Ragıp BAYRAKTAR, Lale BAYRAKTAR, Mehmet DAĞISTAN, Osman DEMİRALAY

ELİSA Tekniğinde Kullanılacak Reagentlerin Hazırlanması

Son zamanlarda soluble antijen veya antikorların enzim immuoassay ile solid faz hale getirilip teşhis . edilmesinde büyük gelişmeler kaydedilmiştir . immunoassay, diğer immunokimyasal tekniklerin en önemlilerinden biri olup aynı zamanda diğer tekniklerden daha az komplike, daha kolay ve daha çabuktur. ELiSA olarak bilinen enzim aracılığı ile antijen-antikor ölçülmesi tekniğine dayanan bu test Engvall ve arkadaşları , Van Weemen ile Schuurs tarafından bulunup geliştirilmiştir . (11 ,23) ELiSA yöntemi ile antikorların veya antijenlerin saptanmasında ortam ya antikor yada antijen ile kaplanarak duyarlaştırılır ve üzerine konan antijen veya antikor ile reaksiyona girer, üzerine ilave edilen enzim işaretli antiimmunoglobulin ve substrat ile renk değişimi şeklinde substratın parçalanma oranına göre test sonuçlandırılır ve mikropleytte oluşturulan bu reaksiyonda bir pleyt üzerinde çok sayıda numune i şlenebilir . Biz bu araştırmada bu test için gerekli olan reagentleri, konjugeyt, substrat ve konjugasyon solüsyonları üretimi olarak ele alıp ürettik vede laboratuvarımıza göre test edip standardizasyonunu gerçekleştirdik.

Anahtar Kelimeler:

ELISA, Reagentler, Ragıp BAYRAKTAR, Lale BAYRAKTAR, Mehmet DAĞISTAN, Osman DEMİRALAY, 1995, Etlik Veteriner Mikrobiyoloji Dergisi

The preparation of.reagents in Elisa techniques

The ELISA assay is an with antigen-antibody reaction system which uses an antibody coupled enzyme with the substrate in directly proportional to the amount of antibody or antigen present in the sample being tested. lmmunoassays are one of the most powerful of all immunochemical techniques. The next stage in the development of enzyme-immunoassays was the linkage of soluble antigensor antibody to an insoluble solid phase in a way in which the reactivity of the immunological component was retanied . This was the basis for the techniques known ELISA (Enzyme Linked lmmunoSorbent Assay) pioneered by Engvall and colleaques quick and easy, yielding information that would be difficult to determine by the othertechniques . (11 ,23) For this assay, a lot of diluents, buffers (coating, washing, substrate and conjugating buffers) antigens , conjugate , substrate and ext. are needed. In this study; these kind of solutions have been prepared, tested and standardized. According to demand, the ELISA reagents will be dispatched to Iaboratory all over the country.

