Analytical and Bioanalytical Chemistry

, Volume 408, Issue 23, pp 6443–6451 | Cite as

Hybridoma as a specific, sensitive, and ready to use sensing element: a rapid fluorescence assay for detection of Vibrio cholerae O1

  • Parichehr Zamani
  • Reza H. SajediEmail author
  • Saman Hosseinkhani
  • Mehdi Zeinoddini
Research Paper


Over the last decade, isolation and purification of monoclonal antibodies, for diagnostic analysis, have been carried out using the hybridoma expression system. The present study describes a novel example of a detection system using hybridoma cells containing antibody against O1 antigen directly for V. cholerae diagnosis, which is a major health problem in many parts of the world, especially in developing countries. This method has advantages such as simplicity, ease of process, and it does not require manipulation of hybridoma cell. For this approach, an efficient amount of fluorescence calcium indicator, fura 2-AM, was utilized, which emitted light when the intracellular calcium concentration increased as result of antigen binding to specific antibody. More reliable results are obtained via this method and it is considerably faster than other methods, which has the response time of less than 45 s for detection of V. Cholerae O1. Also, the limit of detection was computed to be 50 CFU/mL (<13 CFU per assay). In addition, no significant responses were observed in the presence of other bacteria with specific hybridoma or other cell lines exposed to V. cholerae O1. Furthermore, this method was successfully applied to V. cholerae O1 detection in spiked environmental samples, including water and stool samples without any pretreatment. All results reveal that hybridoma cells can provide a valuable, simple, and ready to use tool for rapid detection of other pathogenic bacteria, toxins, and analytes.


Vibrio cholerae O1 Hybridoma Fura 2-AM Calcium increasing Fluorescence emission Monoclonal antibody 



