Flow injection chemiluminescent immunoassay with para-phenylphenol and sodium tetraphenylborate as synergistic enhancers

Analytica Chimica Acta 485 (2003) 57–62

Flow injection chemiluminescent immunoassay with para-phenylphenol and sodium tetraphenylborate as synergistic enhancers Jian-Xin Luo1 , Xiu-Cen Yang∗ West China School of Pharmacy, Sichuan University, Chengdu 610044, PR China Received 18 November 2002; received in revised form 12 March 2003; accepted 24 March 2003

Abstract A new approach of flow injection chemiluminescent immunoassay (FICLIA) with synergistic enhancement was developed. The synergistic action was significant in the chemiluminescence (CL) system of luminol–H2 O2 –HRP with two enhancers, para-phenylphenol (PPP) and sodium tetraphenylborate (NaTPB). The relative CL intensity was 128.84% stronger (compared to NaTPB alone) and 122.66% stronger (compared to PPP alone) using both enhancers than any one alone (both P < 0.001). While the present approach was applied to the determination of rabbit IgG as a model analyte, satisfactory results were obtained in the dynamic range of 2–60 ␮g l−1 with a detection limit of 0.68 fmol per injection and a precision of 4.7–9.3%, and the recoveries were 92.5–99.4%. The results of rabbit IgG determination accorded with those of radio immunoassay (RIA). The lifetime of the immuno-column was considerably long: one column was applied to more than 200 determinations in 1 month without significant loss of activity. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Flow injection analysis; Chemiluminescent analysis; Immunoassay; Synergistic action; para-Phenylphenol; Sodium tetraphenylborate

1. Introduction For a long period of time, radio immunoassay (RIA) has been the most sensitive assay, which was first introduced in 1959 [1]. However, strong efforts have been undertaken in regard to the replacement of radioactive substances in immunoassays by other detection approaches. The enzyme immunoassay (EIA) ∗ Corresponding author. E-mail address: [email protected] (X.-C. Yang). 1 Present address: Chengdu Medical College, The Third Military Medical University, Chengdu 610083, PR China.

was introduced in 1971 [2,3], which, in terms of sensitivity, was for the first time a match for the RIA. In the EIA, several enzymes, alkaline phosphatase (AP), ␤-d-galactosidase and horseradish peroxidase (HRP) were often used as labels. The detection of these labels can be carried out in various ways, including photometric, fluorimetric or chemiluminometric techniques after enzymatic conversion of a suitable substrate into a detectable product. Despite the success of the enzyme-linked immunosorbent assay (ELISA), its end use is limited due to the requirement of trained persons and long assay time [4]. These limitations have sparked the combination of

0003-2670/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0003-2670(03)00392-1

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immunoassay with flow injection assay (FIA), which was introduced by Ruzicka in 1975 [5], and then the new analytical technique of flow injection immunoassay (FIIA) [6–8] was developed, which combines the advantages of rapidity, precision, automation and high selectivity. The FIIA technique has become an immensely powerful analytical method in which both the competitive assay and the sandwich assay approaches can be applied. However, classical FIIA technique failed to satisfy the requirements for trace analysis due to its low sensitivity. In recent years, the use of chemiluminescent and bioluminescent analysis has become attractive in many fields because of its high sensitivity, wide linear range and simple instrumentation [9–12]. In order to further increase the assay sensitivity, the chemiluminescent enhancers such as para-phenylphenol (PPP), para-iodopenol, etc., are widely adopted. Furthermore, Kricka and Ji [13] have found the synergistic action of 1,1 -biphenyl-4-yl-boronic acid and 4- iodophenol in the horseradish peroxidase (HRP)-catalyzed oxidation of luminol. Recently, the synergistic action of para-phenylphenol and sodium tetraphenylborate (NaTPB) in the luminol–H2 O2 –HRP system has been found in our laboratory, which obviously improved the sensitivity of analysis. Although its sensitivity is very high, chemiluminescent analysis could give unsatisfactory results because the decay of the emitted light is rapid and affected by the coexisted species so that the precision and specificity of the assay is lower than other methods. In order to overcome these disadvantages, the approach of flow injection chemiluminescent immunoassay (FICLIA) [14–19] has been developed by combining chemiluminescent analysis with the techniques of flow injection analysis and immunoassay, thus all the advantages of high sensitivity, high precision, high speed and high selectivity are combined. FICLIA has no radioactive wastes, and is versatile and flexible and may therefore be utilized to many different applications. This article describes a new approach of flow injection chemiluminescent immunoassay for determination of rabbit IgG as a model analyte with para-phenylphenol and sodium tetraphenylborate as synergistic enhancers. As a result, the assay sensitivity, precision, speed and other analytical characteristics have been improved.

