免疫检测信号放大抗体偶联物的制备及应用*

目的: 以聚赖氨酸(polylysine,PL)为骨架提高辣根过氧化物酶(horseradish peroxidase,HRP)与羊抗兔IgG的连接数量,比较几种化学偶联剂的偶联效果,通过免疫检测技术对其灵敏度进行检测和比较。方法: 对HRP与PL、聚合物HRP-PL与N-琥珀酰-S-乙酰乙酸(N-succinimidyl-S-acetylthioacetate,SATA)和2-亚氨基硫烷(Traut’s)两种试剂、羊抗兔IgG与琥珀酰亚胺基4-(N-马来酰亚胺甲基)环己烷-1-羧酸酯[succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate,Sulfo-SMCC]、活化后羊抗兔IgG及HRP-PL进行摩尔比的优化;对偶联物IgG-PL-HRP及商品化二抗分别进行斑点免疫印迹、ELISA和免疫组化,并计算偶联物IgG-PL-HRP的检测放大倍数。结果: 当PL与HRP摩尔比为1∶5,HRP-PL与Traut’s摩尔比为1∶15,羊抗兔IgG与Sulfo-SMCC摩尔比为1∶30,羊抗兔IgG与HRP-PL摩尔比为1∶10时,反应效率较高;商品化二抗及偶联物IgG-PL-HRP在斑点免疫印迹实验中的最低检测限分别为2.5 μg和312.5 ng,最大稀释倍数分别为50和100倍;在ELISA实验中的最大稀释倍数分别为5 000和20 000倍;在免疫组化实验中偶联物IgG-PL-HRP的检测特异性及强度均大于商品化二抗。结论: 成功合成抗体偶联物IgG-PL-HRP,且免疫检测信号放大倍数为商品化二抗的3~7倍,这对后续免疫诊断及生物学的研究具有重要意义。     

Electrochemical modulation of antigen-antibody binding

The label-free amperometric detection of a rabbit IgG antigen by an anti-rabbit IgG antibody is achieved by observing the electrochemistry at a glassy carbon electrode modified with antibody entrapped in an electrodeposited polypyrrole membrane. In a flow injection apparatus the electrode is pulsed between -0.2 and +0.4 V versus Ag/AgCl. The pulsing of the electrode switches the polypyrrole membrane between the oxidised and reduced states. When antigen is injected into the flow stream a change in current is observed at the electrode despite the antigen or antibody being redox inactive at the potentials employed. It is proposed that this current is due to a change in the flux of ions into and out of the polypyrrole matrix during a pulse when the poly-anionic antigen is present. The immunoreaction was reversible because the 200 ms pulse at each potential was too short to allow secondary bonding forces (hydrogen bonding and hydrophobic forces) which are responsible for the strength of the antibody-antigen complex to be established. The consequence of the reversibility of the antigen-antibody binding is a low apparent affinity constant but an easily regenerated recognition interface.     

A two-step antibody strategy for surface plasmon resonance spectroscopy detection of protein-DNA interactions in nuclear extracts

Surface plasmon resonance (SPR) spectroscopy has emerged as a powerful alternative to conventional biochemistry methods for studying protein-DNA interactions that involve recombinant proteins of known identity. There are, however, limited demonstrations of SPR detection of protein-DNA bindings in crude samples, e.g., cell extracts, where the challenge is to detect and identify specific DNA binding protein(s) among other protein components in a physiological setting. We have developed a two-step antibody approach for an SPR study of estrogen receptor α (ERα)-DNA interactions, in which nuclear extracts prepared from MCF-7 breast cancer cells were used as the source of ERα protein. Following the binding of nuclear extracts to surface-immobilized estrogen response elements, rabbit anti-ERα antibody followed by a secondary antibody (goat anti-rabbit IgG) were applied to recognize the bound ERα and amplify the signals, respectively. Through a series of experiments, we have demonstrated that the magnitude of the binding signals from the secondary antibody reflects the affinity by which ERα binds to different DNA sequences. The detection sensitivity is determined by the amount of nuclear extracts and the concentration of primary antibody used. The sequence specificity of the nuclear ERα measured using the two-step antibody approach is in agreement with that measured for recombinant ERα protein (using receptor binding signals).     