This work was supported by the research council of Tarbiat Modares University and Ministry of Sciences, Researches, and Technology, Iran.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Maragos CM. Recent advances in the development of novel materials for mycotoxin analysis. Anal Bioanal Chem. 2009;395(5):1205–13.CrossRefGoogle Scholar
  2. 2.
    Pandey S. Hybridoma technology for production of monoclonal antibodies. Hybridoma. 2010;1(2):017.Google Scholar
  3. 3.
    Kramer K, Hock B. Recombinant antibodies for environmental analysis. Anal Bioanal Chem. 2003;377(3):417–26.CrossRefGoogle Scholar
  4. 4.
    Milstein C. The hybridoma revolution: an offshoot of basic research. Bioessays. 1999;21(11):966–73.CrossRefGoogle Scholar
  5. 5.
    Zamani P, Sajedi RH, Hosseinkhani S, Zeinoddini M, Bakhshi B. A luminescent hybridoma-based biosensor for rapid detection of V. cholerae upon induction of calcium signaling pathway. Biosens Bioelectron. 2016;79:213–9.CrossRefGoogle Scholar
  6. 6.
    Petrovick MS, Harper JD, Nargi FE, Schwoebel ED, Hennessy MC, Rider TH, Hollis MA. Rapid sensors for biological-agent identification. Lincoln Lab J. 2007;17(1):63–84.Google Scholar
  7. 7.
    Rider TH, Petrovick MS, Nargi FE, Harper JD, Schwoebel ED, Mathews RH, Blanchard DJ, Bortolin LT, Young AM, Chen J. AB cell-based sensor for rapid identification of pathogens. Science. 2003;301(5630):213–5.CrossRefGoogle Scholar
  8. 8.
    Wilson HA, Greenblatt D, Poenie M, Finkelman F, Tsien R. Crosslinkage of B lymphocyte surface immunoglobulin by anti-Ig or antigen induces prolonged oscillation of intracellular ionized calcium. J Exp Med. 1987;166(2):601–6.CrossRefGoogle Scholar
  9. 9.
    Takahashi N, Nemoto T, Kimura R, Tachikawa A, Miwa A, Okado H, Miyashita Y, Iino M, Kadowaki T, Kasai H. Two-photon excitation imaging of pancreatic islets with various fluorescent probes. Diabetes. 2002;51 Suppl 1:S25–8.CrossRefGoogle Scholar
  10. 10.
    Regehr W, Tank D. Selective fura-2 loading of presynaptic terminals and nerve cell processes by local perfusion in mammalian brain slice. J Neurosci Methods. 1991;37(2):111–9.CrossRefGoogle Scholar
  11. 11.
    Wang L, Wang R, Kong B-W, Jin S, Ye K, Fang W, Li Y. B cells using calcium signaling for specific and rapid detection of Escherichia coli O157: H7. Scientific reports. 5;2015:10598.Google Scholar
  12. 12.
    Robb M, Nichols JC, Whoriskey SK, Murphy JR. Isolation of hybridoma cell lines and characterization of monoclonal antibodies against cholera enterotoxin and its subunits. Infect Immun. 1982;38(1):267–72.Google Scholar
  13. 13.
    Colwell RR, Kaper J, Joseph SW. Vibrio cholerae, Vibrio parahaemolyticus, and other vibrios: occurrence and distribution in Chesapeake Bay. Science. 1977;198(4315):394–6.CrossRefGoogle Scholar
  14. 14.
    Islam MS, Drasar BS, Sack RB. The aquatic flora and fauna as reservoirs of Vibrio cholerae: a review. J Diarrhoeal Dis Res. 1994;87–96.Google Scholar
  15. 15.
    Gubala AJ. Multiplex real-time PCR detection of Vibrio cholerae. J Microbiol Methods. 2006;65(2):278–93.CrossRefGoogle Scholar
  16. 16.
    Kay BA, Bopp CA, Wells JG. Isolation and identification of Vibrio cholerae O1 from fecal specimens. Vibrio cholerae and cholera: molecular to global perspectives. Washington: ASM Press; 1994. p. 3–25.Google Scholar
  17. 17.
    Indrawattana N, Promptmas C, Wat-Aksorn K, Soontornchai S. Real-time monitoring of DNA hybridization for rapid detection of Vibrio cholerae O1. Anal Methods. 2014;6(19):7634–9.CrossRefGoogle Scholar
  18. 18.
    Khemthongcharoen N, Wonglumsom W, Suppat A, Jaruwongrungsee K, Tuantranont A, Promptmas C. Piezoresistive microcantilever-based DNA sensor for sensitive detection of pathogenic Vibrio cholerae O1 in food sample. Biosens Bioelectron. 2015;63:347–53.CrossRefGoogle Scholar
