Spectrophotometric-dual-enzyme-simult Aneous-assay In One Reaction Solution (SDESA) And Its Application To ELISA

To enhance analysis efficiency of enzymatic analysis, an absorbingstrategy is simultaneous assay of multiple enzymes in one reaction solution.This strategy has been proposed for ELISA, but no reports of this strategyinitiate the reactions of multiple enzymes as ELISA labels and quantifytheir substrates/products concomitantly in the same reaction solutions. Forspectrophotometric-dual-enzyme-simultaneous-assay in one solution（SDESA）, all reactions of label enzymes on chromogenic substrates aresimultaneously initiated in one solution and concomitantly monitored in thesame one solution. The feasibility of SDESA requires a group of properenzymes of strong specificity together with an equal number of suitablechromogenic substrates and special methods to resolve the overlappedabsorbance of chromogenic substances and process interference with theactions of label enzymes from involved substances for the quantificationsensitivity and limit of each component comparable to those via separateassay, respectively. Hence, SDESA-ELISA is investigated in this work.1. SDESA requires the following:（a） Enzyme A acts on Substrate A to release Product A bearing the longest difference absorbance peak （λ_A） muchlarger than that of Product B （λ_B） formed by Enzyme B action on SubstrateB; λ_B is close to the longest isoabsorbance wavelength of Product A andSubstrate A （λ₀）;（b） absorbance at λ_Aand λ₀is quantified via swiftalternation of detection wavelengths and corrected on the basis ofabsorbance additivity;（c） inhibition/activation on either enzyme by anysubstance is eliminated;（d） Enzyme A is quantified via an integrationstrategy if levels of Substrate A are lower than the Michaelis constant.SDESA was applied to simultaneous assay of γ-glutamyltransferase（GGT） and lactate-dehydrogenase （LDH）. Chemometrics of SDESA wastested with γ-glutamyltransferase and lactate-dehydrogenase ofcomplicated kinetics. γ-Glutamyltransferase releases p-nitroaniline （PNA）from γ-glutamyl-p-nitroaniline （GGPNA） with λ₀at344nm and λ_Aclose to405nm, lactate-dehydrogenase consumes reduced nicotinamidedinucleotide （NADH） bearing λ_Bat340nm. Kinetic analysis of reactioncurve yielded lactate-dehydrogenase activity free from inhibition byp-nitroaniline; the linear range of initial rates of γ-glutamyltransferase viathe integration strategy, and that of lactate-dehydrogenase after interferenceelimination, was comparable to those by separate assays, respectively; thequantification limit of either enzyme by SDESA at25-fold higher activityof the other enzyme remained comparable to that by a separate assay.2. To test potential application to enzyme-linked-immunoabsorbent assay （ELISA）, SDESA of alkaline phosphatase （ALP） andβ-D-galactosidase （BGAL） as ELISA labels, respectively, were examined.ALP releases4-nitro-1-naphthol from4-nitronaphthyl-1-phosphate with λ₀at405nm and λ_Aat458nm, β-D-galactosidase releases4-nitrophenol fromβ-D-（4-nitrophenyl）-galactoside with λ_Bat405nm. No interference fromsubstrates/products made SDESA of β-galactosidase and ALP simple forELISA of penicillin G and clenbuterol in one well，and the quantificationlimit of either hapten was comparable to that via a separate assay. Hence,SDESA is promising; phosphate or glycosides of4-nitro-1-naphthanol andesters or glycosides of4-nitrophenol are suitable pair of substrates, but anyproper pair of label enzymes should have sufficient specific activities in thesame buffer; the availability of a proper pair of label enzymes may makeSDESA-ELISA a practical approach of enhanced efficiency.3. Common glycosidases have compatible buffers and consistentoptimum pH, and tolerate negligible interference from substrates andproducts. The hydrolysis of β-D-（4-nitro-1-naphthyl）-galactopyranoside byβ-D-galactosidase action yields4-nitro-1-naphthol with λ₀at400nm and λ_Aaround458nm; the action of α-D-glucosidase onα-D-（4-nitrophenyl）-glucopyranoside yields4-nitrophenol with λ_Bat400nm. These spectral properties made SDESA of β-D-galactosidase andα-D-glucosidase applicable to ELISA by concurrently measuringabsorbance at405and450nm via swift alternation of detection wavelengths. Indeed, by using penicillin G-modified β-D-galactosidase andclenbuterol-modified α-D-glucosidase as tracers, competitive ELISA ofpenicillin G and clenbuterol in one reaction well via SDESA gave resultsconsistent with those by separate assay; precision and recovery of eitherhapten via SDESA were comparable to those via separate assay,respectively.4. Furthe screening of pairs of label enzymesFor favorable performance, the following criteria are applied to asuitable pair of chromogenic substrates and a proper pair of label enzymesof SDESA-ELISA.（a） One chromogenic substrate should have the longestisoabsorbance wavelength （absorptivity equal to that of its chromogenicproduct） equal or close to405nm and its chromogenic product has thelongest difference absorbance peak close to450or490nm, while thechromogenic product of the other chromogenic substrate should have thelongest difference absorbance peak close to405nm, so that thequantification sensitivity and limit of either chromogenic product on aconventional microplate reader can be comparable to those via separateassay under optimal conditions, respectively.（b） A proper pair of labelenzymes should have sufficient activity on a predetermined pair ofchromogenic substrates in the same buffer and tolerate negligibleinterference from substances involved in SDESA for robustnes of dataprocessing. In general, a pair of label enzymes with compatible optimum buffers is preferable. In either Tris-HCl or PBS buffer, ACHE both havefavorable activity on the acetylthiocholine. The SDESA of ECAP andACHE in50mmol/L pH8.5Tris-HCl and that of BGAL and ACHE in100mmol/L pH7.4PBS showed negligible interference from substancesinvolved in SDESA for robustnes of data processing. Therefore, theSDESA-ELISA with the pair of ECAP and ACHE or the pair of BGAL andACHE as label enzymes is promising.Hence, molecular engineering of one glycosidase acting on itssubstrate of4-nitro-1-naphthol and another glycosidase acting on itssubstrate of4-nitrophenol for high enough activities can make ELISA viaSDESA a practical new immunoassay platform with ever-enhancedefficiency and satisfactory sensitivity.