Anti-human Immunoglobulin G Antibody Research Articles

Highly-Sensitive Polymer Optical Fiber SPR Sensor for Fast Immunoassay

A new type of human immunoglobulin G (IgG) sensors based on the surface plasmon resonance (SPR) in the low refractive index (RI) plastic optical fiber (POF) and an antibody immobilization method is presented. A 50-nm-thick gold film was formed on the polished D-shaped fiber surface by magnetron sputtering. The RI response of the POF sensor is 30 049.61 nm/RIU, which is 26.5 times higher than that of single mode fiber (SMF) SPR sensors. The proposed SPR biosensor can be developed by simple and rapid modification of the gold film with 11-mercapto undecanoic acid (MUA). Upon immobilization of the goat anti-human IgG antibody, the resonance wavelength shifts by 11.2 nm. The sensor can be used to specifically detect and quantify the human IgG at concentrations down to 245.4 ng/mL with the sensitivity of 1.327 7 nm per µg/mL, which offers an enhancement of 12.5-fold compared to that of the conventional SMF based SPR sensors. The proposed device may find the potential applications in the case of use at the point of care.

Testing of Diamond Electrodes as Biosensor for Antibody-Based Detection of Immunoglobulin Protein with Electrochemical Impedance Spectroscopy

To control the increasing virus pandemics, virus detection methods are essential. Today’s standard virus detections methods are fast (immune assays) or precise (PCR). A method that is both fast and precise would enable more efficient mitigation measures and better life comfort. According to recent papers, electrochemical impedance spectroscopy (EIS) has proven to detect viruses fast and precise. Boron-doped diamond (BDD) was used as a high-performance electrode material in these works. The aim of this work was to perform an initial test of BDD-based EIS for biosensing. As an easily available standard biomaterial, human immunoglobulin G (IgG) was used as analyte. Niobium plates were coated via hot-filament activated chemical vapor deposition with polycrystalline diamond, and doped with boron for electrical conductivity. An anti-human IgG antibody was immobilised on the BDD electrodes as a biosensing component. Four different analyte concentrations up to 1.1 µg per litre were tested. During EIS measurements, both impedance over frequency curves and Nyquist plot demonstrated no clear sign of a change of the charge transfer resistance. Thus, no positive statement about a successful biosensing could be made so far. It is assumed that these issues need to be investigated and improved, including the relation of BDD electrode size to electrolyte volume, termination of the BDD electrodes (H, O) for a successful functionalisation and EIS frequency range. The work will be continued concerning these improvement issues in order to finally use virus materials as analyte.

Paper-Based Microfluidic Devices: Low-Cost Platforms for Rapid Biochemical Detection.

We developed low-cost, portable paper-based diagnostic devices for detection of human immunoglobulin M (IgM) and immunoglobulin G (IgG) in serum without any sample preparation. These devices can be used to help identify presence of diseases, used to provide rapid results (<5 minutes), readily used by untrained personnel, employed in austere environments, configured to obtain multiplexed assays, and easily disposed of. We successfully accomplished colorimetric detection of human IgG and human IgM using a sandwich-style assay within the microfluidic paper device via vertical flow immunoassay configuration. The reaction zone in the wax printed paper layer is a small circular pattern. Gold nanoparticles conjugated with anti-human IgG and IgM antibodies have been used for colorimetric detection of IgG or IgM by naked eye. Colorimetric signal can be precisely quantified through implementation of image analysis software which can be developed as an app for a smartphone. The size of the device is 2 cm × 2 cm × 1 mm. Colorimetric detection of human IgG was accomplished at 100 fg/mL concentration using a gold nanoparticle-conjugated anti-human IgG antibody. The developed platform has a dynamic range of IgM and IgG concentrations between 0.1 pg/mL and 100 μg/mL. These devices provided a color readout in <5 minutes using 20 µL of serum. We also demonstrated that the devices show a significant degree of ruggedness and temperature stability as they were able to provide satisfactory results (detection of 0.1 pg/mL IgG) after 14 days of long stability and shelf-life experiment at an elevated temperature of at least 50 ˚C-the shelf life can be as long as 180 days under ambient conditions for detection of 100 µg/mL IgG. Because of the inherent simplicity of the device operation and their ease of use, there is no variation between samples and users of the device. This low-cost approach enables multiplexing with >1 measurement performed in parallel at the same time. We anticipate that because of the sensitivity, specificity, ease of use, and overall reliability, this approach will become a standard for diagnosis of diseases and health conditions.

