of Enhanced Chemiluminescence: Immunochemical Applications of this Reaction. S. B. Vlasenko, A. A. Arefyevt, A. D. Klimov*, B. B. Kim, E. L. Gorovits, A. P.
JOURNAL OF BIOLUMINESCENCE AND CHEMILUMINESCENCE VOL 4 164-176
An Investigation on the Catalytic Mechanism of Enhanced Chemiluminescence: Immunochemical Applications of this Reaction S. B. Vlasenko, A. A. Arefyevt, A. D. Klimov*, B. B. Kim, E. L. Gorovits, A. E. M. Gavrilova and A. M. Yegorov
Division for Chemical Enzymology, Department of Chemistry, M. V. Lomonosov Moscow State University,
USSR ‘A. N. Bach Institute of Biochemistry, USSR Academy of Sciences, Moscow, USSR ‘Institute for Energy Problem of Chemical Physics, USSR Academy of Sciences, Moscow, USSR
The mechanism of peroxidase-catalysed oxidation of luminol by H202 was studied. The stopped-flow technique was used t o measure the rate constants for the reactions between the oxidized forms of peroxidase with luminol and the following substrates: p-iodophenol, p-bromophenol, p-clorophenol, o-iodophenol, rn-iodophenol, luciferin, and 2-iodo-6hydroxybenzothiazole. The correlation between kinetic parameters and the degree of enhancement was established. The effect of charged synthetic polymers and specific antibodies on the peroxidase activity in the enhanced chemiluminescent reaction was also studied. The close approach of an effector molecule t o the active site of the enzyme was found t o inhibit the enhanced chemiluminescent reaction. Novel homogeneous methods of luminescent immunoassay (LIA) for (1) antibodies t o insulin, (2) insulin and (3)antibodies t o trinitrophenyl group are proposed on the basis of regulatory facilities of the enhanced chemiluminescent reaction. Based on the enhanced chemiluminescent reaction a peroxidase flow-injection assay was developed and successfully tested in the flow-injection enzyme immunoassays for human IgG and for thyroxin (T4). The immunoassay proposed has a detection limit of IO-’M for IgG and lO-”M for T4, the overall time of the assay being 5-15 min. Keywords: Enhanced cherniluminescence; peroxidase; enzyme irnmunoassay; flow-injection analysis; IgG; T4
by a low light intensity and a high level of background reactions. These limitations were Horseradish peroxidase (EC 1 . 1 1.1.7) is widely basically overcome by finding out the effect of used as a marker in the enzyme immunoassay increasing light intensity on simultaneous perox(EIA). Having a wide substrate specificity, idase-catalysed oxidation of aminophthalhydraperoxidase catalyses a group of reactions produc- zides and some easily oxidi~ingsubstrates (White1983; Kricka et d.,1988). The use of ing light. Application of such reactions is limited head et d., 0884-3996189/0301h4-13$06.50
0 1989 by John Wiley 6i Sons. Ltd
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the enhanced chemiluminescent reaction in the solid-phase EIA allows one to achieve the requirements close to those of an ideal detection system, since a high light intensity with a long steady-state region and a low level of background reactions provide a low detection limit and a high specificity of enzyme determination. The complex nature of multistep light-emitting oxidation reactions is typical of the majority of chemiluminescent and bioluminescent processes. The mechanism of such reactions is of considerable interest, first of all because of the search for optimal conditions for assaying substances used as labels, but also for improving the time and concentration parameters of the immunochemical analysis and regulation of the chemiluminescent processes for obtaining homogeneous procedures for LIA. The mechanism of the chemiluminescent reaction of the peroxidase-catalysed oxidation of luminol was studied for the first time by Cormier and Prichard (1968). Recent investigations on the chemiluminescence mechanism of luminol and other cyclic hydrazides by the pulse-radiolysis method (Lind et al., 1983) provided substantial understanding of the mechanism of peroxidasecatalysed luminol oxidation (Lundin and Hallander, 1987). Unfortunately, understanding of the basic principles of enhancer action still remains elusive. It was postulated, that certain radical enhancers may react with luminol to form luminol radicals, thus increasing the overall rate of luminol radical production (Lundin and Hallander, 1987). Elucidation of the enhancement mechanism is still difficult because of the absence of quantitative kinetic data. In this work we report on the experimental data of the stopped-flow study of the kinetics of the enzyme-catalysed oxidation of luminol and substrate-enhancers (halophenols, luciferin, and 2iodo-6-hydroxybenzothiazole). The correlation between kinetic parameters and the degree of enhancement of light emission for easily oxidizing substrates was established. In order to study the paths to homogeneous LIA we investigated the regulation of chemiluminescent oxidations by both the specific effectors (antibodies) and nonspecific effectors (charged polymeric molecules). Advantages of the detecting system based o n the enhanced chemiluminescent reaction are exemplified by the optimization of flow-injection assay of human IgG and T4.
