Evaluation of Rapid IgG4 Test for Diagnosis of Gnathostomiasis

Human gnathostomiasis is a parasitic disease caused by Gnathostoma nematode infection. A rapid, reliable, and practical immunoassay, named dot immuno-gold filtration assay (DIGFA), was developed to supporting clinical diagnosis of gnathostomiasis. The practical tool detected anti-Gnathostoma-specific IgG4 in human serum using crude extract of third-stage larvae as antigen. The result of the test was shown by anti-human IgG4 monoclonal antibody conjugated colloidal gold. The sensitivity and specificity of the test were both 100% for detection in human sera from patients with gnathostomiasis (13/13) and from healthy negative controls (50/50), respectively. Cross-reactivity with heterogonous serum samples from patients with other helminthiases ranged from 0 (trichinosis, paragonimiasis, clonorchiasis, schistosomiasis, and cysticercosis) to 25.0% (sparganosis), with an average of 6.3% (7/112). Moreover, specific IgG4 antibodies diminished at 6 months after treatment. This study showed that DIGFA for the detection of specific IgG4 in human sera could be a promising tool for the diagnosis of gnathostomiasis and useful for evaluating therapeutic effects.     

人IgG标记金纳米棒并用于抗人IgG抗体的检测

目的：研究金纳米棒（GNRs）IgG生物学标记及其在抗人IgG检测中的应用。方法：利用种子生长法制备GNRs,用巯基十一酸（MUA）对GNRs端头邀111妖晶面进行化学修饰,MUA提供的羧基可与人IgG结合;抗人IgG与活化的GNRs反应引起GNRs表面等离子体共振（SPR）特征变化,通过读取SPR值判断免疫反应的结果。结果：合成了不同长径比（AR）的GNRs,成功地将人IgG标记于GNRs（AR=3.7）的端头;利用标记后的GNRs对抗人IgG进行检测,其SPR最大吸收峰发生9nm红移,检测灵敏度可达纳摩尔量级。结论：基于人IgG-抗人IgG免疫反应建立了GNRs用于免疫检测的方法,为GNRs用于免疫检测进而研制免疫传感器奠定了基础。     

Multiple labeling of antibodies with dye/DNA conjugate for sensitivity improvement in fluorescence immunoassay

Fluorescence immunoassays are widely used in life science research, medical diagnostics, and environmental monitoring due to the intrinsically high specificity, simplicity, and versatility of immunoassays, as well as the availability of a large variety of fluorescent labeling molecules. However, the sensitivity needs to be improved to meet the ever-increasing demand in the new proteomics era. Here, we report a simple method of attaching multiple fluorescent labels on an antibody with a dye/DNA conjugate to increase the immunoassay sensitivity. In the work, mouse IgG adsorbed on the surface of a 96-well plate was detected by its immunoreaction with biotinylated goat anti-mouse antibody. A 30 base pair double-stranded oligonucleotide terminated with biotin was attached to the antibody through the biotin/streptavidin/biotin interaction. Multiple labeling of the antibody was achieved after a fluorescent DNA probe was added into the solution and bound to the oligonucleotide at high ratios. By comparison with fluorescein-labeled streptavidin, the assay with the dye/DNA label produced up to 10-fold increase in fluorescence intensity, and consequently about 10-fold lower detection limit. The multiple labeling method uses readily available reagents, and is simple to implement. Further sensitivity improvement can be obtained by using longer DNAs for antibody labeling, which can incorporate more fluorescent dyes on each DNA.     

Biomolecule-functionalized magnetic nanoparticles for flow-through quartz crystal microbalance immunoassay of aflatoxin B<Subscript>1</Subscript>

A flow-through quartz crystal microbalance (QCM) immunoassay method has been developed based on aflatoxin B₁ antibody (anti-AFB₁)-functionalized magnetic core-shell Fe₃O₄/SiO₂ composite nanoparticles (bionanoparticles) in this study. To construct such an assay protocol, anti-AFB₁, as a model protein, was initially covalently immobilized onto the Fe₃O₄/SiO₂ surface, and then the functionalized nanoparticles were attached to the surface of the QCM probe with an external magnet. The binding of target molecules onto the immobilized antibodies decreased the sensor’s resonant frequency, and the frequency shift was proportional to the AFB₁ concentration in the range of 0.3–7.0 ng/ml. The regeneration of the developed immunosensor was carried out via attaching or detaching the external magnet from the detection cell. In addition, the selectivity, reproducibility, and stability of the proposed immunoassay system were acceptable. Compared with the conventional ELISAs, the proposed immunoassay system was simple and rapid without multiple labeling and separation steps. Importantly, the proposed immunoassay method could be further developed for the immobilization of other antigens or biocompounds.     

