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Polarographic Enzyme-Immunoassay for TraceHepatitis B Surface AntigenJingqiu Xu a , Junfeng Song a & Wei Guo aa Institute of Electroanalytical Chemistry ,Northwest University Xi'an , 710069, Shaanxi, P.R. ChinasPublished online: 16 Aug 2006.

To cite this article: Jingqiu Xu , Junfeng Song & Wei Guo (1996) PolarographicEnzyme-Immunoassay for Trace Hepatitis B Surface Antigen, Analytical Letters,29:4, 565-573, DOI: 10.1080/00032719608000421

To link to this article: http://dx.doi.org/10.1080/00032719608000421

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ANALYTICAL LETTERS, 29(4), 565-573 (1996)

POLAROGRAPHIC ENZYME-IMMUNOASSAY FOR TRACE

HEPATITIS B SURFACE ANTIGEN

Keywords Hepatitis B Surface Antigen(HBsAg),serum,Enzyme-Linked

Immunosorbent Assay(ELISA), Polarographic Immunoassay

Jingqiu Xu, Junfeng Song and Wei Guo

Institute of Electroanalytical Chemistry, Northwest University, Xi'an, 71 0069,

Shaanxi, P. R. China

Abstracts A polarographic enzyme-immunoassay for Hepatitis B Surhce

Antigen(Hl3sAg) has k e n established, in which horseradish peroxidase(HRF') is used

as the labeled enzyme, o-phenylenediamine(0PD) as the substrate, and the enzyme-

generated product,2,2'-diaminoazobenzene (DAA), is detected by linear-potential scan

polarography. Under optimal conditions, the second derivative current of DAA is

linear with the concentration of HBsAg from 0.1 to 5 ng/mL. The correlation

coefficient(r) is 0.9994. The detection l i t is O.OSng/mL and the relative standard

deviation is 6.7%(8 replicates). The sensitivity of the assay is about 20-fold higher

565

Copyright 0 1996 by Marcel Dekker. Inc.

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566 XU, SONG, AND GUO

than that of ELISA. The assay has been successfully applied for minute determination

of HBsAg in both human serum and the negative control serum from ELISA kits.

Introduction

Electrochemical techniques have inherent advantages over other analytical methods

in terms of simplified procedure, high sensitivity. wide detection range and so on,

which would be useful as the detection approach of immunoassay, but its sensitivity

is usually lower when the labels are directly detected by voltammetry and

potentiometry. To date, the most sensitive electrochemical immunoassay for HBsAg

reported in literature is an immunoelecrode assay.The instantaneous potential

response of the immunoelectrode to Hl3sAg is observed on an oscillograph,and the

response is linear with the concentration of HBsAg in the range 20-320ngImL.

However, the regeneration of the biosensor is difticult '. It is known that some

enzymes for special substrates have the h c t i o n of both chemical amplification and

the change from an electroinactive substrate to electroactive product. Such h c t i o n

makes it possible to further enhance the sensitivity of an electrochemical

immunoassay when the electrochemical approaches are coupled with enzyme

immunoassay. For example, Heineman et a1 2,3 developed enzyme immunoassay with

amperometric detection. They utilized alkaline phosphate as the labeling enzyme and

voltammetrically measured the anodic current of the enzyme-generated products:

phenol at 750mV or p-aminophenol at 380 mV.Detection limits for digoxin are

50pg/mL and 30pg/mL, respectively.

Horseradish peroxidase (HRP) is widely used as the label enzyme due to its good

stability and cheap cost. The most sensitive chromogen for HRP is considered to be

o-phenylenediamine(0PD). The enzyme-generated product, 2,2'-

diaminombenzene(DAA),is electroactive and can be measured by polarography. We

established a polarographic immunoassay for trace HBsAg using H R P as labeled

enzyme and OPD as the substrate. The detection limit is O.OSng/mL, which is superior

to that of Radio Immunoassay(RIA), 3-4ng/mL4, and EIA, 2.4ng/mL5.

