Establishment Of Magnetic Particles-ELISA And I-PCR Technique For Detecting F1 Antigen Of Yersinia Pestis

ObjectivePlague is a natural focus infection disease and a severely hazardous amphixenosis whichinfects homothermal animal, especial rodents. It is caused by Yersinia pestis and flea is thevector. There had been 3 plague pandemics in the history and caused thousand millions ofdeaths. Epidemic focuses of Yersinia pestis are distributed all over the world. At presentplague epidemic has shown characteristis, like increasing epidemic areas, rising cases,outbreak with interval, spreading to city and areas with intensive population, obviouslyupgrading numbers of main host and re-emerging animal plague. In China, 456 cases werefound in six provinces including Yunnan, Tibet, Qinghai, Gansu, Xinjiang and Neimengguduring 1978-1997, and caused 105 deaths with the mortality of 23.03ï¼…. 350 cases werefound in 1988-1997 and were 3.3 times higher than 106 cases in 1978-1987. Patients weremainly distributed in areas with woodchuck and Rattus flavipectus. During the Tenth FiveYears Plan, 206 cases were found in 43 counties of 7 provinces and animal plague was shownin 278 counties of 14 provinces including 13 new counties. It is very important of rapid diagnosis for plague. Traditional diagnosis should have 4 procedures including microscopicexamition, culture, bacteriophagum schizolysis experiment and animal experiment. It needslong time and tedious steps and can not satisfied the clinical application. With thedevelopment of immunological techniques and so on, new diagnosis methods for variousspecial antigens, virulence factors and antibody were found, such as IndirectHemagglutination Assay(IHA), Enzyme Linked Immunosorbent Assay(ELISA), DOT-ELISA,Radioimmuno Precipitation Test, (RIP) and so on. At present, many proteins and factors wereselected to be the marker of diagnosis, such as pgm,pst,Yops,F1 antigen, V antigen and soon. F1 antigen is a special antigen of Yersinia pestis, and its protective effect has beenconfirmed. F1 antigen is capsule substance and located outside of Yersinia pestis. It is codedby plasmid with of 61-65MD molecular weight. Purified F1 antigen from recombiantedEscherichia coli shows protective effect in mouse experiment, it protected mouse fromattacking of Yersinia pestis with strong toxicity. Detection for F1 antigen and antibodybecame a hot spot of diagnosis of plague.Magnetic Particles were made by Zhao xu-dong et al according to the characteristic ofpolymerization in certain macromolecular chemical materials, immunoaffinity technique andmagnetological principle. Magnetic Particles allergized by antigen(or antibody) can be usedin screening and purification of corresponding antibody (or antigen). Concentration of certainantibody (or antigen) from liquid medium can increase sensitivity of detection. Separationand purification of some proteins, cytokines and nucleinic acid by magnetic adsorb particlehad been reported abroad. In China, separation and detection of tumor cells by magneticparticles made abroad had been currently tried by several persons, but no report aboutproduction of Magnetic Particles and application for detection and purification of antigen orantibody is found up to now.Immuno-PCR(immuno polymerase chain reaction, I-PCR) is a detective technique formicroamount antigen set by Sano T etal in 1992. The basic priciple is combining a knownbiotin labeled DNA to antigen-monoclonal antibody compounds, which amplified by PCR,and judged by electrophoresis. It is a kind of modified ELISA, which enzyme reaction wasreplaced by amplification of reproted DNA by PCR. This technique has characteristics ofhigh specificity of antigen-antibody reaction and high sensitivity of PCR. Study of detection of plague by Immuno-PCR has not been found.This study want to make Magnetic Particles and monoclonal antibody to F1 antigen ofYersinia pestis, establish detective methods of Magnetic Particles-ELISA and Immuno-PCRfor F1 antigen of Yersinia pestis, and compare the