vitro were equal to 0.88, 0.64 and 0.39, respectively. The sensitivity of the NT in vivo and in vitro was 0.25 IU/ml, that of the PHT 0.01-0.005 IU/ml, and that of the ELISA 0.01-0.02 IU/ml Perfringens antitoxin. To perform the NT, not less than 1 ml blood serum is required, while for the PHT and ELISA, 0.1-0.05 ml. Provided hyghly purified anatoxin is used for preparing the erythrocyte diagnosticum Perfringens, and polysterene plates are sensitized in performing the ELISA, all the reactions are specific. While titrating human blood sera containing type A Perfringens antitoxin, use in the PHT may be made of type A Perfringens rabbit antiserum as reference.     

Quantitation of pulmonary surfactant protein SP-B in the absence or presence of phospholipids by enzyme-linked immunosorbent assay

We have developed an enzyme-linked immunosorbent assay (ELISA) that uses polyclonal or monoclonal anti-surfactant protein SP-B antibodies to quantitate purified SP-B in chloroform/methanol and in chloroform/methanol extracts of whole pulmonary surfactant at nanogram levels. This method has been used to explore the effect of the presence of different phospholipids on the immunoreactivity of SP-B. Both polyclonal and monoclonal antibodies produced reproducible ELISA calibration curves for methanolic SP-B solutions with protein concentrations in the range of 20-1000 ng/mL. At these protein concentrations, neither dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, nor phosphatidylcholine or phosphatidylglycerol from egg yolk had significant effects on the binding of antibodies to SP-B up to protein-to-lipid weight ratios of 1:20. Coating of ELISA plates with SP-B concentrations higher than 1 microg/mL produced a substantial decrease in the binding of antibodies to the protein that was prevented by the presence of negatively charged but not zwitterionic phospholipids. Characterization of the secondary structure of SP-B by far-UV circular dichroism showed that phospholipids induced pronounced changes on the conformation of SP-B when the solvent was evaporated and dry lipid-protein films were formed, a necessary step to expose protein to antibodies in ELISA. Under these conditions, negatively charged lipids, but not zwitterionic ones, induced a marked decrease on the ellipticity of SP-B that would be associated with a conformation that is significantly more exposed to antibodies.     

Development of an ultrasensitive immunoassay using affinity maturated antibodies for the measurement of rodent insulin

The measurement of plasma insulin is important for clinical diagnosis of diabetes and for preclinical research of metabolic diseases, especially in rodent models used in drug discovery research for type 2 diabetes. Fasting immunoreactive insulin (F-IRI) concentrations are used to calculate the homeostasis model assessment ratio (HOMA-R), an index of insulin sensitivity. However, even the most sensitive commercially available enzyme-linked immunosorbent assay (ELISA) kits cannot measure the very low F-IRI concentrations in normal rats and mice. Therefore, we sought to develop a new rodent insulin ELISA with greater sensitivity for low F-IRI concentrations. Despite repeated efforts, high-affinity antibodies could not be generated by immunizing mice with mouse insulin (self-antigen). Therefore, we generated two weak monoclonal antibodies (13G4 and 26B2) that were affinity maturated and used to develop a highly sensitive ELISA. The measurement range of the sandwich ELISA with the affinity maturated antibodies (13G4m1 and 26B2m1) was 1.5 to 30,000 pg/ml, and its detection limit was at least 10 times lower than those of commercially available kits. In conclusion, we describe the development of a new ultrasensitive ELISA suitable for measuring very low plasma insulin concentrations in rodents. This ELISA might be very useful in drug discovery research in diabetes.     