Molecular evolution of IgG subclass among nonhuman primates: implication of differences in antigenic determinants among Apes

The cross-reactivity of five different rabbit polyclonal antibodies to human IgG and IgG subclass (IgG1, IgG2, IgG3, and IgG4) was determined by competitive ELISA with nine nonhuman primate species including five apes, three Old World monkeys, and one New World monkey. As similar to those previously reported, the reactivity of anti-human IgG antibody with plasma from different primate species was closely related with phylogenic distance from human. Every anti-human IgG subclass antibody showed low cross-reactivity with plasma from Old World and New World monkeys. The plasma from all apes except for gibbons (Hylobates spp.) showed 60 to 100% of cross-reactivity with anti-human IgG2 and IgG3 antibodies. On the other hand, chimpanzee (Pan troglodytes andPan paniscus) and orangutan (Pongo pygmaeus) plasma showed 100% cross-reactivity with anti-human IgG1 antibody, but gorilla (Gorilla gorilla) and gibbon plasma showed no cross-reactivity. The chimpanzee and gorilla plasma cross-reacted with anti-human IgG4 antibody at different reactivity, 100% in chimpanzee and 50% in gorilla, but no cross-reactivity was observed in orangutan and gibbon plasma. These results suggest the possibilities that the divergence of “human-type” IgG subclasses might occur at the time of divergence ofHomo sapience fromHylobatidae, and that the molecular evolution of IgG1 as well as IgG4 is different from that of IgG2 and IgG3 in great apes, this is probably caused by different in development of immune function in apes during the course of evolution.     

Detection of human asialo-α1-acid glycoprotein using a heterosandwich immunoassay in conjunction with the light addressable potentiometric sensor

Highly specific detection of human ₁-acid glycoprotein (AGP) and asialo-₁-acid glycoprotein (asialo-AGP) was made possible by use of a sandwich immunoassay. The glycoproteins were sandwiched between biotinylated and fluoresceinated polyclonal rabbit anti-human AGP antibodies. Additionally, asialo-AGP could be distinctly detected, apart from AGP, via the formation of a heterosandwich immunoassay using biotinylated polyclonal rabbit antihuman AGP and the lectin, fluoresceinated ricin toxin. Streptavidin was added to the formed immunocomplexes and the immunocomplexes captured on a biotinylated nitrocellulose membrane. The signal generator, urease conjugate of an anti-fluorescein antibody, was then bound to the complex on membrane. The rate of pH change under microvolume conditions (0.6 µl) was monitored using a silicon chip-based, light addressable potentiometer sensor. Results indicated that AGP and asialo-AGP can be detected to the 2 pg level when two antibodies are used to form the immunocomplex. Asialo-AGP can be detected down to 250 pg when the heterosandwich immunoassay is used; this assay exhibited no response up to 10 ng for native AGP or asialofetuin. Both immunoassays can be used to quantify the level of AGP and asialo-AGP in solution. Although the assay presented is very specific for AGP, asialo-AGP and terminal galactose, it is readily adaptable for the detection of any glycoprotein and terminal carbohydrate (or branched structure) by use of a protein-specific antibody and various lectins.     

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).     

Functional properties of proteins immobilized on albumin microspheres

Bovine serum albumin (BSA) microspheres with an average diameter of 12.5 micron were prepared by crosslinking of BSA molecules with glutaraldehyde in the presence of polymethylmethacrylate dissolved in chloroform-toluene. Trypsin and anti-human IgG antibody were immobilized onto their surfaces by the glutaraldehyde-activation method. The catalytic activity and storage stability of the immobilized trypsin were satisfactorily high. The enzyme immunoassay (EIA) method using BSA-microspheres as a solid phase has a high sensitivity (the minimum concentration of detectable antigen in the sample: 0.2 ng/ml) and a wide concentration range (final concentration 0.027-3000 ng/ml) for the detection of human IgG.     

Lanthanide chelates as new fluorochrome labels for cytochemistry

Anti-rabbit IgG labeled with a new fluorescent europium chelate was used to localize rabbit IgG to human smooth muscle myosin in a histological section. The antibody labeled with the europium chelate could be viewed with a conventional fluorescence microscope with a steady-state light source. This result encourages the development of a time-resolved fluorescence microscope, because a significant improvement in the signal-to-noise ratio can be anticipated.     

