Generation of diagnostic anti-teliospore antibodies for development of specific immunodiagnostic format for detection of Karnal bunt (Tilletia indica) of wheat

Rabbit antisera were generated against four preparations of teliospores of Tilletia indica the fungus responsible for Karnal bunt in wheat having titre ranging from 1:2500 to 1:10,000. The suitability of four solubilising agents (I–IV) used for extraction of teliospores/spore proteins was assessed, highest protein extractability was observed with solubilising agent I (0.5% sodium dodecyl sulphate). The maximum immunoreactivity in enzyme linked immunosorbent assay (ELISA) was observed with the teliosporic protein(s) extracted using solubilising agent I followed by agent II, IV and III. Further, the results of western blotting with proteins from different fungal pathogens using anti-intact teliospores antibody showed one unique immunoreactive band of 28 kDa with teliosporic protein of T. indica only. The potential diagnostic antibody was generated against the characterised protein (28 kDa) of teliospore wall's antigen of T. indica. The developed diagnostic antibody and solubilising agent I could therefore be employed for development of specific and rapid immunodiagnostic format for detection of Karnal bunt.     

Ultrasound enhanced detection of individual meningococcal serogroups by latex immunoassay

AIMS: To examine A, C, Y, and W135 Neisseria meningitidis serogroup characterisation by ultrasonic standing wave enhanced latex agglutination tests (USELATs) of clinical samples. In addition, to determine USELAT enhancement of detection sensitivity for the individual antigens compared with conventional card latex agglutination tests (LATs). METHODS: Wellcogen (Abbott Murex), Slidex meningite kit 5 (bioMerieux), and Pastorex (Sanofi) kits and beads coated in house with antibodies to Y and to W135 alone were tested. Positive control antigens consisted of A and C polysaccharide preparations and the Pastorex Y/W135 kit sample. The limiting concentrations of antigen detection were determined by USELAT and by LAT. Thirty five clinical samples (plasma), previously characterised by the polymerase chain reaction (PCR) and culture, were tested by USELAT and, when sample volume allowed, by LAT. RESULTS: USELAT enhancement of control antigen detection ranged from 16 to 128 fold for the different latex systems. Enhancements for the different control antigens were comparable between kits. USELAT tests of clinical (A/C/Y/W135) samples (n = 15) with the Wellcogen (A/C/Y/W135) and Slidex meningite (A/C/Y/W135) kits showed comparable specificities. A set (n = 22) of Y and W135 samples gave 18, 19, and 17 positive results for Wellcogen (A/C/Y/W135), Pastorex (A/C/Y/W135), and in house beads (Y/W135), respectively. Positive USELAT PCR and culture results were concordant. A typical sensitivity for the commercial kits was 80% (Wellcogen). CONCLUSIONS: USELAT identified serogroups for 80% of samples, whereas LATs identified only 40%. The USELAT detection of the A, C, Y, and W135 antigen serogroups showed comparable enhancement for the kits tested. The commercial availability of latex beads coated with antibody to the Y and W135 serogroups would expedite their identification.     

A one-step two-particle latex immunoassay for the detection of Salmonella typhi endotoxin

A simple and rapid test (LIMM, short for latex immunoassay) is described for detecting Salmonella typhi endotoxin. It involves the simultaneous binding of the antigen by two types of reagent particles contained in a micro-tube: an indicator latex particle coated with a monoclonal antibody specific for the O-9 determinant on the endotoxin, and a magnetic bead coated with another monoclonal antibody specific for a different O-determinant. At the end of-the test, the magnetic beads are sedimented by use of a magnet, and the result is read based on the turbidity of the indicator latex suspension. Compared to a similar assay developed previously which uses only a single particle reagent (i.e., a tube agglutination system), LIMM was found to be slightly more sensitive especially when using short (less than 30 min) incubation times, and was at all times easier to read. The sensitivity of LIMM, in fact, increased with increasing time of incubation. When compared to the sensitivity (25 ng/ml) of a conventional slide latex agglutination test performed using the same indicator latex reagent, this parameter was 0-, 4.9-, 12.5- and 28.7-fold better after 5, 15, 30 and 60 min of incubation in the LIMM.     