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TRACE HEPATITIS B SURFACE ANTIGEN 567

Experimental

Apparatus

Polarographic measurements were performed using a Model JP-2 Linear-Potential

Scan Oscillopolarograph(Chengdu Instrument Factory) equipped with a three-

electrode system, which includes a dropping mercury working electrode, a Ag/AgCl

reference electrode and a platinum wire a d a r y electrode. The three-electrode

system is small enough to be directly inserted into the well the microtitre plate. The

scan rate of the potential is 25OmV/s.

Incubations for both the immune reaction and the enzyme-generated reaction were

carried out in a Model HH. W21 .Cr420 incubator(Guangd0ng Shantou Instrument

Factory).

Reagents

HBsAg EIA Kits were purchased fiom Shanghai Kehua Biological Technology

Co.. The kit includes: 12 well(x8) microtitre plates precoated by anti-HBs,

horseradish peroxidase(HRP)-conjugated anti-HBs, negative control serum, positive

control serum and phosphate buffer saline (PBS). 5ng/mL of HBsAg quality control

serum was purchased fiom Shanghai Biological product Institute of Hygiene Ministry.

All chemicals used are of analytical-reagent grade. 0-phenylenediamine was

purchased fiom Beijing Chemical Factory, H202 was purchased fiom Linan Chemical

Factory(Zhejiang Province), Na2HP0,, NaOH and citric acid were purchased fiom

Xi'an Chemical Reagent Factory. All solutions were prepared by a routine method

using doubly distilled water.

Procedure of the sandwich immunoassay

To each well of the microtiter plate, add 50uL of test serum and 50uL of the

conjugates successively. After incubation for l h at 37" C,aspirate and wash each well

( ~ 5 ) in PBS(pH 7.4). Thereafter, add 1 5 W of the substrate solution of O.O37%(v/v)

hydrogen peroxide(H202) 3.7mM 0-phenylenediamine(0PD) in 0.2M phosphate-

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568 XU, SONG, AND GUO

0.1M citrate buffer solution(pH 5.0) and incubate the plate at 37 "C for 15 min. Add

25 uL of 2 M NaOH solution, measure the second derivative peak current of enzyme-

generated product DAA, with peak potential -0.78+0.02V, by a hear-potential scan

oscillopolarograph.

Results and Discussion

Optimization of enzyme immune reaction conditions

Heterogeneous double antibody sandwich assay is of high sensitivity and specificity.

In this assay, the immune reaction scheme was adopted and the commercial HBsAg

kit was directly used. The conditions of the immune reaction and enzyme-generated

reaction were controlled according to the procedure recommended by conventional

ELISA.

Optimization of polarographic detection conditions

The suitable pH range for the HRP-generated reaction is 5.0-5.56, hence 0.2M

phosphate-0.1M citrate buffer solution(pH5.0) is used as the medium of the enzyme-

generated reaction. It is necessary to stop the reaction, using H,SO, solution as

quencher. But in acidic medium, the reduction wave of product DAA could split up

into two peaks and the peak current is rather low. In alkaline medium ,the reduction

wave of DAA is stable and sensitive. The peak current increases and the peak

potential shifts into a negative direction with an increase of the medium pH. The peak

current was maximal and stable when the pH > 7.45 ( Fig. 1 ). At pH = 10, the peak

potential of DAA is -0.78V, being far fiom the double wave of oxygen reduction. In

favor of polarographic detection, 25uL of NaOH solution ranging fi.om 0.5 to 4 M

were applied to stop the enzyme-generated reaction. Experimental results showed that

1.5 to 4 M NaOH solution can completely stop the reaction. So 25 uL of 2M NaOH

solution is used to stop the HRP-generated reaction and to adjust the medium pH to

about 10.

