Study On Real Time Immuno-Polymerase Chain Reaction For Some Polycyclic Aromatic Hydrocarbons

Polycyclic aromatic hydrocarbons(PAHs) are a large group of organic compounds with two or more fused aromatic rings.PAHs are formed mainly as a result of pyrolytic processes,especially the incomplete combustion of organic materials during industrial and other human activities,such as processing of coal and crude oil,combustion of natural gas,including for heating,combustion of refuse, vehicle traffic,cooking and tobacco smoking,as well as in natural processes such as carbonization.Due to the widespread distribution of PAHs in the environment and the carcinogenic and mutagenic nature of PAHs,there has been increased concern in their detection and monitoring in recently years.The U.S.Environmental Protection Agency(EPA) has identified 16 unsubstituted PAHs as priority pollutants,which are monitored routinely for regulatory purposes.Four PAHs studied in the paper are all in the list.So,sensitive and reliable analytical methods are needed to evaluate the presence of PAHs at very low concentration in environmental sample,especially these mentioned above.At present,the most common methods used for analysis of PAH are gas chromatography(GC) and high-performance liquid chromatography (HPLC).But these assays require preconcentration of analyte and are relatively time consuming and difficult to perform on-site.In contrast,immunoassays are typically very sensitive and readily adapted to analytes for which an appropriate antibody is available.The IPCR method,first described by Sano et al.in 1992,combines the well-established ELISA methodology with the signal amplification power of the PCR.A number of research applications describe the advantages of the method,that is,in particular,its high sensitivity and good quantification capabilities due to the great linearity and compatibility with established ELISA protocols.The detection was shown to be 100 to 1000 fold more sensitive than conventional Enzyme-linked immunosorbent assay(ELISA).Since its development,the method has been applied to detect antigens associated with cancer,autoimmune diseases,pathogenic bacteria and bacterial toxin.But no environmental pollutants have been detected by IPCR.Further development in the technology and instrumentation used for the signal detection of IPCR has resulted in the development of Real-Time IPCR(RT-IPCR). However,increased use of RT-IPCR has shown that the method displays improved statistical validation of accuracy over IPCR.Inter-assay error is typically 5-10%vs 15-20%for IPCR.To date,only a few mono-or polyclonal antibody-based immunoassays for PAHs have been reported.And no mono-or polyclonal antibody-based immunoassays for PAHs have been reported.While no paper have reported about the determination of PAHs using RT-IPCR.In this paper,three methods were developed for the determination of trace PAHs in the environment by RT-IPCR assay.The work is summarized as follows:(1) Synthesis of artificial antigens:PAHs are small molecules,which can not be directly used for rabbit immunization.They have to combine with carriers.Haptens should be synthesized and conjugated to protein for rabbit immunization.A carboxyl was inducted to the PAHs(Anthracene,Phenanthrene,Fluoranthene,) molecule by Friedel-Crafts acylation reaction and PAHs butanoic acid,y-oxo-was obtained as the hapten.As to Naphthalene,2-naphthoxy acetic acid was bought as the hapten.The haptens were conjugated to the carrier proteins(BSA,OVA) with the modified active ester method or with the mixed acid anhydride method to form immune antigens and coating antigens.In this way the artificial antigen of PAHs were obtained.Structures of the products were characterized by IR,UV spectra,Mass spectra and 1H NMR.(2) Preparation of polyclonal antibodies:Male New Zealand white rabbits were immunized with the mixture of immune antigens and Freund adjuvant.The immune response consisted of an initial primary response followed by a secondary immune response.After the third immune response,the rabbits were bled from the ear vein and the sera were tested for titer by the agar diffusion test and the tube agglutination reaction test.In tube agglutination reaction test,there were visible precipitation reaction;in agar double-diffusion experiment,there were clear deposit lines.The antiserum from the rabbit was purified by the method of octanoic acid ammonium sulfate two-step precipitation, Sephadex G-25 and DEAE cellulose.In this way the specific and affintive pAbs of PAHs have been yielded.