Development of colloidal gold-based immunochromatographic assay for the rapid detection of medroxyprogesterone acetate residues

One-step membrane-based competitive colloidal gold-based immunoassays in lateral-flow formats for the rapid detection of medroxyprogesterone acetate (MPA) were developed. Nitro-cellulose membrane strip was separately coated with goat anti-rabbit IgG (control line) and MPA hapten-OVA conjugate (test line). Anti-MPA polyclonal antibody labelled with colloidal gold particles was firstly incubated with MPA. A positive reaction as a result of the remaining antibody-gold conjugate combining with antigen coated on the membrane was obvious by visual detection, with detection limits for lateral flow of 5 µg/kg for detecting MPA standard solution, and the limit of detection was 10 µg/kg for detecting the MPA spiked in swine urine and liver. The assay time for test was less than 5 min, suitable for rapid testing on-site.     

Characterization of antibody labelled colloidal gold particles and their applicability in a sol particle immuno assay (SPIA)

This study describes the characterization of antibody labelled colloidal gold particles and their applicability in a sol particle immuno assay (SPIA) to quantify murine monoclonal antibodies of all IgG isotypes. Two physical methods (transmission electron microscopy (TEM) and dynamic light scattering (DLS], were used to obtain information about particle size and morphology of the gold sols, but only with DLS antibody labelling could be detected. In addition, electrophoretic methods like agarose electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed also that antibody labelling was successful. The biological activity of the antibody gold conjugates was determined by using them in a SPIA and as an electrondense marker in an immunogold labelling procedure to visualize meningococcal surface exposed outer membrane proteins labelled with monoclonal antibodies. The SPIA was applicable to determine murine monoclonal antibodies of all IgG isotypes with a sensitivity of 20-80 ng/ml and a co?ffici?nt of variation of 6.7 +/- 2.2%.     

Antigen-binding radioimmunoassays for human IgG antibodies to bovine beta-lactoglobulin

Colloidal gold particles coated with rabbit anti-HCG (Au-(anti-HCG)) were used in a spectrophotometric ‘sandwich’ SPIA for HCG. The effects of several reagent properties and assay variables were investigated in order to optimize the assay. Best results were obtained with 55 nm particles. Optimum assay conditions involved a sample incubation period of 2 h followed by aspiration of the sample and two washings. A suitable Au-(anti-HCG) concentration corresponded to a value of $\text{A}{}_{\mathrm{<mtext>540</mtext><mtext>nm</mtext>}}{}^{\mathrm{<mtext>1</mtext><mtext>cm</mtext>}}\text{= 2.0}$. The steepest dose-response curves were obtained with an Au-(anti-HCG) incubation period of 16 h at 37°C. The test procedure was suitable for preparing dose-response curves for HCG in buffer, urine and serum. The practical measuring range for HCG dissolved in these media was 30–250 IU/l. The within-run coefficient of variation varied between approx. 4 and 12%. The detection limit for HCG (in buffer) was about 1 IU/l.     

Development of colloidal gold-based immunochromatographic assay for the rapid detection of medroxyprogesterone acetate residues

One-step membrane-based competitive colloidal gold-based immunoassays in lateral-flow formats for the rapid detection of medroxyprogesterone acetate (MPA) were developed. Nitro-cellulose membrane strip was separately coated with goat anti-rabbit IgG (control line) and MPA hapten-OVA conjugate (test line). Anti-MPA polyclonal antibody labelled with colloidal gold particles was firstly incubated with MPA. A positive reaction as a result of the remaining antibody-gold conjugate combining with antigen coated on the membrane was obvious by visual detection, with detection limits for lateral flow of 5 µg/kg for detecting MPA standard solution, and the limit of detection was 10 µg/kg for detecting the MPA spiked in swine urine and liver. The assay time for test was less than 5 min, suitable for rapid testing on-site.     