Evaluation of monoclonal antibodies against Plasmodium vivax sporozoites for ELISA development

Nine monoclonal antibodies (MAbs) developed against Plasmodium vivax (Grassi & Feletti) salivary gland sporozoites were evaluated for use in an enzyme-linked immunosorbent assay (ELISA), using sporozoites developed in Anopheles dirus Peyton & Harrison An. gambiae Giles and An.maculatus Theobald. Four of the antibodies were unsuitable due to the low sensitivity of the resulting assays or the requirement for high concentrations of capture antibody. An additional two MAbs were rejected because they resulted in assays with high background absorbance, attributed to self-binding. Of the three remaining MAbs, the use of Navy vivax sporozoite (NVS) 3 resulted in an ELISA with the highest sensitivity and the lowest concentration requirement for capture antibody. Assay sensitivity varied with sporozoite strain indicating possible quantitative epitope heterogeneity. None of the MAbs cross-reacted with the heterologous sporozoites tested by immunofluorescence antibody assay (IFA). The IFA activity was not an indicator of ELISA sensitivity. The use of MAb NVS 3 in a standardized ELISA method resulted in an assay 10 times more sensitive than reported previously for P. vivax sporozoites, with a detection limit of fewer than 100 sporozoites per mosquito.     

Methods of enhancing the specificity of immunoenzyme test systems for the determination of human IgE

The sensitivity and specificity of an enzyme immunoassay (EIA) system for the determination of total IgE in humans increases if polystyrene plates are sensitized with swine gamma-globulin and the conjugate is prepared with the use of sheep gamma-globulin obtained from the commercial preparation of sheep antiserum to human IgE. Such system becomes even more specific and sensitive if sheep antibodies to human IgE, purified by affinity chromatography, are used both for the sensitization of polystyrene plates and for the preparation of the conjugate. This conjugate is necessary for the development of EIA systems intended for the determination of specific human IgE-antibodies to various allergens.     

Monoclonal Antibodies for Detection and Serotyping of Cucumber Mosaic Virus

Two monoclonal antibodies (MAbs) to cucumber mosaic virus (CMV) were selected from a panel of MAbs for use in the direct DAS (double antibody sandwich)-ELISA. Two different test procedures were developed: an ELISA with polyclonal and monoclonal antibodies (mixed ELISA) for the routine detection of CMV and a MAb-ELISA with two MAbs directed against different epitopes for the specific detection of the N serotype which is prevalent in GDR. The conventional two-step incubation of plates precoated with IgG was compared with simultaneous incubation of test sample and labelled antibody (one-step incubation). The mixed ELISA proved to be more sensitive than the direct DAS-ELISA with polyclonal antisera in detecting CMV in crude sap of infected plants. On the other hand, the MAb-ELISA could be used for serotyping of CMV isolates which is important in epidemiological investigations and in resistance breeding. Both the two-step and the one-step procedures gave similar results with some advantages of the latter procedure. One-step incubation is not only time-saving but seems to be also more sensitive with regard to the detection limit. However, care must be taken to circumvent the hook-effect occurring at high virus concentrations.     

The influence of the type of immunosorbent on rabies antibody EIA; advantages of purified glycoprotein over whole virus

Two types of in vitro assay (enzyme immunoassay and sero-neutralization test) for the titration of rabies antibodies were used to assay sera from mice and humans immunized with cell culture vaccines or neural tissue vaccines. Enzyme immunoassays (EIA) were performed in plates sensitized with whole virus, purified glycoprotein or purified nucleocapsid. Neutralizing antibody titres were determined by the rapid fluorescent focus inhibition test (REFIT) and by an in vitro seroneutralization test including a rapid enzyme immunotitration of intracellular antigens (REITICA). The results obtained with sera of immunized mice and humans showed that (1) cell culture vaccines mainly induced the synthesis of antiglycoprotein neutralizing antibodies; and (2) neural tissue vaccines induced a high synthesis of antinucleocapsid non-neutralizing antibodies and a more or less important synthesis of antiglycoprotein antibodies depending on the origin of the tissue used for their preparation. Consequently, it was emphasized that when using EIA, the antibody titration must be run in glycoprotein-coated plates rather than in whole virus-coated plates to appreciate correctly the immunizing potency of a rabies vaccine, especially neural tissue vaccine.     

