A Study On Detection Of Specific CAg And DNA Segment Of Toxoplasma And Its Application

Toxoplasmosis is one kind of zoonosis originated from toxoplasma gondii. Distributed all over the world extensively, this disease infects persons and animals easily. This worm was found by Nicolle and Manceaux in 1908 and the first case was reported in 1920. Before 1949 no report of toxoplasmosis had been found in China. In 1955 Yu Enshu firstly dissociated toxoplasma from the rabbit and cat. In 1964 Xie Tianhua reported the case with toxoplasmosis at the first time. In 1979 Xu Bingkun reported one case with toxoplasmosis and dissociated toxoplasma. Up till now, there are about 5-10 hundred million people infected by toxoplasma. According to the records toxoplasmosis is seldom and not more than 150 cases in our country. But the number of toxoplasmosis cases which are not found potentially and actually scattered in the clinical internal, surgery, gynecology, paediatrics, ophthalMogy, obstetrics and neurology, etc. may be 3.9 million. This worm can transmit vertically and lead to dead birth, spontaneous abortion and even the brain's coloboma or severe mental retardation of the foetus, etc. At the same time it becomes one of the important death causes of immunologic deficiency disease, such as AIDS, etc.Objectives: To make out McAb of toxoplasma, to develop stable magnetic powder ball with abroad purpose and plenty of complexated protein in order to purify toxoplasma antibody and to concentrate antigen in serum; then to explore the best conditions for detection of specific CAg of toxoplasma with mono-multitude antibody ELISA and ABC-ELISA, and for detection of its specific nucleic acid with multiple PCR in order to carry out epidemiological investigation and take test on pregnant women or psychopath so as to accord to further control program.Methods: McAb was prepared with routine methods. The magnetic powder ball was aggregated. After stirring magnetic powder, styrene, methyl benzene and fumaric acid into xylene, put into the dispersing fluid, then stir, wash and dissociate in high speed in 80-90℃affected by the accelerant. Add toxoplasma AG (McAb) to magnetic powder ball PBS mixed liquor and set 48h to allergic magnetic powder ball in 4℃affected by carbodiimide. Put toxoplasma antibody (antigen) waiting for purificated into allergic magnetic powder ball, shake 1h in the rocking bed under 37℃, wash, acid dissociate and dialyse by glycinate pH2.5～2.8. Antigen and antibody were concentrated.The matrix method was used to search for the conditions for detection of toxoplasma CAg with mono-multitude antibody ELISA and ABC-ELISA. The primer of toxoplasma guard gene was devised according to the B1 gene sequence, and the most suitable conditions including cycle temperature, frequency and Mg+ concentration were also explored in lab. The integrated analysis and contrast were carried out after the samples of 10 specific groups were tested including pregnant women, psychopath, blood donors, raisers of dogs and cats, etc.Results: The lowest CAg concentration for mono-multitude antibody ELISA was 0.25μg/ml and for ABC-ELISA was 0.031μg/ml. 1g sensitized magnetic powder ball by antigen(antibody) can purify 1.2mg antigen (or 1.9mg antibody) . The purified antigen (antibody) was electrophoresed and no protein with impurity was found. The both tested results with mono-double antibody ELISA and ABC-ELISA were positive after 0.03μg/ml antigen was concentrated with the ball. No across reaction with the other parasites was found. The least tested amount of DNA was 0. 1 pg. The primer for detection of specific toxoplasma DNA did not multiply DNA of rat, people, pneumocystis carinii, plasmodium and cryptosporidium. 2052 people of 9 target groups were investigated in 6 counties with 2009 people as control ones. The average positive rate was 3.9% (80/2052) with mono-double ELISA, which was lower than 4.83 % (97/2009) of control group. The results with ABC-ELISA and PCR were opposite. However, no significant differences were found.(x~2=2.105, P=0.147),(x~2=0.582, P=0.446)和(x~2=3.342, P=0.068). The results of blood donors were little bit higher than that of control group. (x~2=7.709, P=0.005 ),(x~2=5.046, P=0.025 ) and (x~2=10.703, P=0.001) . The positive rate of dog raisers and cat raisers was higher than that of the control group but no significant differences were found. The positive rate of psychopath was markedly higher than that of the control group. Statistics significance was found when mono-double ELISA was used. (x~2=4.545, P=0.033 ) . The communicated rate in different districts was quite different. In Huaiyang County, the lowest rate was 0.22 % (8/655) and the highest rate was 6.71 % (51/760). The average rate was 7 % (185/4661). There was a significant difference compared the both rates with the average one.(x~2=12.393, P<0.001) and (x~2=11.794, P=0.001) .Conclusion: The secretary McAb by cell series of hybrid nubble prepared in this test beared high effectiveness and high speciality. The magnetic powder ball was the ideal carrier for purification and concentration of antibody and antigen. The clinical diagnosis on toxoplasmosis should apply at least 2 kinds immuno-methods combined with clinical appearances. 3 established methods of mono-multitude antibody ELISA, ABC-ELISA and multiple PCR were credible, specific and sensitive for detection of CAg and DNA segment of toxoplasma and it can be applied to clinical diagnosis and epidemiological investigation in a large scale. It was found in the investigation in specific people group that the positive rate of psychopath was markedly higher than that of the control group so it showed that toxoplasma might be one of the causing factors of psychosis and it should be paid attention to. The positive rate of blood donors was markedly higher than that of the control group. The positive rate of HIV in blood donors was markedly higher than that of the residents in Henan province. Toxoplasmosis control should be strengthened in such groups. The epidemic prevention of pet should be strengthened since the infected rate of dog raisers and cat raisers was higher than that of control group. The relative program should be worked out in the different districts with different infected rate in the prevention.