Luminol (‘Reachim’, USSR) was purified as described by Kalenichenko et ul. (1982). p-Iodophenol, p-bromophenol, p-clorophenol, o-iodophenol, rn-iodophenol, luciferin, 2-iodo-6hydroxybenzothiazole and poly-N-ethyl-4vinylpiridinium bromide (PEVP) were synthesized and purified in the Chemistry Department of Moscow State University. Horseradish peroxidase (specific activity 820 IU/mg, R Z = 2.9-3.0) was purchased from Biolar, USSR. Sodium periodate and Tween 20 were obtained from Merck, West Germany. All other rezgents were analytical grade. The antiserum to human IgG, horseradish peroxidase, T4 and trinitrophenyl group were obtained by immunizing rabbits with proteins or conjugate BSA-hapten. The insulin antiserum was obtained by the immunization of guinea-pigs. IgG fraction was prepared from t h e serum by either fractionation with (NH4)2S04or precipitating twice by polyethylene glycol, m.w. 6000 (Serva, West Germany) followed by passage through a DEAE-Toyopearl 650 (Toyo Soda, Japan) column. The IgG-peroxidase conjugate and insulinperoxidase conjugate were obtained by the periodate method (Nakane and Kawaoi. 1974). The conjugates were purified on a Toyopearl HW 55 column. The conjugate between peroxidase and antibodies to T4 was additionally purified by the affinity chromatography on a column with the BSA-T4 conjugate immobilized on CNBrSepharose. The peroxidase conjugate with trinitrobenzenesulphonic acid (TNBSA) (Serva, West Germany) was obtained according t o t h e method proposed by Ugarova el ul. (1078).
Measurement of the rate constants by stopped-flow technique
Stopped-flow model RA-401 Union Giken (Japan) was used. The measurements were made at the isobestic point of the native peroxidase and compound I, h = 426nm (Cormier and Prichard. 1968). The peroxidase concentration in solution was ca 10-”M. When the kinetics of reduction of
compound I into compound 11 was studied, the hydrogen peroxide concentration used was much higher than that of the enzyme (usually M), whereas the kinetics of reduction of compound I1 into peroxidase was studied at equal initial concentrations of the enzyme and hydrogen peroxide. Variation of concentrations of the reductants (luminol, 0-,m-,p-iodophenols, p bromophenol, p-clorophenol, luciferin, 2-iodo6-hydroxybenzothiazole) was limited, on the one hand, by realization of the meudo-firstorder conditions in the substrate and inability to follow processes faster than the ‘dead time’ of the stopped-flow device, on the other. The measurements were made at constant temperature (25°C).
A procedurefor investigationof the regulation of the enhanced chemiluminescent reaction by specific antibodies and positively charged synthetic polymers
To study the inhibition of the peroxidase activity of the conjugate peroxidase-TNBSA by antibodies to the enzyme and the effect of antibodies to hapten on this process. the conjugate, antibodies to the enzyme and hapten were incubated for 5min in Tris-HCI buffer (0.05M, pH 8.5, 0.05% Tween 20). The interactions between peroxidase and specific antibodies, as well as control experiments with the serum IgG fraction without antibodies to the enzyme, were carried out under the same conditions. To study the effect of PEVP on the peroxidase activity of the conjugate peroxidase-insulin the conjugate and PEVP were incubated for 5 min in Tris-HCI buffer (0.005 M, pH 8.5). Peroxidase was used instead of the conjugate in the control experiment. The interactions between the conjugate, PEVP and antibodies to insulin, as well as the interaction between the conjugate, PEVP, antibodies to insulin, and insulin, were studied under the same conditions. The enhanced chemiluminescent reaction was initiated after the incubation step by simultaneous addition of solutions of luminol, luciferin, and hydrogen peroxide for the peroxidase-TNBSA conjugate and the solutions of luminol, piodophenol and hydrogen peroxide for the
S. B. VLASENKO ETAL.
peroxidase-insulin conjugate. Determinations of the maximal chemiluminescence intensity were performed on a luminometer (LKB-1251, LKBWallac, Finland).
