Dipstick ELISA Research Articles

Embryo select assay: a non-invasive, dipstick elisa strip assay to identify the most competent embryo from the cohort

Embryo select assay: a non-invasive, dipstick elisa strip assay to identify the most competent embryo from the cohort

Kinetics ofFasciola giganticaAntibody Response in Naturally Infected Bovine Ensuing Levamisole and Oxyclozanide Combination Therapy

A study was conducted to determine the kinetics of Fasciolagigantica infection induced antibody response against oxyclozanide-levamisole in naturally infected bovine of South Gujarat, India. Faecal analysis on before, 1st, 4th, 8th, 11th and 37th days post treatment (DPT) recorded 67.14, 34.29, 5.71, 0 and 0% parasites infected bovines. Either parasite(s) positive indigenous cattle or buffalo become parasite(s) free by 8th DPT but a lag period of 4 days had been recorded in the exotic cattle. The trematode parasites infected animals take more time to clear the infection with the used drugs, it's become 100% negative on 11th DPT while the nematodes parasites infected animals successfully clear the infection by 4th DPT and maintain it until the end of experiment. The 5 animals which showed reactivity in dipstick ELISA test following natural infection with F. gigantica using parasites specific diagnostic antigen, cathepsin L cysteine proteinase (CP), was subjected to indirect plate ELISA assay, the mean anti-CP antibody titer was recorded at its maximum at pretreatment in treated group positive animal when probed with CP antigen. Thereafter, a declining trend of antibody response was recorded in these naturally infected bovines which reached to undetectable level on 90th DPT in response to anthelmintic therapy while untreated animals (negative and positive animals) showed almost similar pattern of antibody response during the experiment. A significant decrease in % eosinophilia count was also observed at 37th DPT in response to treatment.

An aptamer-based dipstick assay for the rapid and simple detection of aflatoxin B1

A rapid and simple dipstick assay based on an aptamer has been developed for the determination of aflatoxin B1 (AFB1). The dipstick assay format was based on a competitive reaction of the biotin-modified aptamer specific to AFB1 between target and cy5-modified DNA probes. Streptavidin and anti-cy5 antibody as capture reagents were immobilized at test and control lines on a membrane of the dipstick assay. After optimization, the limit of detection for the dipstick assay was 0.1 ng/ml AFB1 in buffer. The method was confirmed to be specific to AFB1, and the entire process of the assay can be completed within 30 min. Aqueous methanol (20%) provided a good extraction efficiency, and the matrix influence from corn extracts was successfully reduced through 2-fold dilution. The results of AFB1 analysis for corn samples spiked with known concentration of AFB1 by the dipstick assay and ELISA showed good agreement. The cut-off value of the dipstick assay for corn samples was 0.3 ng/g AFB1. Therefore, the dipstick assay is first reported and considered as a rapid, simple, on-site and inexpensive screening tool for AFB1 determination in grains as well as a corn.

Seromonitoring of Newcastle Disease in Backyard Poultry by Indigenously Developed User-friendly tool: Dipstick ELISA

Seromonitoring of Newcastle Disease in Backyard Poultry by Indigenously Developed User-friendly tool: Dipstick ELISA

Evaluation of dipstick–ELISA using 28 kDa Fasciola gigantica cathepsin l cysteine proteinase (FgCL3) for serodiagnosis of fasciolosis in naturally infected goats

In the present investigation 100 goats presented for slaughter were used to evaluate the 28kDa cathepsin l cysteine proteinase (FgCL3) dipstick–ELISA for the diagnosis of fasciolosis. Presence of Fasciola gigantica worms in liver at the time of slaughter was taken as a gold standard for the evaluation of the assay. Faeces, blood and liver were taken from all slaughtered goats. Biochemical parameters and coprological examinations using sedimentation technique were also taken into consideration. Total serum proteins, albumin, mean values of aspartate aminotransferase and alkaline phosphatase of positive group were not significantly different from those of the negative group. The diagnostic sensitivity of dipstick–ELISA was higher than coprological detection of fluke eggs. However, specificity was equal for both the tests. The accuracy of dipstick–ELISA in detecting circulatory antibodies during the course of disease was higher than coproscopic examination. In conclusion, the FgCL3 antigen dependent dipstick–ELISA was more reliable and has better scope for field application in the endemic areas.