2. Experimental 2.1. Reagents and solutions 1,1-Carbonyldiimidazol (CDI) and 3-aminopropyltriethoxysilane were from Flucka. Controlled pore glass (CPG, 520 Å pore size, 100–120 mesh), glycine, luminol, sodium tetraphenylborate, para-phenylphenol, horseradish peroxidase were purchased from Sigma. Rabbit IgG, goat anti-rabbit IgG antibody, horseradish peroxidase labeled goat anti-rabbit IgG antibody were purchased from Cosmo Bio (Tokyo, Japan). The regeneration buffer (pH 2.2) was 0.2 mol l−1 glycine, chemiluminescence (CL) buffer was 0.02 mol l−1 sodium borate at pH 8.5, and the equilibration buffer was 0.01 mol l−1 phosphate-buffered saline (PBS, pH 7.4) containing 1% bovine serum albumin (BSA) and 0.05% sodium azide. The 5 × 10−5 g ml−1 antigen (rabbit IgG) standard solution and the 1×10−5 g ml−1 horseradish peroxidase stock solution were prepared using equilibration buffer. More diluted antigen was prepared daily from its standard solution, and more diluted horseradish peroxidase daily from the stock solution of horseradish peroxidase using equilibration buffer. The 1 × 10−2 mol l−1 luminol stock solution was prepared as described previously [20]. The synergistically enhanced chemiluminescent mixture was prepared using chemiluminescence buffer on the very day of use. All other chemicals were of reagent grade, and water used was doubly distilled. 2.2. Instrumentation The schematic diagram of the apparatus is shown in Fig. 1. The main sections consisted of an intelligent flow injection analyzer (IFIS-A), an immuno-column, a sensitive chemiluminescence detector and a recorder (Spring Com, China). The IFIS-A contained two peristaltic pumps (A and B) which were used to deliver the sample, reagents and carrier buffer streams, respectively, a multi-port valve (eight ways) equipped with two sample loops (A and B) for introducing samples and reagents to the immuno-column, and an intelligent control center for the procedure of assay. The immuno-column (5 mm × 40 mm) was filled with controlled pore glass linked goat anti-rabbit IgG (CPG-Ab, 500 mg). All connecting tubing in

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Fig. 1. Schematic diagram of a manifold used for the flow injection immunoassay determination.

the flow system consisted of 0.5 mm i.d. PTFE tubing. 2.3. Preliminary optimization of the chemiluminescent mixture The chemiluminescent mixture consisted of luminol, H2 O2 , PPP and NaTPB. Their concentrations were screened and preliminarily optimized by the method of uniform design as shown in Table 1. Their chemiluminescence intensity at 8 × 10−7 g l−1 of HRP was determined and the data analyzed by the statistical method of multiple linear regression to obtain the optimum concentrations of the components of the chemiluminescent mixture.

2.4. Preparation of immuno-column The procedures for preparation of immuno-column used in this work is based on a similar procedure as described previously [21–24] with some modifications. At first, the CPG beads were silanized with 3-aminopropyltriethoxysilane [21], and activated using CDI as a linker [22,23]. Then the goat anti-rabbit IgG antibodies were immobilized covalently onto the CDI-activated CPG beads [24]: 500 mg of CDI-activated CPG beads was suspended in 1 l of 0.1 mol l−1 sodium borate buffer, pH 8.5, containing 5 mg of goat anti-rabbit IgG antibody, and the suspension was slowly shaken for 2 days at 4 ◦ C after degassing with ultrasonic vibration for 5 min.

Table 1 The uniform design and results of determination Test number

1 2 3 4 5 6 7

Concentration of factors (mmol l−1 )

Relative CL intensity (mean ± S.D.)

A

B

C

D

10 20 30 40 50 60 70

2 4 6 1 3 5 7

20 50 10 40 9 30 60

50 40 30 20 10 9 60

A, B, C and D stand for luminol, H2 O2 , PPP, NaTPB, respectively.

