Goat Anti-human Immunoglobulin G Research Articles

A novel method for simultaneous analysis of specific platelet antibodies: SASPA

Glycoprotein (GP)-specific platelet antibodies can cause allo-immune and auto-immune thrombocytopenia. The specific detection of relevant antibodies is a prerequisite for diagnosis and treatment. Here, we describe an improved method based on simultaneous detection of various platelet-specific immunoglobulin G (IgG) and IgM antibodies. Bead populations with distinct fluorescence intensities, coated with monoclonal antibodies specific for mouse heavy chain isotypes, were used for the simultaneous immobilization of platelet-GP [IIb/IIIa, Ib/IX, human leucocyte antigen (HLA) class I, or Ia/IIa, CD32, GPIV or CD109, Ib/IX, HLA class I]. In order to detect human IgG and IgM antibodies simultaneously, phycoerythrin- and fluorescein isotiocyanate-conjugated goat anti-human IgG and IgM were added. On this basis, the abundance of six distinct antibodies (three anti-GP, each with subclasses IgG and IgM) were simultaneously analysed without cross-reaction by flow cytometry. For evaluation, sera and platelets from 169 patients with platelet-binding and/or platelet-associated antibodies were investigated. The monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay was performed in parallel as reference test. The simultaneous analysis of platelet-specific antibodies (SASPA) assay was able to detect all platelet-specific IgG and IgM that were also recognized by MAIPA with a comparable sensitivity. SASPA proved to be a rapid and reliable assay that required less platelets than other methods. This method has the potential to pave the way for new investigations of platelet-specific antibodies.

Dominant antibody responses to Fucα1-3GalNAc and Fucα1-2Fucα1-3GlcNAc containing carbohydrate epitopes in Pan troglodytes vaccinated and infected with Schistosoma mansoni

The development of the humoral anti-glycan immune response of chimpanzees, either or not vaccinated with radiation-attenuated Schistosoma mansoni cercariae, was followed during 1 year after infection with S. mansoni. During the acute phase of infection both the vaccinated and the control chimpanzees produce high levels of immunoglobulin G (IgG) antibodies against carbohydrate structures that are characteristic for schistosomes carrying the Fucα1-3GalNAc and Fucα1-2Fucα1-3GlcNAc motifs, but not to the more widespread occurring structures GalNAcβ1-4GlcNAc, GalNAcβ1-4(Fucα1-3)GlcNAc, and Galβ1-4(Fucα1-3)GlcNAc (Lewis x). In addition, high levels of IgM antibodies were found against the trimeric Lewis x epitope. Apparently, the schistosome-characteristic carbohydrate structures are dominant epitopes in the anti-glycan humoral immune response of the chimpanzees. All chimpanzees showed an increase in the level of antibodies against most of the carbohydrate structures tested directly after vaccination, peaking at challenge time and during the acute phase of infection. With the exception of anti-F-LDN antibody responses, the anti-carbohydrate antibody responses upon schistosome infection of the vaccinated animals were muted in comparison to the control animals. Index Descriptors and Abbreviations: Anti-carbohydrate antibodies, glycoconjugates, humoral immune response, infection, Schistosoma mansoni, synthetic glycan epitopes, trematode, vaccination, irradiated cercariae. Circulating cathodic antigen (CCA), Fucα1-3GalNAc(Fucα1-3) 0/1GlcNAcαGal (F-LDN(-F) 0/1), goat anti-human IgG (GaHuIgG), goat anti-human IgM (GaHuIgM), Hepes-buffered saline (HBS), immunoglobulin (Ig), keyhole limpet haemocyanin (KLH), GalNAcβ1-4GlcNAc (LDN), GalNAcβ1-4(Fucα1-3)GlcNAc (LDN-F), GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (LDN-DF), Galβ1-4(Fucα1-3)GlcNAc (Lewis x), peripheral blood mononuclear cells (PBMCs), response units (RUs), surface plasmon resonance (SPR).

Adsorption of Native and Hydrophobically Modified Human Immunoglobulin G on Polyethylene Solid Films: Specific Recognition of Adsorbed Layers

The in situ adsorption/desorption studies of 14C-labeled human immunoglobulin G (IgG) were performed at the polyethylene (PE)/aqueous solution interface. The results reveal that the adsorbed protein molecules were predominantly packed in the end-on orientation. Desorption experiments showed that upon rinsing with buffer only minute fractions of adsorbed IgG could be removed from the PE surface. Both the competitive and the sequential adsorption measurements from the binary mixtures of hydrophobic IgG modified by attachment of 19 caprylic chains (19C8-IgG) and [14C]IgG confirm that native IgG strongly adhered to the PE films. The specific recognition ability of adsorbed human IgG layers was studied by using specific (goat anti-human) IgG and nonspecific (goat anti-rabbit) IgG. It was shown that the prevailing effect of the interaction of [14C]IgG adsorbed onto PE with the specific antibody was the increase in the surface radioactivity, which was attributed to the formation of a more densely packed layer of IgG molecules at the PE surface. It was also demonstrated that, whereas the specific antibody preadsorbed onto PE films retained the recognition ability relative to human IgG, its hydrophobic modification resulted in a substantial decrease in this ability.