PDF

___

ALLEN, J .C., KUNKEL, H.G. 1963: Production and purification of chicken lmmunoglobulins. Science. 139: 229-241.
ASHORA, P ., KROHN, K. 1986: Washing of ELI SA plates with running tap water. Journal of immunological Methods. 88: 141-142.
AVRAMEAS, S., TERNYNCK, T. , GUESDON, J.L.1978: Coupling of Enzymes to Antibodies and Antigens . Scand. J.lmmunol. 8: 7-23 .
BARBARA WiLSON,M., NAKANE, P.K.1978: Recent Developmentsin the periodate method of conjugating Horseradish Peroxidase (HRPO) to antibodies . lmmunofluorescense and related Staining Techniques. Elsevier/NorthHolland Biomedical Press . 1: 215-234.
BENEDICT, A.A ., BROWN, J.R., HERST, R.T . 1962: The temporal synthesis and some chromatographic and ultracentrifugal characteristic of chicken antibodies . J .Exper.med. 90 : 399-411 .
BRADFORD. M .M. 1976: A rapid and sensitive method for the q ua ntitation of m icrog ram q u antites of protein atilizing the prin ciple of proteind ye binding. Analytical Biochemistry. 72 :248-254.
BOORSMA, D.M .. STREEFKERT, J .G. 1978: lmproved method for seperation of peroxidase conjugates . lmmunofluorescense and related staining techniques . Elsevier/North Holland Biomedical press . 225-235
CASE, J .T. and ARDANS, A.A. 1985: Non spesific Reactions in an Enzyme Linked lmmunosorbent Assay caused by binding of lmmunoglobulins in situ to Egg-Propagated lnfectious Bronchitis virus. Avian Dis. 30: 149-153.
DIETLIND, H .. HERWART,A. 1986: Evolution of low molecular weight lmmunoglobulins, Dev. and Comp. lmmunology. 10: 379-380
ENGVALL,E., CARLSSON, H.E. 1976: Enzyme lmmunoassay ELiSA and EMIT. Methods in Enzymology. 70 : 419-438.
ENGVALL, E. , CARLSSON, H.E. 1976: Enyzme-linked lmmunosorbent Assay, ELiSA. First International symposium on lmmunoenzymatic techniques INSERM Symposium No:2: 135-182.
HARLOW,E., LANE,D. 1988: Antibodies. A laboratory manual. Cold Spring Harbor Laboratory. 1: 319-359, 553-592.
HATFIELD POL YTECHNIC, Division of Biological and Environmental Sciences, 28-31 MARCH 1988. Malecular Biology update in USA. 125-142.
HEBERT, G.A. , PATRICIA, L.P ., PITTMAN ,B. 1973: Determination of the optimal Ammonium Sulphate concentration for the fractionation . of Rabbit Sheep, Horse and Goat Antisera. Applied Micro. biology. 25: 26-36
JENSENIUS, J.C. , ANDERSEN, 1. , HAU, J., CRONE, M. and KOCH, C. 1981 : Eggs: Conveniently package0 Antibodies. Methods for purification of YOLK lgG. Journal of lmmunulogical Methods. 46: 63-68
LESLIE, G.A. , CLEM, L.W. 1969: Phylogeny of lg structure and function lll. lg of the chicken. J. Exp. Med. 130:1337-1339.
LOWRY, O.H. 1973: A simplified method forthe Quantative Assay of smail Amounts of Protein in Biologic Material. Analytical Biochemistry. 51 :654- 655.
MARQUARDT, W.W., SNYDER, D.B. and SCHLOTTHOBER, B.A. 1981 : Detection and Quantification of Antibodies to lnfectious Bronchitis virus by ELISA. Avian Dis 25:713-722.
MOCKETT, A.P.A. and DARBYSHIRE, J.H. 1981 : Comparative studies with anELISA for antibodies avian lnfectious Bronchitis virus. Avian Pathology. 1: 10-10.
MOCKETT, A.P.A and COOK, J.K.A. 1986: The detection of specific lgM to lnfectious Bronchitis virus in chicken serum using an ELiSA. Avian Path. 15: 437-446.
MONREAL, G., BAUER, H.J., and WIEGMANN, J.1985: Comparasion of the in h ibition test and Agar gel precipitation test for detection of antibodies to Avian lnfectious Bronchitis virus. avian Pathology. 14: 421-431 .
NAKANA, P.K., KAWAOI, A.1974: Peroxidase-labeled Antibody a new method of conjugation . The Journal of Histochemistry and Cytochemistry. 22: 1084-1091 .
NANDAPALAN,N., WILCOX, G.E., PENHALE, W.J. 1983: Production of antisera to the heavy chains of chicken immunoglobulins. Br. Vet. J. 139: 501-506.
NICHOLAS, R.A.J ., REED, N.E., WOOD, G.W., HEBERT, C.N., MUSKETI, J.C., THORNTON, DH.1985: Detection of antibodies againts infectious bursal disase virus: a comparison of three serological methods. Res. in. vet. Science. 38: 189-192.
PHYLAXIA Veterinary Research Institute, Hungary. 1987: Viral products methods booklet. 30-78.
SMART, I.J ., and GRIX, D.C. 1985: Measurement of Antibodies to infectious Avian Encephelomyelitis virus by ELiSA. Avian Pathology. 14: 341- 352.
SPECTOR, T. 1978: Refinement of the Coomassie Blue method of Protein Quantitation. Analytical Biochemistry 86: 142-146.
SYNDER, D.B., MARQUART, W. W., MALLINSON, E.T., ALLEN, D.A. SAVAGE, P.K. 1985: An Enzyme linked immunosorbent assay method for the simultaneous measurement of antibody titer to multiple viral, bacterial or protein antigens. Veterinary lmmunology and lmmunopathology. 9: 303-317.
SNYDER, D.B., MARQUART, W.W., MALLINSON, E.T., RUSSEK, L. 1982: Rapid Serological Profiling by Enzyme Linked lmmunosorbent Assay. 1. Measurement of Antibody Activity Titer Against Newcastle Disease Virus in a single serum dilution. Avian Dis. 27: 161-170.
SNYDER, D.B., MARQUART, W.W., MALLINSON, E.T., SAVAGE, P.K. and ALLEN, D.C. 1985: Rapid Serological Profiling by Enzyme Linked immunosorbent Assay. lll. Simultaneous measurement of Antibody titers to lnfectious Bronchitis, lnfectious Bursal Disease and Newcastle Viruses in a single serum dilution. Avian Dis. 28: 12-24.
SNYDER, D.B., MARQUART, W.W., MALLINSON, E.T., RUSSEKCOHEN, E., SAVAGE, P.K. and ALLEN, D.C . 1985: Rapid Serological Profiling by Enzyme-Linked lmmunosorbent Assay. IV: Association of lnfectious Sursal Disease Serology with Broiler Flock Performance. Avian Dis: 30: 139-148.
ViLLEGAS,P. 1987: Application of ELiSA. Avian virus disease, Laboratory of Veterinary Medicine, University of Georgia. 48-51.
VOLLER,A. BIDWEL,D.E and BARTLETT,A. 1979: Types of Assay and the applications of ELiSA. The Enzyme Linked lmmunosorbent Assay (ELi SA) 1. st. Edition,The Zoological Society of London. 3-8 , 10-58.
VOLLER,A. BIDWEL,D.E and BARTLETT,A. 1976: Microplate Enzyme lmmunoassays for the Immunodiagnosis of virus infections. Manual of Clinical lmmunology. 69: 506-512.
VUNAKiS, H.V., LANGONE, J .J. 1980: Prodution of reagent antibodies. lmmunochemical Techniques. Methodsin Enzymology. 70: 120-141 .
WEYBRIDGE Central Veterinary Laboratory, U.K. 1988: Viral Products Control Seetion Method Sheets. 3-70.
WILSON, M.B. and NAKANE, P.K. , 1978: Recentdevelopments in the periodate method of conjugating Horse Radish Peroxidase (HRPO) to antibodies. lmmunofluorescense and related staining techniques. Elsevier/North Holland Biomedical Press. 225-235.