  19. 19.
    Yamazaki W, Seto K, Taguchi M, Ishibashi M, Inoue K. Sensitive and rapid detection of cholera toxin-producing Vibrio cholerae using a loop-mediated isothermal amplification. BMC Microbiol. 2008;8(1):94.CrossRefGoogle Scholar
  20. 20.
    Adams LB, Henk MC, Siebeling RJ. Detection of Vibrio cholerae with monoclonal antibodies specific for serovar O1 lipopolysaccharide. J Clin Microbiol. 1988;26(9):1801–9.Google Scholar
  21. 21.
    Hasan JAK, Bernstein D, Huq A, Loomis L, Tamplin ML, Colwell RR. Cholera DFA: an improved direct fluorescent monoclonal antibody staining kit for rapid detection and enumeration of Vibrio cholerae O1. FEMS Microbiol Lett. 1994;120(1/2):143–8.CrossRefGoogle Scholar
  22. 22.
    Wang D, Xu X, Deng X, Chen C, Li B, Tan H, Wang H, Tang S, Qiu H, Chen J. Detection of Vibrio cholerae O1 and O139 in environmental water samples by an immunofluorescent-aggregation assay. Appl Environ Microbiol. 2010;76(16):5520–5.CrossRefGoogle Scholar
  23. 23.
    Heidelberg J, Heidelberg K, Colwell R. Bacteria of the γ-subclass proteobacteria associated with zooplankton in Chesapeake Bay. Appl Environ Microbiol. 2002;68(11):5498–507.CrossRefGoogle Scholar
  24. 24.
    Alam M, Sultana M, Nair GB, Sack RB, Sack DA, Siddique A, Ali A, Huq A, Colwell RR. Toxigenic Vibrio cholerae in the aquatic environment of Mathbaria, Bangladesh. Appl Environ Microbiol. 2006;72(4):2849–55.CrossRefGoogle Scholar
  25. 25.
    Schauer S, Sommer R, Farnleitner AH, Kirschner AK. Rapid and sensitive quantification of Vibrio cholerae and Vibrio mimicus cells in water samples by use of catalyzed reporter deposition fluorescence in situ hybridization combined with solid-phase cytometry. Appl Environ Microbiol. 2012;78(20):7369–75.CrossRefGoogle Scholar
  26. 26.
    Chakraborty S, Alam M, Scobie HM, Sack DA. Adaptation of a simple dipstick test for detection of Vibrio cholerae O1 and O139 in environmental water. Front Microbiol. 2013;4:320.Google Scholar
  27. 27.
    George CM, Rashid M-U, Sack DA, Bradley Sack R, Saif-Ur-Rahman KM, Azman AS, Monira S, Bhuyian SI, Zillur Rahman KM, Toslim Mahmud M. Evaluation of enrichment method for the detection of Vibrio cholerae O1 using a rapid dipstick test in Bangladesh. Tropical Med Int Health. 2014;19(3):301–7.CrossRefGoogle Scholar
  28. 28.
    Nato F, Boutonnier A, Rajerison M, Grosjean P, Dartevelle S, Guenole A, Bhuiyan NA, Sack DA, Nair GB, Fournier JM. One-step immunochromatographic dipstick tests for rapid detection of Vibrio cholerae O1 and O139 in stool samples. Clin Diagn Lab Immunol. 2003;10(3):476–8.Google Scholar
  29. 29.
    Thiramanas R, Jangpatarapongsa K, Tangboriboonrat P, Polpanich D. Detection of Vibrio cholerae using the intrinsic catalytic activity of a magnetic polymeric nanoparticle. Anal Chem. 2013;85(12):5996–6002.CrossRefGoogle Scholar
  30. 30.
    Sharma A, Baral D, Rawat K, Solanki PR, Bohidar HB. Biocompatible capped iron oxide nanoparticles for Vibrio cholerae detection. Nanotechnology. 2015;26(17):175302.CrossRefGoogle Scholar
  31. 31.
    Barzamini B, Moghbeli M, Soleimani NA. Vibrio cholerae detection in water and wastewater by polymerase chain reaction assay. Int J Enteric Pathog. 2014;2(4):e20997.CrossRefGoogle Scholar
  32. 32.
    Yadava JP, Jain M, Goel AK. Detection and confirmation of toxigenic Vibrio cholerae O1 in environmental and clinical samples by a direct cell multiplex PCR. Water SA. 2013;39(5):611–4.CrossRefGoogle Scholar
  33. 33.