Polyaniline–dacron composite as solid phase in enzyme linked immunosorbent assay for Yersinia pestis antibody detection

Polyaniline (PANI) was chemically synthesized on a dacron disk surface and an antigen (F1 fraction) obtained from Yersinia pestis was covalently fixed onto this composite via glutaraldehyde. The enzyme linked immunosorbent assay (ELISA) or rapid ELISA procedure detected immunoglobulin G (IgG) anti-F1 fraction in human serum employing this derivative. The appropriate conditions for carrying out the test were established as an antigen concentration of 2 μg/PANI–dacron disk, peroxidase labeled goat anti-human IgG conjugate diluted 4000 times, and a serum dilution of 1:100. The PANI–dacron disks showed greater antigen retention than conventional poly(vinyl chloride) plates and less antibody unspecific adsorption. © 2001 John Wiley & Sons, Inc. J Biomed Mater Res 56: 257–260, 2001

Detection of biomolecules in the near-infrared spectral region via a fiber-optic immunosensor

The design, development, and application of a fluorescent fiber-optic immunosensor (FFOI) procedure for the detection of antibody/antigen binding within the near-infrared (NIR) spectral region is reported. The technique was developed through the combined use of fiber-optics, semiconductor laser excitation, fluorescence detection, NIR dye, and immunochemical techniques. The antibody is immobilized on the FFOI's sensing tip and utilized as a recognition component for trace amounts of specific antigen. The FFOI is constructed to utilize antibody sandwich technique. Three individual immunoassays are reported. The first two assays utilize the FFOI and NN382, a commercial NIR dye, for the detection of human immunoglobulin G (IgG). In these assays, goat anti-human IgG antibody (GAHG) is immobilized on the sensitive terminal of the FFOI followed by the exposure of the antibody-coated terminal to human IgG. The probe is then introduced to GAHG labeled with NN382, generating a signal. The third assay utilizes the FFOI for the detection of trace amounts of Legionella pneumophila serogroup 1 (LPS1). In this assay, rabbit anti-LPS1 antibody is immobilized on the sensitive terminal of the FFOI followed by exposure to LPS1. The antigen-coated probe is then treated with monoclonal anti-LPS1 antibody followed by incubation with GAHG labeled with NN382. The assays are optimized to detect the corresponding antigen via the NIR–FFOI. Typical measurements are performed in 10–15 min. A 780-nm semiconductor laser provides the excitation of the immune complex and the resulting emission is detected by a 820-nm silicon photodiode detector. The intensity of the resulting fluorescence is directly proportional to the concentration of the antigen. Solutions of IgG and LPS1 with concentrations as low as 10 −11 M and 0.5 ng/ml, respectively, have been detected with a minimum interference.

Biotransformation of halothane, enflurane, isoflurane, and desflurane to trifluoroacetylated liver proteins: association between protein acylation and hepatic injury.

In susceptible patients, halothane, enflurane, isoflurane, and desflurane can produce severe hepatic injury by an immune response directed against reactive anesthetic metabolites covalently bound to hepatic proteins. The incidence of hepatotoxicity appears to directly correlate with anesthetic metabolism catalyzed by cytochrome P450 2E1 to trifluoroacetylated hepatic proteins. In the present study, we examined whether the extent of acylation of hepatic proteins in rats by halothane, enflurane, isoflurane, and desflurane correlated with reported relative rates of metabolism. After pretreatment with the P450 2E1 inducer isoniazid, five groups of 10 rats breathed 1.25 minimum alveolar anesthetic concentration (MAC) of halothane, enflurane, isoflurane, or desflurane in oxygen, or oxygen alone, each for 8 h. Immunochemical analysis of livers harvested 18 h after anesthetic exposure showed tissue acylation (greatest to least) after exposure to halothane, enflurane, or isoflurane. Reactivity was not different between isoflurane as compared to desflurane or oxygen alone. An enzyme-linked immunosorbent assay showed halothane reactivity was significantly greater than that of enflurane, isoflurane, desflurane, or oxygen, and that enflurane reactivity was significantly greater than desflurane or oxygen. Sera from patients with a clinical diagnosis of halothane hepatitis showed antibody reactivity against hepatic proteins from rats exposed to halothane or enflurane. No reactivity was detected in rats exposed to isoflurane, desflurane, or oxygen alone. These results indicate that production of acylated proteins may be an important mediator of anesthetic-induced hepatotoxicity.

