Microchimica Acta

, Volume 172, Issue 1–2, pp 233–239 | Cite as

A real-time immuno-PCR method for detecting 3,3′,4,4′-tetrachlorobiphenyl

  • Han-Yu Chen
  • Hui-Sheng Zhuang
Original Paper


A reliable and sensitive direct competitive real-time immuno-PCR (rt-IPCR) assay was developed for the determination of coplanar 3,3′,4,4′-tetrachlorobiphenyl (PCB 77). PCB77 butanoic acid (γ-oxo-PCB77A) was synthesized as the hapten of PCB77, and a mixed anhydride reaction was used to couple the PCB77A to ovalbumin to form artificial coating antigen. The active ester method was used to couple the PCB77A to bovine serum albumin to form the artificial antigen. Male New Zealand white rabbits were immunized with immune antigen to obtain polyclonal antibodies (pAbs), with which, a novel rt-IPCR assay for determination of PCB77 was described. Using the optimized assay, the linear range for the determination of PCB77 is 10.0 fg mL−1 to 1 ng mL−1 with a correlation coefficient of 0.98 and a detection limit of 1.5 fg mL−1. The rt-IPCRs were tested for their cross-reactivity profiles using 4 selected congeners and 4 Aroclor products. The assays were highly specific for congeners but less specific for Aroclor1248. The rt-IPCR results for soil samples correlated well with the concentrations of PCBs obtained by GC/MS. Recovery was lower or higher with agitation but would still be acceptable for use in an on-site field test to provide rapid, semi-quantitative, and reliable test results for making environmental decisions.


Immunoassay Real time immuno-PCR Coplanar polychlorinated biphenyls Limit of detection 



We acknowledge the financial support by the National Natural Science Foundation of China (project no. 20677008), the science and technology commission of Shanghai municipality in China (key project of fundamental research no. 09JC1407600).