Overexpression of immunoglobulin G prompts cell proliferation and inhibits cell apoptosis in human urothelial carcinoma

Only B lymphocytes can express immunoglobulins according to the traditional immunological theories, and the expression of immunoglobulin G (IgG) messenger RNA (mRNA) and protein was found in certain human cancer cells recently. However, the expression pattern of IgG and its possible role in human urothelial carcinoma are still elusive. In this study, we investigated the expression of IgG in two human urothelial carcinoma cell lines, T24 and BIU-87, and in 56 cases of clinical urothelial carcinoma tissues. The mRNA of IgG was positively detected by in situ hybridization and reverse transcription PCR; furthermore, IgG protein was also positively detected by immunohistochemistry and Western blot. Moreover, blockade of tumor-derived IgG by either antihuman IgG antibody or antisense oligonucleotides increased cell apoptosis and inhibited cell growth in bladder cancer cell lines in vitro, and antihuman IgG antibody could suppress the growth of xenotransplant tumor in vivo. In addition, either antihuman IgG antibody or antisense oligonucleotides enhanced the sensitivity to mitomycin C in bladder cancer cell line T24. Furthermore, blockade of IgG in bladder cancer cell T24 resulted in upregulation of cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase. Our results indicated that bladder cancer cells were capable of expressing IgG, and blockade of IgG expression induced cell apoptosis through activation of caspase-dependent pathway. A novel potential targeted therapy for bladder cancer will be possibly developed based on these data.Electronic supplementary materialThe online version of this article (doi:10.1007/s13277-013-0717-z) contains supplementary material, which is available to authorized users.

Optimization and Validation of a Multiplex, Electrochemiluminescence-Based Detection Assay for the Quantitation of Immunoglobulin G Serotype-Specific Antipneumococcal Antibodies in Human Serum

Pneumovax 23 consists of a mixture of highly purified capsular polysaccharides (Ps) from 23 of the most prevalent serotypes of Streptococcus pneumoniae. Testing of vaccine immunogenicity has been historically performed on the enzyme-linked immunosorbent assay (ELISA) platform, validated to measure immunoglobulin G (IgG) antibodies to all 23 serotypes included in Pneumovax 23. In order to significantly improve the throughput of this form of testing, we have developed and validated a direct binding electrochemiluminescence (ECL)-based multiplex assay that can measure the antibody response in human serum to eight serotypes within a single microtiter well. The pneumococcal (Pn) ECL assay is based on the Meso Scale Discovery (MSD) technology which utilizes a Sulfo-Tag-labeled anti-human IgG antibody that emits light upon electrochemical stimulation. The Pn ECL assay exhibits a wide dynamic range and provides the ability to read concentrations down to the minimum reported concentration in the Merck ELISA (0.1 microg/ml). Cross-reactivity assessment using type-specific monoclonal antibodies showed no cross talk between antigen spots within a well. By use of the WHO Pn sample reference panel, the results obtained with the Pn ECL assay were compared to the results obtained with the international Pn ELISA. The results for the Pn ECL assay satisfied the WHO-recommended acceptance criterion for concordance for all seven serotypes with published Pn ELISA values, and the overall correlation (r value) across the seven serotypes was 0.994. The MSD methodology has great potential to be extremely useful for simultaneously quantitating IgG responses to several Pn serotypes while conserving serum volumes and laboratory testing time.

Microchannel chips for the multiplexed analysis of human immunoglobulin G–antibody interactions by surface plasmon resonance imaging