863.26 1002.38 836.33 939.69 791.23 869.92 1226.19

± ± ± ± ± ± ±

33.83 40.56 30.57 38.35 35.16 29.32 58.59

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The coupling yield of the antibody was determined by measuring the absorbance of the supernatant at 280 nm before and after the coupling reaction, respectively. After washing the beads with PBS (pH 7.4) three times to remove the excessive antibodies, the CPG-Abs were packed into an immuno-column (Ø 5 mm × 40 mm), and then, the column was stored at 4 ◦ C in PBS (pH 7.4) until being used.

the equilibration buffer (PBS, pH 7.4) at a flow rate of 0.6 ml min−1 by pump B (1 min), then, the procedures went into the next cycle. The total time required for one cycle (three determinations of CL) is 8 min. All the above procedures were carried out automatically.

2.5. Analytical procedures

3.1. Optimization of the chemiluminescent mixture

Before the immunoassay of rabbit IgG, the apparatus was first used for optimizing the concentrations of the components of the chemiluminescent mixture, without using the immuno-column. Two hundred microliters of the mixture containing various concentrations of luminol, H2 O2 , PPP and NaTPB was carried rapidly by pump A to loop A (30 s). At the same time, 100 ␮l of 8 × 10−7 g l−1 HRP was carried to loop B by pump B, and then, the mixture mixed with HRP in the chemiluminescence buffer at a flow rate of 0.3 ml min−1 by pump B after turning the valve to allow the chemiluminescent reaction to take place (2 min). The relative chemiluminescence intensity was automatically detected and recorded and the procedures repeated automatically three times for each assay. The total time required for one cycle is 2.5 min. While the apparatus was used for the immunoassay of rabbit IgG, 100 ␮l of the rabbit IgG was taken rapidly (5 s) to loop A by pump A, then to the immuno-column packed with goat anti-rabbit IgG antibodies on CPG by PBS (pH 7.4) at a flow rate of 1.0 ml min−1 using pump B (30 s). After stop flow 1 min for immunoreactions of IgG with goat anti-rabbit IgG antibody, the unlinked rabbit IgG was washed away to waste (30 s). Then, HRP-labeled goat anti-rabbit IgG antibodies were subjected to the same procedure as rabbit IgG. After the unlinked labeled antibodies were washed to waste, 200 ␮l of the chemiluminescent mixture was carried rapidly to loop B by pump A (5 s), and then to the immuno-column at a flow rate of 0.3 ml min−1 by pump B with the chemiluminescence buffer after turning the valve (1 min). The relative chemiluminescence intensity was automatically detected and recorded and these steps repeated three times. At last, regeneration buffer (pH 2.2) was carried to immuno-column at a flow rate of 0.6 ml min−1 by pump A to remove the immunocomplex (1 min), and

In our laboratory, the synergistic action of two enhancers, NaTPB and PPP, in luminol–H2 O2 –HRP system was discovered. Using both enhancers, the relative chemiluminescence intensity was 128.84% stronger (compared to NaTPB alone) and 122.66% stronger (compared to PPP alone) compared to any one alone (both P < 0.001) at 8 × 10−7 g l−1 of the HRP (see Table 2). NaTPB is a new chemiluminescent enhancer introduced by Zhang in 1998, and its specialities were discussed in their paper [20]. It was noted that the CL intensity varied with the concentrations of the components in the synergistically enhanced chemiluminescent system. We screened the concentrations of those components and then optimized them using the uniform design with four factors and seven levels. The data of determination were dealt with statistical analysis using multiple linear regression. The equation of linear regression obtained was

3. Results and discussion

Y = 625.6594 + 21.8149A − 6.0719B +33.1100C + 48.6266D, F = 803.3517

r = 0.9997,

(P < 0.001).

The optimized concentrations of the components of the mixture in the CL system were as follows: luminol at 5 × 10−4 mol l−1 , H2 O2 at 5 × 10−3 mol l−1 , PPP Table 2 The enhancement capacity of different enhancers (n = 10) Test number

Enhancer

Relative CL intensity (mean ± S.D.)

A B C

PPP NaTPB NaTPB–PPP

556 ± 56 541 ± 43 1238 ± 98a

a

P < 0.01 vs. A, P < 0.01 vs. B.