Detection and quantification of proteins using self-excited PZT-glass millimeter-sized cantilever

A composite self-excited PZT-glass cantilever (4mm in length and 2mm wide) was fabricated and used to measure the binding and unbinding of model proteins. A key feature of the cantilever is that its resonant frequency is dependent on its mass. The fabricated cantilever has mass change sensitivity in liquid of 7.2 x 10(-11)g/Hz. Resonant frequency change was measured as protein reacted or bound with the sensing glass cantilever surface. Protein concentrations, 0.1 and 1.0mg/mL, which resulted in nanogram mass change were successfully detected. The mass change sensitivity gave a total mass change of 54+/-0.45 ng for the binding of anti-rabbit IgG (biotin conjugated) to rabbit IgG immobilized cantilever and the subsequent binding of captavidin. The unbinding of anti-rabbit IgG and captavidin gave a total mass change of 54+/-1.70 ng. Fluorescence based assays showed the combined mass of both proteins in the released samples was 54+/-2.24 ng. The binding kinetics of the model proteins is modeled as first order. The initial binding rate constant of anti-rabbit IgG to rabbit IgG was 1.36+/-0.02(min(mg/mL))(-1). The initial binding rate constant of captavidin to biotinylated anti-rabbit IgG was (2.57 x 10(-1))+/-0.003(min(mg/mL))(-1). The significance of the results we report here is that millimeter-sized PZT-actuated glass cantilevers have the sensitivity to measure in real-time protein-protein binding, and the binding rate constant.     

Solid-Phase Immunoassays for Quantitation of Antibody to Bovine White Matter Proteolipid Apoprotein

Abstract: Two solid-phase immunoassays have been developed for quantitation of antibodies to bovine white matter proteolipid apoprotein. Conditions were established for optimal specific antibody binding. Water-soluble proteolipid apoprotein was bound to microtiter plates and plates were incubated with test serum. Goat anti-rabbit IgG conjugated with horeseradish peroxidase was used as the second antibody for an enzyme-linked immunospecific assay and ¹²⁵I-labeled protein A for a radioimmunoassay. Both procedures have been used to follow the time course of anti-proteolipid antibody production in rabbits and to compare different immunization protocols.     

Long-period gratings in photonic crystal fiber as an optofluidic label-free biosensor

Using long-period gratings (LPG) inscribed in photonic crystal fiber (PCF) and coupling this structure with an optically aligned flow cell, we have developed an optofluidic refractive index transduction platform for label-free biosensing. The LPG-PCF scheme possesses extremely high sensitivity to the change in refractive index induced by localized binding event in different solution media. A model immunoassay experiment was carried out inside the air channels of PCF by a series of surface modification steps in sequence that include adsorption of poly(allylamine hydrochloride) monolayer, immobilization of anti-rat bone sialoprotein monoclonal primary antibody, and binding interactions with non-specific goat anti-rabbit IgG (H+L) and specific secondary goat anti-mouse IgG (H+L) antibodies. These adsorption and binding events were monitored in situ using the LPG-PCF by measuring the shift of the core-to-cladding mode coupling resonance wavelength. Steady and significant resonance changes, about 0.75 nm per nanometer-thick adsorbed/bound bio-molecules, have been observed following the sequence of the surface events with monolayer sensitivity, suggesting the promising potential of LPG-PCF for biological sensing and evaluation.     

An immunocytochemical approach to cell kinetics automation.

Antibodies specific for 5-bromodeoxyuridine (BrdUrd) can be used to measure labeling indices in an automated system by image analysis. The antibody, used with an indirect immunoperoxidase technique, will detect de novo DNA synthesis subsequent to growing the cells for various time intervals in 5-bromodeoxyuridine-containing medium. Asynchronously growing CHO cells were pulsed with 3H-5-bromodeoxyuridine, fixed, denatured and then stained with anti-bromouridine antiserum. Peroxidase-coupled goat anti-rabbit IgG was used as the secondary antibody, and slides were stained with diaminobenzidine. Cells which are positive display a reticular pattern indicative of replicating chromatin. "Labeling indices" were generated by scanning the nuclei by TV image analysis. The percentage of labeled cells by the immunocytochemical technique correlates well with that found by autoradiography. Some of the applications of this automated method include cell kinetics and analysis of S-phase by pattern recognition technique.     