We report the multiplexed, simultaneous analysis of antigen-antibody interactions that involve human immunoglobulin G (IgG) on a gold substrate by the surface plasmon resonance imaging method. A multichannel, microfluidic chip was fabricated from poly(dimethylsiloxane) (PDMS) to selectively functionalize the surface and deliver the analyte solutions. The sensing interface was constructed using avidin as a linker layer between the surface-bound biotinylated bovine serum albumin and biotinylated anti-human IgG antibodies. Four mouse anti-human IgG antibodies were selected for evaluation and the screening was achieved by simultaneously monitoring protein-protein interactions under identical conditions. Antibody-antigen binding affinities towards human immunoglobulin were quantitatively compared by employing Langmuir adsorption isotherms for the analysis of SPRi responses obtained under equilibrium conditions. We were able to identify two IgG samples with higher affinities towards the target, and the determined binding kinetics falls within the typical range of values reported in the literature. Direct measurement of proteins in serum samples by SPR imaging was achieved by developing methods to minimize nonspecific adsorption onto the avidin-functionalized surface, and a limit of detection (LOD) of 6.7 nM IgG was obtained for the treated serum samples. The combination of SPR imaging and multichannel PDMS chips offers convenience and flexibility for sensitive and label-free measurement of protein-protein interactions in complex conditions and enables high-throughput screening of pharmaceutically significant molecules.

Direct Binding of Human NK Cell Natural Cytotoxicity Receptor NKp44 to the Surfaces of Mycobacteria and Other Bacteria

Our previous studies demonstrated that Mycobacterium bovis bacillus Calmette-Guérin (BCG) can directly interact with human NK cells and induce the proliferation, gamma interferon production, and cytotoxic activity of such cells without the need for accessory cells. Thus, the aim of the present study was to identify the putative receptor(s) responsible for the recognition of BCG by human NK cells and potentially involved in the activation of NK cells. To this end, we first investigated the surface expression of three NK cell-activating receptors belonging to the natural cytoxicity receptor (NCR) family on highly purified human NK cells upon in vitro direct stimulation with BCG. An induction of the surface expression of NKp44, but not of NKp30 or NKp46, was observed after 3 and 4 days of in vitro stimulation with live BCG. The NKp44 induction involved mainly a particular NK cell subset expressing the CD56 marker at high density, CD56(bright). In order to establish whether NKp44 could directly bind to BCG, whole BCG cells were stained with soluble forms of the three NCRs chimeric for the human immunoglobulin G (IgG) Fc fragment (NKp30-Fc, NKp44-Fc, NKp46-Fc), followed by incubation with a phycoerythrin (PE)-conjugated goat anti-human IgG antibody. Analysis by flow cytometry of the complexes revealed a higher PE fluorescence intensity for BCG incubated with NKp44-Fc than for BCG incubated with NKp30-Fc, NKp46-Fc, or negative controls. The binding of NKp44-Fc to the BCG surface was confirmed with immunogold labeling using transmission electron microscopy, suggesting the presence of a putative ligand(s) for human NKp44 on the BCG cell wall. Similar binding assays performed on a number of gram-positive and gram-negative bacteria revealed a pattern of NKp44-Fc binding restricted to members of the genus Mycobacterium, to the mycobacterium-related species Nocardia farcinica, and to Pseudomonas aeruginosa. Altogether, the results obtained indicate, for the first time, that at least one member of the NCR family (NKp44) may be involved in the direct recognition of bacterial pathogens by human NK cells.

The preparation and characterization of poly( o-phenylenediamine)/gold nanoparticles interface for immunoassay by surface plasmon resonance and electrochemistry

The poly- o-phenylenediamine (P oPD) nonconducting film and gold nanoparticles (AuNPs) were combined to fabricate AuNPs/P oPD film, which is used as a novel biocompatible interface for the immobilization of antibody and develop a simple and sensitive label-free immunoassay for the detection of the related antigen (human immunoglobulin G (IgG)). Surface plasmon resonance (SPR) and electrochemical methods were used to provide the real-time information about the polymer film growth, assembling of various sizes of gold nanoparticles, anti-human IgG antibody (anti-hIgG) immobilization and the antigen–antibody interaction. The microstructures of the P oPD and AuNPs/P oPD films were characterized by atomic force microscopy (AFM). These results demonstrated that AuNPs were uniformly dispersed on the porous surface of P oPD film, which formed a nano-structure biocompatible AuNPs/P oPD interface. The use of gold nanoparticles and P oPD film could enhance the immunoassay sensitivity and anti-nonspecific property of the resulting immunoassay electrode. Additionally, the reproducibility and preliminary application of anti-hIgG/AuNPs/P oPD/Au electrode for SPR detection of hIgG was also evaluated.