Flow-injection enzyme immunoassayfor human IgG and T4
A Tecator FIA 5020 instrument equipped with two pumps and a double-injection valve was used for assaying IgG (Fig. l(a)) and T4 (Fig. l(b)). When IgG was analysed, phosphate buffer (0.01 M, pH 7.4, 0 . 1 4 M NaCl, 0.05% Tween 20) was fed via polyethylene tubing (1 and 1 ‘) using a peristaltic pump 1, and the sample 200pl and 200 pl of the rabbit peroxidase-lgG conjugate was injected with a double-valve injector 3. Through an additional valve 4 (4m long) the point A was reached in one minute by the solutions of antigen and antibody-peroxidase conjugate. A polystyrene bead 6 (Abbott, 6mm diameter) with the immobilized rabbit antibodies to human IgG was placed in a reaction cell. When the antigen sample reached the bead, pump 1 was stopped. After switching on pump 1 , the bead was washed by the buffer contained in valve 4. When the conjugate sample reached the bead, pump 1 was stopped for the second time. After the incubation, the immunosorbent was washed by the buffer to remove the unreacted conjugate. Then the bead was transferred from the reaction cell 5 to the cell 7 of luminometer LKB-1251 and the enhanced chemiluminescent reaction was initiated by simultaneous addition of the solutions of luminol, p-iodophenol, and hydrogen peroxide. While assaying T4, 3 0 ~ 1of both the T4 solution to be analyzed and the peroxidase-IgG conjugate were introduced by the two-channel microinjector 3 in two flows 1 and 1 ’ of Tris-HC1 buffer (0.05 M, pH 8.5, 0.05% Tween 20). A precise adjustment of connecting tubes provided simultaneous mixing of the two flows at point A . The presence of the additional loop 4 was a requirement of continuous monitoring. At the time when the zone of the reaction of the sample with the conjugate was in the centre of the loop, pump 1 was switched off. After switching on the pump, the reaction zone consisting of a mixture of the reacted and unreacted labelled antibodies was placed o n column 5 with the immobilized BSA-T4
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INJECTOR * ’ ” 1‘
FA KCATOR-5020 Figure 1. FIA set-up for chernilurninescent sandwich enzyme immunoassay for human IgG (a) and pseudohomogeneous enzyme irnmunoassay for T4 (b)
and the pump was again switched off. After the incubation the flow was combined (at point B) with solutions of luminol, p-iodophenol (2), and hydrogen peroxide (2’) and was driven into a flow cell 6 of a luminometer LKB-1250. Pumps were switched off and chemiluminescence intensity was measured for 3 min.
RESULTSAND DISCUSSION Mechanism of the peroxidase-catalysed oxidation of luminol
The scheme for the reaction of the peroxidasecatalysed oxidation o f luminol was first suggested
S . B. VLASENKO ETAL.
by Cormier and Prichard (1968), though at that time the secondary reactions of luminol radicals had yet to be studied. The detailed investigation of luminol chemiluminescence and of other cyclic phthalhydrazides by impulse radiolysis enabled this gap to be filled (Lind et al., 1983). The experimental test and mathematical modelling of the scheme, together with the secondary reactions of luminol radicals, have been recently conducted (Lundin and Hallander, 1987). We have also analysed the kinetic data within the limits of the reactions analogous to the suggested one.
(10) A' (11)
AH-+H+ pK', = 6.35  A'+
AP+N2 + h ~ 2.2 x 105c-' I21
native enzyme compound 1 and I 1 luminol different ionic forms of a luminol radical diazaquinone different ionic forms of peroxide adduct 3-aminophthalate su bst ra t e-en hancer radical product of oxidation of substrate-enhancer
*Constants werc obtained in prcscnt work [ 11 = Cormier and Prichard. 1968. 121 Lind ('I a l . , 1983.