Host immunoglobulin G titre and antibody activity in haemolymph of the tick, Ornithodoros moubata

Immunoglobulin G (IgG) in tick haemolymph was analysed immunochemically and biochemically for its antigenicity, antibody activity and relative concentration in a soft tick, Ornithodoros moubata (Murray) sensu Walton 1962 (Acari: Argasidae). Ouchterlony immunodiffusion tests showed that haemolymph from a tick engorged on rabbit IgG (or human IgG) through an artificial membrane, reacted with anti-rabbit IgG (anti-human IgG) but not with anti-human IgG (anti-rabbit IgG). This indicates that haemolymph of the fed tick contains IgG with a similar antigen specificity to host blood IgG. IgG from tick haemolymph was demonstrated by enzyme immunoassay to have the same antibody activity as ingested IgG. The IgG concentration in tick haemolymph was measured by a quantitative single immunodiffusion test. Changes of IgG titre after a bloodmeal were correlated with IgG activity, which was low for 5 days after a bloodmeal and then suddenly increased. The IgG titre reached a maximum 7 days post-engorgement, and remained high for over 4 months during and after oviposition. 125I-labelled IgG was injected into the tick haemocoel to determine the persistence of IgG in the haemolymph. Recovery of labelled IgG was low at 1 and 3 days, and high at 5, 8 and 16 days after engorgement. The data suggest that IgG in haemolymph disappears quickly soon after engorgement possibly by degradation and/or absorption (adhesion to tissues).     

Establishment of a specific radioimmunoassay for bovine interferon tau

A radioimmunoassay (RIA) was developed for quantification of bovine interferon (bIFN) tau, conceptus secretory protein, which allows for the maintenance of the corpus luteum during early pregnancy. A cDNA coding bIFN tau was derived from cultured trophoblast cells (TBs). Recombinant (r) bIFN tau was produced in a baculovirus expression system with two different viruses. The RIA was a double-antibody competitive binding assay that used anti-bIFN tau antiserum (raised in rabbits) as the primary antibody, a radioiodinated derivative of bIFNtau as the radioactive tracer, and goat anti-rabbit IgG as the secondary antibody. The antibody did not cross-react with rbIFN alpha, recombinant human IFN beta or recombinant ovine IFN tau. The correct recovery of amounts of rbIFN tau indicated good accuracy. Serially concentrated TB conditioned media, paralleled the standard curve for bIFN tau. The intra-assay and inter-assay coefficients of variation at bIFN tau levels of 7.8 and 15.6 ng/mL were 7.1 and 8.1%, and 11.0 and 8.5%, respectively. bIFN tau was directly detected in uterine flushings obtained from cows at Day 16 of pregnancy. In summary, this assay was suitable for the measurement of bIFN tau.     

Fluorescence resonance energy transfer between two quantum dots with immunocomplexes of antigen and antibody as a bridge.

In this study, 573 nm quantum dots (QDs)-rabbit IgG-goat anti-rabbit IgG-638 nm QDs immunocomplexes were prepared, utilizing antigen-antibody interaction. 573 nm-emitting QDs were conjugated to antigen (rabbit IgG) and 638 nm-emitting QDs were conjugated to antibody (goat anti-rabbit IgG) via electrostatic/hydrophilic self-assembly, respectively. The mutual affinity of the antigen and antibody brought two kinds of QDs close enough to result in fluorescence resonance energy transfer (FRET) between them; the luminescence emission of 573 nm QDs was quenched, while that of 638 nm QDs was enhanced. The luminescence emission of 573 nm QDs could be recovered when the immunocomplexes were exposed to the unlabelled rabbit IgG antigen. The FRET efficiency (E) and the distance between the donor and the acceptor were calculated.     

