Immunoenzyme demonstration of viruses and virus-specific antibodies by using conjugates based on gene engineering-produced beta-lactamase

Enzyme immunoassays for the detection of viral antigens and virus-specific antibodies in biological samples have been described. Molecular complexes of antibodies and beta-lactamase (penicillinase) have been used as anti-specific conjugates. To synthesize the conjugate, the enzyme obtained with the aid of genetic engineering has been used. Enzyme immunoassays have been tested for the indication of the influenza virus and virus-induced specific antibodies. Enzyme immunoassays were shown to possess certain advantages (e.g., the use of simple and nontoxic substrate) along with the sensitivity identical to that of other methods, employing peroxidase-based conjugates.     

Protein microchips: use for immunoassay and enzymatic reactions

Different proteins such as antibodies, antigens, and enzymes were immobilized within the 100 x 100 x 20-microm gel pads of protein microchips. A modified polyacrylamide gel has been developed to accommodate proteins of a size up to 400,000 daltons. Electrophoresis in the microchip reaction chamber speeded up antigen-antibody interactions within the gel. Protein microchips were used in immunoassays for detection of antigens or antibodies, as well as to carry out enzymatic reactions and to measure their kinetics in the absence or presence of an inhibitor. A protein microchip can be used several times in different immunoassays and enzymatic kinetic measurements.     

Detection of HSV type-1 and type-2 IgG using an in vitro PCA based homogeneous immunoassay

Enzyme immunoassays (EIAs) are widely used in the clinical laboratory and research institutes for the detection of biologically relevant analytes. Almost all EIAs are heterogeneous in nature and require multiple steps of process. In contrast, homogeneous immunoassays (HA) offer a simplified one-step approach with a number of potential advantages over contemporary heterogeneous EIAs such as higher throughput and greater clinical utility. Utilizing TEM-1 β-lactamase as a reporter enzyme, we have developed HAs based on in vitro protein fragment complementation (PCA) for the detection of antibodies and potentially be used for antigens or other biomarkers. In this proof-of-principle study we demonstrate the successful in vitro differentiation of anti-herpes simplex virus (HSV) type-1 and type-2 Immunoglobulin G (IgG) in human serum with high sensitivity and specificity.     

Heterogeneous enzyme immunoassay.

During the past 10 to 15 years immunoassays have gained acceptance as the methods of choice in the diagnosis of a number of disease states. At present the immunodiagnostic techniques employed range from radioimmunoassay for haptens through immunofluorescence for autoimmune diseases to complement fixation for viral infections. All of these assays have their own individual limitations such as: safety, short shelf life and sensitivity. The development of enzyme immunoassays, in particular enzyme linked immunosorbent assay (ELISA), has led to a substantial literature which offers the view that enzyme immunoassays provide a safe, sensitive and specific alternative to standard methods for the detection of antibodies or antigens. The application of heterogeneous enzyme linked immunosorbent assays for the quantitation of haptens, macromolecular antigens and antibodies is reviewed.     

基于上转换纳米材料的免疫检测技术应用

免疫分析法具有简便、快速、准确等特点，广泛应用于医学、食品、环境等领域检测，将免疫分析方法与纳米材料相结合可以提高免疫分析的性能。与传统纳米材料相比，上转换纳米颗粒（upconversion nanoparticles，UCNPs）具有光稳定性好、发光寿命长和狭窄及可调整的发射带等优秀的光学性质，与免疫分析相结合可显著降低背景噪声，提高分析灵敏度。本文简要介绍了UCNPs的发光机制，对UCNPs的合成和表面修饰方法进行了总结，并详细论述荧光共振能量转移、内滤效应、磁分离技术、上转化连接免疫吸附技术和上转换免疫层析技术五种基于UCNPs的免疫检测技术，最后对该技术所面临的挑战和前景进行总结和展望，以期为UCNPs免疫检测技术的发展提供理论指导。     

