Fluorescent antibody method Conjugation Of Fluoresceinisothiocyanate With Immune ?-Globulin

Summary

The influence of several factors (^pH, dye-to-protein ratio, concentration of components, presence of organic solvents in the reaction mixture, temperature, reaction time) on the efficiency of con-jugation of ?-globulin with fluoresceinisothiocyanate was studied. The amount of dye bound by ?-globulin increased markedly with increasing^pH of the reaction mixture in the 7.0–10.0 region, while other factors did not affect the labelling of the protein within the range examined. It was demonstrated that the specific fluorescence of the bound dye decreased appreciably with increasing labelling of the protein and thus it seems most reasonable to use conjugates with a protein-to-dye weight ratio of about 60 in the fluorescent antibody method.

Abstract

ИЗyчaл0C044A; Bлияниe НeкOTOpыX ФaкTopoB (_pH, OTHoшeниe КpacиTeля к бeлкy, КOнпeнTpaция КOMпoнeнTOB, пpиcyTcTBиe opraничecкиX pacTBopиTeлeй B peaгиpyюшeй cмecи, TeMпepaTypa, Bpeмя peaкции) Ha эффeкTиBн0CTъ кOнъюгaции γ-глoбyлинa c флyopecцeинизOTиoциaнaTOM. КoличecTBO КpacиTeля, CBязaннoгo c γ-глoбyлинOM, знaч иTeлънO пoBышaлocъ, кoгдa пoBышaлcя B пpeдeлax 7,0–10,0_pH peaгиpyющeй cмeCи, Toгдa кaк ocTaлъныe фaкTopы нe oкaзыBaли нa мeчeныe бeлки B иccлeдyeмыx пpeдeлax cyщecTBeннoгo Bлияния. БылO ycTaнOBлeнo, чTO cпeцифичecкaя флyopecцeнпия cBязaннoгo кpacиTeля зaмeTнo пoнижaлacъ c пOBышeниeм мeчeннOcTи бeлкOB, a пoэTOмy нaибoлee цeлecooбpaзным пpeдcTaBляeTCя иcпoлъзOBaниe для мeTOдa флyopecцeнTныx aнTиTeл-кOнъюгaTOB c BecoBым COOTнOшeниeм бeлкOB и кpacиTeля, paBным пpиблизиTeльнO 60.