C-terminal glyoxylyl peptides for sensitive enzyme-linked immunosorbent assays

Summary A model peptide antigen derived from HCV Core protein was modified by a hydrophilic glyoxylamide arm using a tartaramide-based linker and used in an enzyme-linked immunosorbent assay (ELISA) on hydrazide plates. A comparative study with the standard non-covalent adsorption procedures demonstrated a large gain in sensitivity for the detection of antibodies in HCV-positive sera.     

Triple-site antigen capture ELISA for human myoglobin can be more effective than double-site assay

Using a panel of monoclonal antibodies against human myoglobin (Mb), we have shown that the sensitivity of antigen-capture ELISA can be significantly increased by simultaneous immobilization of two cooperating capture monoclonal antibodies on a solid phase. This method ("triple-site ELISA") uses three monoclonal antibodies to different epitopes of the same antigen (two capture/one tracer) unlike the traditional double-site assay using one capture and one tracer monoclonal antibody. We developed double- and triple-site ELISA for Mb by varying the capture and tracer monoclonal antibodies. Triple-site assays showed 4-6-fold increase in sensitivity compared to the double-site assays. A model for this effect is suggested; according to the model, in triple-site ELISA, high-affinity cyclic configurations can be formed by an antigen, two capture monoclonal antibodies, and the surface of the solid phase.     

Development of a solid-phase immunoenzyme method for determining the antibody level in the blood sera of persons vaccinated with influenza vaccine

To control the effectiveness of vaccination against influenza, the optimum conditions for making the enzyme-linked immunosorbent assay (ELISA) with a view to determine the level of anti-influenza antibodies in human blood sera have been established. The kinetics of influenza virus adsorption in the wells of ELISA polystyrene plates and the kinetics of the interaction between the immobilized antigen and species-specific peroxidase-labeled antibodies have been studied. The method has been shown to be more sensitive than the hemagglutination inhibition test in the determination of seroconversion in persons immunized with influenza vaccine.     

Monoclonal antibodies against hepatitis B virus surface antigen: preparation and characterization

Hybridomas secreting HBsAg antibodies were obtained by fusing murine myeloma cell line P3-X63-Ag8 to spleen cells of BALB/c mice sensitized with HBsAg. The surface antigen used for immunization of mice was prepared by purification from pooled human plasma specimens. Resulting monoclonal antibodies were detected by the SPRIA method. Clones producing highest anti-HBs titres were used to prepare mouse ascitic fluids. Monoclonal antibodies in ascitic fluid reached a titre of 10(6) to 10(7) at a protein concentration of 1 mg per ml. Two of the prepared monoclonal antibodies, HBS-01 and HBS-02, both belonging to IgG1 subclass of immunoglobulins, were selected for further study in order to assess their potential useability in the commercial ELISA kit. The pI values for HBS-01 ranged from 6.60 to 6.85, for HBS-02 from 5.6 to 6.1. In solid phase ELISA test the use of HBS-01 antibody improved accuracy of the assay by increasing its detection sensitivity for HBsAg subtypes adw and ayw in the reference serum; this sensitivity was evidently much better than that seen with the commercially available rabbit polyclonal anti-HBsAg antibody. The monoclonal antibody HBS-01 is specific to the determinant "a", which makes it suitable for use in ELISA test aimed at HBsAg detection. The antibody HBS-02 showed a markedly better reaction with HBsAg subtype adw than subtype ayw and can thus be used with advantage for their discrimination.     