Detection of Clostridium botulinum neurotoxin type A using immuno-PCR

AIMS: An immuno-polymerase chain reaction (immuno-PCR) has been developed for the sensitive detection of antigens, which greatly extends the detection limits of immunoassays. In the current study, the method was applied to the detection of Clostridium botulinum neurotoxin type A (BTx-A). METHODS AND RESULTS: Anti-BTx-A antibody-DNA conjugates were synthesized using a heterobifunctional cross-linker reagent to covalently link the reporter DNA and the antibodies. The antibody-DNA conjugates with antigens were amplified by PCR, and dose-dependent relationships for each analyte were demonstrated. Detection limits of immuno-PCR for BTx-A (3.33 x 10(-17) mol) exceeded the conventional enzyme-linked immunosorbent assay (3.33 x 10(-14) mol) by a 1000-fold enhancement in detection sensitivity. CONCLUSION: Detection of BTx-A antigens by immuno-PCR demonstrated 100% sensitivity and 100% specificity in 100-fold magnitude below the detection limit of ELISA. SIGNIFICANCE AND IMPACT OF THE STUDY: It is concluded that the immuno-PCR method could be used to detect a very low level of BTx-A for clinical diagnosis.     

A novel erythrocyte-based immunoassay for simultaneous detection of both antimycobacterial antibody response and mycobacterial antigen in human serum samples of pulmonary tuberculosis and a control group of patients using 'a single probe'

A modified passive hemagglutination using double aldehyde stabilized cells (tanned sheep erythrocytes treated with glutaraldehyde and pyruvic aldehyde) was evaluated for detection of both antimycobacterial antibodies and circulating mycobacterial antigens simultaneously in human serum samples from patients with pulmonary tuberculosis (n=40) and a control group (n=44). Double aldehyde stabilized cells sensitized with an optimum dose of 200 microg mL(-1) of sonicate extract of Mycobacterium tuberculosis antigens was used as single probe to detect both antibodies and antigen, respectively, by passive hemagglutination and passive hemagglutination inhibition. The sensitivity limit of passive hemagglutination inhibition was determined to be 280 ng mL(-1) using a dose-response curve. Sensitivity of passive hemagglutination and passive hemagglutination inhibition, respectively, was 90% and 52.5%, and specificity was 91% and 100%. Although passive hemagglutination and passive hemagglutination inhibition need further evaluation, these erythrocyte-based immunoassays are potentially advantageous, especially as double aldehyde stabilized sensitized cells could be used as a single probe for detection of both antibodies and antigen. In addition, erythrocyte-based immunoassays are rapid, simple and cost-effective with a high degree of sensitivity.     

Electrochemical arrays coupled with magnetic separators for immunochemistry

Electrochemical methods are increasingly applied to immunoassays, because they overcome problems associated with other modes of detection. In particular, with respect to conventional immunoassays, electrochemical immunosensors show versatility, reliability, and fast analysis time. In immunosensor strategy, the antigen or antibody can be immobilized directly onto the surface of the electrochemical transducer that will finally be used to reveal the amount of the affinity reaction. However, the use of the electrode surface as a solid phase as well as an electrochemical transducer presents some problems: a shielding of the surface by biospecifically bound antibody molecules can cause hindrance in the electron transfer, resulting in a reduced voltammetric signal. Thus, as an alternative solid phase, magnetic beads because of their low toxicity and high biocompatibility have gained much attention in chemistry, associated with various analytical techniques, due to their suitability for immobilization of biomolecules. Magnetic micro- or nanobeads can be separated easily and quickly by magnetic forces and will be used together with bioaffine ligands, e.g., antibodies or proteins with a high affinity to the target. The special advantages of magnetic separation techniques are the fast and simple handling of a sample vial and the opportunity to deal with large sample volumes without the need for time-consuming centrifugation steps. This also makes biomagnetic separation ideal for automated assay/analysis systems which will play a very important role in the near future. This review presents some examples of immunochemical assay developed using magnetic beads as a solid phase coupled with electrochemical detection techniques, in particular, using electrochemical arrays as transducers. Applications related to static measurements, together with in-flow detection systems are presented.     