Directional surface plasmon-coupled emission: application for an immunoassay in whole blood

We present a new approach for performing fluorescence immunoassay in whole blood using fluorescently labeled anti-rabbit immunoglobulin G (IgG) on a silver surface. This approach, which is based on surface plasmon-coupled emission (SPCE), provides increased sensitivity and substantial background reduction due to exclusive selection of the signal from the fluorophores located near a bioaffinity surface. This article describes the effect of an optically dense sample matrix, namely human whole blood and serum, on the intensity of the SPCE. An antigen (rabbit IgG) was adsorbed to a slide covered with a thin silver metal layer, and the SPCE signal from the fluorophore-labeled anti-rabbit antibody, binding to the immobilized antigen, was detected. The effect of the sample matrix (buffer, human serum, or human whole blood) on the end-point immunoassay SPCE signal was studied. It was demonstrated that the kinetics of binding could be monitored directly in whole blood or serum. The results showed that human serum and human whole blood attenuate the SPCE end-point signal and the immunoassay kinetic signal only approximately two- and threefold, respectively, as compared with buffer, resulting in signals that are easily detectable even in whole blood. The high optical absorption of the hemoglobin can be tolerated because only fluorophores within a couple of hundred nanometers from the metallic film contribute to SPCE. Excited fluorophores outside the 200-nm layer do not contribute to SPCE, and their free space emission is not transmitted through the opaque metallic film into the glass substrate. We believe that SPCE has the potential of becoming a powerful approach for performing immunoassays based on surface-bound analytes or antibodies for many biomarkers directly in dense samples such as whole blood with no need for washing steps.     

In vitro effect of biogenic amines on the hormone content of immune cells of the peritoneal fluid and thymus. Is there a hormonal network inside the immune system?

Immune cells contain different hormones and hormone-like molecules, such as insulin, endorphin, triiodothyronine (T3) histamine, serotonin. In earlier in vitro experiments insulin down-regulated histamine, serotonin and T3 content of thymus cells. Now we studied the effect of biogenic amines on the endorphin, T3, serotonin and histamine content of rat peritoneal and thymic cells. Cells were obtained from male rats of 100g body weight. 100 ng/ml serotonin or 300 ng/ml histamine was added for 30 min. After that the cells were prepared for flow cytometric analysis with antibodies to endorphin, T3, histamine and serotonin as primary antibodies and anti-rabbit IgG as secondary antibody. Finishing the measurements the cells were also studied by confocal microscopy. T3 concentration (binding of anti-T3 antibody) increased in peritoneal mast cells after serotonin treatment and in the monocyte-macrophage-granulocyte group after histamine treatment. Thymocytes' T3 content radically decreased after both treatments. Serotonin and histamine treatment also radically reduced the amine content of each other. Endorphin level was resistant to hormonal treatments. The results call attention to a possible hormonal network inside the immune system in which hormones produced by the immune cells themselves can influence each other.     

A nonisotopic estrogen receptor-based assay to detect estrogenic compounds

We have used the ligand binding domain of the recombinant human estrogen receptor (hER) to develop a nonisotopic assay for detection of estrogenic compounds. The assay is based on competition of the estrogenic ligand with 17beta-estradiol for binding to the receptor, which leaves 17beta-estradiol free to bind to an anti-17beta-estradiol antibody. Unbound anti-17beta-estradiol antibody then binds to immobilized 17beta-estradiol-protein conjugate (to which hER is unable to bind for steric reasons), and is detected by an enzyme-labeled anti-rabbit IgG antibody. We used the assay to detect estrogenic compounds (mainly members of the flavonoid group of plant polyphenols) in a variety of commonly consumed plant foods.     

