A Rapid Immunochromatography Test Format For Aeromonas Caviae Detection

Aeromonas caviae is a pathogenic bacteria that can infect various farmed fish and cause great economic loss in aquaculture.For adapting to the development of fish culture,and for convenient detecting of this bacteria,gold-immunochromatography assay(GICA)was established for detect the pathogen A.caviae.The conjugate release pad which sprayed with gold conjugate,nitrocellose membrane which sprayed with polyclonal antibody and goat-anti-mouse IgG on the capture line,sample pad and absorbing pad were assembled to the rapid test strip.Sensitivity was tested using specimens contained A.caviae.The preliminary feasibility study of this method was described as following:high specificity with no cross reaction with 7 pathogenic bacteriaes of aquaculture;the sensitivity limit was 10⁶ cfu.ml^-1;resultion will take place were read within 5 minutes.The method of detecting A.scaviae with GICA strips was rapid,accurate,reliable,sensitive and suitable for into using.There were a series of experiments and the methods and results were as follows:(1)Hybridoma technology was used to produce mAbs against A.caviae,by fusing of mouse Sp2/0 myeloma cells and spleen cells of BALB/c mouse immunized by inactivated A.caviae.The positive clones which produced mAbs against A.caviae were screened by enzyme linked immunosorbent assay(ELISA).After cloning,three strains of hybridoma named respectively 0E10,1C4 and 1F10 stably secreting specific mAbs against A.caviae were obtained.Identification of IgG subgroup showed that 0E10 was the IgG1,1C4 and 1F10 were the IgG2a.Hydroperitoneum of the antibodies were and 2.56×10^-4～5.12×10^-5mensurating by ELISA.Bacteria cross-reaction showed that all the mAbs did not have cross-reaction with A.hydrophil, A.sobria,Vibrio Anguillarum,Edwardsiella tarda,Vibrio vulnificus and V. alginolyticus,showing that these mAbs all have high specificity and could be used to detect A.caviae.The mAbs against A.caviae might also be useful tool for studying biological properties of A.caviae.(2)In this article,20nm colloidal gold particles are prepared and be conjugated with A.caviae monoclonal antibodies,and it is sprayed on the conjugated pad of a porous nitrocellulose membrane.The conjugate pad is stable and specificity.(3)The polyclonal antibody of A.caviae and goat anti-mouse IgG are sprayed on test capture line and control capture line respectively.(4)The conjugate release pad which sprayed with gold conjugate,nitrocellose membrane which sprayed with polyclonal antibody and goat-anti-mouse IgG on the capture line,sample pad and absorbing pad were assembled to the rapid test strip.(5)The trip was used as follow:the sample is added to the sample pad and then passed along the membrane by capillary action.According to the principle of competitive inhibitory immunochromatography,if the sample contain A.caviae,an antibody(polyclonal antibody)-antigen(A.caviae)-antibody(conjugated monoclonal antibody)reacts and forms visible lines in the test capture line;if the sample not contain A.caviae,a color line in the test capture line will be invisible.By this method, a rapid immunochromatographic assy is developed to detect A.caviae.10⁶ cfu.ml^-1of A.caviae is detected in less than 5 min.In conclusion,the experiments were produced mAbs against A.caviae,selected mAb 1F10 to conjugate with gold particles.The gold-based rapid tests were established for detect the pathogen A.caviae.The gold-immunochromatography assay(GICA)trips can be useful in the detecting of this bacteria and its infection,and the method were established first time. 属性不符...