Ferrocenemonocarboxylic-HRP@Pt nanoparticles labeled RCA for multiple amplification of electro-immunosensing

A multiple amplification immunoassay was proposed to detect alpha-fetoprotein (AFP), which was based on ferrocenemonocarboxylic-HRP conjugated on Pt nanoparticles as labels for rolling circle amplification (RCA). Firstly, the capture antibody (anti-AFP) was immobilized on glass carbon electrode (GCE) deposited nano-sized gold particles. After a typical immuno-sandwich protocol, primary DNA was immobilized by labeling secondary antibody, which acted as a precursor to initiate RCA. The products of RCA provide large amount of sites to link detection DNAs, which were labeled by signal probes (ferrocenemonocarboxylic) and horseradish peroxidase (HRP). Moreover, the enzymatic amplification signals could be produced by the catalysis of HRP and Pt nanoparticles with the addition of H?O?. These lead to multiple amplification signals monitoring by electrochemical instrument and further resulted in high sensitivity of the immunoassay with the detection limit of 1.7 pg/mL.     

Homogeneous immunoenzyme analysis of human immunoglobulin G using horseradish peroxidase

The author studied the steady-state kinetics of cooxidation of 4-aminoantipyrine (AAP) with phenol and its derivatives--alpha-naphthol, o- and m-acetylaminophenols--by horseradish peroxidase and its conjugate with human immunoglobulins IgG (HRP-IgG). When phenol and AAP were used as peroxidase substrates, anti-human IgG antibodies stimulated the HPR-IgG enzyme activity in the presence of excess H2O2. A homogeneous enzyme immunoassay of human IgG was developed on the basis of this stimulating effect. The kinetics of the interactions between immunological reagents was studied. In the presence of 3% polyethylene glycol 6000, a complete antibody-antigen interaction proceeds at 37 degrees for 15-20 min. The sensitivity of the enzyme immunoassay is 350 ng/ml IgG, and the dynamic detection range is 0.35-15.0 mg/ml.     

Sensitive immunoassay of Listeria monocytogenes with highly fluorescent bioconjugated silica nanoparticles probe

In this paper, a sensitive immunoassay method was proposed for Listeria monocytogenes detection by using highly fluorescent bioconjugated nanoparticles probe. (FITC-IgG)-doped fluorescent silica nanoparticles (fsNPs) firstly were synthesized by a microemulsion method and characterized by TEM and fluorescent spectra. Then the prepared fsNPs were conjugated with polyclonal rabbit anti-L. monocytogenes antibody (pAb) and used as indicator probe. A sandwich-type immune affinity reaction between polyclonal rabbit anti-L. monocytogenes antibody coated onto microplate wells, target bacteria and the fsNPs-antibody conjugates subsequently was conducted to detect target L. monocytogenes and assemble the indicator probe onto the wells. The target L. monocytogenes was measured by the fluorescent signals of the assembled indicator probes. Under the optimal conditions, the calibration graph of fluorescent intensity is proportional to the amount of target bacteria over the range of 50-10,320 CFU/mL with a detection limit of 50 CFU/mL. The proposed method has been successfully applied to detect L. monocytogenes in food samples offering the advantages of sensitivity, simplicity, and stability.     

Fluorescent IgG fusion proteins made in E. coli

Antibodies are among the most powerful tools in biological and biomedical research and are presently the fastest growing category of new bio-pharmaceutics. The most common format of antibody applied for therapeutic, diagnostic and analytical purposes is the IgG format. For medical applications, recombinant IgGs are made in cultured mammalian cells in a process that is too expensive to be considered for producing antibodies for diagnostic and analytical purposes. Therefore, for such purposes, mouse monoclonal antibodies or polyclonal sera from immunized animals are used. While looking for an easier and more rapid way to prepare full-length IgGs for therapeutic purposes, we recently developed and reported an expression and purification protocol for full-length IgGs, and IgG-based fusion proteins in E. coli, called “Inclonals.” By applying the Inclonals technology, we could generate full-length IgGs that are genetically fused to toxins. The aim of the study described herein was to evaluate the possibility of applying the “Inclonals” technology for preparing IgG-fluorophore fusion proteins. We found that IgG fused to the green fluorescent proteins enhanced GFP (EGFP) while maintaining functionality in binding, lost most of its fluorescence during the refolding process. In contrast, we found that green fluorescent Superfolder GFP (SFGFP)-fused IgG and red fluorescent mCherry-fused IgG were functional in antigen binding and maintained fluorescence intensity. In addition, we found that we can link several SFGFPs in tandem to each IgG, with fluorescence intensity increasing accordingly. Fluorescent IgGs made in E. coli may become attractive alternatives to monoclonal or polyclonal fluorescent antibodies derived from animals.     

