Indian Journal of Public Health Research & Development

Open Access Open Access  Restricted Access Subscription Access
Open Access Open Access Open Access  Restricted Access Restricted Access Subscription Access

A Pilot Project to Develop in Vivo Detection of Ricin through Fluorescence Sandwich ELISA Technique


Affiliations
1 Department of Biochemistry Saraswathi Institute of Medical Sciences Hapur, Panchsheel nagar, U.P., India
2 Department of Biochemistry, University College of Medical Sciences Delhi, India
3 Division of Pharmacology and Toxicology, Defence Research & Development Establishment, Gwalior, India
     

   Subscribe/Renew Journal


Aims & Objective: In the present study our main objective was to develop a fluorescence sandwich ELISA assay for in vivo detection of ricin from plasma of mice after intoxication. Ricin (also called RCA-II or RCA60), one of the most potent toxins and documented bioweapons, is derived from castor beans of Ricinus communis.

Material & Method: The assay was performed in plasma obtained from ricin treated and control mice after 30 minutes of exposure. The assay utilizes an antiricin rat polyclonal antibody to adsorb ricin from plasma of ricin treated mice. The rabbit polyclonal antiricin antibody is then used to form a sandwich, and anti-rabbit FITC-conjugate allows development of fluorescence. The developed fluorescence was read at an excitation-emission wavelength of 485/528nm. The graph was drawn by plotting the ricin concentration against the fluorescence (rf) values.

Result: We were able to detect more than 19.5ng/ml of ricin in assay buffer, 28ng/ml in diluted plasma (1:20480) at the dose of 10mg/kg and 27ng/ml of diluted Plasma (1:80) at the dose of 5mg/ kg body weight. The coefficient of variation ranged from 22% for ricin in assay buffer, 9% for 5mg/ kg body weight dose plasma, 14% for 10mg/kg body weight dose plasma and 4% for non treated control plasma.

Conclusions: The detection of ricin from serum/plasma of individuals exposed to ricin will be quite difficult at lethal dose, estimated for humans to be 1 to 10μg per kg body weight following inhalation or injection. Hence, more efforts are needed for the detection of ricin at its lethal exposure dose.


Keywords

Ricin, In vivo Detection, Fluorescence Sandwich ELISA
Subscription Login to verify subscription
User
Notifications
Font Size


  • Greenfield RA, Brown BR, Hutchins JB, Iandolo JJ, Jackson R, et al. Microbiological, biological, and chemical weapons of warfare and terrorism. Am J Med Sci. (2002), 323:326–40.

  • Balint GA. Ricin: the toxic protein of castor oil seeds. Toxicology (1974), 2:77–102.

  • Franz DR, Jaax NK. Ricin Toxin. Medical Aspects of Chemical and Biological Warfare; Chapter (1997), 32:631–642.

  • Doan LG. Ricin: mechanism of toxicity, clinical manifestations, and vaccine development. J Toxicol Clin Toxicol (2004), 42:201–208.

  • Audi J, Belson M, Patel M, Schier J, Osterloh J. Ricin poisoning: a comprehensive review. Jama (2005), 294:2342–2351.

  • Lin TT, Li SL. Purification and physicochemical properties of ricins and agglutinins from Ricinus communis. Eur J Biochem (1980), 105:453–459.

  • Nagler-Anderson C. Man the barrier: Strategic defences in the intestinal mucosa. Nat Rev Immunol (2001), 1:59–67.

  • Knigh B. Ricin-A potent homicidal poison. British Medical Journal (1975), 1:350-351.

  • Crompton R, Gall D. Georgi Markov-Death in a pellet, Medico-legal J (1980), 48:48-62.

  • Shea D, Gottron F. Ricin: Technical Background and Potential Role in Terrorism, Congressional Research Service, The Library of Congress, updated February (2004), accessed on October 28, 2008.

  • Lubelli C, Chatgilialoglu A, Bolognesi A, Strocchi P, Colombatti M and Stirpe F. Detection of ricin and other ribosome-inactivating proteins by an immunopolymerase chain reaction assay. Analytical Biochemistry (2006), 355:102-109.

  • Pradhan S, Kumar O. Detection of Ricin by Comparative Indirect ELISA Using Fluorescence Probe In Blood And Red Blood Cells. J Med CBR Def (2009), 7:508-518.