The given model satisfactorily describes a number of experimental dependencies; however, some results are not explained by this scheme. This fact points to the incompleteness of the kiOF H+ HZO2 model and the necessity f o r its correction. p K , = 11.58 The dominant reactions leading to chemiluminescence are the reactions (1)-(9), (12)-( 13) AT + A7 i I l t + A -t AH('diazaquinone' channel) and (1)-(S), (lo)-( 1 1 ) . 2k(, = 5.0 x 10XM-'c'-' ( 2 ) (12)-( 13) ('superoxide' channel). The contribution of the 'superoxide' channel to the resulting A + 1 4 0 ; --+ (A021I)signal o f chemiluminescencc is shown to be h l = 5.2 X 10 M - I c - ' (21 significant only at very low peroxidase concentrations ( oiodophenol > m-iodophenol; p-iodophenol > p-bromophenol > p-clorophenol; 2-iodo-6hydroxybenzothiazole > luciferin. Similar dependencies for substituted iodophenols are probably associated with a facile loss of electron by the substrate molecule and with better stabilization of the radical formed by a para-substituted molecule compared with ortho- and meta- ones. For ~~
various halophenols the order found can also be accounted for in terms of structural features of a molecule provided by two halo atoms at a para position. One can speculate that the iodo-substituted derivative loses an electron most readily, giving the most stable intermediate radical, followed by bromo- and chloro- derivatives. Luciferin and 2-iodo-6hydroxybenzothyazole, estimated by the rate constants of the enzymatic steps, are close to p-iodo- and p-bromophenol. For peroxidase analysis by enhanced chemiluminescence, the following conditions are typical: [H202] > [S-] [AH-]. In the steady-state conditions of general intermediates (E, E , . EZ. A:), taking into consideration that k L % k 3 , k4 9 ks and k l . [H202] 9 k2 . [AH-], k4 . IS-], the rate of luminol radicals generation in the absence and in the presence of substrate-enhancer can be obtained thus:
(2,2 + -
. k s . [ A H - ] - [El,,
As is followed from the scheme, ICL
Using the values o f the estimated constants k4 and kS for different substrates the degree of enhancement (f) in the presence of enhancer were calculated (Table 1). The reason for acceleration -
Table 1. Rate constants of oxidation of substrates at the enzymatic steps
p-iodophcnol p-bromophenol p-clorophenol o-iodophenol m-iodophenol lucifcrin 2-iodo-6-hydroxyhcnzothiazolc
(MI f 0.12) x (8.2') + 0.21) x (2.42 f 0. 14) x (3.60 f 0.12) x (1.14 +_ 0.44) x (1.25 f 0.14) x (3.41 5 0.1s) x
10' 10" 10"
lo" 10" 10'
(3.41 +_ om) x (5.73 f 0.34) x (3.62 + 0.21) x (3.52 f 0.16) x (4.36 i 0.15) x (8.96 0.62) x (1.15 +_ O M ) x
8.2 14.5 10' 66.9 loJ 102.3 10' 26.7 io5 13.0 IO(~ 20.7
4.7 2.9 1.0 0.x
S. B. VLASENKO ETAL.
is obvious. During mutual oxidation of luminol and substrate-enhancer El and E2 react generally with enhancer (as k,.[AH-] G k 4 . [ S - ] and k3.[AH-] 4 k 5 . [ S - ] , [AH-] [S-I), and as k4/k5 < k2/k3,so stationary concentration of the highly active form El in the presence of enhancer is larger than in their absence. The increase of E , concentration leads to the increase of luminol radicals generation rate (GA = (k2 . [El] + k , . [E2]) [AH-]). The estimated values of enhancement correlate with experimentally observed values for the studied number of substrates. Although, the estimated values of enhancement are much less in quantity than experimental ones (Kricka et uf., 1988). This fact proves that there are some other mechanisms of enhancement, perhaps, related to the reactions of enhancer radicals with luminol or intermediates of its oxidation. There are n o experimental data for such reactions at the present. The enhanced chemiluminescent reaction, because of its physicochemical parameters, corresponds to a greater extent to the requirements of immunoassay systems (Kricka et uf., 1988). Both the multicomponent nature of the system and the presence of the large number of elementary steps generating light emission makes the system sensible to fine regulatory effects and opens the way to homogeneous LIA techniques.