Detection of IgG and IgM in Sera from Canines with Blastomycosis using Eight Blastomyces dermatitidis Yeast Phase Lysate Antigens

The objective of this study was to compare the efficacy of eight Blastomyces dermatitidis yeast phase lysate antigens (T-58: dog, Tennessee; T-27: polar bear, Tennessee; ERC-2: dog, Wisconsin; B5894: human, Minnesota; SOIL: soil, Canada; B5896: human, Minnesota; 48089: human, Zaire; 48938: bat, India) in the detection of the immunoglobulins IgG and IgM in serum specimens from canines with blastomycosis. An indirect enzyme-linked immunosorbent assay (ELISA, peroxidase system) was used to analyze sera collected during four different intervals post-infection. The yeast lysate antigen 48938 was a reactive antigen for the detection of both IgG (mean absorbance value range: 1.198–2.934) and IgM (mean absorbance value range: 0.505–0.845). For the same sera, antigen T-27 was also effective in the detection of IgG (mean absorbance value range: 0.904–3.356) and antigen 48089 was useful for the detection of IgM (mean absorbance value range: 0.377–0.554). The yeast lysate antigen B5894 proved to be a poor antigen for the detection of both IgG and IgM (mean absorbance value ranges: 0.310–0.744 for IgG, 0.025–0.069 for IgM). Inherent variations in yeast lysate antigens such as these may be utilized to develop improved immunoassay procedures for the specific detection of IgG or IgM in cases of blastomycosis.     

Fluorescent IgG fusion proteins made in E. coli

Antibodies are among the most powerful tools in biological and biomedical research and are presently the fastest growing category of new bio-pharmaceutics. The most common format of antibody applied for therapeutic, diagnostic and analytical purposes is the IgG format. For medical applications, recombinant IgGs are made in cultured mammalian cells in a process that is too expensive to be considered for producing antibodies for diagnostic and analytical purposes. Therefore, for such purposes, mouse monoclonal antibodies or polyclonal sera from immunized animals are used. While looking for an easier and more rapid way to prepare full-length IgGs for therapeutic purposes, we recently developed and reported an expression and purification protocol for full-length IgGs, and IgG-based fusion proteins in E. coli, called “Inclonals.” By applying the Inclonals technology, we could generate full-length IgGs that are genetically fused to toxins. The aim of the study described herein was to evaluate the possibility of applying the “Inclonals” technology for preparing IgG-fluorophore fusion proteins. We found that IgG fused to the green fluorescent proteins enhanced GFP (EGFP) while maintaining functionality in binding, lost most of its fluorescence during the refolding process. In contrast, we found that green fluorescent Superfolder GFP (SFGFP)-fused IgG and red fluorescent mCherry-fused IgG were functional in antigen binding and maintained fluorescence intensity. In addition, we found that we can link several SFGFPs in tandem to each IgG, with fluorescence intensity increasing accordingly. Fluorescent IgGs made in E. coli may become attractive alternatives to monoclonal or polyclonal fluorescent antibodies derived from animals.     

Magnetic nanobead-based immunoassay for the simultaneous detection of aflatoxin B1 and ochratoxin A using upconversion nanoparticles as multicolor labels

A novel and sensitive immunoassay for the simultaneous detection of aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) in food samples was developed by using artificial antigen-modified magnetic nanoparticles (MNPs) as immunosensing probes and antibody functionalized upconversion nanoparticles (UCNPs) as signal probes. NaY_0.78F₄:Yb_0.2, Tm_0.02 and NaY_0.28F₄:Yb_0.7,Er_0.02 UCNPs were prepared and functionalized, respectively, with immobilized monoclonal anti-AFB₁ antibodies and anti-OTA antibodies as signal probes. Based on a competitive immunoassay format, the detection limit for both AFB₁ and OTA under optimal conditions was as low as 0.01 ng mL⁻¹, and the effective detection range was from 0.01 to 10 ng mL⁻¹. The proposed method was successfully applied to measure AFB₁ and OTA in naturally contaminated maize samples and compared to a commercially available ELISA method. The high sensitivity and selectivity of this method is due to the magnetic separation and concentration effect of the MNPs, the high sensitivity of the UCNPs, and the different emission lines of Yb/Tm and Yb/Er doped NaYF₄ UCNPs excited by 980 nm laser. Multicolor UCNPs have the potential to be used in other applications for detecting toxins in the field of food safety and other fields.     