(3) Preparation and purification of biotinylated reporter DNA:The reporter DNA with biotin was a 103-base pair sequence from the pUC19 Vectors.The reporter DNA was generated by PCR as follows.The forward primer, M13/pUC sequencing as G TAA AAC GAC GGC CAG T,was biotinylated at the 5' end to generate a biotiny group to the DNA.The reverse primer,M13/pUC reverse sequencing as CAG GAA ACA GCT ATG AC was also biotinylated at the 5' end to generate a biotiny group to the DNA.All regents were added to PCR tube as described by the DNA PCR kit handbook.The PCR conditions were:hold 94℃for 4 min;30 cycles of 94℃for 20 s,55℃for 20 s,and 72℃for 20s.The 72℃step was extended to 3 min in the final cycle.The reporter DNA was purified and retrieved by UNIQ-10 PCR DNA Extraction Kit.The DNA was quantified by UV absorbency and checked qualitatively using agarose gel.(4) Preparation of biotinylated polyclonal antibodies:The specific and affmitive pAbs yielded in our own laboratory were biotinylated using biotinamido-caproate-N-hydroxysuccinimide ester(BNHS) as reported.The biotinylated antibody was then purified on a PD-10 gel filtration column(Amersham Biosciences).(5) Preparation of biotinylated PAHs hapten:Haptens synthesized by Friedel-Crafts acylation reaction from four PAHs were biotinylated using biotinamido-caproate-N-hydroxysuccinimide ester(BNHS) as reported.The biotinylated haptens were then purified on a PD-10 gel filtration column(Amersham Biosciences).(6) Direct competitive Real-time Immuno-PCR(RT-IPCR) assay:Direct competitive Real-time Immuno-PCR(RT-IPCR) assay for the determination of four PAHs in environment was first developed.Coating antigens adsorbed to the PCR tubes treated with glutaraldehyde,is used to competing with the PAHs in combining biotinylated pAbs.Avidin is used as a bridge between the biotinylated pAbs and the biotinylated 103 bp reporter DNA.The reporter DNA is amplified and measured by RT-PCR under the optimize procedure.Standard curves of the four PAHs were produced.Cross-reactivity,recovery rates and detection limit were observed.Some environmental samples were analyzed with satisfactory results.(7) Indirect competitive Real-time Immuno-PCR(RT-IPCR) assay:Indirect competitive Real-time Immuno-PCR(RT-IPCR) assay for the determination of four PAHs in environment was first developed.Coating antigens adsorbed to the PCR tubes treated with glutaraldehyde,is used to competing with the PAHs in combining pAbs.Biotinylated goat anti-rabbit IgG were added in combining pAbs.Avidin is used as a bridge between the biotinylated goat anti-rabbit IgG and the biotinylated 103 bp reporter DNA.The reporter DNA is amplified and measured by RT-PCR under the optimize procedure.Standard curves of the four PAHs were produced.Cross-reactivity,recovery rates and detection limit were observed.Some environmental samples were analyzed with satisfactory results.(8) Antibody-Coated Real-time Immuno-PCR(RT-IPCR) assay:Antibody-Coated Real-time Immuno-PCR(RT-IPCR) assay for the determination of four PAHs in environment was first developed.Glutaraldehyde treated PCR tubes were adsorbed by Coating pAbs specific for PAHs,with which biotinylated PAHs hapten and antigen(Ag) PAHs are competing in combining. Avidin is used as a bridge between the biotinylated PAHs hapten and the biotinylated 103 bp reporter DNA.The reporter DNA is amplified and measured by RT-PCR under the optimize procedure.Standard curves of the four PAHs were produced. Cross-reactivity,recovery rates and detection limit were observed.Some environmental samples were analyzed with satisfactory results.(9) HPLC optimize and Comparison of the four methods:The chromatographic fractionation conditions for the separation of the four PAHs were optimized according to national Standard method of HPLC.Standard curves of the four PAHs were produced.Detection limit were observed.Some environmental samples were analyzed with satisfactory results.Comparison of the four methods was taken and their advantages and disadvantages were discussed.