Enzyme-linked immunosorbent assay (ELISA): a practical tool for rapid diagnosis of viruses and other infectious agents.

Tissue culture techniques are inadequate to diagnose some viral infections. Thus, solid-phase immunoassays have been developed for the direct detection of viral antigens in clinical specimens. While radioimmunoassays (RIA) have attained widespread use, solid-phase enzyme-linked immunosorbent assays (ELISA) offer a number of advantages over RIA systems. ELISAs can be established with approximately the same sensitivity as radioimmunoassays without utilizing unstable, gamma-emitting isotopes. However, before ELISA systems can obtain widespread usage, a number of aspects of the test must be optimized. These include the preparation and use of reagents, the nature of the solid phase, the choice of enzyme, and the enzyme-antibody conjugation method. With the solving of these problems, ELISA should attain widespread usage for rapid diagnosis of a large number of infectious agents.     

An immunochemical assay model system for the sensitive detection of pyruvate dehydrogenase complex (PDHc) and its decarboxylating subunit pyruvate dehydrogenase (E1).

An immunochemical enzyme immunoassay model system was developed and compared for maximum sensitivity with a radioimmunoassay method and the classic enzyme activity method for the detection of pyruvate dehydrogenase complex (PDHc) and its decarboxylating subunit, pyruvate dehydrogenase (E1), isolated from Escherichia coli. Cross-linked large molecular weight antibody-enzyme conjugate systems are compared with heterobifunctional singular antibody conjugates substituted with high levels of horseradish peroxidase. Both polyclonal and monoclonal antibodies generated to the Escherichia coli PDHc and E1 antigens were used to develop a double-antibody sandwich microtiter plate enzyme-linked immunosorbent assay. It is demonstrated that a double sandwich immunochemical assay system can be quantitative for PDHc, can detect PDHc in crude cell lysates and has levels of sensitivity of 2.0.10(-16) mol for the detection of PDHc. This assay model system provides specific antibody selection criteria and coupling methods needed to select specific antisera that cross-react with human PDHc. This rapid and sensitive immunochemical assay method clearly demonstrates that sensitive mass assay systems can be developed for the detection of PDHc. Different from Western blot, this methodology could be used to generate mass assays which could be applied to the rapid detection of mammalian antigens (employing the corresponding antibodies) implicated in a number of pyruvate dehydrogenase deficiencies associated with human disorders.     

Europium Nanoparticle-Based High Performing Immunoassay for the Screening of Treponemal Antibodies

Treponema pallidum subspecies pallidum (Tp) is the causative agent of syphilis which mainly spreads through sexual contact, blood transfusion and perinatal route. In order to curtail the spread of the infection and to clinically manage the disease, timely, accurate and reliable diagnosis is very important. We have developed an immunoassay for the detection of treponemal antibodies in human serum or plasma samples. In vivo biotinylated and non-biotinylated versions of the recombinant antigen were designed by the fusion of three Tp-specific antigens namely Tp15, Tp17 and Tp47. These fusion antigens were expressed in E. coli and purified using single-step metal affinity chromatography. Biotinylated fusion antigen immobilized on streptavidin coated plate was used to capture the treponemal antibodies and the non-biotinylated antigen coated on europium nanoparticles was used as tracer. Assays with two different incubation times of 10 min and 1 h were developed, and following the incubation the europium fluorescence was measured using time-resolved fluorometry. The developed time-resolved fluorometric (TRF) immunoassays were evaluated with in-house and commercial serum/plasma sample panels. For well-established treponemal antibodies positive or negative samples, the sensitivity of TRF immunoassay with 10 min incubation time was 97.4%, and of TRF immunoassay with 1 h incubation time was 98.7%, and the specificities of both the TRF immunoassays were 99.2%. For the samples with discordant results with the reference assays, both the TRF immunoassays showed better specificity than the Enzygnost syphilis enzyme immunoassay as a screening test. The two different incubation times did not have any significant effect on the signal to cutoff (S/Co) ratios obtained with the two immunoassays (p = 0.06). Our results indicate that the developed immunoassay with a short incubation time of 10 min has the potential to be used in clinical laboratories and in blood-bank settings as a screening test for treponemal antibodies.     