A new strategy for electrochemical immunoassay based on enzymatic silver deposition on agarose beads

A novel, sensitive electrochemical immunoassay in a homogeneously dispersed medium is described herein based on the unique features of agarose beads and the special amplified properties of biometallization. The immunochemical recognition event between human immunoglobulin G (IgG) and goat anti-human IgG antibody is chosen as the model system to demonstrate the proposed immunoassay approach. Avidin-agarose beads rapidly react with the biotinylated goat anti-human IgG antibody to form agarose beads-goat anti-human IgG conjugate (agarose bead-Ab). Agarose bead-Ab, alkaline phosphatase conjugated goat anti-human IgG antibody (ALP-Ab) and the human IgG analyte are mixed to form sandwich-type immunocomplex followed by the addition of the enzymatic silver deposition solution to deposit silver onto the surface of proteins and agarose beads. The silver deposited are dissolved and quantified by anodic stripping voltammetry. The influence of relevant experimental variables was examined and optimized. The logarithm of the anodic stripping peak current depended linearly on the logarithm of the concentration of human IgG in the range from 1 to 1000 ng/ml. A detection limit as low as 0.5 ng/ml human IgG was attained by 3 σ-rule. The R.S.D. of the approach is 9.65% for eight times determination of 10 ng/ml human IgG under same conditions. Optical microscope and TEM graphs were also utilized to characterize agarose beads and silver nanoparticles formed.

DETECTION OF CRYPTOSPORIDIUM ANTIBODIES IN SERA AND ORAL FLUIDS USING MULTIPLEX BEAD ASSAY*

For the first time, a multiplex bead assay (MBA) was used to assay oral fluid and serum specimens for immunoglobulin G (IgG) antibodies to specific Cryptosporidium parvum antigens that were coupled to polystyrene beads. Recombinant C. parvum 17- and 27-kDa antigens (r17 and r27, respectively) both linked with glutathione-S-transferase (GST) fusion proteins, native 17-kDa antigen, and GST alone were each coupled to microspheres that could be differentiated based on variable amounts of internally incorporated red fluorescent dye. Initial and follow-up serum and oral fluid specimens from a 1997 cryptosporidiosis outbreak in Spokane, Washington, were incubated with the coupled beads. Antibodies bound to the coupled beads were detected using biotinylated monoclonal anti-human IgG antibody and streptavidin-labeled r-phycoerythrin. Fluorescence intensity was measured by flow cytometry. For the 3 C. parvum antigens, the median of the mean fluorescence intensity (MFI) was significantly higher (P < 0.03) in the initial specimens than in the follow-up specimens. No significant change in IgG responses to GST in oral fluids or serum specimens was observed. For all Cryptosporidium antigens, the MFI in the initial serum specimens correlated with the MFI in the initial oral fluid specimens (P < 0.001, r > 0.673). For the recombinant antigens used in the MBA, the MFI correlated with the response as measured by an enzyme-linked immunosorbent assay that used r17 and r27 expressed without the GST fusion partner (P < 0.001, r > 0.854). MBA using sera or more conveniently collected oral fluids, especially from children, may be an option for immunodiagnosis of C. parvum infection and for prospective epidemiological studies designed to monitor infection risk.

Interaction of human immunoglobulin G with CD16 on natural killer cells: ligand clearance, FcgammaRIIIA turnover and effects of metalloproteinases on FcgammaRIIIA-mediated binding, signal transduction and killing.

Human natural killer (NK) cells express low-affinity Fc immunoglobulin G (IgG) receptor (FcgammaRIIIA/CD16). The binding of monomeric IgG (mIgG) and F(ab')(2) fragments of 3G8 anti-CD16 monoclonal antibody (mAb) to FcgammaRIIIA was investigated by flow cytometry. Over 90% of NK cells bound endogenous IgG, and during incubation at 37 degrees C, the FcgammaRIIIA occupancy decreased slowly. Approximately 90% of NK cells bind mIgG or F(ab')(2) fragments of 3G8 anti-CD16 mAb. The calculated half-time (T(1/2)) of in vitro mIgG dissociation from FcgammaRIIIA was 130 min. By cross-linking the mIgG ligand with F(ab')(2) fragments of anti-human IgG antibody, the T(1/2) decreases to 85 min. In kinetics study, it has been shown that (125)I-mIgG bound to FcgammaRIIIA is slowly released in the culture supernatant, maybe eluted at acid pH, or partially internalized and degraded. The binding of IgG to FcgammaRIIIA was increased by 53.8% on cells cultured in the presence of RU36156, a matrix metalloproteinase (MMP) inhibitor. Furthermore, an increase in phosphorylation of Lyn tyrosine kinase, after cross-linking of mIgG-FcgammaRIIIA complex, was observed on NK cells treated with RU36156. When the FcgammaRIIIA was occupied by mIgG, the capacity of NK cells to kill K562 target cells was decreased by RU36156, because the MMP inhibitor protects CD16 from proteolysis. Our data demonstrate that binding of mIgG to human NK cells is followed by ligand dissociation and/or internalization, enzymatic degradation and exocytosis. The RU36156 MMP inhibitor protects FcgammaRIIIA from cleavage, augments NK-cell activation and may interfere in their killing capacity.