Dipstick Format of an Improved ELISA for On-Site Atrazine Monitoring in Water in Pakistan

A dipstick format was developed for on-site atrazine monitoring in water samples of different origins. It was derived from an in-house-developed ELISA based on polyclonal antibodies that also cross-react with hydroxyatrazine (30%) and terbuthylazine (17%). Test reagents were evaluated for temperature and pH stabilities and rapidity for field applications. Reagents performed well within a temperature range of 20-30 degrees C and were tolerant to alkaline pH (up to 8.5) of the assay buffering system. Tracer incubation time could be reduced to 40 min. Bovine serum albumin addition (1%) in the assay buffer improved assay performance, giving 50% B/B0 (IC50) of 65 ng/L and the lowest LOD of 2 ng/L at 90% B/B0 (IC10). The dipstick ELISA format was standardized on a membrane support. Nylon membrane, positively charged, was superior to PVDF for qualitative or semiquantitative analysis regarding color intensity and stability. Tracer incubation time was further reduced to 30 min with a lowest LOD of 0.1 microg/L. For real sample screening with dipsticks, acceptable results were obtained for water. Significant correlation was found between dipstick and plate ELISA results. Validation using GC with a nitrogen-phosphorus detector and HPLC indicated that dipstick signals in aged water samples, which were mainly due to hydroxyatrazine, were significantly above European Commission regulations of 0.1 microg/L. However, dipsticks were superior, fast, and cost-effective.

Production and Characterization of Monoclonal Antibody Specific to Recombinant Dengue Multi-Epitope Protein

Monoclonal antibodies against novel dengue recombinant protein were produced following immunization of Balb/c mice with recombinant dengue multi-epitope protein (r-DMEP) expressed in Escherichia coli vector and purified in a single-step chromatography system. Antigenicity of r-DMEP was evaluated by dot enzyme immunoassay. Mice were immunized intraperitoneally with five doses each of 100 microg of this novel antigen at 1-week intervals and a final intravenous booster dose prior to the fusion. Hybridomas resulted from fusion of myeloma cells and splenocytes using PEG-1500 as an additive. Selection of the hybrids was done using HAT medium, and the hybrids thus selected were finally screened qualitatively and quantitatively by dot and plate immunoassays, respectively. Five antibody secretory hybrid clones exhibited specific reactivity against r-DMEP by dot-ELISA, whereas a lone clone was found to be cross-reactive with Japanese encephalitis virus (JEV). Monoclonal antibodies (MAbs) specific to r-DME protein recognized the envelope and non-structural epitopes by Western blot analysis. These MAbs were further checked for their diagnostic efficacy using dengue suspected clinical samples and found overall sensitivity and specificity for DRDE dipstick ELISA. MAb-based dipstick ELISA results were 85%, 75% and 85%, 90%, respectively.

Simultaneous direct detection of toxigenic and non-toxigenic Vibrio cholerae from rectal swabs and environmental samples by sandwich ELISA

A mAb-based simple, specific and rapid two-tip dipstick ELISA was developed for simultaneous detection of toxin- and non-toxin-producing strains of Vibrio cholerae, and for direct detection of V. cholerae from rectal swabs of patients and from environmental water samples. Rabbit polyclonal antibodies and murine mAbs were raised against recombinant protein (r-protein) antigens of cholera toxin B (CtxB) and outer membrane protein W (OmpW). Rabbit polyclonal antibodies to both r-proteins were coated individually onto the tips of nitrocellulose (NC) membranes of a two-tipped NC dipstick as capture antibodies and a mixture of two mAbs was used for the detecting antibodies. The test was found to be specific for V. cholerae strains O1, O139, non-O1 and non-O139, and did not show any cross-reaction to closely related bacterial strains. The test was evaluated on rectal swabs collected at the bedside of 75 hospitalized diarrhoeal patients and on 50 environmental water samples after enrichment for 4 h in alkaline peptone water. The mAb two-tip dipstick ELISA detected V. cholerae in 52/75 rectal swabs and 2/50 environmental water samples for CtxB antigen, and in 1/50 environmental water samples for the non-toxin OmpW antigen of V. cholerae within 1.5 h. These findings were identical to those observed using PCR and conventional culture methods. Thus, this mAb-based two-tip dipstick ELISA could be used for early and reliable simultaneous detection of toxigenic and non-toxigenic strains of V. cholerae from clinical and environmental water samples.