Nylon bead enzyme-linked immunosorbent assay for detection of sub-picogram quantities of Brucella antigens.

An indirect sandwich enzyme-linked immunosorbent assay, using antibody covalently coupled to nylon beads, has been adapted for the detection of Brucella antigens. Optimum conditions were achieved by incubation of 1 ml of reaction mixture with a single bead, and by minimizing nonspecific interactions through the use of beads coated with purified bovine antibodies, preabsorption of third layer rabbit antibodies with normal bovine serum, and treatment of beads with normal goat serum before addition of the goat anti-rabbit enzyme conjugate. Beta-galactosidase was selected for use with clinical samples primarily because of low levels of endogenous enzyme in bovine leukocytes. Use of a fluorogenic substrate enhanced sensitivity 20-fold. Under these conditions, 100 fg of solubilized crude lipopolysaccharide or 8 to 10 Brucella cells was detectable in a fixed volume of 1 ml. A system was also devised for concentrating antigen which permitted ready detection of 2 pg of lipopolysaccharide in a volume of 50 ml (40 fg/ml). Attempts to detect lipopolysaccharide in the presence of concentrated serum or plasma were unsuccessful, but 10 brucellae added to a suspension of leukocytes from 100 ml of normal bovine blood were easily measured.     

Latex immunoassay for rapid detection of rotavirus.

A latex agglutination (LA) test was evaluated for the detection of human rotaviruses in stool specimens. Both antiserum and immunoglobulin G (IgG)-sensitized latex particles were used, with IgG-coated beads being more sensitive for human rotavirus antigen detection. Latex beads sensitized with anti-simian-SA-11 IgG were stable for at least 8 months when stored at 4 degrees C. The sensitivity of the test was compared with that of the Rotazyme (Abbott Laboratories, Diagnostics Div., North Chicago, Ill.) test. The least number of particles detected was 9.0 X 10(5) particles by the LA test versus 4.5 X 10(5) particles by the Rotazyme test. When 10 stool specimens were serially diluted for antigen endpoint determinations, the geometric mean titer by the LA test was 592 versus 1,280 by the Rotazyme test. Forty-three stool samples positive by the Rotazyme test were all positive by the LA test, and no false negative results were detected. Unconfirmed false positive reactions ranged between 8 and 24%. The LA test for rotavirus antigen detection is direct, easy to perform, sensitive, quick, and may have application for use in diagnostic laboratories, emergency rooms, and physician's offices.     

In vitro study of the phagocytic processes in splenic granulocytes of the tench (Tinca tinca, L.).

The different stages of the phagocytic process by splenic granulocytes of Tinca tinca were studied. Adherence capacity to both endothelium and tissue substrate, mobility rate, the phagocytosis capacity for both cells (Candida albicans) and inert particles (latex beads), candidicide power, and capacity of digestion measured by nitroblue tetrazolium (NBT) reduction were evaluated in splenic granulocytes of healthy adult tench. The capacity of adherence to nylon fiber was possessed by 51% of the granulocytes. The percentage capable of adherence to smooth plastic surfaces rose with incubation time. Casein, an effective chemoattractant, increased the random mobility of the granulocytes. Phagocytosis was greater for opsonized C. albicans than for nonopsonized. However, the number of phagocytosed yeast cells destroyed by the granulocytes did not depend on whether or not the C. albicans had been previously opsonized. The phagocytosis indices and the percent phagocytosis of latex beads were greater than those obtained for the phagocytosis of C. albicans in the absence of serum. Finally, the metabolic activity in these cells following the digestion of ingested material showed a 148 ± 31% stimulation. The results show that splenic cells of tench have the capacity to make a phagocytic response against both cells (C. albicans) and inert particles (latex beads).     

Preparation of urine samples for use in commercial latex agglutination tests for bacterial antigens.

