Gıdalarda Antibiyotik Kalıntılarının Saptanması için Enzim İmmunoassay Geliştirilmesi

Year 2016, Volume: 9 Issue: 2, 122 - 126, 01.06.2016

Ulaş Acaröz Damla Arslan-acaröz Zeki Gürler

Abstract

Veteriner hekimlikte tedavi ve koruyucu amaçla çok sayıda farmakolojik madde kullanılmaktadır. En yaygın ve bilinçsiz kullanılanlarının başında ise antibiyotikler yer almaktadır. Antibiyotikler, hastalıkların tedavisinde önemli rol oynamalarına rağmen yanlış kullanımlarına bağlı olarak et, süt, yumurta, bal gibi hayvansal kökenli gıdalarda kalıntı sorununa neden olmaktadır. Antibiyotik kalıntısı içeren gıdalar halk sağlığı açısından tehlike oluşturmaktır. Halk sağlığını koruma amacıyla, Avrupa Birliği hayvansal kökenli gıdalarda farmakolojik aktif maddeler için maksimum kalıntı limitleri belirlemiştir. Günümüzde gıdalarda antibiyotik kalıntılarının tespiti için geliştirilmiş mikrobiyal inhibisyon testleri, immunoassayler ve kromatografik metotlar mevcuttur. Enzim-linked immunosorbent assay (ELISA) metodu laboratuvarlarda yaygın olarak kullanılmakta, güvenilir sonuçlar vermekte ve kısa sürede çok sayıda örnekle çalışma imkanı sunmaktadır. Bu metodun geliştirilmesinde dikkat edilmesi gereken en önemli aşama immunojenin sentezidir. Antibiyotikler küçük moleküler ağırlığa sahip olduğundan genellikle immun yanıt oluşturamazlar. İmmun yanıt oluşturabilmek için antibiyotik yeterli büyüklükte bir proteine bağlanarak immunojen elde edilir. Antibiyotiğin proteine bağlanması çoğunlukla karbodiimid, glutaraldehid ve aktif ester gibi metotlar ile gerçekleştirilir. Hangi metodun kullanılacağı ilgili antibiyotiğin kimyasal yapısına bağlıdır. Sentezlenen immunojen ile deney hayvanları immunize edilerek, spesifik antikorlar elde edilir. Daha sonra metot optimize edilerek çeşitli gıda maddeleri için geliştirilir.

Keywords

Hayvansal Kökenli Gıdalar, Antibiyotik Kalıntısı, Enzim-Linked İmmunosorbent Assay, Metot Geliştirme.

Enzyme Immunoassay Development for the Detection of Antibiotic Residues in Foods

Abstract

In veterinary medicine, a large number of pharmacological agents are used for prophylactic purpose and treatment. Antibiotics are in the first place among most commonly and inappropriately used pharmacological agents. Although antibiotics play a crucial role in the treatment of the illnesses, misuse of them causes residue problem in the food of animal origin such as milk, eggs, meat, and honey. The food containing antibiotic residues endanger public health. To protect the public health, maximum residue limits for pharmacologically active substances in foods of animal origin have been established by the European Union. To detect the antibiotic residue in foodstuff, several methods such as microbial inhibition tests, immunoassays and chromatographic methods have been established. An enzyme-linked immunosorbent assay (ELISA) method is widely used in a laboratory gives reliable results and offers to work with a large number of samples in a short time. The most important step in the development of this method is the immunogen synthesis. Antibiotics are generally not able to stimulate the immune response because of their low molecular weight. To stimulate the immune response, the immunogen is synthesized by conjugating antibiotics to a protein of sufficient size. The conjugation of antibiotic to protein is mostly performed using some methods such as carbodiimide, glutaraldehyde, and the active ester. Conjugation method is chosen depending on the chemical structure of used antibiotics. Specific antibodies are generated by immunizing the experimental animals with the obtained immunogen. Then, methods are optimized and developed for various foodstuff.