Silver-enhanced lateral flow immunoassay for highly-sensitive detection of potato leafroll virus

Rapid non-laboratory screening of plants for pathogenic viruses crucially influences crop yields in modern agricultural technologies. The aim of this study was to develop a highly-sensitive lateral flow immunoassay (LFIA) for rapid detection of potato leafroll virus (PLRV), an infectious agent of one of the most widespread potato diseases. The proposed LFIA combines the formation of sandwich immune complexes with gold nanoparticles (GNP) as labels and silver enhancement. The enhancement stage was realized using mixture of silver lactate and hydroquinone and subsequent addition of chloride-containing buffer to stop silver reduction. LFIA with silver enhancement was 15 times more sensitive (detection limit 0.2?ng/mL; 15?min) compared with conventional LFIA (detection limit 3?ng/mL; 10?min). The enhanced LFIA detected PLRV in leaves’ extracts of infected potato in dilutions higher than enzyme-linked immunosorbent assay.     

Competitive immunochromatographic assay for the detection of the organophosphorus pesticide EPN

An immunochromatographic assay (ICA) based on competitive antigen-coated format and colloidal gold label was developed for the detection of the organophosphorus insecticide O-ethyl O-4-nitrophenyl phenylphosphonothioate (EPN). The ICA test strip consisted of a membrane with a detection zone, a sample pad and an absorbent pad. The membrane was separately coated with EPN hapten-OVA conjugate (test line) and anti-mouse IgG (control line). As the competition is between the migrating analyte and the immobilised analyte hapten for the binding sites of the migrating antibody–colloidal gold (Ab–CG) conjugate, this study suggests that the relative migration speed between the two migrating substances is a critically important factor for the sensitive detection by competitive ICA. Based on this criterion, a nitrocellulose (NC) membrane was chosen as the most suitable for EPN ICA. The detection limit of the ICA for EPN standard and EPN spiked into agricultural samples were 10^?2 and 5×10^?2 µg mL^?1, respectively.     

The selection and optimization of the detection system for self-contained multiplexed dot-immunoassay

This study performed a comparative estimation of the detection systems with the use of conjugates based on peroxidase, alkaline phosphatase, colloidal gold, and the amplification system “Super-CARD” for multiplex dot-immunoassay of antibodies. The results of the study show that the sensitivity of the detection system with colloidal gold was approximately 8 times higher than that of the system with amplification “Super-CARD”, 30 times higher than that of the system with conjugate of alkaline phosphatase, and 250 times higher than the sensitivity of the system with the peroxidase conjugate. Gold immunosols limit the direct detection of human IgG of 10 pg with dynamic range of optical signal change from 5 ng to 10 pg. This limit corresponds to a range of concentrations of IgG from 2.5 μg/mL to 5 ng/mL and covers the range of specific IgG concentrations, to which a typical natural immune response leads.

The most typical reasons for aggregate formation during obtainment of colloidal gold and the binding of colloidal gold with biocomponents were explored. The method of minimization of particle clusters formation was suggested as well as the method for increasing stability of diluted preparations of probe by means of sedimentation of aggregates from the ready-made product.     

The immunogold-silver staining approach in the study of lymphocyte subpopulations in transmission electron microscopy.

The potential of immunogold-silver staining has been evaluated in immunoelectron microscopic studies of human normal peripheral blood lymphocyte subpopulations. The cells were labeled, before being embedded in resin, using 5 nm colloidal gold particles and this was followed by silver enhancement. The use of colloidal gold particles permits detection of small amounts of antigen; the silver intensification forms a sphere of heavy metal around the gold granule giving rise to an ultrastructural marker which can be easily seen even at low magnification. The ultrastructural details of the cells were well preserved and there was no significant background staining. The major advantage of the present IGS technique is that it permits a rapid and simultaneous evaluation of both the immunophenotype and the ultrastructural characteristics of cells.     