The use of latex agglutination (LA) tests for bacterial antigen detection in urine specimens is hindered by troublesome reactions such as nonspecific agglutination. Therefore, procedures such as boiling or membrane filtration of urine specimens are often used before LA testing. We discovered that the composition of the membrane filter used in filtration has a marked effect on the performance of an LA test used for detection of Haemophilus influenzae type b antigen. False-positive LA reactivity was common in urine specimens from pediatric patients that were processed by membrane filtration through certain filters; furthermore, such reactivity also occurred in LA tests for antigens other than those of H. influenzae. A protein present in urine at low concentrations appeared to be responsible for these phenomena.     

Application of Cloth-Based Enzyme Immunoassay for the Characterization of Monoclonal Antibodies to Salmonella Lipopolysaccharide Antigens

The specificity, detection limit, and stability of twelve anti-Salmonella monoclonal antibodies (Mabs) were evaluated by cloth-based enzyme immunoassay (CEIA) and polymyxin-cloth based enzyme immunoassay (p-CEIA). Using the p-CEIA, five Mabs were found to react with cholate extracted lipopolysaccharide (IPS) antigens of all 44 Salmonella strains representing 19 different serogroups examined, with the exception of the one strain of serogroup-O tested. These five Mabs did not react with cholate extracts of any of 16 Gram-positive or Gram-negative non-Salmonella bacteria tested. The detection limit of purified S. typhimurium LPS antigen in the p-CEIA was approximately 40 ng for four of the Mabs and approximately 20 ng for the other Mab. Four of the five Mabs were stable during storage at 22°C-23°C for 24 h. These four Mabs are potentially useful for the immunodetection of Salmonella in foods and other samples.     

A two-particle turbidometric latex immunoassay for the detection of specific IgM antibodies

A simple two stage assay using latex particles as a reaction indicator has been developed for the detection of IgM antibodies to Trichinella spiralis. In the first stage, magnetic polystyrene beads (Dynabeads) coated with T. spiralis antigen were incubated for 30 min with the test serum. After washing, in the second stage, the assay was developed for 1 h using anti-mu-coated latex particles. After sedimentation of the Dynabeads the turbidity of the resultant latex suspension was measured spectrophotometrically at a wavelength of 400 nm. A decrease in turbidity of more than 20% from that of the control, unreacted, suspension was considered positive. Using an IgM phosphorylcholine-binding monoclonal antibody which was reactive with T. spiralis, the sensitivity of the assay was determined to be 110 ng/ml of antibody. This was 20-fold less than the sensitivity achieved in an indirect enzyme-linked immunosorbent assay (ELISA). When the assay was applied to sera obtained from CBA/N or BALB/c mice, which were either normal or immunized against T. spiralis, the expected results were obtained with titers up to 1/640 observed, and confirmed (r = 0.93, P less than 0.001) in the ELISA.     

Latex agglutination test based on single-chain Fv recombinant antibody fragment

Recombinant antibodies have been proposed as invaluable tools for various therapeutic and diagnostic purposes. Here, we describe the development of a novel latex agglutination test (LAT) using single-chain Fv recombinant antibody fragment for the detection of K99(+) enterotoxigenic Escherichia coli strains. For the production of a single-chain Fv antibody fragment (scFv) against the major colonization factor (FanC) of K99 antigen, the scFv gene was integrated into a bacterial expression vector under the control of T7 promoter. After high-level expression of soluble scFv (approximately 50 mg/l) in flask cultivation of E. coli DE3 and purification, scFv was immobilized on different latex particles, and then, these sensitized beads were used in LAT. Results obtained with our latex reagents revealed that the recombinant antibody-coated particles were able to give a good agglutination signal with purified antigen, intact cells displaying this protein and clinical specimens. The strength of agglutination of scFv-coated beads for antigen was comparable to that of polyclonal anti-K99-coated particles. However, the assay proved to be simple and rapid, similar to conventional LATs, and owing to more convenient and economical production of recombinant antibodies, they can be considered as a useful reagent for replacing monoclonal antibodies in LATs.     