Immunolocalization of prophenoloxidase among hemocytes of the silkworm, Bombyx mori

Silkworm (Bombyx mori) hemocytes were fixed immediately after collection. Thin sections of the hemocytes were stained by an indirect immunogold staining method using rabbit anti-prophenoloxidase/IgG as a primary antibody and colloidal gold coated with goat anti-rabbit/IgG as a secondary antibody. Electron micrographs of the sections revealed that only plasmatocytes and oenocytoids have prophenoloxidase both in cytoplasm and nucleus whereas granulocytes, spherulocytes and prohemocytes do not have appreciable amounts of the proenzyme. Cytoplasmic inclusions of oenocytoids also contain the proenzyme. A wide variety of concentrations of prophenoloxidase was observed among oenocytoids. Plasmatocytes appeared to have less prophenoloxidase than any oenocytoids. Once materials in the granules of granulocyte were discharged into the plasma and formed coagula, they cross-reacted with antiprophenoloxidase/IgG, suggesting that prophenoloxidase was trapped in the coagula by unknown mechanisms. This observation is discussed in relation to the dispute concerning the presence of prophenoloxidase or phenoloxidase in the granulocyte.     

Immunohistochemical localization of barley stripe mosaic virions in infected wheat cells

Barley stripe mosaic virus particles were localized in ultrathin sections with colloidal gold-labeled specific IgG or antiserum followed by gold-labeled goat anti-rabbit IgG. On the average, 1.5 gold particles were attached per virus rod. A statistical analysis of counts of gold and virus particles showed that the staining procedure was highly reproducible from experiment to experiment and after several independently prepared colloidal gold solutions. The procedure should be useful for the intracellular localization of any protein to which an antibody can be prepared.     

Microfluidic systems integrated with two-dimensional surface plasmon resonance phase imaging systems for microarray immunoassay

This study reports a microfluidic chip integrated with an arrayed immunoassay for surface plasmon resonance (SPR) phase imaging of specific bio-samples. The SPR phase imaging system uses a surface-sensitive optical technique to detect two-dimensional (2D) spatial phase variation caused by rabbit immunoglobulin G (IgG) adsorbed on an anti-rabbit IgG film. The microfluidic chip was fabricated by using micro-electro-mechanical-systems (MEMS) technology on glass and polydimethylsiloxane (PDMS) substrates to facilitate well-controlled and reproducible sample delivery and detection. Since SPR detection is very sensitive to temperature variation, a micromachine-based temperature control module comprising micro-heaters and temperature sensors was used to maintain a uniform temperature distribution inside the arrayed detection area with a variation of less than 0.3 degrees C. A self-assembled monolayer (SAM) technique was used to pattern the surface chemistry on a gold layer to immobilize anti-rabbit IgG on the modified substrates. The microfluidic chip is capable of transporting a precise amount of IgG solution by using micropumps/valves to the arrayed detection area such that highly sensitive, highly specific bio-sensing can be achieved. The developed microfluidic chips, which employed SPR phase imaging for immunoassay analysis, could successfully detect the interaction of anti-rabbit IgG and IgG. The interactions between immobilized anti-rabbit IgG and IgG with various concentrations have been measured. The detection limit is experimentally found to be 1 x 10(-4)mg/ml (0.67 nM). The specificity of the arrayed immunoassay was also explored. Experimental data show that only the rabbit IgG can be detected and the porcine IgG cannot be adsorbed. The developed microfluidic system is promising for various applications including medical diagnostics, microarray detection and observing protein-protein interactions.     

THE EFFECT OF LABELING INTENSITY,ESTIMATED BY REAL-TIME CONFOCAL LASER SCANNING MICROSCOPY,ON FLOW CYTOMETRIC APPEARANCE AND IDENTIFICATION OF IMMUNOCHEMICALLY LABELED MARINE DINOFLAGELLATES1

Two different fluorescein isothiocyanate (FITC) conjugates were used to analyze the effect of labeling intensity on the flow cytometric appearance of marine dinoflagellates labeled with antibodies that specifically recognized the outer cell wall. Location of the labeling was revealed by epifluorescence and real-time confocal laser scanning microscopy using an anti-rabbit IgG/FITC-conjugated secondary antiserum. Flow cytometric measurements showed that cells of Prorocentrum species labeled this way could not always be distinguished from unlabeled cells. The labeling intensity increased several times when a biotinylated anti-rabbit IgG secondary antiserum was used in combination with a streptavidin/FITC conjugate. Flow cytometry indicated that the labeling intensity had increased 50%, which resulted in an improved separation of clusters of labeled and unlabeled cells.     

