Development Of Colloidal Gold Labeled Immunochromatographic Strip Test For Vibrio Vulnificus

BACKGROUND AND OBJECYIVE:Vibrio vulnificus (V.vulnificus,Vv) is a species of gramnegative motile curve bacterium, which is the fifth group of Vibrio bacteria.It can be isolated from oysters, clams, mussels, and fish, as well as from sediment and plankton. Based on phenotypic characteristics and host criteria, V. vulnificus isolates have been grouped into three biotypes (Ⅰ,Ⅱ,Ⅲ).And type I is the major pathogen of human wound infection and primary septicemia. V. vulnificus infection can cause serious human infections and sepsis. The mortality rate is as high as 50%. Primary V. vulnificus infection rate showed a trend of increasing year by year. In China, the first reported case of V. vulnificus infection was in 1990.And after that, there were sporadic cases every year around coastal regions. These diseases share the characteristics that the bacteria multiply extremely rapidly in host tissues resulting in extensive tissue damage and high fatality rate. So, Lack of full understanding of the disease which would lead to incorrect diagnosis in early period of disease or make misdiagnosed as hypersensitivity and used large doses of hormones is the main cause of high mortality. However because of the diarrhea and initial symptoms caused by Vibrio vulnificus exhibed no specific, and thus the most reliable laboratory detection can efficiently assist clinical diagnosis. So, it is very important to establish a rapid and convenient detection method to diagnose vibrio vulnificus infection and provide effective evidence for food safety. The purpose of this research was to establish a rapid and simple diagnostic strip for V. vulnificus, which based on the specific immunochromatographic lateral-flow and colloidal gold tracing.METHOD:1.Production of anti- V. vulnificus polyclonal antibody(1)The standard strain ofⅤ. vulnificus is ATCC27562 which is purchased from the Chinese Academy of Sciences Institute of Microbiology collection management center. Gram stained and observed under light microscopy aget recovery culture. Finally, crossed inoculated on Marie Broth2216 agar plate and collected the V. vulnificus antigen.(2) Anti-Ⅴ. vulnificus polyclonal antibodies were obtained from rabbits immuned by V. vulnificus. The polyclonal antibodies were collected and purificated by caprylicacid-ammonium sulfate precipitation. Then concentration of purified PcAb was detected by BCA protein concentration determination. SDS-PAGE were used to research the purity of PcAb, through the comparison with PcAb before purify. The antigenicity of recombination proteins were detected by ELISA. PMSF were used respectively for separating and withdrawing the major outer manbrane proteins (MOMP) of V. vulnificus.Its constituent were analyzed by using SAD-PAGE. Western-blot was used to detect antibody against MOMP.2 Preparation of antibody-colloidal gold conjugate(1)Colloidal gold nanoparticles were prepared by controlled reduction of gold chloride with different concentration of trisodium citrate.And then, the nanoparticles were scan by spectrophotometer between 400nm～700nm. UV-spectrophotometer showed the peak and the absorbance. Observe the color, transparency and stability of different diameter of colloidal gold. And choose the optimal diameter to conjugate the antibody.(2) Different volumes K2CO3 were used to adjust the pH value. To each tube, antibody was added. Then take the absorbance of each tube at 520nm and 580nm. The ratio of A520nm and A580nm is directly related to the stability of antibodu-colloidal gold conjugate. To determine the minimum amount of antibody conjugate needed to stabilize colloidal gold solution, different volumes of antibody was added in different tubes. The absorbances at 520 and 580nm were measured on the UV-spectrophotometer. The antibody-colloidal gold conjugate was then prepared under the optimal conditions. The gold particles was blocked by BSA and centrifuged for purify. The various factor and conditions of the conjugate were explored, and the optimal reaction conditions of the assay were ascertained. 