Immunochromatographic Test Strip For Detection Research Articles

Development of an immunochromatographic test strip to detect pneumolysin by monoclonal antibody capture method

BackgroundThe currently available methods for the detection of Streptococcus pneumoniae are labour-intensive, and involve time-consuming steps needing skilled technicians to perform the assays. Therefore, we developed a rapid lateral flow based immunochromatographic test strip for detection of S. pneumoniae using pneumolysin using monoclonal antibody capture approach. Methodology − The strip was designed by assembling a sample pad, conjugation pad, nitrocellulose membrane and adsorbent pad supported by a plastic backing card. Optimization of control spot was done by using three different concentrations of monoclonal antibody (0.1 µg, 0.25 g and 0.5 µg). Monoclonal antibody coupled with gold nanoparticles was immobilized on conjugate pad. Recombinant pneumolysin at three different concentrations (10 ng/ml, 50 ng/ml and 100 ng/ml) was used. Diluted pneumolysin was poured onto the sample pad which migrated through the conjugate pad containing gold antibody conjugate and the antigen–antibody complex migrated to the test spot to bind to the polyclonal antibody. The remaining free antibody bound to the control spot to visualize a control spot. Results: Control spot was optimized at 0.5 µg of mouse IgG. Of three different concentrations of Pneumolysin, the spot was best visualized at 50 ng/ml Pneumolysin concentration. Conclusion: Our ICT based device could detect a commercially available recombinant Pneumolysin of S. pneumoniae. Future study is directed to validate the strip for detection of Pneumolysin using biological fluids of patients (particularly children) infected with S. pneumoniae.

Evaluation of Animal Rabies Surveillance System, Ekiti State, Nigeria, 2012-2017

Evaluation of Animal Rabies Surveillance System, Ekiti State, Nigeria, 2012-2017

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin.

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

Developing a novel immunochromatographic test strip with gold magnetic bifunctional nanobeads (GMBN) for efficient detection of Salmonella choleraesuis in milk

To date, immunochromatographic test strip has been widely applied in the rapid detection of foodborne pathogens. The label of an immunochromatographic test strip is the key factor for rapid detection. In this study, gold magnetic bifunctional nanobeads (GMBN) were selected as a novel label to prepare immunochromatographic test strip for detection of Salmonella choleraesuis. The strip was systematically developed and optimized. In the procedure of conjugation between GMBN and anti-S. choleraesuis monoclonal antibodies, the pH of system was 6 and 150 μg of antibody was added in 200 μL of GMBN (5 mg/mL). Twenty microliter of the antibody-GMBN complex was used to capture S. choleraesuis. The capture efficiency of S. choleraesuis was 82.4%. Furthermore, the S. choleraesuis-antibody-GMBN complex could be used to detect S. choleraesuis in immunochromatographic test strip. Results showed that the test strip was specific and rapid. The sensitivity of the strip was 5 × 105 CFU/mL. The immunochromatographic test strip could be applied in food samples (whole milk) and the entire detection time was 20 h.

Development of an immunochromatographic test strip for detection of cholera toxin.

Because cholera toxin (CT) is responsible for most of the symptoms induced by Vibrio cholerae infection, detection of CT is critical for diagnosis of the disease. In this study, we constructed an immunochromatographic test strip for detection of CT (CT-IC) with polyclonal antibodies developed against purified recombinant whole CT protein. The detection limit of the CT-IC was 10 ng/mL of purified recombinant CT, and it could detect the CT in culture supernatant of all 15 toxigenic V. cholerae isolates examined, whereas no false-positive signal was detected in all 5 nontoxigenic V. cholerae isolates examined. The specificity of the CT-IC was examined with recombinant heat-labile toxin (LT), which shares high homology with CT, and it was revealed that the minimum detection limit for LT was 100 times higher than that for CT. In addition, lt gene-positive enterotoxigenic Escherichia coli (ETEC) was examined by CT-IC. The false-positive signals were observed in 3 out of 12 ETEC isolates, but these signals were considerably faint. The CT-IC did not develop false-positive signals with all 7 V. parahaemolyticus isolates. These results showed the high specificity of CT-IC and the feasible use of it for the detection and surveillance of toxigenic V. cholerae.

Development of a colloidal gold immunochromatographic test strip for detection of lymphocystis disease virus in fish

To develop a gold immunochromatographic test strip for on-site rapid detection of lymphocystis disease virus (LCDV). Monodispersional colloidal gold and gold-labelled anti-LCDV monoclonal antibody (McAb) 2D11 were prepared and characterized by UV-visible spectroscopy and transmission electron microscopy. Gold-labelled probe was used as the detection antibody, and goat anti-mouse IgG at the control line and anti-LCDV McAb 1A8 at the test line of the test strip served as the capture antibody. The positive results could be easily judged by the presence of a red test line with naked eye within 10min. The test strip, in good agreement with enzyme-linked immunosorbent assay and dot-blotting in sensitivity and LCDV detection, gave a detection limit of 1μgml(-1) of LCDV and was stable for 6months at room temperature and 12months at 4°C. The test strip was specific, simple and convenient for rapid detection of LCDV presenting good stability and reproducibility. This ready-to-use test strip allows on-site rapid detection of LCDV in fish without the requirement of specialized equipments and professional personnel, which could augment the practical application for diagnosis of LCDV even in disadvantage areas.