Visual detection of IS6110 of Mycobacterium tuberculosis in sputum samples using a test based on colloidal gold and latex beads

The IS6110 sequence was detected visually in sputum samples of tuberculosis patients using a bi-probe system. One of the probes was an oligonucleotide conjugated to colloidal gold particles, complementary to one end of the target strand. The other probe was an oligonucleotide conjugated to latex beads complementary to the other end of the target strand. In a reaction mix, these two probes bind to the target strand, and the latex beads are then separated by filtration. Bound latex beads have gold colloid particles at the other end of the target strand. These gold colloid particles were made visible to the naked eye by silver autometallography on the 'invisible' colloidal gold particles. The lower detection limit was 50 ng of genomic DNA of Mycobacterium tuberculosis. This new test, together with conventional PCR, was performed on DNA extracted from sputum samples of suspected tuberculosis patients. The new test was simple to perform, the results were visible to the naked eye, and the test was highly specific, as even single point mutations in the target strand sequence could be differentiated. The test could be useful in field-level laboratories because it requires no sophisticated equipment.     

Development of a lateral flow dipstick immunoassay for the rapid detection of glycyrrhizic acid

A sensitive antibody-based lateral flow dipstick was developed for glycyrrhizic acid (GL) detection. The stick consisted of a sample pad, a conjugate pad, membrane and an absorbent pad. The membrane was coated with two capture reagents, GL-BSA conjugate and goat anti-mouse antibodies, forming a test line and a control line, respectively. The conjugate pad was saturated with colloidal gold particles coated with affinity purified monoclonal anti-GL antibody. The visual detection limit was 20–50 ng ml^?1 of GL and the reaction time was 10 min. The dipstick was used for the detection of GL in roots, leaves and stems of liquorice plant samples. The results were compared with those obtained using an indirect competitive enzyme-linked immunosorbent assay (icELISA). After sample extraction with tap water for 30 min at room temperature, it also can be used for identifying liquorice roots and measuring their quality. The dipstick assay proved to be a sensitive and rapid tool for quality control of liquorice herbs.     

Development of a lateral flow dipstick immunoassay for the rapid detection of glycyrrhizic acid

A sensitive antibody-based lateral flow dipstick was developed for glycyrrhizic acid (GL) detection. The stick consisted of a sample pad, a conjugate pad, membrane and an absorbent pad. The membrane was coated with two capture reagents, GL-BSA conjugate and goat anti-mouse antibodies, forming a test line and a control line, respectively. The conjugate pad was saturated with colloidal gold particles coated with affinity purified monoclonal anti-GL antibody. The visual detection limit was 20-50 ng ml^-1 of GL and the reaction time was 10 min. The dipstick was used for the detection of GL in roots, leaves and stems of liquorice plant samples. The results were compared with those obtained using an indirect competitive enzyme-linked immunosorbent assay (icELISA). After sample extraction with tap water for 30 min at room temperature, it also can be used for identifying liquorice roots and measuring their quality. The dipstick assay proved to be a sensitive and rapid tool for quality control of liquorice herbs.     

Silver-enhanced colloidal gold metalloimmunoassay for Schistosoma japonicum antibody detection

A silver-enhanced colloidal gold metalloimmunoassay has been proposed for the determination of Schistosoma japonicum antibody (SjAb) in rabbit serum. The adult worm antigen of S. japonicum (SjAg) was adsorbed passively on the walls of a polystyrene microwell and then reacted with the desired SjAb. The colloidal gold-labeled goat anti-rabbit IgG secondary antibody was adsorbed on the walls of the polystyrene microwells through the reaction with SjAb, followed by the silver enhancement process, dissolution of silver metal atoms in an acidic solution, and determination of dissolved silver ions by anodic stripping voltammetry (ASV) at a glassy-carbon electrode. Assay conditions were optimized, including the reaction time of SjAg with SjAb, the interaction of SjAb with the colloidal gold-labeled secondary antibody, the dilution ratio of the colloidal gold-labeled secondary antibody and the silver enhancement time. The integration of the anodic stripping peak current depended linearly on the SjAb logarithmic concentration over the range of 6.4 ng/ml to 100 microg/ml. A detection limit as low as 3.0 ng/ml SjAb was achieved, which was better than the piezoelectric body acoustic wave sensor (detection limit of 7.2 microg/ml) and the renewable amperometric immunosensor (detection limit of 0.36 microg/ml). Rabbit serum samples with various degrees of infection were analyzed, and the results demonstrate that the proposed method meets the requirements of clinical analysis.     

