Development of a colloidal gold-based lateral flow dipstick immunoassay for rapid qualitative and semi-quantitative analysis of artesunate and dihydroartemisinin.

BACKGROUND: Artemisinin-based combination therapy (ACT) plays an indispensable role in malaria control and elimination. However, the circulation of counterfeit, substandard drugs has greatly threatened malaria elimination campaigns. Most methods for the analysis of artemisinin and its derivatives require expensive equipment and sophisticated instrumentation. A convenient, easy-to-use diagnostic device for rapid evaluation of the quality of artemisinin drugs at the point-of-care is still lacking. In this study a lateral flow dipstick immunoassay was developed for qualitative and semi-quantitative analysis of artesunate (ATS) and dihydroartemisinin (DHA) in anti-malarial drugs. METHODS: This assay was based on a monoclonal antibody (mAb) raised against ATS. ATS-bovine serum albumin and goat anti-mouse IgG, used as the test capture reagent and the control capture reagent, were coated on the nitrocellulose membrane to form the test line and control line, respectively. The conjugate pad was saturated with the gold-labelled anti-ATS mAb. RESULTS: The indicator range of the dipsticks, defined as lowest concentration of the target analytes between which the test line was not visible, were 100-200 and 200-500 ng mL(-1) for ATS and DHA, respectively. No competitive inhibition was observed up to 5,000 ng mL(-1) of quinine, chloroquine diphosphate salt, primaquine phosphate, pyrimethamine, lumefantrine, amodiaquine, piperaquine tetraphosphate tetrahydrate or pyronaridine tetraphosphate. Semi-quantitative analysis of ATS and DHA in commercial drugs and raw drug materials with the dipsticks produced result agreeable with those determined by high performance liquid chromatography (HPLC). Storage test showed that the indicator range for artemisinins remained unchanged after a week at 37 °C and increased four-folds after six months of storage at 4 °C or ambient temperature. CONCLUSIONS: The new selected mAb 3D82G7 with high avidity and broad cross reactivity for artemisinins was used to develop and optimize a dipstick immunoassay for qualitative and semi-quantitative analysis of ATS and DHA in anti-malarial drugs. The semi-quantitative analysis of ATS and DHA in commercial drugs and raw drug materials, and the specificity test of the artemisinin-related drugs both proved the accurate performance of the developed dipsticks for semi-quantitation of ACT samples. The dipstick may be used as a point-of-care device for identifying substandard and counterfeit ATS- and DHA-containing anti-malarial drugs.

Development of a new sensitive immunostrip assay based on mesoporous silica and colloidal Au nanoparticles.

A new competitive immunostrip assay was developed to detect human serum albumin (HSA) in urine sample with use of conjugated monoclonal antibody gold nanoparticles (mAb-AuNPs) and mobile crystalline material (MCM)-41-HSA bioconjugate. To prepare the immunostrip, the colloidal AuNPs with an average particle diameter of 20 nm, was synthesized, labeled with antibody and applied on the conjugate pad as the detection reagent. Then, HSA was attached to the MCM-41 mesoporous nanoparticles and immobilized to a nitrocellulose membrane as the test line. In the optimized investigational conditions, the immunostrip could detect HSA in a high linear range (from 1 to 200 µg/ml) and low detection limit (ng/ml). The reliability of the testing procedure was examined by performing the immunostrip test with 30 urine samples and comparing the results with those obtained via immunoturbidimetry. The immunostrip was adequately sensitive and accurate for a rapid screening of HSA in the urine. This new strategy for competitive immunostrip design can be used and developed for other antigen based immunostrip assay.

Development of a serotype colloidal gold strip using monoclonal antibody for rapid detection type Asia1 foot-and-mouth disease.

BACKGROUND: In this study, we developed a rapid, one step colloid gold strip (CGS) capable of specifically detecting type Asia1 foot-and-mouth disease virus (FMDV). We have produced two monoclonal antibodies (mAb) to type Asia1 FMD (named 1B8 and 5E2). On the test strip, the purified 1B8 labelled with the colloidal gold was used as the detector, and the purified 5E2 and goat anti-mouse antibodies were wrapped onto nitrocellulose (NC) membranes as the test and the control line, respectively. The rapid colloidal gold stereotype diagnostic strip was housed in a plastic case. RESULTS: In specificity and sensitivity assay, there was no cross-reaction of the antigen with the other type of FMD and SVDV. The detection sensitivity was found to be as high as 10(-5) dilution of Asia1/JSL/05 (1 × 10(7.2)TCID(50)/50 µL). There was excellent agreement between the results obtained by CGS and reverse indirect hemagglutination assay (RIHA), and the agreement can reach to 98.75%. CONCLUSION: We developed colloidal gold strips that have good qualities and does not require specialized equipment or technicians. This method provided a feasible, convenient, rapid, and effective for detecting type Asia1 FMDV in the fields.