Keywords

Foods of animal origin, Antibiotic residue, Enzyme-linked immunosorbent assay, Method development

References

• Aalipour F, Mirlohi M, Jalali, M. Determination of antibiotic consumption index for animal originated foods produced in animal husbandry in Iran, 2010. J Environ Health Sci and Eng. 2014; 12(1): 42.
• Acaröz U. Entwicklung und Anwendung von generischen monoklonalen Antikörpern zum Nachweis von Chinolon-Antibiotika in Lebensmitteln. Doctoral Dissertation, Universitätsbibliothek der Ludwig-Maximilians-Universität, Müchen, 2015.
• Bittencourt MS, Martins MT, de Albuquerque FG, Barreto F, Hoff R. High-throughput multiclass screening method for antibiotic residue analysis in meat using liquid chromatography-tandem mass spectrometry: a novel minimum sample preparation procedure. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2012; 29(4): 508-516.
• Bremus AC. Optimierung und Evaluierung von Enzymimmuntests zum Nachweis von Cephalosporin-Antibiotika in Milch. Doctoral Dissertation, Ludwig-Maximilians-Universität, München, 2012.
• Burkin MA, Galvidis IA. Development of a competitive indirect ELISA for the determination of lincomycin in milk, eggs, and honey. Journal Agr And Food Chem. 2010; 58(18): 9893-9898.
• Campbell GS, Mageau RP, Schwab B, Johnston RW. Detection and quantitation of chloramphenicol by competitive enzyme-linked immunoassay. Antimicrob Agents And Chemother. 1984; 25(2): 205-211.
• Chen Y, Wang Z, Wang Z, Tang S, Zhu Y, Xiao X. Rapid enzyme-linked immunosorbent assay and colloidal gold immunoassay for kanamycin and tobramycin in swine tissues. J Agri Food Chem. 2008; 56(9): 2944-2952.
• EC. Commission Regulation (EU) No 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. Off J Eur Communities. 2010; L 15: 1–72.
• Engvall E, Perlmann P. Enzyme-linked immunosorbent assay, ELISA. 3. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin antigen-coated tubes. J Immunol. 1971; 109: 129-135.
• Galarini R, Diana F, Moretti S, Puppini B, Saluti G, Persic L. Development and validation of a new qualitative ELISA screening for multiresidue detection of sulfonamides in food and feed. Food Control. 2014; 35(1): 300-310.
• Gan SD, Patel KR. Enzyme immunoassay and enzyme-linked immunosorbent assay. J Invest Dermatol. 2013; 133(9): e12.
• Howard SJ, Catchpole M, Watson J, Davies SC. Antibiotic resistance: global response needed. The Lancet Infect Dis. 2013; 13(12): 1001-1003.
• Huet AC, Charlier C, Tittlemier SA, Singh G, Benrejeb S, Delahaut P. Simultaneous determination of (fluoro) quinolone antibiotics in kidney, marine products, eggs, and muscle by enzyme-linked immunosorbent assay (ELISA). J Agri Food Chem. 2006; 54(8): 2822-2827.
• Kim YJ, Cho YA, Lee HS, Lee YT, Gee SJ, Hammock BD. Synthesis of haptens for immunoassay of organophosphorus pesticides and effect of heterology in hapten spacer arm length on immunoassay sensitivity. Analytica Chimica Acta. 2003; 475(1): 85-96.
• Laxminarayan R, Duse A, Wattal C, Zaidi AK, Wertheim HF, Sumpradit N, Vlieghe E, Hara GL, Gould IM, Goossens H, Greko C, So A, Bigdeli M, Tomson G, Woodhouse W, Ombaka E, Peralta AQ, Qamar FN, Mir F, Kariuki S, Bhutta ZA, Coates A, Bergstrom R. Antibiotic resistance-the need for global solutions. The Lancet Infect Dis. 2013; 13(12): 1057-1098.
• Le T, Yi SH, Zhao ZW, Wei W. Rapid and sensitive enzyme-linked immunosorbent assay and immunochromatographic assay for the detection of chlortetracycline residues in edible animal tissues. Food Addit Contam Part B. 2011; 28(11): 1516-1523.