  • Weil CS. Tables for convenient calculation of median-effective dose, (LD50 or ED50) and instructions in their use. Biometrics (1952), 8:249– 263.

  • Endo Y, Tsurugi K. The RNA N-glycosidase activity of ricin A-chain. The characteristics of the enzymatic activity of ricin A-chain with ribosomes and with rRNA. J Biol Chem. (1988), 263:8735–8739.

  • Olsnes S. The history of ricin, abrin and related toxins. Toxicon (2004), 44:361–370.

  • Lord JM, Roberts LM, Robertus JD. Ricin: structure, mode of action, and some current applications. Faseb J (1994), 8:201–208.

  • Godal A, Olsnes S and Pihl A. Radioimmunoassay of abrin and ricin in blood. J. Toxicol. Environ. Health (1981), 8:409-417.

  • Poli MA, Rivera VR, Hewetson JF and Merill GA. Detection of ricin by colorimetric and chemiluminescence ELISA. Toxicon (1994), 32:1371-1377.

  • Shyu RH, Chiao DJ, Liu HW and Tang SS. Monoclonal antibody based enzyme immunoassay for detection of ricin. Hybrid Hybridol (2002a), 21:96-73.

  • Shyu RH, Shyu HF, Liu HW and Tang SS. Colloidal gold based immunochromatographic assay for detection of ricin. Toxicon (2002b), 40:255-258.

  • Rubina AY, Dyukova VI, Dementieva EI, Stomakhin AA, Nesmeyanov VA, Grishin EV and Zasedatelev AS. Quantitative immunoassay of biotoxins on hydrogel based protein microchips. Anal. Biochem (2005), 340:317-329.

  • Cook DL, Jonathan D and Griffiths GD. Retrospective identification of ricin in animal tissues following administration by pulmonary and oral routes. Toxicology (2006), 223:61-70.

  • Malcolm A and Brian P. A sensitive immunosorbent assay for the detection of ricin in blood, Defence Science and Technology Organisation (DSTO), (1997), Report No. DSTOTR- 0572.


Abstract Views: 669

PDF Views: 0




  • A Pilot Project to Develop in Vivo Detection of Ricin through Fluorescence Sandwich ELISA Technique

Abstract Views: 669  |  PDF Views: 0

Authors

Poonam Kachhawa
Department of Biochemistry Saraswathi Institute of Medical Sciences Hapur, Panchsheel nagar, U.P., India
Kamal Kachhawa
Department of Biochemistry, University College of Medical Sciences Delhi, India
Om Kumar
Division of Pharmacology and Toxicology, Defence Research & Development Establishment, Gwalior, India

Abstract


Aims & Objective: In the present study our main objective was to develop a fluorescence sandwich ELISA assay for in vivo detection of ricin from plasma of mice after intoxication. Ricin (also called RCA-II or RCA60), one of the most potent toxins and documented bioweapons, is derived from castor beans of Ricinus communis.

Material & Method: The assay was performed in plasma obtained from ricin treated and control mice after 30 minutes of exposure. The assay utilizes an antiricin rat polyclonal antibody to adsorb ricin from plasma of ricin treated mice. The rabbit polyclonal antiricin antibody is then used to form a sandwich, and anti-rabbit FITC-conjugate allows development of fluorescence. The developed fluorescence was read at an excitation-emission wavelength of 485/528nm. The graph was drawn by plotting the ricin concentration against the fluorescence (rf) values.

Result: We were able to detect more than 19.5ng/ml of ricin in assay buffer, 28ng/ml in diluted plasma (1:20480) at the dose of 10mg/kg and 27ng/ml of diluted Plasma (1:80) at the dose of 5mg/ kg body weight. The coefficient of variation ranged from 22% for ricin in assay buffer, 9% for 5mg/ kg body weight dose plasma, 14% for 10mg/kg body weight dose plasma and 4% for non treated control plasma.

Conclusions: The detection of ricin from serum/plasma of individuals exposed to ricin will be quite difficult at lethal dose, estimated for humans to be 1 to 10μg per kg body weight following inhalation or injection. Hence, more efforts are needed for the detection of ricin at its lethal exposure dose.


Keywords


Ricin, In vivo Detection, Fluorescence Sandwich ELISA

References