A Rapid Intraoperative Parathyroid Hormone Assay Based on the Immune Colloidal Gold Technique for Parathyroid Identification in Thyroid Surgery.

Objective: A novel immunochromatographic test strip method was developed to detect tissue parathyroid hormone (PTH) using the immune colloidal gold technique (ICGT). The accuracy and application value of this method for intraoperative parathyroid identification were evaluated. Methods: Serum samples were collected to measure PTH by both ICGT and electrochemiluminescence immunoassay (ECLIA). Patients who underwent unilateral and total thyroidectomy were enrolled to evaluate the feasibility and clinical efficacy of rapid intraoperative identification of parathyroid glands via PTH determination using ICGT. Two sample preparation methods, fine needle aspiration (FNA) and tissue block homogenate (TBH), were used for PTH-ICGT analysis. Results: Bablok analysis showed a linear relationship between the serum PTH measurements obtained by ICGT and ECLIA. Non-parathyroid tissues had much lower PTH concentrations (14.8 ± 2.1 pg/ml, n = 97) detected by ICGT, compared to the parathyroid gland tissues (955.3 ± 16.1 pg/ml, n = 79; P < 0.0001), With biopsy results as the standard, ICGT showed higher diagnosis rates as compared with direct visual inspection, for identifying both parathyroid glands (97.4 vs. 78.2%) and non-parathyroid tissues (100 vs. 68.9%). The cut-off values for parathyroid identification by FNA and TBH methods were 63.99 and 136.30 pg/ml, respectively. The detection time was 2 min by TBH method for in vitro tissue detection and 6 min by FNA method for in situ tissue detection, both of which were faster than traditional intraoperative cryopathological examination (usually >30 min). Intraoperative application of ICGT method was associated with higher postoperative serum calcium and blood PTH levels at 1 and 3 months as well as a lower incidence of postoperative transient hypocalcemia, as compared with direct visual inspection. Conclusion: PTH-ICGT assay shows high potential as a rapid, novel alternative for intraoperative parathyroid identification.

Development of a colloidal gold-based lateral-flow immunoassay for the rapid simultaneous detection of clenbuterol and ractopamine in swine urine.

A multianalyte lateral-flow immunochromatographic technique using colloidal gold-labeled polyclonal antibodies was developed for the rapid simultaneous detection of clenbuterol and ractopamine. The assay procedure could be accomplished within 5 min, and the results of this qualitative one-step assay were evaluated visually according to whether test lines appeared or not. When applied to the swine urines, the detection limit and the half maximal inhibitory concentration (IC(50)) of the test strip under an optical density scanner were calculated to be 0.1 +/- 0.01 ng mL(-1) and 0.1 +/- 0.01 ng mL(-1), 0.56 +/- 0.08 ng mL(-1), and 0.71 +/- 0.06 ng mL(-1), respectively, the cut-off levels with the naked eye of 1 ng mL(-1) and 1 ng mL(-1) for clenbuterol and ractopamine were observed. Parallel analysis of swine urine samples with clenbuterol and ractopamine showed comparable results obtained from the multianalyte lateral-flow test strip and GC-MS. Therefore, the described multianalyte lateral-flow test strip can be used as a reliable, rapid, and cost-effective on-site screening technique for the simultaneous determination of clenbuterol and ractopamine residues in swine urine.

Mast cell-specific gangliosides and FcepsilonRI follow the same endocytic pathway from lipid rafts in RBL-2H3 cells.

Recent studies have shown that, in mast cells, membrane microdomains rich in cholesterol and glycosphingolipids called lipid rafts play an important role in FcepsilonRI signaling. The present study demonstrates that, in RBL-2H3 cells following stimulation, the mast cell-specific gangliosides associated with FcepsilonRI are internalized from lipid rafts along with the receptor. When the cells are labeled with iodinated antibodies against the gangliosides or against FcepsilonRI and the cell components are then fractionated on Percoll density gradients, in stimulated cells the gangliosides are internalized with the same kinetics as FcepsilonRI and at 3 hr are present in the dense lysosome fraction. Using transmission electron microscopy, with antibody against the gangliosides conjugated to horseradish peroxidase and antibody against FcepsilonRI conjugated to colloidal gold, it was possible to demonstrate that the gangliosides and FcepsilonRI are internalized in the same coated vesicles. At 5 min, the gangliosides and FcepsilonRI can be identified in early endosomes and at 3 hr are found together in acid phosphatase-positive lysosomes. This study demonstrates that the mast cell-specific gangliosides are internalized from lipid rafts in the same vesicles and traffic intracellularly with the same kinetics as FcepsilonRI. This study contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Metallographic in situ hybridization.