Recombinant antigen-based dipstick elisa for the diagnosis of leptospirosis in dogs

A recombinant LipL32 antigen-based dipstick elisa was developed as a screening test for the detection of leptospiral antibodies in serum samples from dogs. The antibodies were detected by a change...

Production, purification and characterization of recombinant dengue multiepitope protein

Dengue is an acute mosquito-borne viral disease of humankind. Dengue fever, dengue haemorrhagic fever and dengue shock syndrome have become global public health problems in recent years. rDME-G (recombinant dengue multiepitope protein that can specifically detect IgG) was produced in a 5-litre fermenter in Escherichia coli for use in diagnosis. The culture was induced with 1 mM isopropyl beta-D-thiogalactoside and cells were further grown for 4 h before harvesting. After fermentation, dry cell weight resulted in approx. 16.2 g/l. The rDME-G protein was purified from inclusion bodies using affinity chromatography. The final yield of purified rDME-G protein from fermentation resulted in approx. 168 mg/l of pure biologically active rDME-G protein. The purity of rDME-G protein was checked by SDS/PAGE analysis and the reactivity of this protein was further determined by Western blotting. The purified protein was used to develop an in-house dipstick ELISA and tested using a panel of 60 patient sera characterized using the commercially available tests for detection of dengue antibody. We compared our results with IgG-capture ELISA (Pan-Bio, Windsor, QLD, Australia) and rapid IC (immuno-chromatography) test (Pan-Bio). By using rDME-G protein as an antigen, in the dipstick ELISA, the results were in excellent agreement with commercial rapid IC test and IgG capture ELISA. These results show that the product has a promising potential to be used for diagnosis of dengue in both laboratory- and field-based detection systems with minimum cost and a high degree of sensitivity and specificity.

Antigenic Characterization of Outer Membrane Protein of Aeromonas sobria Isolated from Goldfish (Carassius auratus L.)

Aeromonas sobria, along with A. hydrophila, has frequently been reported as a causative agent of motile aeromonas septicemia (MAS) in fish and other aquatic organisms. Currently, there are no precise tools to diagnose or vaccinate against this disease. The aim of the present study was to fractionate and characterize the outer membrane protein (OMP) antigen of A. sobria by sero- logical and cellular techniques so as to identify immunoreactive molecules that might be useful in preparing immunodiagnostic and/or immunoprophylactic tools against A. sobria infection in goldfish. Eight fractions were isolated from the crude OMP antigen using sephacryl S-200 and DEAE-cellulose chromatography. The highest seroreactivity was observed in the gel-permeated protein G1 which had an optical density (OD) of 0.72 nm, higher even than that of the crude OMP antigen, 0.63 nm. The serodiagnostic potential of G1 was assessed by using dip-stick ELISA. The in vitro goldfish lympho-proliferation ability of the fractionated antigen, G1A3, was higher than of all the other fractionated antigens and the crude OMP. Therefore, fractionated antigen G1 (molecular wt 42-67 kDa) and G1A3 (molecular wt 45-47 kDa) should be further studied in immunodiagnostic and/or immunoprophylactic preparations for A. sobria infection.

Reemergence of dengue virus type-3 (subtype-III) in India: Implications for increased incidence of DHF & DSS

BackgroundDengue virus infection has recently taken endemic proportion in India implicating all the four known dengue serotypes. There was a major dengue outbreak in northern India including Delhi in October- December, 2003 and again in 2004. We have carried out a detailed investigation of the 2004 outbreak by Serosurveillance, RT-PCR, nested PCR, virus isolation and genotyping. We also report the molecular epidemiological investigation of these outbreaks.ResultsThe serological investigation of 162 suspected serum samples using an in-house dengue dipstick ELISA revealed 11%-IgM, 51%-IgG and 38%-both IgM and IgG antibody positivity. The RT-PCR analysis revealed presence of dengue RNA in 17 samples. Further subtyping and genotyping by nested PCR and nucleotide sequencing of C-prM gene junction revealed the association of subtype III of dengue virus type 3 in the outbreak.ConclusionThe sudden shifting and dominance of the dengue virus serotype-3 (subtype III) replacing the earlier circulating serotype-2 (subtype IV) is a point of major concern and may be attributed to increased incidence of DHF and DSS in India.

