Say Paraan na Colorimetry Tan Colorimetry Panamegley na ELISA

(1) pangipapatnag na kolor

Say Chromogenic ELISA so unor a kundang ed incubation reaction, diad sayan panaon et say enzyme so man-catalyze ed andiay kolor ya substrate pian makapawala na kolor iran produkto. Say temperatura tan oras na reaksion so siansia nin mangaapekta ed idakel na kolor. Diad sakey a panaon, say negatibon abut et nayarin mansiansian andiay kolor, tan say positibon abut et diad ilalabas na panaon tan say kolor to et napaaligwas. Say manepeg ya itagey na temperatura et makatulong pian maples so itatakken na kolor. Diad quantitative determination, say temperatura na reaksion tan say panaon kayari na pangiyarum na substrate et nepeg ya susto unong ed saray totontonen. Say kolor ya nadetermina ed kalidad et nayarin gawaen ed temperatura na kuarto, say oras et kaslakan ya ag kaukolan ya istrikton kontrolen, no maminsan unong ed positibon kontrol a bobon tan negatibon kontrol a bobon so kolor na situasyon et nayarin matukoy pian napatikeyan odino napalaknab so panaon na reaksion, nipanpanaon a panangukom.

Say kolor na OPD substrate et kaslakan ya ag-onaaralem kayari na 20-30 minutos ya reaksion ed temperatura ed paway odino 37 degree, insan say panaon na reaksion et nayarin naparukey pian napalaknab so background value. Say OPD substrate liquid et automatikon manguman so kolor to no nipaarap ed liwawa. Say reaksion na kolor et nepeg ya gawaen ed ambilunget. No asumpal lay reaksion na kolor, walay iyarum ya solusyon pian nasumpal so reaksion. Kayari na OPD product ya nasumpal ed sulphuric acid, say kolor to et manguman manlapud orange yellow ya anggad brown yellow.

Say TMB et ag apektado na liwawa tan nayarin iyan ed operating table ed temperatura na kuarto pian nanengneng so resulta na side reaction. Balet, pian naseguro so inkapekder na resulta na eksperimento, maabig no basaen iray resulta diad dugan panaon. Kayari na HRP ya panagtambal, say kolor na TMB et akasabi na manga 40 minutos, insan kalkalnan kinmapuy, tan sigpot lan kinmupas ya anggapoy kolor to kayari na 2 oras. Say TMB et mansumpal ed nanduruman solusyon. Saray enzyme inhibitor a singa say sodium azide tan sodium dodecyl sulfate (SDS) et nayarin mamatunda ed reaksion. Sayan klase na terminator et makapangiter met na kolor ya asul ed andukey a panaon (12-24 oras), sakey a maong ya terminator parad pakanengnengan. Niarum ni, saray nanduruman acidic terminator et manguman ed asul ya magmaliw ya duyaw, no say espisipikon wavelength (450nm) et nayarin usaren pian nabasa so absorption value.

(2) colorimetric

Antis na colorimetry, nepeg ya usaren so malinis ya absorbent paper pian pamaraan so likidon akapeket ed leksab na plato, insan say plato et nepeg ya iiyan ed colorimetric frame na enzyme standard colorimeter. Diad test ya walay soft plate bilang carrier, say plato et nepeg ya iiyan ed estandarten 96-hole frame antis ya nagawaan so panangikompara na kolor. Antis ya idagdag so substrate solution parad kolor, putoten so gilig na soft plate ya malinis, pian say plato et sigpot ya onyurong ed frame na yurongan.

No colorimetry, say zero point et nepeg ya i-adjust nin unona panamegley na distilled water, tan say substrate hole (say hole ya walay substrate solution ya inyarum ya anggapoy antokaman ya reaksion) tan say blank hole (say hole ya walay normal ya saline odino diluent imbes ya say specimen bilang interon proseso) et nepeg ya sukaten tan basaen pian nirekord so reagent status na sayan test. Insan saray blankon abut et nayarin usaren pian na-calibrate so zero point panamegley na distilled water, tan say absorbance na saray holes ed tagey et nepeg ya ibawas ed absorbance na saray blank holes, insan kalkulaen.

Say panangibalikas na resulta na colorimetric et opical density (OD) nensaman, balet say absorbence (A) et inusar unong ed saray totontonen, ya walaan na parehon kabaliksan. Say kaslakan ya paraan na pangibalikas et say pangisulat na absorption wavelength ed leksab a kawanan a suyok na letra A. Singa bilang, say absorption wavelength na OPD et 492nm, tan say paraan na pangibalikas et "A492nm" odino "OD492nm".

(3) enzyme standard colorimeter

Say enzyme standard colorimeter et tatawagen ya enzyme standard instrument, ya kaslakan et say photometer ya mansukat na absorbance na resulta na ELISA. Base ed nanduruman klase na solid carrier, wala ray espisyal ya desinyo parad plato, bead tan melag ya test tube. Dakel a kompanya na reagent so mangiiter na mipara ya instrumento na enzyme label. Saray manunan indeks na performance na enzyme plate instrument et: kapeles na panagbasa, inkasusto na panagbasa, pakaulit, grado tan nasukat a laknab, inka-linear tan arum nira. Say babasaen na maong ya microplate meter et kaslakan ya 0.001, ya say inkasusto to et ±1% tan say pakaulit to et anggad 0.5%. Singa bilang, no say A value na A hole et 1.083, sirin say tuan A value na hole no ikompara ed dagem et nepeg ya 1.083±0.01 (1.073~1.093). No paulit-ulit ya sukaten, say A value et nepeg ya 1.083±0.05 (1.078~1.088). Say sukat na enzyme plate analyzer et manduruma unong ed performance na enzyme plate analyzer. Say limitasyon na kaslakan ya enzyme plate analyzer et 0.000~2.000, balet say limitasyon na balon enzyme plate analyzer et pinalaknab ya anggad 2.900, odino mas atagey ni. Say balor ya onlampas ed tagey a limitasyon na sukat et mabetbet ya ipapanengneng na "*" odino "over" odino arum niran simbolo. Nepeg ya imanoen so pandumaan na nasukat a laknab tan say linear a laknab. Say linear range et mabetbet ya abeba nen say nasukat ya range. Singa bilang, say nasukat ya range na sakey ya enzyme plate instrument et 0.000~2.900, bangta say linear range to et 0.000~2.000 labat, ya nepeg ya imanoen no manggagawa na standard curve ed quantitative ELISA.

Say enzyme label instrument et ag nepeg ya iiyan ed silong na agew odino mabiskeg ya liwawa. Say temperatura na kuarto et nepeg ya 15~30℃legan na operasyon. Say instrumento et nepeg ya painitan na 15 anggad 30 minutos antis ya usaren, pian mas malet so resulta na panagbasa.

No sukaten so A value, say sensitibon absorption peak na produkto so nepeg ya pilien. Singa bilang, say OPD et manguusar na wavelength ya 492nm. Say unonan pankanawnawa et diad sankaabigan ya wavelength (W1) tan say komaduan pankanawnawa et diad insensitive wavelength (W2). Say posisyon na ELISA plate et ag-onalis ed baetan na duaran sukat. Singa bilang, say OPD et manguusar na 492nm bilang W1 tan 630nm bilang W2, tan say pinal ya asukat ya A value et say pandumaan na duara (W1-W2). Say dual wavelength reading et makabawas ed liwawa ya sengegan na saray gasgas odino marka na gamegamet ed pananginan.