    Alam M, Sultana M, Nair GB, Siddique A, Hasan NA, Sack RB, Sack DA, Ahmed KU, Sadique A, Watanabe H, Grim CJ, Huq A, Colwell RR. Viable but nonculturable Vibrio cholerae O1 in biofilms in the aquatic environment and their role in cholera transmission. Proc Natl Acad Sci. 2007;104(45):17801–6.CrossRefGoogle Scholar
  34. 34.
    Leal N, Sobreira M, Leal‐Balbino T, Almeida A, Silva M, Mello D, Seki LM, Hofer E. Evaluation of a RAPD‐based typing scheme in a molecular epidemiology study of Vibrio cholerae O1, Brazil. J Appl Microbiol. 2004;96(3):447–54.CrossRefGoogle Scholar
  35. 35.
    Di Pinto A, Ciccarese G, Tantillo G, Catalano D, Forte VT. A collagenase-targeted multiplex PCR assay for identification of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus. J Food Prot. 2005;68(1):150–3.Google Scholar
  36. 36.
    Boyd EF, Moyer KE, Shi L, Waldor MK. Infectious CTXΦ and the vibrio pathogenicity island prophage in Vibrio mimicus: evidence for recent horizontal transfer between V. mimicus and V. cholerae. Infect Immun. 2000;68(3):1507–13.CrossRefGoogle Scholar
  37. 37.
    Choopun N, Louis VR, Huq A, Colwell RR. Simple procedure for rapid identification of Vibrio cholerae from the aquatic environment. Appl Environ Microbiol. 2002;68(2):995–8.CrossRefGoogle Scholar
  38. 38.
    Nhung PH, Ohkusu K, Miyasaka J, Sun XS, Ezaki T. Rapid and specific identification of 5 human pathogenic Vibrio species by multiplex polymerase chain reaction targeted to dnaJ gene. Diagn Microbiol Infect Dis. 2007;59(3):271–5.CrossRefGoogle Scholar
  39. 39.
    O'Hara CM, Sowers EG, Bopp CA, Duda SB, Strockbine NA. Accuracy of six commercially available systems for identification of members of the family Vibrionaceae. J Clin Microbiol. 2003;41(12):5654–9.CrossRefGoogle Scholar
  40. 40.
    Panicker G, Myers ML, Bej AK. Rapid detection of Vibrio vulnificus in shellfish and Gulf of Mexico water by real-time PCR. Appl Environ Microbiol. 2004;70(1):498–507.CrossRefGoogle Scholar
  41. 41.
    Senachai P, Chomvarin C, Namwat W, Wongboot W, Wongwajana S, Tangkanakul W. Application of tetraplex PCR for detection of Vibrio cholerae, V. parahaemolyticus, V. vulnificus, and V. mimicus in cockle. SE Asian J Trop Med Public Health. 2013;44:249–58.Google Scholar
  42. 42.
    Zeinoddini M, Saeedinia AR, Sadeghi V. Rapid detection of Vibrio cholerae using hexaplex PCR assay. J Police Med. 2014;3(2):78–84.Google Scholar
  43. 43.
    Sungkanak U, Sappat A, Wisitsoraat A, Promptmas C, Tuantranont A. Ultrasensitive detection of Vibrio cholerae O1 using microcantilever-based biosensor with dynamic force microscopy. Biosens Bioelectron. 2010;26(2):784–9.CrossRefGoogle Scholar
  44. 44.
    Lipp EK, Rivera ING, Gil AI, Espeland EM, Choopun N, Louis VR, Russek-Cohen E, Huq A, Colwell RR. Direct detection of Vibrio cholerae and ctxA in Peruvian coastal water and plankton by PCR. Appl Environ Microbiol. 2003;69(6):3676–80.CrossRefGoogle Scholar
  45. 45.
    Rivera ING, Chun J, Huq A, Sack RB, Colwell RR. Genotypes associated with virulence in environmental isolates of Vibrio cholerae. Appl Environ Microbiol. 2001;67(6):2421–9.CrossRefGoogle Scholar
  46. 46.
    Mukherjee P, Ghosh S, Ramamurthy T, Bhattacharya MK, Nandy RK, Takeda Y, Nair GB, Mukhopadhyay AK. Evaluation of a rapid immunochromatographic dipstick kit for diagnosis of cholera emphasizes its outbreak utility. Jap J Infect Dis. 2010;63(4):234–8.Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Parichehr Zamani
    • 1
  • Reza H. Sajedi
    • 1
    Email author
  • Saman Hosseinkhani
    • 1
  • Mehdi Zeinoddini
    • 2
  1. 1.Department of Biochemistry, Faculty of Biological SciencesTarbiat Modares UniversityTehranIran
  2. 2.Department of Bioscience and BiotechnologyMallek Ashtar University of TechnologyTehranIran

Personalised recommendations