• Lim WS, Kim DH, Jin SY, Choi YS, Lee SH, Huh HJ, Chae SL, Lee Y. A case of fixed drug eruption due to doxycycline and erythromycin present in food. Allergy Asthma Immunol Res. 2013; 5(5): 337-339.
• Lipman NS, Jackson LR, Trudel LJ, Weis-Garci F. Monoclonal versus polyclonal antibodies: distinguishing characteristics, applications, and information resources. ILAR Journal. 2005; 46(3): 258-268.
• Märtlbauer E. Enzymimmuntests für antimikrobiell wirksame Stoffe. Enke Verlag, Stuttgart, Deutschland, 1993.
• inac o DG, Sa nchez-Baeza F, Marco MP. Molecular modeling assisted hapten design to produce broad selectivity antibodies for fluoroquinolone antibiotics. Anal Chem. 2012; 84(10): 4527-4534.
• ullukçu H. Toplum Kökenli İnfeksiyonlarda Olgularla Akılcı Antibiyotik Kullanımı. ANKEM Derg. 2013; 27(2): 111-112.
• Reig M, Toldrá F. Patents for ELISA tests to detect antibiotic residues in foods of animal origin. Recent Pat Food Nutr Agric. 2011; 3(2): 110-114.
• Ritter MA. Polyclonal and monoclonal antibodies. In Diagnostic and Therapeutic Antibodies. Humana Press. 2000; pp. 23-34.
• Spinks CA, Wyatt GM, Lee HA, Morgan MRA. Molecular modeling of hapten structure and relevance to broad specificity immunoassay of sulfonamide antibiotics. Bioconjugate chemistry, 1999; 10(4): 583-588.
• Strasser A, Usleber E, Schneider E, Dietrich R, Bürk C, Märtlbauer E. Improved enzyme immunoassay for group-specific determination of penicillins in milk. Food and agricultural immunology. 2003; 15(2): 135-143.
• Suryoprabowo S, Liu L, Peng J, Kuang H, Xu C. Development of a Broad Specific Monoclonal Antibody for Fluoroquinolone Analysis. Food Analytical Methods. 2014; 7(10): 2163-2168.
• Thirumala-Devi K, Mayo MA, Hall AJ, Craufurd PQ, Wheeler TR, Waliyar F, Subrahmanyam A, Reddy DVR. Development and application of an indirect competitive enzyme-linked immunoassay for aflatoxin M1 in milk and milk-based confectionery. J Agri Food Chem. 2002; 50(4): 933-937.
• Tijssen P. Practice and theory of enzyme immunoassays. Elsevier, Amsterdam, Holland, 1985.
• Uçan US, Aras Z, Zorlutuna M. Detection of canine brucellosis by a rapid agglutination test using Rhizobium tropici as antigen. Revue Med Vet. 2010a; 161: 51-56.
• Uçan US, Aras Z, Semacan A. Serodiagnosis of Brucella canis infection in dogs by a dipstick enzyme immunoassay. Eurasian J Vet Sci. 2010b; 26(2): 109-112.
• Van Weemen BK, Schuurs A. The influence of heterologous combinations of antiserum and enzyme-labeled estrogen on characteristics of estrogen enzymeimmunoassays. Immunochemistry 1975; 12: 667-670.
• Wang Z, Zhu Y, Ding S, He F, Beier RC, Li J, Jiang H, Feng C, Wan Y, Zhang S, Kai Z, Yang X, Shen J. Development of a monoclonal antibody-based broad-specificity ELISA for fluoroquinolone antibiotics in foods and molecular modeling studies of cross-reactive compounds Anal Chem. 2007; 79(12): 4471-4483.
• Wang Z, Mi T, Beier RC, Zhang H, Sheng Y, Shi W, Zhang S, Shen J. Hapten synthesis, monoclonal antibody production and development of a competitive indirect enzyme-linked immunosorbent assay for erythromycin in milk. Food Chem. 2015; 171: 98-107.
• Wise R, Hart T, Cars O, Streulens M, Helmuth R, Huovinen P, Sprenger M. Antimicrobial resistance: Is a major threat to public health. BMJ: British Med J. 1998; 317(7159): 609-610.
• Yüksek N. Etlerde antibiyotik kalıntılarının aranması üzerinde çalışmalar. J Fac Vet Med. 2001; 20: 85-90.
• Zheng N, Wang J, Han R, Xu X, Zhen Y, Qu X, Sun P, Li S, Yu Z. Occurrence of several main antibiotic residues in raw milk in 10 provinces of China. Food Addit Contam Part B. 2013; 6(2): 84-89.