Lack of specific binding of anticardiolipin antibodies to intact platelets.

The aims of this study were to investigate whether anticardiolipin antibodies (aCL) bind to intact (resting or activated) platelets in vitro. Suspensions of resting, activated (with a mixture of thrombin and collagen) and freeze-thawed platelets from healthy subjects were incubated with either affinity-purified aCL or pooled normal human immunoglobulin G (IgG). Platelet-bound IgG was measured by flow cytometric analysis of platelets incubated with a fluorescein-conjugated polyclonal goat anti-human IgG. There was no significant binding of IgG aCL to intact resting or activated platelets, while significant specific binding to freeze-thawed platelets was demonstrated. These results question the theory that aCL bind/activate intact platelets in vivo.

Fluorescence immunological determination of immunoglobulin G in human serum by high-performance liquid gel-permeation chromatography

A high-performance liquid gel-permeation chromatographic method is described for the determination of human serum immunoglobulin G (IgG) by separating the fluorescent immuno complex from the free fluorescence-labeled antibody. Fluorescence-labeled antibody used in this study was fluorescein isothiocyanate (FITC)-labeled Fab fragment goat anti-human IgG (anti-IgG Fab). Immuno complexes and antibody of different molecular sizes can be separated. FITC-labeled anti-IgG Fab was added to the serum and the mixture is passed through the column. An immuno complex separates as well-delineated peak in the column void volume, and was measured by the fluorescence of the column eluate (Ex=490nm, Em=520nm). The total analysis time for a serum sample was approximately 15min. The minimum detection limit was 25 mg/dl. The relative standard deviation was below 2% (peak area). The results of the HPL-GPC analysis correlate well with those obtained by laser nephelometric assay (r=0.992).

Determination of human serum immunoglobulin G using flow injection analysis with rate turbidimetric detection.

An immunological reaction between human serum immunoglobulin G (IgG) and goat anti-human IgG was investigated using a fully automated stop-flow merging zones flow injection analysis manifold. Turbidimetric detection at 340 nm was used to monitor the rate of reaction. A sampling rate of 40 samples per hour and a precision of 2.0–6.8% RSD (relative standard deviation) were obtained for a range of human serum standards. Serum samples and a human reference serum were analysed and their IgG concentrations interpolated from a second-order fit of the calibration data. The consumption of expensive antiserum was less than 1 µl per assay.

A solid-phase chemiluminescence immunoassay for determination of IgG in eluates of psoriatic scales

A solid-phase chemiluminescence immunoassay for determining immunoglobulin G (IgG) in eluates of psoriatic scales is described. Polystyrene balls coated with protein A bound the IgG present in the eluates and in human sera. The bound IgG was then detected by a luminol-coupled goat anti-human IgG conjugate, characterized for antibody, protein and luminol content. This method correlated well with radial immunodiffusion in determining IgG levels in human sera ( r = 0.97). The sensitivity of the assay was 0.5 μg IgG. Eluates prepared from psoriatic scales treated sequentially with buffers of decreasing pH all contained IgG when tested by this method. pH 2 eluates, however, contained more IgG than pH 3 eluates, suggesting the presence of specifically bound antibodies.

DEAE-Affi-Gel Blue chromatography of human serum: Use for purification of native transferrin

Human serum was subjected to chromatography on DEAE-Affi-Gel Blue which combines ion-exchange and pseudo-ligand-affinity chromatography in a 0.02 m phosphate buffer, pH 7.0. All serum proteins were bound with the exception of transferrin, IgG (immunoglobulin G) and trace amounts of IgA. After a second step of Sephadex G-100 gel chromatography, or affinity chromatography against goat anti-human IgG F(ab′) 2 coupled to AH-Sepharose 4B, IgG and IgA were removed. The transferrin obtained was homogeneous and of high yield (>80%), and was unaltered as judged by analyses of molecular weight, isoelectric point, iron-binding capacity, antigenicity, and ability to bind to high-affinity specific cellular receptors. Thus, DEAE-Affi-Gel Blue chromatography may be used as the basis for a simple, rapid, two-step method for the purification of large amounts of native transferrin from serum.

Magnetisable solid-phase fluoroimmunoassay of human immunoglobulin G in serum

A simple fluoroimmunoassay is described for the determination of immunoglobulin G (IgG) in serum. It employs IgG labelled with fluorescein and magnetisable polyacrolein/iron oxide particles covalently linked to goat anti-human IgG serum. Diluted patient sample or standard and fluorescein-labelled IgG are incubated for 30 min with solid-phase antibody, the antibody-bound and free antigen separated simply by means of a magnet and the fluorescence in the supernate (free fraction) determined, which directly reflects the IgG concentration of the standard or sample. Correlation studies with two other immunological methods showed good agreement.