Metallographic methods, in which a target is visualized using a probe or antibody that deposits metal selectively at its binding site, offers many advantages for bright-field in situ hybridization (ISH) detection as well as for other labeling and detection methods. Autometallographically enhanced gold labeling procedures have demonstrated higher sensitivity than conventional enzyme chromogens. Enzyme metallography, a novel procedure in which an enzymatic probe is used to deposit metal directly from solution, has been used to develop bright-field ISH methods for HER2 gene determination in breast cancer and other biopsy specimens. It provides the highest level of sensitivity and resolution, both for visualizing endogenous gene copies in nonamplified tissues and for resolving multiple gene copies to allow copy enumeration in amplified tissues without the need for oil immersion or fluorescence optics. An automated enzyme metallography procedure, silver ISH, has been developed for use in slide-staining instruments. Metallographic staining also provides excellent results for immunohistochemistry and may be combined with other staining procedures for the simultaneous detection of more than one gene or combinations of genes and proteins.

Development of immunochromatography strip-test using nanocolloidal gold-antibody probe for the rapid detection of aflatoxin B1 in grain and feed samples.

An immunochromatography (ICG) strip test using a nanocolloidal gold-antibody probe was developed and optimized for the rapid detection of aflatoxin B1 (AFB1). A monoclonal antibody specific to AFB1 was produced from the cloned hybridoma cell (AF78), coupled with nanocolloidal gold, and distributed on the conjugate pad of the ICG strip test. The visual detection limit of the ICG strip test was 0.5 ng/ml, and this method showed a cross-reaction to aflatoxin B2, G1, and G2. In total, 172 grain and feed samples were collected and analyzed by both the ICG strip test and HPLC. The results of the ICG strip test showed a good agreement with those obtained by HPLC. These results indicated that the ICG strip test has a potential use as a rapid and cost-effective screening tool for the determination of AFB1 in real samples and could be applied to the preliminary screening of mycotoxin in food and agricultural products, generating results within 15 min without complicated steps.

A sensitive immunochromatographic assay using colloidal gold-antibody probe for rapid detection of fumonisin B1 in corn.

A sensitive immunochromatographic assay (ICA) using a colloidal gold-antibody probe for the rapid detection of fumonisin B1 (FB1) in corn samples was developed. The colour density of the test line correlated with the concentration of FB1 in the range 2-40 ng ml(-1) by the assay, and the detection limit for FB1 was 2 ng ml(-1). The linear range for FB1 was 50-1000 µg kg(-1), and the visual limit detection of the test was 1000 µg kg(-1) in corn samples. The ICA to detect FB1 is sensitive, specific and rapid. Specific anti-FB1 monoclonal antibody (mAb) and FB1-ovalbumin (FB1-OVA) conjugate antigen were prepared. FB1 mAb, labelled with colloidal gold, was used as the probe on the immunochromatographic strip. FB1-OVA and goat-anti-mouse IgG were coated onto a nitrocellulose (NC) membrane as test lines and control lines, respectively. FB1 in samples will competitively combines the FB1 mAb with the FB1-OVA in an NC membrane and the results are directly observed by the colour of the detection and quality control lines. The concentrations of FB1 mAb labelled with colloidal gold, detecting antigen and goat anti-mouse IgG, were optimised. The results indicate that the test strip is specific for FB1, with no cross-reactivity to other toxins. The strip assay for FB1 was simple, only needing one step without complicated assay performance and expensive equipment, and the total time for visual evaluation was less than 10 min. A survey of 24 corn samples from Hefei, China, was performed with the test strip and HPLC, and the detection results showed that the developed ICA and the HPLC were in excellent agreement. Hence, the developed ICA can be used as a method for rapid detection of FB1 in corn samples.

Preparation of Colloidal Gold Particles and Conjugation to Protein A/G/L, IgG, F(ab')2, and Streptavidin.