Monoclonal antibody-based quantitation of poly(ethylene glycol)-derivatized proteins, liposomes, and nanoparticles

Covalent attachment of poly(ethylene glycol) (PEG) molecules to drugs, proteins, and liposomes is a proven technology for improving their bioavailability, safety, and efficacy. Qualitative and quantitative analysis of PEG-derivatized molecules is important for both drug development and clinical applications. We previously reported the development of a monoclonal IgM antibody (AGP3) to PEG. We now describe a new IgG1 monoclonal antibody (E11) to PEG and show that it can be used in combination with AGP3 to detect and quantify PEG-derivatized molecules. Both antibodies bound the repeating subunits of the PEG backbone and could detect free PEG and PEG-modified proteins by ELISA, immunoblotting, and flow cytometry. Detection sensitivity increased with the length and the number of PEG chains on pegylated molecules. Both antibodies also efficiently accelerated the clearance of a PEG-modified enzyme in vivo. A sandwich ELISA in which E11/AGP3 were employed as the capture/detection antibodies was developed to detect PEG-modified proteins at concentrations as low as 1.2 ng/mL. In addition, the ELISA could also quantify, in the presence of 10% fetal bovine serum, free methoxy-PEG20,000, PEG2,000-quantum dots, and PEG2,000-liposomes at concentrations as low as 20 ng/mL (1.0 nM), 1.4 ng/mL (3.1 pM), and 2.4 ng/mL (3.13 nM phospholipids), respectively. Finally, we show that the sandwich ELISA could accurately measured the in vivo half-life of a PEG-modified enzyme. These antibodies should be generally applicable to the qualitative and quantitative analysis of all PEG-derivatized molecules.     

The standardization of a 'spot-test' ELISA for the rapid screening of sera and hybridoma cell products II. The determination of binding capacity, binding ratio and coefficient of variation of different ELISA plates in sandwich and indirect ELISA

The different commercially available enzyme-linked immunosorbent assay (ELISA) plates were compared for their binding capacity for purified foot-and-mouth disease virus antigen or IgG, their binding ratio (a measure of the efficiency with which positive and negative serum samples may be distinguished), and their coefficients of variation within a plate, between plates and between batches of plates. No one plate could be described as having ideal characteristics, and the choice of ELISA plate depends on the use to which the ELISA is being put. For our purposes, viz. a 'spot-test' which rapidly and efficiently detects specific antibody when the levels of that antibody are low (hybridoma culture supernatants) or when the antibody is contaminated with other 'interfering' proteins (high concentrations of serum), we found that most of the PVC plates and, of the polystyrene plates, the Nunc Immunoplate I and Dynatech M129B plates performed well. The lowest coefficients of variation were obtained using Nunc Immunoplate I, Dynatech M129B and Falcon 3912 plates.     

Oriented Immobilization of Anti-Pneumolysin Tagged Recombinant Antibody Fragments

Recombinant antibodies such as Fab and scFv are monovalent and small in size, although their functional affinity can be improved through tag-specific immobilization. In order to find the optimum candidate for oriented immobilization, we generated Fab and scFv fragments derived from an anti-pneumolysin monoclonal antibody PLY-7, with histidine and cysteine residues added in diverse arrangements. Tagged antibody fragments scFv-Cys7-His6, His6-scFv-Cys7, and Fab-Cys7 lost considerable affinity for the antigen; however, Fab-His6, Fab-Cys1, and scFv-His6-Cys1 were able to detect immobilized antigen, revealing that the position and number of histidine and cysteine residues are involved differently in the reactivity of antibody fragments. Random and orientated immobilizations were carried out using conventional polystyrene and commercial surface-pretreated ELISA plates. The best orientation performance was obtained with Fab-Cys1-biotin on streptavidin-coated plates with increased signal levels of 62%, while oriented immobilization of Fab-His6 and scFv-His6-Cys1 on nickel- and maleimide-coated plates failed to improve the ELISA sensitivity.     

The determination of the antibodies and the somatic O-antigen of Salmonella group B by using the complement-bound lysis of liposomes]

A simple and sensitive method for the determination of group B Salmonella O-antigen and specific antibodies to group B Salmonella by means of the complement-dependent lysis of liposomes sensitized with S. typhimurium lipopolysaccharide (LPS) is proposed. The factors affecting the sensitivity of the method during the determination of antibodies and free antigen have been studied. The method permits the determination of soluble LPS antigen in concentrations of 0.5-200 micrograms/ml.