Immunological detection of mycobacterial antigens in infected fluids, cells and tissues by latex agglutination. Animal model and clinical application

We devised an immunoassay for the detection of mycobacterial antigens in cell lysates and in tissue extracts which is based on the agglutination of latex particles coated with anti-Mycobacterium bovis F(ab')2, followed by counting of non-agglutinated particles. Mycobacterium bovis cell lysates were tested and a reference curve was established, having a lower limit of detection of 15-20 Mycobacteria. We were able to detect mycobacterial antigens in cell lysates from bronchoalveolar washings and in spleen and liver lysates obtained from experimentally infected rabbits. Antigens were also detected in ten out of 11 samples obtained from patients with proven tuberculous infection. These samples were readily distinguished from 32 negative control samples after pepsin treatment. In contrast, periodate treatment of samples to destroy carbohydrate, abolished all reactivity. Following gel filtration chromatography we identified three peaks with antigenic properties in samples of all types. The detection of mycobacterial carbohydrate antigens by latex agglutination and particle counting should be a useful adjunct in the diagnosis of tuberculosis.     

Commercial latex agglutination test for rapid diagnosis of group B streptococcal infection in infants.

Although latex agglutination assays for detection of a variety of bacterial antigens in body fluids from patients with systemic infection have been shown to be useful as rapid diagnostic techniques, lack of commercial availability has restricted their application. The Streptex latex test kit for the detection of group B streptococcal (GBS) antigen in admission body fluid specimens was evaluated for sensitivity and specificity in 54 infants with meningitis and in 10 infants with normal cerebrospinal fluid (CSF) parameters. GBS antigen was detected in 22 of 28 (78.6%) CSF specimens by latex agglutination and in 23 of 28 (82.1%) by countercurrent immunoelectrophoresis. Antigen was present in 21 of 28 (latex agglutination) and 19 of 26 (countercurrent immunoelectrophoresis) CSF specimens after the initiation of antimicrobial therapy. Heat-labile factors accounted for nonspecific agglutination reactions with latex suspensions other than group B in 3 of 28 CSF samples from patients with GBS meningitis. These nonspecific reactions were readily eliminated by heating specimens for 10 min at 100 degrees C. Fifteen patients with GBS meningitis had admission serum and urine samples collected in addition to CSF. Antigen was detected by latex agglutination and countercurrent immunoelelectrophoresis in 14 of 15 (93.3%) and 13 of 15 (86.7%) concentrated urine specimens, respectively, and in 12 of 15 (80%) CSF specimens and 4 of 15 (27%) sera by each method. These findings indicate that the Streptex latex test is a rapid, sensitive, and readily available method for detection of GBS antigen in admission body fluid specimens from infants with meningitis.     

Development of a double monoclonal antibody sandwich ELISA test for Verticillium dahliae Kleb.

Verticillium dahliae causes wilt diseases in a wide range of horticultural and field crops in many parts of Turkey. In the Aegean region it has been a serious problem in cotton and vegetables for many years. More recently, it has become a major problem for olive growers in relatively newly established orchards. The fact that the only possible control methods of Verticillium wilt disease is the use of healthy and pathogen free propagating material has directed us to produce monoclonal antibodies against V. dahliae in order to apply rapid, sensitive and reliable serological detection methods. Verticillium spp. isolates were obtained from olive plantations, as well as from cotton and tomato fields in the Aegean and Marmara Regions. All suspected isolates were obtained as V. dahliae after determining microscopic morphological characteristics and pathogenicity tests on cotton seedlings. Immunizing antigens were prepared by three different methods including surface washing system, czapek dox agar and gel filtration methods. BALB/c mice were immunized with each antigenic form. Lymph node, spleen and bone marrow cells were used as sources of B-lymphocytes and 8D2 (IgM) and 7D6 (IgG1) were obtained from the spleen and lymph node fusion. The monoclonal antibodies were purified and immunoglobulin types were identified. 8D2 monoclonal antibody gave positive reaction with the V.dahliae isolates from olive, cotton, tomato and watermelon; however, it didn't give any cross reactivity with other epiphytic fungi. 7D6 antibody displayed cross-reactions with a few fungi. The monoclonal antibody (8D2) was conjugated with horseradish peroxidase (HRP). These monoclonal antibodies were characterized for use in the development of diagnostic kits based on double-monoclonal antibody sandwich ELISA test system for detecting V. dahliae in Turkish isolates. In this test, the first antibody was used as capture antibody and the second one was used for detection of antigens.     