Inhibition of the peroxidase activity by antibodies to the enzyme. The assay of antibodies to hapten It has been shown that peroxidase polyclonal antibodies inhibit the enzyme activity in oxidation of chromogeneous substrates (Conroy and Marruci. 1976). However, the level of inhibition does not usually exceed 50%. We have studied the effect of antibodies on the peroxidase activity in the enhanced chemiluminescent reaction. Fig. 2(a) presents the inhibition of the peroxidase activity by the rabbit antibodies in the simultaneous oxidation of luminol and luciferin by hydrogen peroxide. igG fractions from unspecific serum were used ar a control. If the antibodies are in excess, a complete inhibition is observed. Polyclonal serum probably contains antibodies able to bind in close proximity to the peroxidase active site and to affect the reactions involved in light emission. The antibodies to trinitrophenyl group had no effect o n the peroxidase activity of
the peroxidase-TNBSA conjugate. It was found, however, that addition of antibodies to hapten to the conjugate inhibited by antibodies provided partial restoration of the enzymatic activity (Fig. 2(b)). It is likely that the antibodies to hapten partly substitute the conjugate from the complex with the inhibiting antibodies. Fig. 2(b) is a typical curve of titration of antibodies. To this end, the inhibition of the peroxidase activity by antibodies and its restoration on interaction of the enzyme-hapten conjugate with antibodies to hapten can be used for development of homogeneous LIA of antibodies to hapten or hapten itself based on the competitive mechanism. Inhibition of the enhanced cherniluminescent reaction by positively charged polymers. The assay of insulin and insulin antibodies
The influence of polycations on the chemiluminescent reaction has been studied in the case when the reaction is catalysed by insulin-modified peroxidase. Representative data on the effect of synthetic P E W on the chemiluminescence are presented in Fig. 3(a). In the case of the enhanced chemiluminescent reaction catalysed by the insulin-peroxidase conjugate bearing a large negative charge at pH 8.5 brought about by the antigen and, hence, increasing a polymer concentration it is possible completely to inhibit light emission (Fig. 3(a)). If the process is catalysed by unmodified peroxidase, addition of polycation leads to a minor decrease in chemiluminescence. This effect can be accounted for in terms of electrostatic interactions between a negatively charged conjugate molecule and the polycation as a result of which the active site of the enzyme comes into close proximity with the positively charged polyelectrolyte. A contact between the active zone of the chemiluminescent reaction and PEVP may considerably alter the kinetic parameters of enzymatic and subsequent steps. Addition of low-molecular-weight electrolytes even at low concentrations completely eliminates the inhibition supporting an electrostatic nature of the effect observed. A study of the interaction between the peroxidase-insulin conjugate with insulin antibodies in the presence of polycation has shown that the formation of the specific immune complex eliminates the inhibition. The chemiluminescent signal
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I , V CL 1 -
0 .5 -
CONCENTRATION OF IgG, M Figure 2. Inhibition of peroxidase activity by specific antibody Concentration of enzyme 5 x 10 '' M (a) Restoration of chemilurninescent intensity by antibody to hapten (b) Curves 2 control experiment with nonspecif c IgG
increases proportionally to the concentration of antibodies added (Fig. 3(b)). In the control experiment when nonspecific immunoglobulin was added there was no such an increase. One can
assume therefore that the specific interaction with antibodies competes with the electrostatic binding. The effect investigated may be used for assaying insulin antibodies.