Colloidal gold probes, including protein A/G/L, IgG, F(ab')2, and streptavidin labeled with gold particles, are useful tools to localize antigens in cells and tissues by immunoelectron microscopy (IEM). This chapter describes different methods for the preparation of colloidal gold and conjugation of colloidal gold to protein A/G/L, IgG, and streptavidin.

Preparation of gold colloid monolayer by immunological identification.

The design and initial characterization of the self-assembled gold colloid monolayer by a sandwich structure via the immunological identification are reported. The 13 nm gold colloid nanoparticles and the silicon or quartz substrates have been modified with the mouse polyclonal antibody against hepatitis B virus surface antigen (PAb) and the mouse monoclonal antibody against hepatitis B virus surface antigen (MAb), respectively. They can be linked by a special reaction with their corresponding hepatitis B virus surface antigen (Antigen) as a sandwich structure. Thus, the density of gold nanoparticles self-assembled on the substrate can be readily controlled by the amount of the antigen added. The resulting substrates have been characterized by atomic force microscopy (AFM) and surface-enhanced Raman scattering (SERS) spectroscopy when the gold nanoparticles were modified with SERS-active probe molecules of 4-mercaptobenzoic acid (MBA) after silver enhancement. These data show that the gold nanoparticles are separately fixed onto the substrate and form a uniform monolayer, which possess a set of features that make them very attractive for both basic and applied uses, including roughness, high stability, and biocompatibility.

Development of ELISA and Colloidal Gold-PAb Conjugate-Based Immunochromatographic Assay for Detection of Abrin-a.

When abrin-a was combined with several polyclonal antibodies (PAb), the detection limit could be increased. In this way, a monoclonal antibody (capture) and polyclonal antibody (detection) sandwich enzyme-linked immunosorbent assay (ELISA) and a colloidal gold-PAb conjugate-based immunochromatographic assay for detection of abrin-a were developed. The ELISA had a detection limit of 3.9 ng/mL for abrin-a in standard solution and 7.8 ng/mL in soybean milk, and was more sensitive than polyclonal antibody (capture) and monoclonal antibody (detection) ELISA, which had a detection limit of 15.6 ng/mL. The test strip had a detection range of 50 to 500 ng/mL for abrin-a and a detection limit in standard solution or soybean milk samples of 50 ng/mL. However, the test strip had a reduced detection capability compared with a colloidal gold-monoclonal antibody conjugate-based immunochromatographic assay test strip, which had a lower detection limit of 10 ng/mL. The developed ELISAs and test strip show the specificity towards abrin-a and have no cross-reactivity towards abrin-b, -c, -d, ricin, or the agglutinins from either castor beans or rosary peas.

Development of colloidal gold immunochromatographic strips for detection of Riemerella anatipestifer.

Riemerella anatipestifer is one of the most important bacterial pathogen of ducks and causes a contagious septicemia. R. anatipestifer infection causes serositis syndromes similar to other bacterial infections in ducks, including infection by Escherichia coli, Salmonella enterica and Pasteurella multocida. Clinically differentiating R. anatipestifer infections from other bacterial pathogen infections is usually difficult. In this study, MAb 1G2F10, a monoclonal antibody against R. anatipestifer GroEL, was used to develop a colloidal gold immunochromatographic strip. Colloidal gold particles were prepared by chemical synthesis to an average diameter of 20 ± 5.26 nm by transmission electron microscope imaging. MAb 1G2F10 was conjugated to colloidal gold particles and the formation of antibody-colloidal gold conjugates was monitored by UV/Vis spectroscopy. Immunochromatographic strips were assembled in regular sequence through different accessories sticked on PVC plate. Strips specifically detected R. anatipestifer within 10 min, but did not detect E. coli, S. enterica and P. multocida. The detection limit for R. anatipestifer was 1 × 10(6) colony forming units, which was 500 times higher than a conventional agglutination test. Accuracy was 100% match to multiplex PCR. Assay stability and reproducibility were excellent after storage at 4°C for 6 months. The immunochromatographic strips prepared in this study offer a specific, sensitive, and rapid detection method for R. anatipestifer, which is of great importance for the prevention and control of R. anatipestifer infections.

[Development of colloidal gold immunochromatography assay strip for schistosomiasis diagnosis in domestic animals].