Enhancement of the detection limit for lateral flow immunoassays: evaluation and comparison of bioconjugates

There is an increasing demand for convenient and accurate point-of-care tools that can detect and diagnose different stages of a disease in remote or impoverished settings. In recent years, lateral flow immunoassays (LFIA) have been indicated as a suitable medical diagnostic tool for these environments because they require little or no sample preparation, provide rapid and reliable results with no electronic components and thus can be manufactured at low costs and operated by unskilled personnel. However, even though they have been successfully applied to acute and chronic disease detection, LFIA based on gold nanoparticles, the standard marker, show serious limitations when high sensitivity is needed, such as early stage disease detection. Moreover, based on the lack of comparative information for label performance, significant optimization of the systems that are currently in use might be possible. To this end, in the presented work, we compare the detection limit between the four most used labels: colloidal-gold, silver enhanced gold, blue latex bead and carbon black nanoparticles. Preliminary results were obtained by using the biotin-streptavidin coupling as a model system and showed that carbon black had a remarkably low detection limit of 0.01 μg/mL in comparison to 0.1 μg/mL, 1 μg/mL and 1mg/mL for silver-coated gold nanoparticles, gold nanoparticles and polystyrene beads, respectively. Therefore, as a proof of concept, carbon black was used in a detection system for Dengue fever. This was achieved by immobilizing monoclonal antibodies for the nonstructural glycoprotein (NS1) of the Dengue virus to carbon black. We found that the colorimetric detection limit of 57 ng/mL for carbon black was ten times lower than the 575 ng/mL observed for standard gold nanoparticles; which makes it sensitive enough to diagnose a patient on the first days of infection. We therefore conclude that, careful screening of detection labels should be performed as a necessary step during LFIA development in order to enhance the detection limit in a final test system.     

Enhanced luminescence enzyme immunoassay for factor VIII related antigen.

A sandwich enzyme immunoassay for plasma factor VIII related antigen has been developed which exploits a para-iodophenol enhanced chemiluminescent reaction to detect the horseradish peroxidase label. The assay entailed 15 min incubations with sample and with conjugate and had a detection limit of 0.12 mU. It showed good within batch precision (coefficient of variation = 2.95-5.8%) and results on a series of 57 specimens agreed with results obtained by immunoelectrophoresis (correlation coefficient = 0.97).     

Postembedding immunogold labelling of fixed glutamate: an electron microscopic analysis of the relationship between gold particle density and antigen concentration

A series of glutamate-glutaraldehyde-brain protein conjugates, prepared with different glutamate concentrations, was embedded in resin, cut, and incorporated in a multilayered sandwich which was embedded anew. Ultrathin cross-sections through the sandwich were collected on mesh grids and incubated together with tissue sections in an antiserum recognizing fixed glutamate, followed by a second layer antibody coupled to colloidal gold particles. This model system revealed a roughly linear relationship between the concentration of fixed glutamate (up to 154 mmol/l) and gold particle density. Extrapolated to the accompanying tissue sections this finding implies that a tissue compartment displaying 2n gold particles/microns2 contains twice as many fixed glutamate residues per volume as a compartment with n gold particles/microns2. By use of the model system the concentration of fixed glutamate in presumed glutamatergic nerve terminals in the cerebellum was estimated to be two to three times the average tissue level and about five times higher than the concentration of fixed glutamate in terminals thought to use gamma-aminobutyric acid as transmitter. This study represents a step towards the use of electron microscopic immunocytochemistry as a tool to assess the absolute concentrations of neuroactive amino acids in nerve terminals and other tissue compartments.     