2 4 6 8 x lo-’’ CONCENTRATION OF INSULIN, M
CONCENTRATION OF PEVP, M
1.5 x 10-9
CONCENTRATION OF IgG. M
Figure 3. Chemiluminescence intensity versus (a) concentration of PEVP (for conlugate (1) and for native enzyme (Z)), concentration of peroxidase and conjugate 2 x 10 l 1 M, (b) concentration of antibody to insulin (concentrations of conjugate 2 x 10 ” M and PEVP 8 x 10 ’M, (c) concentration of insulin (concentrations of conlugate 2 x 10 ” M, PEVP 8 x 10 ’M and antibody 7 x 10 ’OM)
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Insulin was introduced into the system when concentrations of the conjugate, polymer, and antibodies produced an intensity of chemiluminescent signal, 60-8096 of that of the free conjugate. Fig. 3(c) shows the plot of the intensity of chemiluminescence against a concentration of the hormone. This plot can form the basis for the competitive chemiluminescent assay of insulin. Homogeneity of the immunochemical steps reduced the analysis time to 7-10 min. Sensitivity of the hormone assay in solution was 2 X 10-" M. Flow-injection immunoassayof human IgG
To optimize the analytical procedures for flowinjection assay of human IgG the kinetic features of the binding of the antigen had to be determined with specific antibodies adsorbed on the surface of polystyrene beads. Absorbtion curves for the antigen on the immunosorbent are presented in Fig. 4(a) and were obtained by varying the time of incubation of the antigen sample from 1 to I0min. At high initial antigen concentrations, saturation is rapidly achieved. At lower antigen concentrations kinetic curves become linear. Fig. 4(b) shows the interaction between labelled antibodies and immobilized antigen-antibody com lex at an initial concentration of IgG of 10-'M and the time of its interaction of with the immunosorbent of 2min. At a high initial concentration of labelled antibodies the system equilibrates within 5 min, thereby lowering the assay time for both steps of the immunoassay sandwich-scheme. Calibration curves for the human IgG assay over a wide concentration range, and at different incubation times for the antigen and immunosorbent are shown in Fig. 4(c). Comparison of the curves shows that a decrease in the incubation time of the antigen shifts the working region o f the curves towards increasing the concentration of the analyte. In particular, incubation for 1 0 and 1 min provide the sensitivity of analysis of lo-" and lO-'M, respectively, since the absolute values of chemiluminescent signals are lowered. In summary, the kinetic study of antigenantibody interactions studied by flow-injection analysis has allowed us to propose a rapid method of chemiluminescent irnmunoassay as exemplified by human IgG. Using t h e advantages of enhanced chemiluminescence we have succeeded in car-
rying out both steps of the sandwich-scheme of the immunoassay in a kinetic fashion, by decreasing the time of the immunochemical steps as well as label detection. The method developed is characterized by a total time of a single IgG determination of 10-15 min and a lower limit of 1 x 10-"M. Flow-injection enzyme immunoassayof T4
We have studied the kinetics of binding of T4 and peroxidase-labelled rabbit antibodies in homogeneous solution. Data are shown in Fig. 5(a). The latter were obtained by varying the incubation time from 0.5 to 10min. To achieve this, pump 1 was switched off when the reaction zone of the sample and the labelled antigen was in the centre of loop L. The homogeneous nature of the process provides faster equilibration compared, for instance, with the solid-phase reaction of IgG and the specific antibodies immobilized on the beads. It takes 2min for the 75% binding at concentration of T4 equal to 5 x lo-" M. The intensity of chemiluminescence versus the time for contact of the flow of the reaction mixture with T4 immobilized on a CNBrSepharose is shown in Fig. 5(b). Some time is necessary to achieve a constant signal level and this depends on the T4 concentration in t h e sample. It takes 2 min to achieve the 80% binding at hormone and conjugate concentrations of 1 x 10-'M and 3 x 10-"M. respectively. Carrying out the immunochemical steps at lower concentrations of hapten analysed leads to the nonequilibrium binding on the column. The possibility for determining the chemiluminescence intensity of specific antigen-antibody interaction, coming from the data presented, allows studies to be made on the kinetic regime of these reactions. The plot of the intensity of chemiluminescence against the T4 concentration at fixed parameters of the analytical procedure (concentration of the antibody-peroxidase conjugate o f 3 x lo-" M. time o f the contact of hapten analysed with the conjugate 2 min, time of incubation of affinity column with the mixture 1 min, time o f incubation of the peroxidase-label with the substrate mixture ( 3 min) is shown in Fig. S(c). A calibration curve involves the T4 concentration range of 1 x 10-"-5 x 10-"'M. The lowest limit of hormone detection is 1 x 10-"'M. In conclusion, a detailed study of the kinetics ol'
10-7 C O H C E W I R A I I O H OF H U H A H I g C , ll
Figure 4. (a) Kinetics of the interaction between antigen and immunosorbent. 1.0 X Plot was obtained for the following initial concentration of antigen (I) 1 0 - 6 M , ( 2 ) 5 01~0 ~ ’ M . ( 3 ) 2 , 5 x 10 7 M , ( 4 ) 1 . 3 x 10-7M.(b)Kineticsofthe interaction between antibody-enzyme conjugate (1.O x lo-’ M) and immobilized antigen-antibody complex. The first step of the analysis carried out with 2 rnin incubation time. Initial concentration of antigen was 1.0 X 10-6M. (c) Standard curves of human IgG in a flow-injection analysis using quantitation of peroxidase label with an enhanced luminescent reaction. The incubation time for the first step of the assay varied as follows: (1) 10 rnin, (2) 5 rnin. (3) 2 min. (4) 1 min. The second step of the analysis carried out with 2-rnin incubation time. Initial concentration of antibody--enzyme conjugate was 1.O x 1 0 ~M
I , CL
1 x 10-10
C O N C E N T R A T I O N OF T4. M
Figure 5. Chemlumlnescence intensity versus (a) incubation time in loop, (b) incubation in affinity column (c) Standard curves of T4 in a flow-injection analysis using quantitation of peroxidase label with an enhanced lumtnescent reaction
rn Z I
S . B. VLASENKO ET AL.
