Goat Anti-human Immunoglobulin M Research Articles

Development and Evaluation of an Electrochemical Biosensor for Detection of Dengue-Specific IgM Antibody in Serum Samples.

Dengue is an arbovirus disease transmitted mainly by Aedes mosquitoes. As dengue shares similar clinical symptoms with other infectious diseases, prompt and accurate diagnosis is pivotal to clinicians’ decisions on appropriate management. Conventional diagnostic tests to detect the dengue-specific IgM antibody are limited in their performance and ease of use. To address these issues, we developed and evaluated a biosensor based on screen-printed carbon electrodes (SPCEs) for the detection of dengue-specific immunoglobulin M (IgM) antibodies. Various optimisations were performed in order to increase the sensitivity and specificity of the biosensor. For optimal and proper orientation of the paratope sites of goat anti-human IgM capture antibodies (GAHICA), various antibody techniques, including passive, covalent, protein A, protein G and streptavidin/biotin systems, were tested on the SPCEs. The assay reagents for the biosensor were also optimised prior to its evaluation. Analytical sensitivity evaluation was carried out using pooled sera, while analytical specificity evaluation was conducted on a panel of six non-dengue serum samples. Subsequently, diagnostic sensitivity and specificity evaluation were performed using 144 reference samples. Electrochemical current signals generated from H2O2 catalysed by HRP-labelled anti-dengue detection antibodies were measured using the chronoamperometric technique. With a limit of detection (LOD) of 106 serum dilution, the analytical sensitivity of the developed biosensor was 10 times higher than commercial ELISA. The analytical specificity of this dengue IgM biosensor was 100%. Similarly, the biosensor’s diagnostic performance was 100% for sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). These findings suggest that the developed biosensor has a great potential to be used to diagnose dengue after seroconversion.

A Highly Sensitive Resonance Scattering Assay for Immunoglobulin M Using Ag(I)–Hydroquinone–Immunonanogold Catalytic Reaction

Ten-nanometer nanogold showed the strongest catalytic effect on the particle reaction between Ag(I) and hydroquinone to form nanosilver particles that exhibited the strongest resonance scattering (RS) peak at 350 nm. The enhanced RS intensity was linear to the nanogold concentration in the range of 30–5,700 nM Au. The nanogold was used to label goat antihuman immunoglobulin M (GIgM) to obtain an immunonanogold probe (AuGIgM) for immunoglobulin M (IgM). Based on the nanogold-labeled immunoreaction between IgM and AuGIgM, centrifugation, and AuGIgM–Ag(I)–hydroquinone nanocatalytic reaction, a highly sensitive and selective immunonanogold-catalytic Ag particle RS assay for 0.2–300 ng mL−1 IgM was proposed, with a detection limit of 0.1 ng mL−1. This assay was simple and sensitive and was applied to assay IgM in serum samples, with satisfactory results.

Rapid Assay of Trace Immunoglobulin M by a New Immunonanogold Resonance Scattering Spectral Probe

Nanogold, 8 nm in size, was used to label goat antihuman immunoglobulin M (GIgM) to obtain a new immunonanogold resonance scattering (RS) probe (Au-GIgG) for quantitation of trace immunoglobulin M (IgM). The Au-GIgG combined with IgM to form nanogold-labeled immunocomplex causes the RS intensity at 580 nm to be enhanced, in pH 4.49 KH(2)PO( 4)-Na(2)HPO(4) buffer and in the presence of polyethylene glycol 6000. The enhanced RS intensity at 580 nm (DeltaI(580 nm)) is proportional to the IgM concentration in the range of 1.5 to 2000 ng/mL, with a lower detection limit of 0.98 ng/mL. The immunonanogold RS assay was used to assay IgM in serum samples, with sensitivity, selectivity, and simplicity.

Comparison of Two Rapid Diagnostic Assays for Detection of Immunoglobulin M Antibodies to Dengue Virus