The use of a measles latex reagent for the determination of measles antibodies and in a specific test for multiple sclerosis

Measles virus antigens covalently linked to latex spheres were used for measuring measles-specific antibodies in a direct agglutination test either in microtitre plates or as a rapid slide-agglutination test. The titres were compared to that obtained by conventional assays. The measles-latex spheres were also used as the antigen for a radio-immuno assay. By incorporating a¹⁴C-radioactive marker into the measles-latex spheres their interaction with lymphocytes from multiple sclerosis and control patients was determined. Lymphocytes from multiple sclerosis patients reacted with a higher percentage of beads at low bead/lymphocyte ratios compared with controls, whereas the reverse was found when the ratio of beads was increased.     

Evaluation of a latex test for rapid detection of pneumococcal antigens in sputum

A latex agglutination test (Directigen) for detection of pneumococcal capsular polysaccharide antigens in sputum was evaluated in comparison to culture and Gram stain using 121 sputum specimens. The sensitivity, specificity and rate of agreement of the latex test compared to culture were 90 %, 79 % and 81 %, respectively. The results suggest that the latex test may be useful for detecting pneumococcal antigen in sputum.     

Evaluation of the interferon-γ ELISPOT-assay for quantification of peptide specific T lymphocytes from peripheral blood

ELISPOT assays for detection of antigen specific HLA class I restricted T lymphocytes have recently been developed. Reliable quantitation of T cell frequencies is crucial for the monitoring of specific cancer vaccines. We have evaluated the accuracy of quantitative results obtained with the IFN-γ ELISPOT assay and both sensitivity and specificity obtained with several pairs of antibodies was compared. The detection system was tested with IFN-γ coupled latex beads and the quantitation of influenza specific T cells with several sets of dilution experiments. The reproducibility of the quantitative results was established. Only one pair of monoclonal antibodies had an acceptable specificity. In a serial dilution, almost 100% of IFN-γ-coated beads could be detected. Furthermore, the number of influenza-peptide specific spots correlated closely with (a) the number of antigen specific T cells derived from an influenza-peptide specific T cell line diluted in PBMC, and (b) the number of CD8+ T cells diluted in autologous CD8-depleted PBMC. In three healthy individuals and three cohorts of healthy volunteers, the number of influenza-peptide specific spots was highly reproducible. The data presented here demonstrate that the frequency of peptide specific CD8+ T cells can be reliably determined from peripheral blood with the IFN-γ ELISPOT assay.     

Detection of toxoplasma IgM antibody by passive latex agglutination reaction

Three major components (designated Sp-1, -2 and -3) of the microsomal pellet of Toxoplasma gondii (Tp) were isolated by Ultragel AcA 44 gel filtration chromatography from the microsomal pellet solubilized with detergents. Of these, Sp-2 proved to be most reactive with anti-Tp antibodies and its reactivity with IgM and IgG antibodies varied with the concentration at which it was used for sensitizing latex particles. Sp-2 antigen reacted with IgM antibody alone when latex particles were sensitized with less than or equal to 100 micrograms of this antigen/mg of particles, and its reactivity with IgG antibody appeared and increased progressively with increasing sensitizing concentrations of this antigen. Based on this finding, a method of direct measurement of anti-Tp IgM antibody in serum by passive latex agglutination has been developed. Polyacrylamide disc gel electrophoretic analysis of Sp-2 antigen in the presence of SDS revealed four constituents of 43, 35, 28 and 14 kDa. All these components reached with both IgM and IgG when tested by immunoblotting.     

