Development Of A Colloidal Gold Immunochromatographic Strip For Rapid Detection Of IgG Antibody To Toxoplasma Gondii In Canine Sera

Toxoplasmosis caused by Toxoplasma gondii is an important zoonosis.The disease can cause reproductive failure in pregnant women and animals,manifested as puny, abnormal embryo,abortion and stillbirths.Toxoplasmosis is ubiquitous among people and animals throughout the world.Human and dogs are it's secondary host.It is reported that human's toxoplasmosis is associated with dog's infection.At present,many methods have been used to detect the antigen or antibody of Toxoplasma gondii,such as parasitological test,indirect hemagglutination test,indirect immunofluorescent antibody test,enzyme-linked immunosorbent assay and polymerase chain reaction.All these methods are unsatisfactory,because these methods are still complex,time-consuming, and requiring special equipment.So,the aim of our research is to establish a rapid, convenient immunodiagnostic assay,which is only using for the diagnosis of toxoplasmosis immunoglobulin G(IgG) and as epidemiological investigation in dogs. The establishment of this method is not only helpful to investigate the infection rate of house dog,but also can block the Toxoplasma gondii dissemination from the dog to humen.So this method is useful to public health.In this reseach,Toxoplasma gondii soluble antigen and excretory-secretory antigen were prepared respectively by using tachyzoite of Toxoplasma gondii to infect Kunming mouse through abdominal cavity infection.Canine anti-Toxoplasma gondii sera were prepared by using excretory-secretory antigen to immunize healthy dogs.Sera of healthy dogs were Collected,then canine IgG from the sera was extracted by(NH₄)₂SO₄ salting-out and SephadexG-200 column chromatography.The rabbit anti-dog IgG antibody was prepared by using the purified canine IgG to immunize rabbits. The titer of the antibody was detected by double agar diffusion method.When the antibody titer was arriving 1:64,rabbit sera were collected,then purified by caprylic acid/ammonium sulfamate salting-out method.Twenty nanorneter colloidal gold was produced by using trisodium citrate reducing aurum chloride,then using colloidial gold to conjugate rabbit anti-dog IgG as pad.The experiment showed that the optimal pH for the conjugation between colloidal gold and antibody is 9.0,the optimal amount of rabbit anti-dog IgG for the conjugation between colloidal gold and antibody is 12μg/ml.Toxoplasma gondii soluble antigen and goat anti-rabit IgG are labled on the nitrocellulose membrane as the test line and the control line respectively. The anti-toxoplasma gondii IgG in the dog's serum will conjugate with rabbit anti-dog IgG and form antigen-antibody compound,which flow on the surface of the nitrocellulose membrane by means of Immunochromatography,the compound will be captured by soluble antigen and goat anti-rabit IgG in the test line and the control line respectively,showing obvious readable red line on the surface of the nitrocellulose membrane.The experiment indicated the optimal test conditions as following:the concentration of soluble antigen was 2.5mg/ml;the concentration of goat anti-rabbit IgG was 0.75mg/ml;colloidal gold-protein compound diluted at ratio of 1:2.Ninety four serum samples by random sampling obtained from pet hospital and six serum samples obtained by artificial infection were tested respectively with Gold Immunochromatography Assay(GICA,our laboratory)and with Indirect hemagglutination test kit(IHA,Lanzhou veterinary research institution).In the 94 sera sample,3 sera were found to be positive with GICA,4 sera were found to be positive with IHA;In the 6 sera sample by artificial infection,4 sera were positive by GICA,and 4 sera were positive by IHA too,and the 4 positive sera were of the same sample.The final result showed that the correspondence rate of the 6 sera sample by artificial infection was 100%and the total correspondence rate,the positive correspondence rate and negative correspondence rate of GICA was 99.0%(99/100),87.5%(7/8) and 98.9%(92/93) respectively compared with IHA.Conclusion:GICA has good correspondence rate with IliA,and GICA has the following advantages:testing result could be obtained within 10～15minutes;the test strip could be used to detect the sample in field and do not require special equipment;and had high sensitivity.So GICA has potential value for immunodiagnosis of canine toxoplasmosis.