Cathepsin L cysteine proteinase in the diagnosis of bovine Fasciola gigantica infection

Cathepsin L cysteine proteinase from Fasciola gigantica was evaluated for its potential in the early prepatent detection of this helminth infection in bovine calves. Five cross-bred bovine calves were experimentally infected with 400 metacercariae/calf and evaluated for anti-cathepsin L antibody response. F. gigantica infection in these calves could be detected 4 weeks post-infection using an ELISA, dipstick ELISA and Western blotting with 100% sensitivity. The antigen was also used to detect F. gigantica field infection in cattle, by screening 256 sera of these animals by an ELISA, which demonstrated an overall infection rate of 26.95%. Preliminary studies showed that F. gigantica cathepsin L cysteine proteinase does not cross-react with Paramphistomum epiclitum, Gigantocotyle explanatum and hydatid cyst antigens. However, extensive studies on the cross-reactivity of this antigen with related helminth parasites of cattle and buffaloes are required, before this antigen can be considered suitable for immuno-diagnosis of fasciolosis in these ruminants.

Immunoreactive antigens of the outer membrane protein of Aeromonas hydrophila, isolated from goldfish, Carassius auratus (Linn.)

Aeromonas hydrophila causes disease under stress conditions or in concert with infection by other pathogens in goldfish. Sero-diagnostic and/or immunoprophylactic tools against Aeromonas infection in goldfish are not available so far. The present study was undertaken to fractionate and characterise the outer membrane proteins (OMP) of A. hydrophila and to identify suitable immunoreactive components. A total of 10 fractions were generated from crude OMP antigens upon gel permeation and subsequent ion-exchange chromatography. One of the fractionated antigens (GPID2), primarily a 57-kDa polypeptide, showed maximum sero-reactivity, even higher than the crude OMP. Suitability of GPID2 antigen for use in diagnostic preparations was assessed by dip-stick ELISA. In vitro goldfish lymphoproliferative ability of fractionated antigen, GPIID2 (primarily a 23-kDa polypeptide) was observed to be higher than all the fractionated antigens as well as crude OMP. It can be concluded that the 57 kDa and 23 kDa polypeptides of the OMP of A. hydrophila, possessing high immunoreactivity, should be given due attention while preparing immunodiagnostic and immunoprophylatic tools against Aeromonas infections in goldfish.

Fasciola gigantica cathepsin-L cysteine proteinase in the detection of early experimental fasciolosis in ruminants

Cathepsin-L cysteine proteinase was purified from Fasciola gigantica regurgitant by two-step alcoholic fractionation, followed by ion-exchange chromatography. The purification strategy was evolved to eliminate other contaminating proteins co-precipitating with the purified proteinase during alcoholic fractionation. The enzyme was stable on long-term storage at -20 degrees C rendering it more suitable for field diagnostic use. The purified cathepsin-L cysteine proteinase was assayed for detection of F. gigantica experimental infection in sheep and buffaloes and could detect infection, as early as 4 weeks post-infection by ELISA, Western blotting and Dipstick ELISA. The 28-kDa cathepsin-L cysteine proteinase seems a promising antigen for the diagnosis of tropical fasciolosis in domestic animals.

LEPTO Dipstick: a rapid and simple method for serodiagnosis of acute leptospirosis

The LEPTO Dipstick assay is a newly developed test for the diagnosis of leptospirosis and uses a broadly reactive antigen for detecting IgM antibodies. The test was evaluated in the Andaman and Nicobar Islands, using 867 serum samples from known cases of leptospirosis and controls. The efficacy of IgM ELISA was also tested for comparison. The LEPTO Dipstick had a sensitivity of 78·7%, a specificity of 88·3% and a positive predictive value of 91·0%. The test had a good level of agreement with the standard criteria for diagnosis using paired microscopic agglutination tests (κ = 0·64). These indices were similar to those of IgM ELISA (sensitivity 78·5%, specificity 87·6%, positive predictive value 90·5%, κ 0·63). Both the LEPTO Dipstick and IgM ELISA had the highest sensitivity during the second, third and fourth weeks of illness (87·6% and 88·2%, respectively). Sensitivities during the first week and after 4 weeks were relatively low but acceptable. The test is very easy to perform and does not require any special skills for its performance. The reagents and dipsticks have a long shelf-life even at room temperature. As the test can be performed without the aid of sophisticated equipment, it is suitable for use at the peripheral level as a rapid screening test for the diagnosis of leptospirosis.