A specific and sensitive double-immunofluorescence method for the demonstration of S-antigen and serotonin in trout and rat pinealocytes by means of primary antibodies from the same donor species

 Immunocytochemical double-labeling methods are important tools in cell and neurobiology. Here we describe a method which is based on double immunofluorescence and allows specific detection of two different antigens located in the same cell compartment by two primary antibodies raised in the same species. As an example, we present the double-immunolabeling method for the S-antigen (SAg), a photoreceptor-specific protein, and the indoleamine serotonin (5HT) in dissociated trout and rat pineal cells immobilized on coversliped and in frozen sections of the trout pineal organ. As a first step, the preparations on the slides or coverslips were sequentially incubated with the first primary antibody (rabbit anti-SAg), the fluorescein-labeled (anti-rabbit) secondary antibody, and then with normal rabbit serum. Meanwhile, the second primary antibody (rabbit anti-5HT) was coupled to a Cy3-labeled secondary (anti-rabbit) antibody in a reaction tube and excess binding sites were quenched with normal rabbit serum. This complex was applied to the specimens after completion of the first (SAg) immunoreaction on the slide. For control experiments, the first (anti-SAg) or the second (anti-5HT) primary antibody were omitted. Most of the rat and trout pinealocytes were double immunolabeled for SAg and 5HT. In the trout, few cells contained SAg or 5HT immunoreaction only. This underlines the selectivity of each immunoreaction. The results show that the method can be used for the analysis of whole cells and tissue sections by means of conventional fluorescence and confocal laser scanning microscopy. Accepted: 20 October 1997     

Development of Dot-ELISA for the detection of human rotavirus antigen and comparison with RNA-PAGE

AIMS: Development of a simple, specific, rapid and inexpensive Dot-ELISA test for diagnosis of rotaviral antigen in stool samples. METHODS AND RESULTS: Hyperimmune rabbit antisera raised against SA-11 (Simian Agent-11) strain was used as primary antibody. The secondary antibody conjugate used was the goat anti-rabbit IgG alkaline phosphatase, and BCIP/NBT solution was used as substrate. Faecal extracts were diluted 10-fold and used for the detection of rotavirus antigen. RNA-PAGE was performed to compare the specificity and sensitivity of the diagnostic tests. Dot-ELISA positive samples were further confirmed by Western blot analysis. CONCLUSIONS: This Dot-ELISA test could be used as an alternative method for diagnosing rotaviral samples in the field. SIGNIFICANCE AND IMPACT OF THE STUDY: The Dot-ELISA test is simple, specific, rapid and cost effective. It is suitable for identifying a large number of samples obtained from epidemiological studies and hence, reducing the death rate of rotavirus-infected patients.     

Determination of cell surface expression of Toll-like receptor 4 by cellular enzyme-linked immunosorbent assay and radiolabeling

Lipopolysaccharide (LPS) is recognized by Toll-like receptor 4 (TLR4) of macrophages triggering production of pro-inflammatory mediators. One of the factors determining the magnitude of responses to LPS, which may even lead to life-threatening septic shock, is the cell surface abundance of TLR4. However, quantitation of the surface TLR4 is difficult due to the low level of receptor expression. To develop a method of TLR4 assessment, we labeled the receptor on the cell surface with a rabbit antibody followed by either anti-rabbit immunoglobulin G–fluorescein isothiocyanate (IgG–FITC) for flow cytometry or with anti-rabbit IgG–peroxidase for a cellular enzyme-linked immunosorbent assay (ELISA). Alternatively, the anti-TLR4 antibody was detected by anti-rabbit IgG labeled with ¹²⁵I. Flow cytometry did not allow detection of TLR4 on the surface of J774 cells or human macrophages. In contrast, application of cellular ELISA or the radiolabeling technique combined with effective blockage of nonspecific binding of antibodies provided TLR4-specific signals. The level of TLR4 on the surface of J774 cells did not change on treatment with 1–100 ng/ml LPS; however, it was reduced by approximately 30–40% after 2 h of treatment with 1 μg/ml LPS. These data indicate that down-regulation of surface TLR4 can serve as a means of negative regulation of cell responses toward high doses of LPS.