A novel sensitive immunoassay method based on the Invader technique

A novel and sensitive immunoassay method has been developed in which the conventional sandwich immunoassay and the highly sensitive DNA detection method, the Invader method, are combined. The signal amplification function of the latter method has been successfully used to enhance the sensitivity of the sandwich immunoassay. The new assay method may be called the Immuno-Invader assay. The assay format involves three important steps: (1) a target antigen is captured and flagged with a biotin-conjugated detection antibody by the sandwich method, (2) streptavidin and a biotin-conjugated oligonucleotide are added to form a complex with the detection antibody, and (3) the oligonucleotide in the complex is detected using the Invader method. The method was applied to the assay of human tumor necrosis factor-α (hTNF-α). Detection limits obtained were 0.1 pg/ml hTNF-α when a luminescent europium chelate was used with a time-resolved measurement mode, and 0.8 pg/ml when fluorescein was used with a normal prompt fluorescence measurement mode. On the other hand, the detection limit of a commercially available hTNF-α enzyme-linked immunosorbent assay that uses horseradish peroxidase was 3.5 pg/ml. These results demonstrate the feasibility and potential of the new assay method for highly sensitive immunoassay.     

Construction of electrochemical flow immunoassay system using capillary columns and ferrocene conjugated immunoglobulin G for detection of human chorionic gonadotrophin

In this paper is reported a miniaturized flow immunoassay system. Ferrocenecarboxylic acid (Fc) conjugated with anti-HCG immunoglobulin G (IgG) antibody (Fc–IgG) was prepared, and used as a novel analytical reagent. The system consists of the immunoreaction section, the capillary column packed with cation exchange resin, and the flow cell for electrochemical detection of Fc–IgG. Antibody–antigen complexes were separated from their free conjugate on the basis of differences in isoelectric point (pI) using a cation exchange capillary column. The assay yielded a linear relationship between signal and HCG concentration in the range 0–2000 mIU/ml. This simple technique enables the assay of HCG within 2 min. The cation exchange capillary column was regenerated by occasional elution with malonate buffer (pH 6.0) containing 0.5 M NaCl, to remove free conjugate. Free conjugate recovered in this manner could be reused up to eight times without significant decreases in the sensitivity of the immunoassay. This electrochemical flow immunoassay requires only minute quantities of serum and generates highly reproducible results.     

Identification of antigenic components of Toxoplasma gondii by an immunoblotting technique

Proteins of Toxoplasma gondii were separated by SDS-polyacrylamide gel electrophoresis with subsequent transfer to a nitrocellulose sheet by electrophoretic blotting. Immunologically reactive polypeptides were detected by human sera with previously known toxoplasma antibody levels. Heavy chain-specific, peroxidase-conjugated anti-human immunoglobulins were used as the indicator antibodies for the separate identification of IgG and IgM reactive polypeptides. IgG toxoplasma antibodies reacted with several antigens of M_r ≈27 000–67 000, while toxoplasma-specific IgM seemed to detect only a few polypeptides. The M_r of 35 000 for the dominating IgM reactive polypeptide was observed.     

The use of CdTe quantum dot fluorescent microspheres in fluoro-immunoassays and a microfluidic chip system.

Polystyrene fluorescent microspheres prepared by deposition of CdTe quantum dots (QDs) are used in an immunoassay in this study. CdTe QDs/polyelectrolyte multilayers on the surface of polystyrene microspheres have been formed by layer-by-layer self-assembly via electrostatic interactions. As a model antigen, rabbit IgG has been bound to the outermost layer of the fluorescent microspheres. The immunoreaction between fluorescent microspheres/rabbit IgG and the corresponding antibody was confirmed by change of the fluorescence spectrum and competitive immunoassay. This approach allowed detection of the antigen (rabbit IgG) in the range 1-500 mg/L, based on the change in the fluorescence intensity of the reporter (fluorescent microspheres/rabbit IgG). A novel microfluidic chip device with a laser-induced fluorescence system was established and used for the detection of fluorescent microspheres in this study.