Inkonzo yokuvuthwa kwe-Antibody Affinity

I-Alpha Lifetech Inc.inokubonelela ngenkonzo yokuvuthwa kwe-antibody affinity ngokusebenzisa ubuchwepheshe bokuguqulwa kunye nokukhetha. Sidla ngokusebenzisa i-scFv njengefomathi ye-antibody kwiinkqubo zenkonzo yokuvuthwa kwe-affinity kunye nenkqubo ye-monovalent display phagemid esetyenzisiweyo ukunciphisa iziphumo ze-avidity ngexesha lokuhlolwa kwe-antigen-binding. Iqela lethu leengcali linokubonelela ngeenkonzo zokuvuthwa kwe-affinity kwi-antibody ye-single-domain.

I-Alpha Lifetech Inc.Iyazingca ngokubonelela ngeenkonzo ezipheleleyo ukuhlangabezana neemfuno ezahlukeneyo zabathengi behlabathi. Injongo yethu kukuqonda nokuhlangabezana neemfuno zabathengi abahlukeneyo kunye nokunceda ngazo naziphi na iingxaki ezizayo nezivelayo kumsebenzi wophando.

Yintoni Ukuvuthwa Kobudlelwane?

Ukuvuthwa kwe-Affinity yinkqubo apho ii-antibodies ziguquka ngokuphindaphindiweyo kwaye zikhethwa ukuze kuphuculwe ukuhambelana kwazo nee-antigens ezithile. Ngokuyintloko, ezifumaneka kwiziko le-germinal le-secondary lymphoid organs, ii-B cells zenza i-somatic hypermutation kunye nokukhetha phantsi kwempembelelo yee-T cells kunye nee-follicular dendritic cells.
Ngexesha lenkqubo yokuvuthwa kwe-affinity, ii-B cells ezine-low-affinity surface immunoglobulin receptors zivuselelwa zi-antigen concounters kwaye zenze ukwanda kunye nokwahlukana. Ngokukhula kwe-affinity ye-somatic hypermutation, utshintsho lwe-nucleotide olungaqhelekanga lwenzeka kwindawo eguquguqukayo ye-immunoglobulin genes, nto leyo ekhokelela ekuvelisweni kwee-antibody variants ezahlukeneyo. Emva kwe-somatic hypermutation, ukhetho oluqhutywa yi-antigen lwenziwa kwii-B cells ezine-mutated immunoglobulin receptors. Ii-B cells ezivelisa ii-antibodies ezine-antigen affinity eyongeziweyo zigcinwa ngokukhethekileyo kwaye zivunyelwe ukuba zande, ngelixa ii-B cells ezine-affinity enciphileyo okanye engatshintshanga zenzeka kwi-apoptosis. Le nkqubo yokuphindaphinda yokuguqulwa kunye nokukhetha ikhokelela ekuvelisweni kwee-B cell clones, ivelisa ii-antibodies ezine-affinity ephezulu kancinci kancinci kwi-antigen.

Inkonzo yokuvuthwa kwe-Antibody Affinity

Iingenelo eziphambili zeendlela ezisekelwe kwi-PCR kukuba utshintsho lujoliswe ngqo kwiqhekeza elikhulisiweyo; izinga leempazamo kulula ukulilawula kwaye indlela ikhawuleza kwaye kulula ukuliseta kwaye ayisebenzisi iikhemikhali eziyingozi. Kuyaziwa ukuba i-Taq DNA polymerase iphindaphinda i-DNA ngokuthembeka okuphantsi, ngenxa yokungabikho komsebenzi wokuhlola uvavanyo we-3' ukuya kwi-5'.I-Alpha Lifetech'sIqonga lobunjineli be-antibody lisebenzisa indlela ye-PCR enokuphosisa ukuguqula ikakhulu iindawo ze-CDR ngexesha lokwakhiwa kwelayibrari engaphantsi. Kwaye sihlala senza utshintsho kwiindawo ezingacwangciswanga ngokupheleleyo kuzo zonke iziqwenga ze-VH kunye ne-VL, ukuze sandise ulwahluko lwezakhi zofuzo lwelayibrari engaphantsi. Emva kokwakhiwa kwethala leencwadi lezakhi zofuzo le-antibody eliguquliweyo yi-PCR enokuphosisa, ukukhethwa kweendidi ze-affinity eziphezulu (ulwalamano lwe-scFv antibodies lunokufikelela kwi-10^8 - 10^10 M) kwenziwa ngokufaka isisombululo okanye kwi-antigen engashukumiyo eneemeko zokuhlamba ezilungiselelwe ukhetho oluxhomekeke kwisantya esingekho semgangathweni.
  