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TRACE HEPATITIS B SURFACE ANTIGEN 569

Fig 1. Effect of pH on peak height (1) and peak potential (2) of DAA sensitivity=0.15

Because of the heterogeneous enzyme immunoassay scheme,the interference from

clinical sample matrix components such as serum protein can be easily removed by

simple aspiration and rinsing. Due to using the precoated and blocked microtitre

plate, a washing PBS buffkr solution without surfactant Tween 20 and BSA could be

employed, avoiding the interference of these substances.In addition, there are

residuary substrate molecules, H202 and OPD, in the microtiter plate well, after the

HRP-generated reaction. Experiments show that O.OS%(v/v) H202 is tolerable, and

the tolerable concentration of H202 exceeded its initial one. However, OPD is

electroinactive over the cathodic potential range. So the product DAA can

quantitatively detected by polarography without interference of residuary substrate

and serum protein798.

Calibration curve

Under optimum conditions described above, the peak current of DAA produced

by the spontaneous reaction between OPD and H202 is too small to be measured.The

low background characteristics are advantageous to improve the sensitivity of the

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570 XU, SONG, AND GUO

assay. The peak current of the enzyme-generated DAA is proportional to the

concentration of HBsAg. The calibration curve was constructed by plotting the peak

current of DAA vs. the HBsAg standard concentration(Fig. 2). The linear range is

over 0.1-5 ng/mL (higher concentrations were not tested), and the linear regression equation is :

I’p = 15 + 1’25 C

where I’p represents the peak current in nA, C represents the concentration of HBsAg

in ng/mL. Correlation coefficient r=0.9994.

Precision and Detection limit

The repeatability of the assay was studied by running 8 replicate assays on 1 ng/mL

of HBsAg in serum. A mean peak current of 140 nA was observed, and the relative

standard derivation was 6.7%.

The detection limit, detined as three times the standard deviation of the blank, was

0.05 ng/mL.

Determination of HBsAg in human serum samples

The HBsAg level in human serum specimens and a negative control serum in the

EIA kit were determined by the proposed assay. These samples were also analyzed

in a hospital laboratory by the conventional ELISA method. Results obtained by these

two assays were summarized in Table I , and are in good agreement, demonstrating

that the proposed method is more accuracy, sensitive and easy to use in clinical

examinations.

Recovery

Recovery tests were performed on two serum samples in which the concentration

of HBsAg had been detected. Known amounts of standard HBsAg serum( 1 ng/mL)

were added prior to the immune reaction step. The results in Table 2 indicated that

satisfactory recoveries were achieved.

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TRACE HEPATITIS B SURFACE ANTIGEN 57 1

. 2 i 50

40'

30 -

20 -

. 0 1 2 3 4 5ng/mL

_I__ Fig. 2. Calibration curve for HBsAg sensitivity=O. 15

Table 1 Determination of HBsAg in Serum

specimen Dilution ratio HBsAg levels

the proposed ELISA** assay( ng/mL.) *

healthy serum 1 no 0 3 no 0 3 no 1.2

patient serum 4 1:1000 5 1:lOOO 6 1:lOOO 7 1:lOOO 8 1 : lOOO

I400 + 1500 + 1500 + 1900 + 2750 +

negative serum in ELISA kit no 0.6

* ** Results were supplied by Shaanxi peoples' hospital. "+" or "-" represents that the

Each specimen is replicated 3 times

HbsAg level is higher or lower than Sng/mL, respectively.

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572 XU, SONG, AND GUO

Table 2 Recovery Test

HBsAg in samples added found recovery average (ndml) ("/.I recovery( YO)

1.2 1 .o

0.88 1 .O 1.7 82 82 1.6 72 1.7 82 1.7 82 1.75 87 2.1 90 90 2.05 85 2.15 95 2.1 90 2.1 90

In conclusion, the proposed method has many advantages that can make it ideal for

use in the clinical lab: it utilizes an inexpensive cost of the used equipment, and high

sensitivity and accuracy of polarographic detection, it is a mature technique with the

wide application of HRP labeling, and it is sensitive and accurate.

Acknowledgement

We greatly appreciate the support of National Science Foundation of China.

References

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TRACE HEPATITIS B SURFACE ANTIGEN 573

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8.

Received: August 8, 1995 Accepted: September 12, 1995

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