Two easy-to-use commercial diagnostic assays, a dipstick enzyme-linked immunosorbent assay (ELISA) (Integrated Diagnostics, Baltimore, Md.) and an immunochromatographic card assay (PanBio, Brisbane, Australia) were evaluated for detection of immunoglobulin M (IgM) antibody to dengue virus with an in-house IgM antibody capture microplate ELISA as a reference assay. The dipstick ELISA was based on the indirect-ELISA format using dengue 2 virus as the only antigen and enzyme-labeled goat anti-human IgM antibody as the detector. The total assay time was 75 min. The immunochromatographic card assay was based on the antibody capture format and separately measured both anti-dengue virus IgM and IgG in the same test. Colloidal-gold-labeled anti-dengue virus monoclonal antibody bound with dengue virus 1 to 4 antigen cocktail was the detector, and anti-human IgM and IgG were the capture antibodies. The total assay time was <10 min. Sera from 164 individuals classified as either anti-dengue virus IgM positive (94) or anti-dengue virus IgM negative (70) in the reference microplate ELISA with a dengue virus 1 to 4 antigen cocktail were tested in the two commercial assays. The dipstick ELISA missed 7 of 94 positive samples, for a sensitivity of 92.6%, while the immunochromatographic card assay missed two positive samples, for a sensitivity of 97.9%. Of the 70 negative samples, four were false positive by the dipstick ELISA and two were false positive in the immunochromatographic card assay, resulting in specificities of 94.3 and 97.1%, respectively. Both commercial assays provide sensitive and specific detection of anti-dengue virus IgM antibody and could prove useful in settings where the microplate ELISA is impractical.

Cytoplasmic IgM in leukemic B cells by flow cytometry

The presence of intracellular cytoplasmic immunoglobulin M (IgM) in leukemic cells from patients with acute lymphocytic leukemia (ALL) and chronic lymphocytic leukemia (CLL) was investigated by flow cytometry. The objective of the study was to develop a reproducible flow cytometric method. A Burkitt's lymphoma-derived B-cell line, Daudi, and a pre-B ALL, Nalm-6, served as prototypes. Normal B cells and cells from patients with chronic myelogenous leukemia (CML) were used as negative controls. Cytoplasmic μ was expressed in 77.3 ± 7.5% ( n = 10) of Nalm-6 cells. CALLA+ ALL and CML cells lacked cytoplasmic μ. The surface-membrane immunoglobulin on the viable B cells was blocked with purified goat anti-human IgM. Subsequently, the B cells were fixed in cold absolute methanol and stained with a fluorescein-conjugated goat anti-human IgM to demonstrate cytoplasmic IgM. After the surface-membrane IgM was blocked, normal B cells had no cytoplasmic IgM (0.3–0.5% positive cells) detectable by flow cytometry. However, the peripheral blood lymphocytes from five patients with CLL and the Daudi cells contained cytoplasmic IgM, ranging from 7.8 to 76.7% and 45.7 to 89.3%, respectively. We conclude that cytoplasmic μ in Nalm-6, CLL, and Daudi cells can be easily and rapidly demonstrated by flow cytometry.

Detection of Japanese encephalitis virus immunoglobulin M antibodies in serum by antibody capture radioimmunoassay.

An assay for detecting human immunoglobulin M (IgM) antibodies to Japanese encephalitis (JE) virus was developed by using the antibody capture solid-phase radioimmunoassay approach (JE IgM ACRIAAA). Heavy-chain-specific goat antihuman IgM was first bound to the wells of a polyvinyl microtiter plate, and successive steps involved sequential binding of test sample IgM, acetone-extracted mouse brain JE antigen, and (125)I-labeled flavivirus hyperimmune human IgG. Among 20 patients hospitalized in Bangkok with clinical diagnoses of acute encephalitis, and with acute flavivirus infections proven by hemagglutination inhibition (HAI) serology, 16 had detectable (positive/negative [P/N] ratio, greater than 3.0) JE IgM ACRIA antibodies in the acute-phase serum specimen, and 19 had such antibodies in the convalescent-phase serum specimen. Convalescent patient sera regularly had higher P/N values than the corresponding acute-phase sera (mean +/- 1 standard deviation = 13.0 +/- 9.3 with acute-phase sera and 25.8 +/- 19.6 with convalescent-phase sera). JE virus-infected patients with HAI serological responses indicative of a primary flavivirus infection had higher JE IgM ACRI P/N responses than did those patients whose serological response indicated past exposure to other flaviviruses. None of 70 serum specimens from healthy Thai adults and children with serum JE HAI antibodies had detectable JE IgM ACRIA activity (P/N ratios all less than or equal to 3.0). Biological false-positives with low P/N ratios (range, 3 to 15) were found in sera from patients with acute or recent infections with flaviviruses other than JE virus but could be differentiated by the fact that these sera gave higher P/N ratios with homologous antigens than with JE virus. False-positive reactions with low P/N ratios (range, 3 to 6) due to serum rheumatoid factor activity were differentiated by testing with control antigen. The JE IgM ACRIA technique permits a rapid, accurate diagnosis of acute JE virus infections in both patients with and those without previous exposure to other flaviviruses.

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.