antigen-antibody interactions by the flowinjection analysis in the system of T4 and specific antibodies has allowed us to optimize the analytical procedure for hormone assay. The creation of this fast and very sensitive method of the assay is made possible by advantages of t h e enhanced chemiluminescent reaction, and the sensitive determination of the peroxidase label which in turn can be done in a short period of time. The flow-injection method of analysis of T4 proposed has definite advantages compared with traditional immunochemical methods of antigen and antibody determination.
REFERENCES Conroy, J. M. and Marruci. A. A . (1976). Immunochcmical studies of horseradish peroxidasc: factors effecting the inhibition of cnzyme activity by spccific antibody. Immunochemislry, 13, 599-603. Cormicr, M. J. and Prichard. P. M. (1968). An investigation of mechanism o f the luminescent peroxidation of luminol hy stopped flow techniques. J . R i d . Chem., M3. 4706-17 14. Kalenichcnko, I. E., Pilipcnko, A . T. and Tkachuk, T. M. (1982). Comparison of diffcrcnt methods of luminol purification for chemilumineccnce assays applications, J. Anal. Chim. (in Russian), 37, 213-215.
Kricka. L. J.. Stott, R. A . W. and Thorpe, G. H. G . (19x8). Enhanced chcmiluminesccncc cnzyme immunoassays. I n Complementary Immunoassays, Collins. W. P. (Ed.), John Wiley. Chichcstcr, pp. lhS17Y. Lind, J . , Mercnyi, G. and Erikscn, T. E. (19x3). Chemilumincsccnce mcchanism of cyclic hydrazidcs such as luminol in aqueous solutions. J . A m . Chem. S o c . , 105, 7655-7661. Lundin, A . and Hallandcr, L. 0. B. (1987). Mechanisms of horscradish peroxidasc catalysed luminol reaction in presence and ahscncc o f various enhancers. In Riolumine.scence and Chemilumine.scence. New' per.spectives. Scholmcrich, J., Andrccscn, R., Kapp, A.. Ernst, M., Woods, W . G . (Eds), John Wilcy, Chichcstcr, pp, 555-558. Nakanc, P. K. and Kawaoi, A . (1974). Pcroxidasc-lahcllcd antibody. A New mcthod of conjugation. J. Hbtochcm. Cyrochern., 22, 1064-1091. Ugarova, N. N., Rojkova, G . D. and Bcrezin, I . V. (197X). Chemical modification of NH,-group of horseradish peroxidax, Riochimia (in Russian), 43, 13X2-13X9. Vlasenko, S . B., Klimov, A . D., Gavrilova, E. M. and Yegorov, A. M. (1987). Investigation of mechanism of peroxidation of luminol. In Chemical Physics of Enzyme Catalysis, Tallinn, p. 37. Abstracts from the International Conference on Chemical Physics of Enzyme Catalysis, Tallinn, USSR, September 21-27, 1987. Whitehead, T . P . , Thorpe, G . H. G . , Carter, T. J . N., Groucutt, C. and Kricka, L. J . (1983). Enhanced luminescence procedure for sensitive determination of peroxidase-labclled conjugates in immunoassay. Nulure, 305, 158-159.