Glycosylation of FcgammaRIII in N163 as mechanism of regulating receptor affinity.

Human FcgammaRIII (CD16) is a low-affinity receptor for immunoglobulin G (IgG). There are two different isoforms of this protein: CD16a (transmembranous, expressed on natural killer cells and on macrophages) and CD16b (glycosylphosphatidylinositol-linked, expressed on neutrophilic granulocytes in two allelic forms NA1 and NA2). Both forms of the protein have a variable glycosylation pattern. The NA1 allele of CD16B has four asparagine (N)-linked glycosylation sites. One of them (N163) is localized in the ligand-binding site of domain II. This site is shared by the NA2 allele and CD16A. To examine the functional role of the glycosylation we mutated the four glycosylation sites of the NA1 allele (N39, N75, N163, N170) into glutamine (Q). HEK293 cells were stably transfected with the single mutants and wild-type CD16 as control. We determined binding of human IgG to transfected cells using immunofluorescence studies with anti-human IgG antibody. Monomeric IgG bound to N163Q transfectants with higher affinity than to other transfectants, showing that glycosylation in N163 influences the affinity of CD16 to its ligand. In addition, preincubation of WT-CD16-transfected cells with Tunicamycin (an inhibitor of N-glycosylation) resulted in an increased binding of monomeric IgG whereas N163Q-CD16-transfected cells remained unaffected. Therefore, glycosylation in N163 is a mechanism of regulating affinity of FcgammaRIII to its ligand IgG.

Immunoglobulins of untreated Graves' patients with or without thyrotropin receptor antibody (determined by porcine thyrocytes) universally elicit potent thyroid hormone-releasing activity in cultured human thyroid follicles.

Thyrotropin receptor antibody (TRAb), comprising thyrotropin binding inhibitor immunoglobulin (TBII) and thyroid-stimulating antibody (TSAb), both of which are conventionally determined using porcine thyrocytes in Japan, is not always positive in patients with untreated Graves' disease. To elucidate whether immunoglobulin G (IgG) obtained from TBII/TSAb-positive (+) or negative (-) Graves' disease patients are responsible for hyperthyroidism, we investigated the thyroid hormone-releasing activity (THRA) of these IgGs in human thyroid follicles in suspension culture, in which bovine thyrotropin (bTSH) is detectable even at 0.1 microU/mL. Human thyroid follicles, obtained from Graves' disease patients by subtotal thyroidectomy, were cultured in serum-free F-12/RPMI-1640 medium supplemented with bTSH or purified Graves' IgGs. After preculturing for 3 days, 125I was added, and after an additional 3 days of culture, 1251 incorporated into the thyroid follicles and organic 125I released into the culture medium (mainly 1251 -T4 + 125I-T3) were counted. Seventy TBII(+)/TSAb( + )-, 3 TBII( + )/TSAb( - )-, and 3 TBII( - )/TSAb( + )- patients with untreated Graves' disease were all positive for THRA, which became undetectable in spontaneous remission obtained after several years of medical treatment. The THRA was equivalent to 0.8-230 microU/mL bTSH. Furthermore, 2 TBII(-)/TSAb(-) patients were significantly positive for THRA. This TBII(-)/TSAb(-)IgG stimulated human thyrocytes to produce cyclic adenosine monophosphate (cAMP), and this was partially inhibited by antihuman IgG antibody. The THRA induced by TBII(+)/TSAb(+) IgGs as well as TBII(-)/TSAb(-) IgG was inhibited by blocking-type TRAb obtained from TBII(+) patients with myxedema. There was a significant correlation between THRA and TSAb. These in vitro findings suggest that all IgGs obtained from untreated Graves' patients (n = 78) elicit potent THRA in human thyroid follicles in suspension culture. Because the TBII(-)/TSAb(-) IgGs can stimulate cAMP production in human but not in porcine thyrocytes, they probably recognize epitope(s) of TSH-binding sites specific to the human thyrotropin (hTSH) receptor. Furthermore, we have demonstrated that the thyroid gland of hyperthyroid Graves' patients is stimulated by IgG(s) equivalent to at least 0.8 microU/mL bTSH (about 5 microU/mL hTSH) in vitro.