Combining RNA-DNA swapping and quantitative polymerase chain reaction for the detection of influenza A nucleoprotein

The detection of proteinaceous antigens generally relies on traditional immunoassays and, more recently, on immuno-PCR (polymerase chain reaction) assays and their derivatives, which do not take advantage of the intrinsic function or binding property of a protein. The RNA-binding nucleoprotein has been shown to be an excellent target for the development of various influenza A diagnostics due to its high antigenicity and the presence of large numbers in the virus. It binds nonspecifically to the sugar-phosphate backbone of RNA as well as to single-stranded DNA (ssDNA) in vitro. We decided to take advantage of this property to develop an ssDNA probe for the detection of nucleoprotein by quantitative PCR (qPCR). We found that recombinant influenza A nucleoprotein from avian H5N1 subtype binds strongest to a 74-base-long ssDNA. Two systems, one comprising an antibody-based nucleoprotein capture surface and the other based on direct nucleoprotein adsorption under denaturing conditions, were developed combining the replacement of RNA bound to nucleoprotein by a discrete ssDNA probe and a qPCR for the detection of nucleoprotein in the low picomolar (pM) range.     

Chromogenic substrates for horseradish peroxidase

Two new detection systems for horseradish peroxidase (HRP) have been developed for the staining of membranes used in immunoassays. These systems use dimethyl or diethyl analogues of p-phenylenediamine with 4-chloro-1-naphthol to generate a blue product or 3-methyl-2-benzothiazolinone hydrazone with 4-chloro-1-naphthol to generate a red product. These reagents offer increased sensitivity and lower background staining than currently available chromogenic detection substrates. In addition, the incorporation of these substrates increases the sensitivity of HRP labels to be comparable to that of alkaline phosphatase with the 5-bromo-4-chloro-3-indolyl phosphate + nitro blue tetrazolium substrate.     

Glass slide models for immunocytochemistry and in situ hybridization

We have developed a model system based on the immobilization of microdroplets (0.1 l volumes) of antigens or of target (sense) oligonucleotides onto aminoalkylsilane-coated glass slides. Oligopeptide antigens need to be vapour-fixed in order to achieve efficient immobilization, while oligonucleotides do not require fixation. Protein antigens, exemplified by rabbit immunoglobulin, may be subjected to liquid fixation. The glass slide models are optically translucent and useful both for in situ hybridization and immunocytochemistry. The slides are compatible with detection systems of both light and fluorescence microscopy and permit measurement of staining intensities by microfluorimetry or computerized microdensitometry. The model systems can be used for comparisons of method sensitivities, for characterizing antibody and probe sensitivities and cross-reactivities, and as internal standards or quality controls for immunocytochemistry and in situ hybridization.     

单核细胞增生李斯特菌检测技术研究进展

单核细胞增生李斯特菌(Listeria monocytogenes,LM)是常见的食源性致病菌之一。目前,在众多单增李斯特菌的检测方法中应用较广的是免疫学检测法、分子学检测法。免疫学检测时间短,操作简单,但该方法依赖高特异性的抗体,会出现假阳性,还需要进一步鉴定检测结果。分子学检测法克服了免疫学检测法不能在种的水平鉴定单增李斯特菌的缺点,省时省力,灵敏度高,但是分子学检测法需要丰富的操作经验,并且不适于现场大批量检测。新兴的代谢学检测法、光谱学检测法、生物传感器等也都有各自的优缺点。本文综合近年最新文献,就单增李斯特菌检测的最新方法、检测进展及未来发展趋势予以分析综述,以期为该菌的检测提供参考。     

