Development And Preliminary Application Of Dot Immunogold Filtration Assay For Rapid Diagnosis Of Brucellosis

Objective:Brucellosis is a highly contagious zoonotic disease caused by Brucella invading In China,Brucellosis is class B infectious disease.Human beings can infect brucellosis through direct contact,digestive tract and respiratory tract.And human beings are generally susceptible to the disease.In recent years,Brucellosis is very serious in China.And about fifty thousand new cases were reported every year.Animal Brucellosis caused huge economic losses.The clinical manifestations of Brucellosis are untypical.It is easy to be confused with influenza,which causes misdiagnosis and delays treatment,finally makes it to be a chronic infection.Then it is difficult to be cured and brings severe mental and financial burden to patient Although some vaccines are used in animal immunization,no vaccine is for human to resist Brucellosis effectively.In order to make the Brucellosis patients can be early diagnosed and get early treatment,a quick and accurate method for detection of Brucellosis is desperately needed.The aim of this paper is to develop a simple,rapid,specific and sensitive dot immunogold filtration assay for the detection of BrucellosisMethodsWe combined BP26 and LPS as detect antigen and used dot immunogold filtration assay to test the specific antibody of Brucella in human serum.The various conditions on the reaction system were optimized,such as antigen preparation,optimal antigen package conditions(dilution buffer,antigen concentration),the optimum conditions of colloidal gold marking(gold colloid dosage,pH value and the minimum amount of labeled Goat anti-human antibody),nitrocellulose membrane,sealing solution,the washing solution,and the best absorbent material ?Then,we detected health human serum samples as the specificity test and brucellosis serum samples as sensitivity test.We also did repeatability and stability test.At the same time,we did the tube agglutination reaction test(SAT)and colloidal gold immunochromatography assay as the control to evaluate our method.Results1.Antigens preparation:BP26 protein was expressed by BL21.After identification and purification,the concentration of BP26 was 1.3mg/ml.And LPS was purchased2.The best condition of antigens coating:using the PB buffer of lOmM with PH 6.8 to dilute the BP26 protein and LPS to 0.5?g/?l and 0.0625?g/?l respectively,and then took 0.3?l for coating3.The best conditions for colloidal gold marking:using the potassium carbonate 10?l of 0.2mol/L to adjust the pH value of colloidal gold solution to the best PH 5.5.Then using 14?g goat anti-human antibody to mark the colloid gold solution4.Reaction system:using 100?l PBST solution with 1%BSA as sealing solution;diluting colloidal gold marker solution 10 times and using 50?l for testing;using 100?l PBST solution with 1%Tween-20 as washing buffer;choosing Whatman BA85 nitrocellulose membrane as filtration membrane;using toilet paper as optimum absorbent material5.Clinical sample test:we used 245 healthy human serum samples as the negative control,125 Brucellosis positive serum samples as the experimental samples.The operation procedure of our method:put card on the table,add 100?l of sealing solution,waiting for its penetration completely;add 50?l of serum samples,waiting for its penetration completely;add 100?l the washing buffer,waiting for its penetration completely;add 50?l of colloid gold marker solution,waiting for its penetration completely;add 100?l of washing buffer,the result can be observed immediately.Finally,the results were as follows:245 healthy serum samples were negative,121 positive results of 125 SAT positive serum samples and 4 negative results.43 serum samples were detected by dot immunogold filtration assay,SAT and colloidal gold immunochromatography assay respectively.Dot immunogold filtration assay was positive in 14 cases and negative in 29 cases,which was consistent with the results of colloidal gold immunochromatography assay.SAT was positive in 5 cases and negative in 38 cases.During the process of operation,the result of DIGFA can be observed by naked eyes immediately,without waiting like colloidal gold immunochromatography assay.So it was more time-saving.This method does not need any special instrument.And it was reproducible,stable and economical.ConclusionWe established a dot immunogold filtration assay for the detection of brucella infection.This method was specific,sensitive,reproducible,stable,simple,economical and can be applied to the grass-roots level medical institutions.