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.
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.
___
- 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.