Development of a rapid dipstick immunoassay for the detection of peanut contamination of food

Polyclonal antisera were raised to conarachin, the 7S globulin of peanut, Arachis hypogea. The antisera were of high titre and were specific for conarachin, showing no significant cross‐reaction with proteins from a range of nuts and legumes, as determined by immunoblotting and ELISA. A dipstick ELISA was developed using these antisera as both the capture and detector elements of the assay. The final steps utilized an avidin‐biotin detection system and tetramethylbenzidine as the substrate. The dipstick assay was highly sensitive, and employed a simple one‐step extraction method. It was able to detect as little as 0.01% (w/w) of peanut in marzipan and 0.1% (w/w) of peanut in chocolate. Roasted nuts were also detected, down to a concentration of 0.1% (w/w) in both foods. The dipstick assay also functioned with a range of foodstuffs, and readily indicated any that contained peanut. This method enables analysts to test, for the first time, for the presence of peanuts in food in a fast and easy‐to‐use manner. The availability of such technology makes the task of monitoring foods for contamination by peanuts readily achievable, providing the industry with an important tool for quality control of raw materials, processes and products. Increased testing will give consumers, particularly those sensitive to this potent allergen, increased assurance as to the safety of the food they eat.

An evaluation of dipstick-dot immunoassay in the detection of antibodies to HIV-1 and 2 in Zimbabwe.

There is a need, in many developing countries, for simple and inexpensive HIV serology tests for use at the district level of health care. The Programme for Appropriate Technology in Health has developed a simple dipstick ELISA to detect antibodies to HIV-1 and 2, at a cost considerably lower than current ELISAs, which requires no specialized washing or reading equipment. In order to evaluate this dipstick under local conditions we used a panel of 546 sera selected from frozen stocks maintained by the Zimbabwe AIDS Prevention Project in Harare, Zimbabwe. Prior to storage, the sera had been tested by Abbott recombinant peptide HIV-1 and 2 ELISA and Enzygnost synthetic peptide HIV-1 and 2 ELISA. The panel included sera that were positive by both (including symptomatics and asymptomatics), negative by both, and sera showing discrepant test results. The panel was not representative of a "normal' batch of sera in Zimbabwe, and in particular included an abnormally high number of sera showing discrepant results. Thawed sera were retested using the Abbott recombinant peptide HIV-1 and 2 ELISA and concurrently with the synthetic peptide ICL-Dipstick ELISA. Both the sensitivity and specificity of the ICL Dipstick exceeded 99% when using sera that were positive or negative in all 3 plate ELISAs as the gold standard. When using sera that gave discrepant results between the two pre-storage ELISAs, most results with the ICL Dipstick concurred with findings from other test systems, including Western blot and p24 antigen detection. Considering the accuracy, low cost and case of operation of the ICL Dipstick ELISA, this test can be recommended for use for the rapid detection of antibodies to HIV at district level in developing countries.

Dot‐Elisa for the rapid detection of gentamicin in milk

A dipstick dot-ELISA for the detection of gentamicin in milk of dairy cattle is reported for the first time. The test is based on a sandwich ELISA using high affinity monoclonal antibodies to gentamicin. Antibodies were adsorbed to nitrocellulose filters, blocked, dried, and stored for several weeks before use. The dipstick ELISA detected gentamicin at a concentration of 0.1 microgram/ml and produced strongly positive results at 0.2 microgram-0.3 microgram/ml. This ELISA is highly specific and no false positives were detected when tested against various aminoglycoside analogs including streptomycin, kanamycin, bekanamycin, amikacin, neomycin, and tobramycin. Further, the elimination in cow milk of gentamicin residues following intramammary administration of the drug was studied in two dairy cattle using dot-ELISA. Milk gentamicin levels were detected at post injection hours up to 120 hr in each of the two dairy cattle. It therefore, appears that gentamicin residues can still be detected in milk after 5 days using dot-ELISA. Based on the simplicity of performance and the economical nature of the test system, dipstick is recommended as a suitable method for wide scale use in field studies and diagnostic laboratories.

Dipstick enzyme immunoassay for rinderpest antibody in cattle

A dipstick enzyme immunoassay (ELISA) has been standardized for the detection of rinderpest antibodies. One hundred and thirty bovine serum samples were analysed by the dipstick ELISA method and the results compared with the conventional plate ELISA method. The sensitivity was found to be similar in both methods. The dipstick ELISA does not require expensive micro-plates and an ELISA reader, and is recommended for use in field laboratories where the qualitative detection of rinderpest antibodies is required.