Enzyme-linked immunosorbent assay for quantitating the humoral immune response to the colonization factor antigen of enterotoxigenic Escherichia coli.

The enzyme-linked immunosorbent assay technique was used to quantitate, in milligrams per milliliter, anti-colonization factor antigen/I (CFA/I) immunoglobulin G (IgG) in acute- and convalescent-phase sera of individuals who experienced diarrhea associated with CFA/I-positive enterotoxigenic Escherichia coli. Purified CFA/I was used as antigen to coat polystyrene Microtiter plate wells for the determination of anti-CFA/I antibody. A reference anti-CFA/I IgG preparation was obtained by affinity chromatography of a high-titered serum with a CFA/I-Sepharose 4B column; IgG was the only class of immunoglobulin detectable in this serum as anti-CFA/I. Goat anti-human IgG conjugated to alkaline phosphatase was used in the enzyme-linked immunosorbent assay. Quantitation of IgG in the reference anti-CFA/I serum was achieved by comparison with a known sample of pure human IgG. Anti-CFA/I in test sera was quantitated by titration with CFA/I-coated Microtiter plate wells in the enzyme-linked immunosorbent assay, using a standard curve obtained with the reference anti-CFA/I serum. Anti-CFA/I IgG in paired sera was determined as percentage of total IgG by using the radial immunodiffusion technique to quantitate total IgG for each test serum. Diarrhea with isolation of CFA/I-positive enterotoxigenic E. coli was associated with a significant rise in serum anti-CFA/I IgG when these values were expressed as either milligrams of IgG per milliliter or as percentage of total IgG, although the response varied quantitatively and nonresponders were detected. None of the matched controls showed an anti-CFA/I IgG response. Further elucidation of the immune response to enterotoxigenic E. coli can now be accomplished by applying these methods to determine the class and specificity of immunoglobulins in external secretions such as saliva and intestinal contents.

Passive immunity to feline leukemia: evaluation of immunity from dams naturally infected and experimentally vaccinated

Antibodies against feline leukemia virus (FeLV) and the feline oncornavirus-associated cell membrane antigen (FOCMA) were transferred from pregnant cats to their suckling kittens. All of these kittens were protected against infection and oncogenesis by virulent FeLV when challenged at 2 weeks of age. Suckling kittens acquired 25 to 100% of maternal virus-neutralizing and FOCMA titers by 3 days of age, and titers underwent linear decay to undetectable levels by 2 to 3 months of age. FOCMA antibody in dams and kittens was identified as immunoglobulin G (IgG) by use of goat anti-human IgG serum, which cross-reacts with feline IgG in the indirect membrane immunofluorescence test for FOCMA antibody. In an attempt to induce protective maternal antibody by vaccination, 10 pregnant cats were immunized by three to five weekly intramuscular injections with purified FeLV inactivated by ultraviolet irradiation. After the course of immunization, neither virus-neutralizing nor FOCMA antibody was detectable in the dams or in 19 kittens born to these cats. When these kittens were challenged with FeLV at 2 weeks of age, 18 of 19 developed persistent viremia and FeLV-related disease.

Cytomegalovirus Specific IgM and IgG Response in Humans Studied by Radioimmunoassay

An indirect solid phase micro-radioimmunoassay (RIA) was adapted for the measurement of anti-cytomegalovirus class-specific immunoglobulin M (IgM) and immunoglobulin G (IgG). Cytomegalovirus (CMV) antigen (Ag) was added to the wells of microtiter plates and desiccated onto the bottom surface of the wells. Serial dilution of human CMV antisera were added and allowed to react with the Ag. The amount of viral antibody (Ab) was determined by measuring the specific binding of 125I-labeled goat anti-human IgG (125I-AHIgG) and/or IgM (125I-AHIgM). The multiple factors which influence the test were determined. Serial samples of sera from CMV-positive patients showed progressive increases in Ab titers on the basis of specific binding of 125I-AHIgG. The titers of IgM class anti-CMV Ab were also determined with the same sera, and enhancement of the titers was demonstrated when the incubation periods of the first Ag-Ab reaction were extended from 1 to 3 hr. The competition between the different Ig classes of Ab for CMV Ag and the involvement of rheumatoid factor (RF) were investigated. The anti-CMV IgG that may compete with the IgM determination was removed by adsorption of sera with Staphylococcus aureus (Cowan 1). The RF that would cause false anti-CMV IgM results was adsorbed with glutaraldehyde-insolubilized IgG. Our results indicate that this RIA is a practical, specific, and reproducible technique for detection of specific IgG and IgM Ab to CMV.