A new immune complex dot assay for detection of rotavirus antigen in faeces

A new immune complex dot assay (ICDA) using immune gold/silver staining is described for the sensitive and rapid detection of rotavirus in cell culture and stool specimens. The method involves spotting preformed antigen-antibody complexes onto nitrocellulose paper, followed by incubation with colloidal gold-labelled secondary antibody and silver enhancement. ICDA was sensitive and specific and detected rotavirus antigens over a wide range of concentrations. It was more sensitive than a conventional immunodot assay (CIDA) and two commercial enzyme immunoassays (EIA) based on testing serial dilutions of a positive stool specimen. Of 26 stool specimens tested ICDA detected rotavirus antigen in 17; 14 were positive by Pathfinder Rotavirus EIA, 16 by Testpack Rotavirus EIA, and direct electron microscopy (DEM) detected only 12. The ICDA offers improved sensitivity over commercial EIAs and DEM.     

A bead-based immunogold-silver staining assay on capillary-driven microfluidics

Point-of-care (POC) diagnostics are critically needed for the detection of infectious diseases, particularly in remote settings where accurate and appropriate diagnosis can save lives. However, it is difficult to implement immunoassays, and specifically immunoassays relying on signal amplification using silver staining, into POC diagnostic devices. Effective immobilization of antibodies in such devices is another challenge. Here, we present strategies for immobilizing capture antibodies (cAbs) in capillary-driven microfluidic chips and implementing a gold-catalyzed silver staining reaction. We illustrate these strategies using a species/anti-species immunoassay and the capillary assembly of fluorescent microbeads functionalized with cAbs in “bead lanes”, which are engraved in microfluidic chips. The microfluidic chips are fabricated in silicon (Si) and sealed with a dry film resist. Rabbit IgG antibodies in samples are captured on the beads and bound by detection antibodies (dAbs) conjugated to gold nanoparticles. The gold nanoparticles catalyze the formation of a metallic film of silver, which attenuates fluorescence from the beads in an analyte-concentration dependent manner. The performance of these immunoassays was found comparable to that of assays performed in 96 well microtiter plates using “classical” enzyme-linked immunosorbent assay (ELISA). The proof-of-concept method developed here can detect 24.6 ng mL^?1 of rabbit IgG antibodies in PBS within 20 min, in comparison to 17.1 ng mL^?1 of the same antibodies using a ~140-min-long ELISA protocol. Furthermore, the concept presented here is flexible and necessitate volumes of samples and reagents in the range of just a few microliters.     

A sol-particle immunoassay for determination of anti-rubella antibodies; development and clinical validation

A sol-particle immunoassay (SPIA) was developed for determination of anti-rubella antibodies. The test is based on antibody-coated gold sol particles, which may agglutinate in the presence of rubella haemagglutinin antigen. The agglutination which gives a decrease in optical density can be inhibited by specific antibodies present in sera incubated with the immunochemical reagents. The test is performed in microtitration plates and results can be read in a plate-reader. Clinical validation studies were performed comparing the anti-rubella SPIA with the haemagglutination inhibition test and with the latex agglutination test. The sensitivity of the SPIA was calculated to be 99%; the specificity of the test was at least 99.5%. The anti-rubella SPIA is highly suitable for large-scale screening to determine the rubella immune-status.     

铜绿假单胞菌胶体金免疫层析法的研制和应用

为建立一种简单、快速、准确的铜绿假单胞菌检测方法,结合抗重组铜绿假单胞菌外膜蛋白单克隆抗体（mAb）、胶体金标记技术,采用双抗体夹心法,建立了检测铜绿假单胞菌的胶体金免疫层析法,并对该方法的特异性、灵敏度和稳定性进行了评价,对临床痰标本进行了检测。结果显示,该胶体金免疫层析法能在3～15min内完成检测,方法的特异性高、稳定性强,检测灵敏度能达到105CFU/mL。与传统细菌培养方法相比,胶体金免疫层析法的相对灵敏度和特异性分别是80%和90.6%,两种方法的总符合率为86%。结论：利用抗铜绿假单胞菌单克隆抗体建立的胶体金免疫层析法,适合现场快速、特异检测铜绿假单胞菌。