OBJECTIVE: To develop a quick and easy colloidal gold immunochromatography assay (GICA) strip for schistosomiasis diagnosis in domestic animals. METHODS: The reconstruction of Streptococcal Protein G (SPG) was designed and its gene was subcloned into plasmid pET-28a(+) to express in Escherichia coli. The recombinant SPG was purified and labeled with colloidal gold. The Schistosoma japonicum soluble egg antigen (SEA) and rSPG were blotted on the nitrocellulose membrane for the test line and control line respectively. The specificity, sensitivity and cross-reaction of the strip method were detected. RESULTS: The rSPG was successfully expressed and purified to label with colloidal gold. The colloidal gold immunochromatography assay strips were assembled and they could detect the sera of S. japonicum infected BALB/c mice, New Zealand white rabbits, buffalo and sheep successfully. Besides, the sensitivity of GICA strip was 100% in the sera of mice and the serum of rabbits with S. japonicum infection. The specificity was 100% in the serum of mice and the sera of rabbits with free of infection. The sensitivity was 100% in the sera of sheep with miracidia of S. japonicum hatching from the stool and the specificity was 88.46% in the sera of sheep without that. The sensitivity was 94.44% in the sera of buffalo with miracidia hatching from the stool and the specificity was 100% in the sera of buffalo without that. The cross-reaction rate was 5.88% in Paramphistomum. CONCLUSION: The GICA strip can successfully detect a variety of S. japonicum infected domestic animals and may be a useful tool for screening on a large scale in the endemic areas.

Colloidal gold conjugated monoclonal antibodies, studied in the BIAcore biosensor and in the Nycocard immunoassay format.

Interactions between immobilized capture monoclonal antibodies (mAbs), analyte molecules and colloidal gold conjugated second monoclonal antibodies have been investigated in the BIAcore biosensor and in the Nycocard immunoassay format. This report focuses on six monoclonal antibodies against human heart myoglobin, although, results with other antigens are also discussed. The BIAcore was used to screen monoclonal antibodies as antigen capture reagents, and for their function as colloidal gold conjugated second antibodies in the Nycocard. Some antibodies with low affinity caused by a rapid antigen dissociation rate, showed high affinity kinetics when used unlabelled or as gold conjugated detector reagents. One gold conjugated mAb with excellent properties in the Nycocard, showed double binding to one epitope, when tested in the BIAcore. The real time visualization of association and dissociation rates was a unique tool in the elucidation of antigen-antibody interactions. Our study confirmed that good antibody candidates selected with the BIAcore must always be tested in their actual conjugation situation before final optimization.

Expression of IGF-I, IGF-I receptor and IGF binding proteins-1, -2, -3, -4 and -5 in human atherectomy specimens.

The molecular and cellular processes that induce rapid atherosclerotic plaque progression in patients with unstable angina and initiate restenosis following coronary interventional procedures are uncertain. We examined primary (de novo) and restenotic lesions retrieved at the time of directional coronary atherectomy for expression of insulin-like-growth factor-I (IGF-I). IGF-I receptor, and five IGF binding proteins (IGFBPs), IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5 in smooth muscle cells (SMCs) using colloidal gold immunocytochemistry. IGF-1, its receptor and binding proteins were not detected in SMCs of normal coronary arteries. IGF-I localized primarily in synthetic smooth muscle cells (sSMCs) in both de novo and restenotic plaques. IGF-I receptor localized on sSMCs and their processes and colocalized with IGF-I. Although morphometric analysis of IGF-I and IGF-I receptor immunoreactivity in sSMCs of de novo and restenotic lesions showed comparable levels of IGF-I (3.2 +/- 1.0 and 2.9 +/- 0.9, respectively). IGF-I receptor was significantly higher in de novo lesions as compared to restenotic lesions (10.7 +/- 2.5 and 4.2 +/- 1.3, P < 0.05, respectively). IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4 and IGFBP-5 localized in the cytoplasm of sSMCs and in the extracellular matrix. Quantitative reverse transcription polymerase chain reaction (QRT-PCR) performed on de novo atherectomy specimens identified mRNA for IGF-I, IGF-I receptor, IGFBP-1, IGFBP-2, IGFBP-4, IGFBP-5 levels and detected mRNA for IGFBP-3. The expression of IGF-I, IGF-I receptor, and IGFBPs in atherectomy plaques suggests that the development of coronary obstructive lesions may be a result of changes in the IGF system.

Enhancement of the effectiveness of electroporation-augmented cutaneous DNA vaccination by a particulate adjuvant.