  1. 1.
    Smart J, Robert CH, Greaves J (1992) Methods for the analysis of persistent chlorinated hydrocarbons in tissues. J Chromatogr B 580:257CrossRefGoogle Scholar
  2. 2.
    Kunisue T, Watanabe M, Subramanian A, Sethuraman A, Titenko AM, Qui V (2003) Accumulation of features of persistent organochlorines in resident and migratory birds from Asia. Environ Pollut 125:157CrossRefGoogle Scholar
  3. 3.
    Fängström B, Strid A, Grandjean P, Weihe P (2005) A retrospective study of PBDEs and PCBs in human milk from the Faroe Islands. Environ Health 4:12CrossRefGoogle Scholar
  4. 4.
    Kannan K, Kajiwara N, Le Boeuf BJ (2004) Organochlorine pesticides and polychlorinated biphenyls in California sea lions. Environ Pollut 131:425CrossRefGoogle Scholar
  5. 5.
    Durand B, Dufour B, Fraisse D, Defour S, Duhem (2008) Levels of PCDDs, PCDFs and dioxin-like PCBs in raw cow’s milk collected in France in 2006. Chemosphere 70:689CrossRefGoogle Scholar
  6. 6.
    Beck V, Roelens SA, Darras VM (2006) Exposure to PCB 77 induces tissue-dependent changes in iodothyronine deiodinase activity patterns in the embryonic chicken. Gen Compar Endocrinol 148:327CrossRefGoogle Scholar
  7. 7.
    Sawatsubashi T, Tsukahara C, Baba K, Ohi E (2008) Development of new-type rapid analysis technology of polychlorinated biphenyls by using liquid chromatographic clean-up material (polyvinyl alcohol gel). J Chromatogr A 1177:138CrossRefGoogle Scholar
  8. 8.
    Rezaei F, Bidari A, Birjandi AP, Assadi Y (2008) Development of a dispersive liquid–liquid microextraction method for the determination of polychlorinated biphenyls in water. J Hazard Mat 158:621CrossRefGoogle Scholar
  9. 9.
    Wang SH, Deng LY, Lin SL, Zhuang HS (2006) Flow injection chemiluminescence for the determination of estriol via a noncompetitive enzyme immunoassay. Microchim Acta 155:421CrossRefGoogle Scholar
  10. 10.
    Chiu YW, Li QX, Karu AE (2001) Selective binding of polychlorinated biphenyl congeners by a monoclonal antibody: analysis by kinetic exclusion fluorescence immunoassay. Anal Chem 73:547CrossRefGoogle Scholar
  11. 11.
    Chuang JC, Miller LS, Davis DB, Van Emon JM (1998) Analysis of soil and dust samples for polychlorinated biphenyls by enzyme-linked immunosorbent assay (ELISA). Anal Chim Acta 376:67CrossRefGoogle Scholar
  12. 12.
    Del Carlo M, Mascini M (1996) Enzyme immunoassay with amperometric flow-injection analysis using horseradish peroxidase as a label. Application to the determination of polychlorinated biphenyls. Anal Chim Acta 336:167CrossRefGoogle Scholar
  13. 13.
    Kim IS, Setford SJ, Saini S (2000) Determination of polychlorinated biphenyl compounds in electrical insulating oils by enzyme immunoassay. Anal Chem Acta 422:167CrossRefGoogle Scholar
  14. 14.
    Tsutsumi T, Miyoshi N, Sasaki K, Maitani T (2008) Biosensor immunoassay for the screening of dioxin-like polychlorinated biphenyls in retail fish. Anal Chim Acta 617:177CrossRefGoogle Scholar
  15. 15.
    Van Emon JM, Chuang JC, Lordo RA (2008) An enzyme-linked immunosorbent assay for the determination of dioxins in contaminated sediment and soil samples. Chemosphere 72:95CrossRefGoogle Scholar
  16. 16.
    Lin YY, Liu GD, Wai CM, Lin YH (2008) Bioelectrochemical immunoassay of polychlorinated biphenyl. Anal Chim Acta 612:23CrossRefGoogle Scholar
  17. 17.
    Liu XY, Nakamura C, Nakamura N, Hirano T (2005) Detection of polychlorinated biphenyls using an antibody column in tandem with a fluorescent liposome column effect of albumin on phospholipase A2-catalyzed membrane leakageJ. Chromatogr A 1087:229CrossRefGoogle Scholar
  18. 18.
    Endoa T, Okuyamab A, Matsubara Y, Nishib K (2005) Fluorescence-based assay with enzyme amplification on a micro-flow immunosensor chip for monitoring coplanar polychlorinated biphenyls. Anal Chim Acta 531:7CrossRefGoogle Scholar
  19. 19.
    Sano T, Smith CL, Cantor CR (1992) Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates. Sci 258:120CrossRefGoogle Scholar
  20. 20.
    Niemeyer CM, Adler M, Blohm D (1997) Fluorometric polymerase chain reaction (PCR) enzyme-linked immunosorbent assay for quantification of immuno-PCR products in microplates. Anal Biochem 246:140CrossRefGoogle Scholar
  21. 21.
    Zhuang HS, Zhou C (2009) Determination of anthracene by real-time immuno-polymerase chain reaction assay. Anal Chim Acta 633:278CrossRefGoogle Scholar
  22. 22.
    Zhou C, Zhuang HS (2009) Determination of fluoranthene by antigen-coated indirect competitive real-time immuno-PCR assay. J Environ Monito 11:400CrossRefGoogle Scholar
  23. 23.
    Safe SH (1994) Polychlorinated biphenyls (PCBs): environmental impact biochemical and toxic responses and implications for risk assessment. Crit Rev Toxicol 24:87CrossRefGoogle Scholar
  24. 24.
    Tomoaki T, Yoshiaki A, Akira O (2006) Application of an ELISA for PCB 118 to the screening of dioxin-like PCBs in retail fish. Chemosphere 65:467CrossRefGoogle Scholar
  25. 25.
    Fránek M, Deng AP, Kolár V (2001) Direct competitive immunoassays for the coplanar polychlorinated biphenyls. Anal Chim Acta 444:131CrossRefGoogle Scholar
  26. 26.
    Fischer C, Fredriksson A, Eriksson P (2008) Neonatal co-exposure to low doses of an ortho-PCB (PCB 153) and methyl mercury exacerbate defective developmental neurobehavior in mice. Toxicology 244:157CrossRefGoogle Scholar
  27. 27.
    Chen HY, Zhuang HS (2009) Development and characterization of new polyclonal antibodies specific for three polychlorinated biphenyls China. Chem Lett 20(4):496Google Scholar
  28. 28.
    Zhou C, Wang QE, Zhuang HS (2008) Determination of phenanthrene by antibody-coated competitive real-time immuno-PCR assay. Anal Bioanal Chem 391:2857CrossRefGoogle Scholar
  29. 29.
    Yu YY, Wang QE, Zhuang HS (2006) Study on the new fluorescence immunoassays for the 2, 4, 6-trichlorophenol in the environmental water. Anal Lett 39:937CrossRefGoogle Scholar
  30. 30.
    Zhang MC, Wang QE, Zhuang HS (2006) A novel competitive fluorescence immunoassay for the determination of dibutyl phthalate. Anal Bioanal Chem 386:1401CrossRefGoogle Scholar
  31. 31.
    Lind K, Kubista M (2005) Development and evaluation of three real-time immuno-PCR assemblages for quantification of PSA. J Immunol Meth 304:107CrossRefGoogle Scholar
  32. 32.
    Joerger RD, Truby TM, Hendrickson ER, Young RM, Ebersole RC (1995) Analyte detection with DNA-labeled antibodies and polymerase chain reaction. Clin Chem 41:1371Google Scholar
  33. 33.
    Mauriz E, Calle A, Manclús JJ, Montoya A, Hildebrandt A, Barceló D, Lechuga LM (2007) Optical immunosensor for fast and sensitive detection of DDT and related compounds in river water sample. Biosens Bioelectron 22:1410CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  1. 1.College of Environmental Science and EngineeringShanghai Jiao Tong UniversityShanghaiChina
  2. 2.Department of Environment and Municipal EngineeringHenan University of Urban ConstructionHenanChina

Personalised recommendations