sensitivity, specificity, stability withSandwich-ELISA, ABC-ELISA and Imuuno-PCR by detecting F1 antigen of Yersinia Pestis.MethodsMagnetic Particles were made by polymerization technique. Magnetic particle, benzenevinyl, methyl benzene, transbutene dioic acid,ρ-xylene were mixed, agitated with high speed,washed and separated in dispersal liquid on the effect of catalyst at 80-90℃, Stability ofMagnetic Particles were observed during 1-6 months stored at -20℃,-5℃and 4℃separately.F1 antigen was added to PBS mixture with carbodiimide and Magnetic Particles, andMagnetic Particles could be allergized after 48h at 4℃. Then antibody to Yersinia pestis wasmixed and shaked with allergized Magnetic Particles for 1h at 37℃, dialysed by glycinate(pH2.5～2.8).Special Monoclonal antibody for F1 antigen of Yersinia Pestis was made using normalmonoclonal technique, and purified by Magnetic Particles sensitized by F1 antigen ofYersinia Pestis. Antibody titer was verified by Indirect Enzyme Linked Immunosorbent Assay.Specificity was certified by antigen of Brucella, Yersinia enterocolitica O₃, O₉, Bacillus typhi,Bacillus comma, Bacterium dysenteriae and Bacillus coli.The optimized concentrations of antigen, antibody and avidin were defined by tests, anddifferent concentrations of F1 antigen of Yersinia Pestis were detected by sandwich-ELISAand ABC- ELISA.Method of Magnetic Particles-ELISA was below, allergized Magnetic Particles by F1McAb of Yersinia Pestis and specimen with F1 antigen were mixed, lightly swirl -shaked for40 min at 37℃, the next procedures were the same as the normal ELISA except for usinglightely swirl-shaked method, and magnet for washing.Compare the detecting results withsandwich-ELISA, ABC-ELISA and I-PCR methods.Upstream primer was biotin labeled M13-20 (Bio-5'-GTA AAA CGG CCA GT-3'),downstream primer was non-biotin labeled M13 (5'-GGA AAC AGC TAT GAC CAT G-3'). Production was 227bp, different concentrations of F1 antigen of Yersinia Pestis were detectedby I-PCR.ResultsMagnetic Particles were successfully made, with diameter between 0.5 and 2.0μm andspecific gravity of 1.07～1.15.When they were stored for 6 months at -5℃and 4℃, there were10ï¼…and 6ï¼…quality decrease separately after putting them into the sulphuric acid.F1 antigen special McAb were purified by Magnetic Particles and the maximum purifiedprotein quantity was 1.9mg/g. Purification capability of Magnetic Particles decreased 5.3ï¼…and 26ï¼…when they were stored for 6 months at -5℃and 4℃.Special McAb to F1 antigen of Yersinia Pestis were obtained, and was IgG1, titre ofwhich was 1:1×10⁶. Cross reactions with Brucella, Yersinia enterocolitica O3,O9 were notfound.The optimized concentrations of polyclonal antibody and monoclonal antibody were 1:1000 and 1: 2000; The lowest detective concentration of Sandwich-ELISA was 0.032μg/ml.The optimized concentration of Avidin was 1μg/ml; The lowest detective concentrationof ABC-ELISA was 0.004μg/ml.The lowest detected concentration of F1 antigen was 0.002μg/ml using MagneticParticles-ELISA.The optimized concentrations of Avidin and biotin labeled-DNA were 10ng/ml and10pg/ml; The lowest detective concentration of was 5pg/ml.Conclusions1. Magnetic Particles were successfully obtained and could be used in purification ofantigen and antibody, as well as immunological diagnosis of disease.2. Monoclonal antibody to F1 antigen of Yersinia Pestis was obtained and had thecharacteristic of high purity and strong specificity after purified by Magnetic Particlessensitized by F1 antigen.3. Diagnosis method of Magnetic Particles-ELISA for detecting F1 antigen of YersiniaPestis was established, and had higher sensitivity, specificity and stability thanSandwich-ELISA and ABC-ELISA.It may be used in plague epidemic investigation in big area after procedures' consummation and standardization.4. I-PCR had high sensitivity with the defect of easily polluted and long time, may beused in detection of very low concentration of antigen in labs after development andmodification of procedures.