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.

Antibodies against macaque monoclonal immunoglobulin G in rheumatoid arthritis.

The presence of rheumatoid factors (RF) in the serum of rheumatoid arthritis (RA) patients is commonly evidenced by agglutination tests: the Waaler-Rose assay, based on the use of human red blood cells (RBCs) coated with rabbit anti-RBC antibodies, and the latex test, which uses latex particles coated with denatured human immunoglobulin G (IgG). The aim of the present study was to characterize the RF able to agglutinate human RBCs coated with macaque antihuman RBC IgG antibodies secreted from macaque-mouse heterohybridomas (two from rhesus monkey and one from crab-eating macaque). Human RBCs coated with macaque monoclonal antibodies (MacMoAbs) were used for agglutination tests and these were carried out in parallel with standard tests (Waaler-Rose and latex agglutination tests) on sera from 82 RA patients, 86 patients with other forms of inflammatory chronic arthritis and 47 healthy human subjects. MacMoAb-coated RBCs identified RF in the sera of 66% patients with RA. By contrast, the frequency of positive sera in other inflammatory diseases was 5% and all 47 healthy controls were negative. Antimacaque IgG antibodies were found to be more specific for RF than standard tests, in the sera of patients with RA.

Rapid evaluation of serum and gingival crevicular fluid immunoglobulin G subclass antibody levels in patients with early-onset periodontitis using checkerboard immunoblotting.

A method was developed to evaluate the presence of immunoglobulin G (IgG) subclass (1-4) antibody to Actinobacillus actinomycetemcomitans, serotype b (strain Y4) in patients with early-onset periodontitis on a single nitrocellulose membrane. Sera from 30 early-onset periodontitis patients and gingival crevicular fluid samples from 2 patients were collected and tested with four different preparations of A. actinomycetemcomitans (Y4). The principle steps of the assay are: a) binding of the bacterial antigen (Y4) and the anti-human IgG antibody (capture antibody) in parallel lanes on nitrocellulose membranes; b) incubation of known concentrations of the IgG subclasses 1, 2, 3 and 4, as well as a dilution of serum and/or gingival crevicular fluid from patients in lanes perpendicular to the antigen lanes; c) incubation of the membranes with the corresponding peroxidase conjugated anti-human IgG subclass secondary antibody; d) detection of positive signals by enhanced chemiluminescence. The blots were evaluated by visual comparison to a series of blots containing known concentrations of IgG subclasses. The method was used to rapidly screen a relatively large number of patient sera and gingival crevicular fluid samples for IgG subclasses in a cost-effective assay. The predominant IgG subclass found in early-onset periodontitis was IgG2.

Immunocytochemical and labelled tracer approaches to uptake and intracellular routing of Immunoglobulin-G (IgG) in the human placenta

Isolated lobules of freshly delivered human term placenta were (a) subjected to an indirect immunoelectron ultracryo method in which the immunoreactivity of endogenous Immunoglobulin-G (IgG) to rabbit anti-human IgG antibody was localized with protein-A-colloidal gold and (b) extracorporeally perfused and human IgG molecules complexed to horseradish peroxidase (HRP) added to the maternal perfusate and the uptake of IgG-HRP over different perfusion durations visualized ultrastructurally by using diaminobenzidine cytochemistry. Immunoreactivity to anti-human IgG antibody was localized all along the apical plasmalemma, in apical coated and uncoated vesicles, in apical and juxtanuclear multivesicular bodies, and in basal vesicles of the syncytiotrophoblast layer of the placenta. The stroma separating the syncytiotrophoblast from the foetal endothelium as well as vesicles within the endothelium were immunoreactive. No immunoreactivity was localized in paracellular clefts of endothelia. A similar distribution of exogenous IgG-HRP was observed for the perfused placentae. When bovine IgG-HRP or HRP alone were used as control tracers no uptake was seen for the former whilst the latter was observed only in early endosomal vesicles of the syncytiotrophoblast. The pattern of localization visualized in both studies is consistent with receptor-mediated uptake of IgG by the syncytiotrophoblast and a vesicular transport of IgG across the foetal endothelium.