3. Development of colloidal gold labeled immunochromatographic strip(1) The colloidal gold labeled immunochromatographic strip consisted of four components:sample application pad, glass fiber membrane, nitrocellulose membrane and absorbent pad. The glass membrane contained a deposited antibody-colloidal gold conjugate in a dry state. The nitrocellulose membrane was used as a solid matrix to immobilize two different antibodies at spatially separated point, One, capture anti-Ⅴ. vulnificus and the other capture anti-IgG rabbit. The absorbent membrane had the role of absorbing liquid medium to provoke a continuous flow of fluid,the membranes were dried at the optimal condition.(2) The sample liquid was added to the immunochromatographic strip at the sample application pad and allowed the liquid to migrate. After 30 min, the test results were evaluated visually or test lines were scanned and measured the PU value. The negative test results in one red line (control lines). The moreⅤ. vulnificus in the sample, the stronger the test lines appear. Evaluation of the specificity, sensitivity and stability of the immunochromatographic strip was analyzed. Sample recovery was analyzed forⅤ. vulnificus using the test strips and ELISA.RESULTS:V. vulnificus ATCC27562 geows in plate yellow white colonies, shiny and shows a typical mucous colony.1. The purified polyclonal anti-Ⅴ. vulnificus were specific and their potency respectively reached 1:25600. The polyclonal anti-Ⅴ. vulnificus was collected and purificated by caprylicacid-ammonium sulfate precipitation. Protein concentration was determined to 12.5 mg/ml. The purified antibody was performed under SDS-PAGE electrophoresis. It showed protein bands on 55KD to 61KD of the gelatin. Western-blot was used to detect antibody againstⅤ. vulnificus MOMP. It showed a well combine between them. Antibody potency respectively reached 1:12800.2. Colloidal gold were prepared through reducing HAuCl4.3H2O by sodium citrate, and it was then characterized by UV-spectra. The diameters of colloidal gold were 54.88nm 36.15nm 36.15nm 17.42nm and 12.74nm, separately. The optimum volume of K2CO3 for antibody conjugating colloidal gold was 9μl, and the concentration of antibody was 36μg/ml. The antibody-colloidal gold was analyzed by UV-spectra, and the results showed that the conjugation was successful. We optimized reaction system for antibody-colloidal gold in which the optimal buffer was boracic acid buffer with0.5%BSA,0.5%PEG20 000,15%saccharose and 0.05%Tween-20.3. We established and optimized the sandwich immunochromatographic strip to detectⅤ. vulnificus. We optimized condition for the strip, in which the optimal captured dilution of anti-Ⅴ. vulnificus was 1:4, and the dilution of anti-IgG rabbits was 1:8. The nitrocellulose membrane was used the 1%BSA liquid to block, and dried at 37℃for 1 hour. The glass membrane contained a deposited antibody-colloidal gold conjugate was dried at 4℃. The sample liquid was added to the immunochromatographic strip at the sample application pad and allowed the liquid to migrate. The negative test results in one red line (control line). The positive test results in two red lines (control line and test line).The more V. vulnificus was in samples; the stronger the test lines appear.The sensitivity of the method was 2×106 CFU/ml. The specificity of the strip was determined using 12 pure-cultured bacteria, including Vibrio Parahaemolyticus, Escherichia coli, Shigella dysenteriae, Staphylococcus haemolyticus, Staphy lococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, K.Peneumoniae, Stenotro phomonas maltophilia, Bacillus aerogenes, Aerobacter cloacae, Proteus mirabilis. It was found that all the 12 strains demonstrated a negative signal. The strips could be kept stable for four months at 4℃. Recovery test demonstrate that both ELISA test and immunochromatographic strip test had a better recovery at level V. vulnificus.CONCLUSIONS:The colloidal gold labeled immunochromatographic strip for detecting V. vulnificus could finished within 20 to 30 min.The detection limit of the method was 2×106 CFU/ml. The strip has no cross-reation with other bacterial strains. The strips could be kept stable for four months at 4℃. These results indicate that the colloidal gold labeled immunochromatographic strips exhibite the method is sensitive, sepicific, simple and rapid for screen Vibrio vulnificus.