DNA vaccines are attracting increased attention due to multiple advantages over conventional vaccines. Attempts to improve these vaccines focus on enhancing DNA delivery and employing novel immunoadjuvants. Electroporation (EP) has emerged as an effective method for delivering DNA vaccines, significantly enhancing humoral and cellular responses. To further improve EP-augmented DNA vaccination, we used micron-size gold particles as a particulate adjuvant. DNA is not bound, or adsorbed, to the particles. Gold particles were coinjected intradermally with plasmid DNA encoding the hepatitis B virus surface antigen (HBsAg) into mice, both in the absence and presence of noninvasive EP. The particles enhanced the percentage of responding animals, and shortened the time for reaching maximal antibody titers by 2 weeks. Subtyping of the produced antibodies revealed a predominantly Th1-like response which did not change significantly with the absence or presence of particles. The particles likely function as an attractant for antigen-presenting cells (APCs), and probably do not affect EP or antigen expression to a significant extent. We conclude that micron-size gold particles injected intradermally together with DNA followed by EP give rise to an accelerated, potent immune response with a strong cellular component. This method may become important for the development of fast-acting therapeutic and prophylactic vaccines.

[The endocytic routes of exogenous antigen in murine dendritic cells and macrophages].

OBJECTIVE: To compare the endocytic routes of exogenous antigen in murine dendritic cells (DC) and macrophages. METHODS: Murine bone marrow-derived DC and peritoneal macrophages were pulsed with HRP-5 nm colloidal gold for 10 minutes and chased for 0-120 minutes in culture medium. Intracellular distribution of 5 nm colloidal gold was explored by means of cellular enzymatic-chemistry of acidic phosphatase and MHC II cellular immunochemistry under electron microscope. RESULTS: After 10 minutes pulse with HRP-5 nm colloidal gold and then 30 minutes of chase, most of HRP-5 nm colloidal gold internalized by DC entered into MHC II positive MIICs, but a little entered into acidic phosphatase-positive lysosomes. In contrast to DC, most of lysosomes of macrophages were accessed by HRP-5 nm colloidal gold, and a small portion of HRP-5 nm colloidal gold entered into MIICs. After 60 minutes of chase, 5 nm colloidal gold could hardly be seen within macrophages, whereas most of 5 nm colloidal gold retained in DC. CONCLUSION: The endocytic route of exogenous antigen in DC apparently differs from that in macrophages. Most of antigens taken by macrophages enter into lysosomes within 30 minutes. In the case of DC, most of internalized antigens enter into MIICs, which may be related to their unique antigen-presenting function. In addition, macrophages seem to have more powerful capacity of scavenging exogenous antigen than DC.

[Use of colloid gold conjugates for identification of actins of various origin]. / Ispol'zovanie kon''ugatov kolloidnogo zolota dlia identifikatsii aktinov razlichnogo proiskhozhdeniia.

Some optimized methods of purifying actin from animal, plant, and bacterial cells were developed. Variants of the preparation of antiactin antibodies are described which use both traditional methods of immunization and phase display technology and antigen adsorption on colloidal gold particles. The conjugates of colloidal gold with phalloidin and heavy meromyosin as well as with antibodies were proposed. It was shown that these markers make it possible to reliably identify actins of different origin by the methods of light, and electron microscopy and dot-analysis.

[Use of specific antibodies, labeled with colloidal gold particles, for the detection of Brucella antigens using dot-immunoassay]. / Ispol'zovanie spetsificheskikh antitel, mechennykh chastitsami kolloidnogo zolota, dlia obnaruzheniia antigenov brutsell metodom dot-immunoanaliza.

The method of dot immunoassay with the use specific antigens, labeled with colloidal gold particles, for the detection of brucellar antigens was developed and tested under laboratory and field conditions. In this work soluble antigens isolated from different Brucella species and corpuscular antigens (13 strains belonging to 7 species of the genus Brucella, most pathogenic for humans and animals in the S- and R-forms) were used. The method was tested in the study of pathological material obtained from sick animals and humans in a farm with unfavorable situation for brucellosis in the Irkutsk Region. The sensitivity of the proposed assay system was found to be high and constituted 10 pg/ml to 1 ng/ml for soluble brucellar antigens and 200 CFU/ml to 13.5 x 10(6) CFU/ml for corpuscular antigens of Brucella S- and R-forms. The specificity of the method was tested with the use of 10 heterologous microorganisms. False positive results were observed only with Yersinia enterocolitica 0:9 at a concentration of 1 x 10(6) CFU/ml due to similarity of their polysaccharide-containing surface antigens. The newly developed dot immunoassay is simple for use, rapid and does not require expensive reagents and equipment.