Hyperphosphatemia in multiple myeloma due to a phosphate-binding immunoglobulin

Hyperphosphatemia (HP) is usually seen in patients with hypoparathyroidism, renal failure, and tumor lysis. The authors described a patient with HP due to a phosphate-binding immunoglobulin (Ig). An 86-year-old woman had serum phosphate levels as high as 4.75 mmol/l, (normal, 0.77 to 1.45 mmol/l). Serum ionized calcium, blood urea nitrogen (BUN), creatinine, and N-terminal parathyroid hormone (PTH) levels were normal, but serum 1,25-dihydroxyvitamin D level was subnormal at less than 12 pmol/l (normal, 36 to 146 pmol/l). Serum total protein was elevated at 105 g/l (normal, 60 to 80 g/l), and additional studies confirmed a diagnosis of immunoglobulin G (IgG) multiple myeloma. Results of in vitro studies using anti-human IgG antibodies showed that the IgG of the patient bound inorganic phosphate. Several isolated case reports have documented spurious HP due to interference of the paraprotein in the routine serum phosphate assay. In only one patient, however, has actual binding of phosphate to a myeloma protein been documented. The studies of the authors document phosphate binding by an IgG paraprotein and suggest that in this setting HP may be of physiologic significance as evidenced by depressed serum levels of 1,25-dihydroxyvitamin D.

Correlation Between Dermal Interstitial Immunoglobulin G and Hypergammaglobulinemia

The diffuse dermal immunofluorescence (DDIF) observed in human skin biopsies that is produced by fluorochrome-conjugated antisera reactive with human immunoglobulin G (IgG) has commonly been viewed in the past as an artifact of direct immunofluorescence microscopy. However, it has been our belief that DDIF, which has been observed in 14% of the biopsies processed in our laboratory, might represent an in vivo phenomenon of excess unbound dermal interstitial immunoglobulin G reflective of elevated serum IgG levels. To examine this hypothesis, we carried out a blinded prospective study of the frequency of DDIF in two groups of age- and sex-matched patients that differed with respect to the presence or absence of serum hypergammaglobulinemia (gamma globulin greater than 2.5 mg/100 ml). The subjective intensity of DDIF and the fluorescein-isothiocyanate (FITC) conjugated anti-human IgG antibody titer at which point DDIF disappeared were both compared with serum gamma globulin levels. Also, the amount of IgG that could be extracted from biopsy specimens by an overnight phosphate-buffered saline (PBS) elution procedure was quantified by solid-phase immunoassay. We noted a trend of increased DDIF intensity with increasing serum gamma globulin levels. In addition, there was a relatively weak but significant positive correlation between DDIF endpoint titers and serum gamma globulin levels (p = 0.05, r = 0.429). The strongest positive statistical correlation observed in the study was between the quantities of IgG that could be eluted from skin biopsies and serum gamma globulin levels (p = 0.01, r = 0.599). These findings suggest that DDIF is a true biologic phenomenon reflective of elevated serum gamma globulin levels and demonstrate that simple overnight elution of unbound dermal IgG can unmask the presence of disease-related, specifically-bound IgG.

Detection of immunoglobulins G and M to rubella virus by time-resolved immunofluorometry

We describe new methods for the detection of immunoglobulin G (IgG) and IgM rubella-specific antibodies in serum. The IgG assay was based on a solid-phase rubella antigen immobilization approach, and the IgM assay was based on the IgM capture assay principle. Both assays used biotinylated antibodies (anti-human IgG and antirubella monoclonal antibody, respectively). The tracer system was based on streptavidin labeled with a fluorescent europium chelate. The final measurements were done by using time-resolved fluorescence. Both assays were thoroughly evaluated with clinical samples and compared successfully with established techniques. We anticipate that these assays are suitable for routine clinical use.