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at 1 × 10−4 mol l−1 , and NaTPB at 8 × 10−5 mol l−1 . After being optimized by the above uniform design, the detection limit for HRP defined as the concentration yielding a signal of 3 S.D. of the blank signal, was found to be 1.9 × 10−18 mol per assay. Currently, the exact mechanism whereby sodium tetraphenylborate or para-phenylphenol enhances the light emission of the HRP-catalyzed oxidation of luminol remains still unclear. In the literature, no suggestions about the mechanism of the synergistic action of chemiluminescent enhancers have been reported up to now. Hence, the mechanism of synergistic action of PPP and NaTPB might be worthy of further study. 3.2. Amounts of antibody and antibody-HRP The amount of antibody immobilized on the immuno-column is an important factor in heterogeneous immunoassay. The sensitivity of assay would not be high because of insufficiency of immune reaction resulted from shortage of antibody or antibody-HRP. However, the assay becomes high-priced while antibody or antibody-HRP is used excessively; besides, the blank signal becomes higher, and assay time longer with larger amounts of antibody-HRP. In this study, the relation between the amounts of CPG immobilized goat anti-rabbit IgG antibody and the relative chemiluminescence intensity emitted by the same concentration of rabbit IgG was explored to obtain the optimized amount of antibody (see Fig. 2). It was shown that 500 mg CPG-Abs and 2000-fold dilution of the antibody-HRP, of which the final concentration of total proteins was 250 ␮g l−1 , were satisfactory.

Fig. 2. Relative CL intensity vs. the amount of CPG.

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3.3. Analytical range and detection limit Under the optimized condition, the dynamic range for determination of rabbit IgG was 2–60 ␮g l−1 , and the obtained regression equation was log[CL] = 1.0384 + 1.1279 log[IgG], r = 0.9941. The detection limit, defined as the concentration yielding a signal of 3 S.D. of the blank signal, was found to be 0.68 fmol per injection. It is 2000 times more sensitive than other determinations without utilization of chemiluminesence, which gave a sensitivity of 1.6 pmol [25], or about 300 times more sensitive than other chemiluminescent assay, which gave a sensitivity of 0.2 pmol [26]. 3.4. Accuracy, precision and lifetime of column To determine the accuracy of the assay, recovery tests and comparison studies were performed. Various amounts of rabbit IgG standard were added to the rabbit serum and, recoveries obtained were 92.5–99.4% with an average recovery of 95.1%. In the comparison studies, 50 solutions containing different concentrations of IgG prepared by diluting the standard solution randomly with 0.02 mol l−1 of PBS at pH 7.4 were determined on the same day by RIA (X) and by the present method (Y), respectively. The average of rabbit IgG concentrations was found to be 8.12 ␮g l−1 by the RIA and 8.09 ␮g l−1 by the present method. The equation of linear regression was Y = 0.1877 + 0.9805X, n = 50, r = 0.9858. The t-test for the correlated data obtained from the two methods showed that they were in good agreement (P > 0.05). The precision of the assay was determined repeatedly using different concentrations of rabbit IgG, and the relative standard deviations (n = 10) were obtained. The precision of the within run (n = 10) was from 4.7 to 6.2% with an average precision of 5.5%, and that of the between run (n = 5) from 7.3 to 9.3% with an average precision of 8.1%. The lifetime of the immuo-column was considerably long: one column was applied to more than 200 determinations in 1 month without significant loss of activity.

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4. Conclusion

References

This work demonstrates that two enhancers, para-phenylphenol and sodium tetraphenylborate, have produced obvious synergistic action in the luminol–H2 O2 –HRP system. The relative chemiluminescence intensity was more than one time stronger using both enhancers than any one alone in optimized chemiluminescent systems, and the detection limit for HRP was 1.9 × 10−18 mol per assay. Owing to the synergistic enhancement, the sensitivity of the chemiluminescent immunoassay for the determination of rabbit IgG as a model analyte was improved about 300 times as compared with other chemiluminescent assay. The results for determination of rabbit IgG accorded with those of RIA (r = 0.9858). The proposed method exhibits all the advantages of three techniques (chemiluminescence monitoring, flow injection analysis and immunoassay): high speed, ease for automation, simplification of operation, high sensitivity, high precision, and absence of radioactive wastes. Besides, loops A and B run at the same time in the analytical procedures, thus the total time of determination of rabbit IgG required for one cycle was as short as 8 min (three determinations of CL). The synergistically enhanced chemiluminescent system can have powerful applications in other immunoassays, e.g. ELISA. While the routine ELISA takes a couple of hours for incubation and the whole assay procedures usually take several hours to 2 days, application of chemiluminescence for monitoring the end-point can generally improve its sensitivity [27].

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Acknowledgements The authors would like to thank the Health Ministry of China for the financial support of this work in the project (no. 94-1219).