鼠抗丙型肝炎病毒ns－5区单克隆抗体的研究

用ＨＣＶ非结构区ｎｓ－５合成多肽抗原交联后免疫小鼠，成功地建立了３株抗ｎｓ－５单克隆抗体（ＭｃＡｂ），经检测这３株ＭｃＡｂ属于同一位点，与其它区域无明显交叉反应，具有高度特异性。ＭｃＡｂ的研制为检测ｎｓ－５抗原奠定了一定基础。     

Immunoglobulin E reactivity to latex antigens in the sera of patients from Finland and the United States

Background: Patients with latex sensitivity and latex antigens from the United States and Finland, two countries where allergic reactions to latex have been widely reported, were evaluated to determine the spectrum of immune responses.Methods: Sera from 27 patients from Finland and 18 from the United States with latex allergy and control sera from nonsensitive individuals were studied for latex-specific IgE antibodies. Four antigen preparations were used: two extracted from gloves and one each extracted from rubber tree sap from Malaysia and India. All 45 patients had skin prick test results that were positive to latex antigens, and all sera were evaluated by enzyme-linked immunosorbent assay (ELISA) with the various antigens.Results: There were considerable differences in the reactivity of patient sera with the different antigens. Only 50% of the sera from patients with latex allergy from Finland demonstrated significant levels of IgE to latex as determined by enzyme-linked immunosorbent assay. These patients showed more reactivity with rubber tree sap antigens than with glove antigens. However, 72% of the patients from the United States demonstrated antibodies to latex, and no marked differences were noted between the antigen extracts.Conclusions: The results indicate that reagents such as rubber tree sap, which contain multiple clinically significant antigenic components, should be included in evaluation of latex allergy and that differences in patient populations may result in serologic variances.     

Unbalanced regulation of the expression of HLA-A and -B antigens in metastatic melanoma cells compared to autologous PBMC: A quantitative analysis of fourteen patients.

The quantitative cell surface expression of the gene products of HLA-A and -B loci is genetically predetermined (following Mendelian inheritance) in a given individual; in addition, the regulation of their expression is tightly coordinated since the ratio of HLA-A and -B antigens expression is constant on different cell types and the expression of HLA-B antigens is lower than that of HLA-A antigens. In view of these considerations and of the potential relevance of the quantitative expression of the gene products of HLA-A and -B loci in antigen presentation and for T cell-based specific immunotherapy, levels of cell surface HLA-A (mAb A131) and -B (mAb YTH) antigens were investigated by flow cytometry on fourteen primary cultures of melanoma cells (analyzed between in vitro passages 5 to 10) derived from unrelated melanoma patients and compared to those obtained with autologous PBMC. All melanoma cells and PBMC investigated were stained by mAb A131 and YTH (samples were considered positive when the mean value of fluorescence intensity with specific mAb was at least double than negative control mAb). The mean values of mean fluorescence intensity obtained for HLA-A and HLA-B antigens were 275±247 and 35±30 on melanoma cells and 1520±490 and 780±340 on PBMC and were both significantly different in a paired test between melanoma cells and PBMC (HLA-A, P=2×10⁻⁶; HLA-B, P=1×10⁻⁶); thus, the expression of HLA-A and -B antigens was 5.5 and 22.1 times lower on melanoma cells than on autologous PBMC. Simple linear regression analysis showed a high correlation (r=0.9; P=1×10⁻⁵) between the mean values of fluorescence intensity observed for HLA-A and -B antigens in PBMC; in contrast, a low correlation (r=0.6; P=1×10⁻²) was found in melanoma cells. Therefore, we calculated the ratio between the mean values of mean fluorescence intensity obtained for HLA-A and -B antigens in melanoma cells and autologous PBMC. The ratio HLA-A vs HLA-B was 10.9±8.0 (range: 2.1 to 32.6) and 2.1±0.7 (range: 1.28 to 3.58) in melanoma cells and PBMC, respectively (p=1×10⁻³, paired t test). Results similar to those obtained with mAb YTH were also obtained with the anti-HLA-B antigens mAb Q6/64 and H2-89-1. Our data, altogether, strongly suggest the existance of an alteration involving the coordinated regulation of the expression of HLA-A and HLA-B loci in melanoma cells.