Izazinzulu kwiI-Alpha Lifetech Inc.Sebenzisa iintlobo ze-mutator ze-Escherichia coli mutAD5TM njengenye yeendlela ezininzi zokuguqula ukuze kungeniswe utshintsho olungacwangciswanga, kwaye ngaloo ndlela kuguqulwe ukuhambelana kunye nokubonakaliswa kweziqwenga ze-antibody eziphinda zihlanganiswe. Iimeko zokukhetha zinokuguqulwa kwiziqwenga ze-antibody ezinamazinga aphezulu emveliso. Iimeko zokukhula kwiiseli ze-mutator ze-Escherichia coli zinokulungiswa ukuze kungeniswe utshintsho olulodwa lwe-random point kwi-kilobase ye-DNA, phantse ilingane notshintsho lwe-codon olunye kwisiqwenga se-scFv. Emva kwemijikelo eliqela yokuguquka, ukuboniswa, kunye nokukhetha, singayiphucula i-affinity constant ukusuka kwi-10^5 - 10^6 M ukuya kwi-10^8 - 10^10 M.

Phucula amaqhinga okukhulisa ubudlelwane

Iindawo ezithile zee-antibodies zinokucwangciswa ngokwe-variance echaziweyo (njengokwenza ngokungacwangciswanga ngokupheleleyo kunye nazo zonke ii-amino acid ezingama-20 okanye ukwenza ngokungacwangciswanga ngokwe-biased kunye nee-amino acids ezikhethiweyo kwiipesenti ezimiselweyo) ukuphucula i-affinity. Sineendlela ezimbini zokuphucula i-affinity ye-antibody: okokuqala, ii-mutations ziziswa kwiindawo ezithintelweyo kwiindawo ezimisela i-complementarity (CDRs) nge-site-directed mutagenesis; okwesibini, ii-mutations ziziswa kwiindawo zonke ze-V-coding nge-random mutagenesis. Ngokusebenzisa ezi ndlela zimbini, i-conserved amino-acid sequences okanye yonke indawo yesakhelo se-antibody iya kutshintshwa zezinye ii-amino acids, ezidibene neetekhnoloji zethu ze-peptide library scanning, zinokunyusa kakhulu i-affinity yee-antibodies ezinomdla ukuya kwinqanaba eliphezulu kakhulu.

Ukudibana kwePhage Display-Antibody

Nje ukuba ilayibrari ye-scFv mutant yakhiwe, iindlela ezimbini zokuvavanya ziyafumaneka: i-biopanning kunye ne-solid-antigen sorting. Kwindawo yokuqala, i-subtractive concentration ye-substrate isetyenziselwa ukwahlula i-high-affinity antibody, njengoko i-low-affinity mutants iya kuhlanjwa, ishiye ii-high-avidity phage particles. Isicwangciso sesibini sisebenzisa i-antigen ebhalwe igama kwisisombululo, ukukhethwa okusekelwe kwi-equilibrium constant (Kd), kunye nokukhethwa okusekelwe kwi-binding kinetics, le ndlela yokukhetha ikhetha ii-antibody variants ezine-Kd ephuculweyo.

Ukuba unemibuzo, nceda ukhululeke ukunxibelelana nathi nangaliphi na ixesha.