Expression of Endogenous RNA C-Type Virus Group-Specific Antigens in Mammalian Cells

Highly sensitive and specific radioimmunoassays are described for quantitation of the intraspecies determinants of several mammalian C-type viral group-specific (gs) antigens. An interspecies (gs-3) immunoassay has been developed which has both the broad reactivity and great sensitivity necessary for detection of C-type viruses where intraspecies gs assays are not available. By using these immunoassays, the expression of endogenous virus-specified gs antigens in mammalian cells of different species has been studied. Whereas mouse gs antigen was clearly detectable in tissue culture cells of several mouse strains, the respective gs antigens of rat, cat, Chinese hamster, woolly monkey, and gibbon ape were not detectable in cells of those species, using assays of comparable sensitivity. Thus, differences exist in the level of endogenous virus expression in cells of different mammalian species.     

Rapid genotyping assays for the identification and differentiation of Yersinia ruckeri biotype 2 strains

Aims: To establish PCR‐based assays for the rapid identification and differentiation of each of four known biotype 2 (BT2) phenotype‐causing alleles in Yersinia ruckeri strains currently circulating in Europe and the United States. Methods and Results: Novel assays were developed relying on detection of mutant allele‐specific changes in restriction enzyme cleavage sites within targeted PCR products. The developed assays were validated against isolates previously genotyped by DNA sequencing. Conclusions: The described methods were specific, rapid and simple to perform and interpret. Significance and Impact of the Study: The developed genotyping assays provide a valuable tool for identification and differentiation of specific BT2 strains of Y. ruckeri. These assays will be critical for the design and validation of new vaccines or other measures meant to control BT2 strains.     

蛋白微阵列研究进展

蛋白微阵列是随着基因微阵列技术发展起来的,用于基因微阵列的制备方法、信号的检测及分析系统,也可用于蛋白微阵列。各种蛋白微阵列基质的发展,提高了蛋白的固定效率。放射性同位数、化学发光、激光共聚焦荧光扫描等技术都已用于微阵列的检测。重组蛋白技术的发展,提高了蛋白微阵列检测的通量和灵敏度。蛋白微阵列具有通量高、使用样品少、重复性好、可定量的特点,使其在生物医药科学研究中得到了广泛应用。本综述了蛋白微阵列的制备及其在免疫检测、医学诊断及蛋白组研究中的应用。     

Immunoenzyme detection of specific brain antigens as a criterion of the permeability of the hemato-encephalic barrier in rats following acute gamma irradiation

The immunoenzyme detection systems for the measurement of the alpha-2 globulin of the brain (alpha 2M) and glial fibrillary acidic antigens (GFAP) were developed. These systems were used for the study of the penetration through hemato-encephalic barrier in rats subjected to gamma radiation. This method is recommended for the indirect evaluation of the hemato-encephalic barrier functional disorders.     

Bacterial surface antigen-specific monoclonal antibodies used to detect beer spoilage pediococci.

Fourteen monoclonal antibodies (Mabs) were isolated that react with surface antigens of Pediococcus beer spoilage organisms, including P. damnosus, P. pentosaceous, P. acidilactici, and unspeciated isolates. Immunoblotting, enzyme immunoassays (EIAs) of protease- and neuraminidase-treated surface antigen extracts, carbohydrate competition EIAs, and cardiolipin EIAs were used to characterize the bacterial antigens involved in Mab binding. Antigen stability in situ was tested by protease treatment or surface antigen extraction of washed bacteria. In most cases, the Mabs bind to Pediococcus surface antigens that appear to be covalently bound cell wall polymers resistant to alteration or removal from the bacterial surface. These bacterial surface antigen reactive Mabs show good potential for rapid, sensitive, and specific immunoassay detection of Pediococcus beer spoilage organisms.