Evaluation of a rapid immunochromatographic test strip for detection of Rabies virus in dog saliva samples

An immunochromatographic test strip for Rabies virus was evaluated with dog saliva samples. The test was initially validated against 237 dogs of known infection status, and then evaluated in the field with 1,290 live dogs. By validation of paired saliva-brain specimens obtained from dogs at necropsy, the saliva strip test was 94.4% specific and 93.0% sensitive when compared to the gold standard fluorescent antibody test (FAT) on brain smears. The sensitivity and specificity of a nested polymerase chain reaction (nPCR) assay using saliva were 100% compared to the FAT results. The performance of strip test with field saliva samples from street dogs had a specificity of 98.7% in comparison to nPCR as the reference method. As the strip test kit can potentially be used outside the laboratory and be applicable as an on-site testing assay, it represents a powerful screening tool for epidemiological surveys and disease control. The test could be useful for the surveillance of rabies in dogs and, in particular, be used to monitor the success of rabies control programs.

A simple and rapid immunochromatographic test strip for detection of pathogenic isolates of Vibrio harveyi

Mouse monoclonal antibodies (MAbs) and rabbit polyclonal antibody (PAb) against Vibrio harveyi were generated from immunization of mice and rabbits with highly virulent isolate of V. harveyi. Two MAbs specific to virulent isolates of V. harveyi were obtained and one of them (VH4) was selected to conjugate with colloidal gold as the detector antibody was laid on a sample pad. Rabbit polyclonal antibody was used as the capture antibody at the test line (T) and goat anti-mouse IgG antibody (GAM) was used as the capture antibody at the control line (C) of nitrocellulose strip. The ready-to-use strip was held in a plastic case and then stored in a desiccated plastic bag. A sample volume of 100 μl of bacterial suspension from various sources mixed with application buffer was applied to the sample chamber at one end of the strip and allowed to flow by chromatography through the nitrocellulose membrane to the other end. In test samples containing virulent isolates of V. harveyi, the bacteria would bind to the monoclonal antibody conjugated with colloidal gold and the resulting complex would be captured by the antibodies at the test line to give a reddish-purple band. Any unbound monoclonal antibody conjugated with colloidal gold moved across the test line would be captured by the GAM and form a band at the control line (C). In sample without V. harveyi or with V. harveyi below the limit (< 10 6 CFU/ml) of detection for the kit, only the control line band was observed. If the test sample was pre-enriched in tryptic soy broth (TSB) for 6 h before application to the strip, the sensitivity would increase to 1–10 CFU/ml which is comparable to that of PCR. This method could be used to detect pathogenic isolates of V. harveyi in pond water or infected shrimp in order to monitor and to reduce the risk of V. harveyi outbreak in the shrimp culture. The beneficial features of this kit are that simple, convenient and quick results (within 15 min) can be obtained without the requirement of sophisticated tools or special equipments and skills.

A simple and rapid immunochromatographic test strip for detection of white spot syndrome virus (WSSV) of shrimp

A simple strip-test kit for white spot syndrome virus (WSSV) detection was developed using monoclonal antibody W29 (against the VP28 structural protein) conjugated with colloidal gold as the detector antibody. A rabbit anti-recombinant VP28F118 (rVP28) protein antibody in combination with a W28 monoclonal antibody was used as the capture complex at the test line (T), and goat anti-mouse IgG antibody (GAM) was used as the capture antibody at the control line (C). For evidence, the ready-to-use strip was kept in a plastic case and stored in a desiccated plastic bag. A sample volume of 100 microl gill homogenate in application buffer was applied to the sample chamber at one end of the strip and allowed to flow by chromatography through the nitrocellulose membrane to the other end. In test samples containing WSSV, the virus bound to the monoclonal antibody conjugated with colloidal gold and the resulting complex was captured by the antibodies at T to give a reddish-purple band. Any unbound monoclonal antibody conjugated with colloidal gold moved across T to be captured by the GAM and formed a band at C. In samples without WSSV or with WSSV below the limit of detection of the kit, only the band at C was seen. This method was 4 times less sensitive than dot blotting, and about 2 000 000 times less sensitive than 1-step PCR. Nonetheless, it could be used to screen individual shrimp or pooled shrimp samples to confirm high levels of WSSV infection or WSSV disease outbreaks. The beneficial features of this kit are that simple, convenient and quick results can be obtained without the requirement of sophisticated tools or special skills.