A sensitive screening test for the simultaneous detection of hepatitis B surface antigen and antibody

Journal of VirologicalMethods, 13 (1986) 245-253

245

Elsevier JVM 00493

A SENSITIVE

SCREENING

OF HEPATITIS

CAROL

TEST FOR THE SIMULTANEOUS

B SURFACE

J. ATHERTON

DETECTION

ANTIGEN AND ANTIBODY

and ELIZABETH

H. BOXALL

Regional Virus Laboratory, East Birmingham Hospital, Bordesley Green, Birmingham, U.K. (Accepted

10 February

A modification which

allows

sensitivity samples

HBsAg

1986)

of the Blood

simultaneous

of the method

Products

assay

Laboratory

of both

has been assessed

hepatitis

radioimmunoassay B surface

and its performance

antigen

for hepatitis and antibody

as a screening

B surface

antigen

is described.

The

test in over 2,500 routine

has been evaluated.

anti-HBs

radioimmunoassay

sensitive

screening

test

INTRODUCTION

The laboratory diagnosis of hepatitis B has become of increasing importance to the clinician. In our laboratory, this has resulted in a continual increase of work since we began screening for hepatitis B surface antigen (HBsAg) and antibody (anti-HBs) in 1970. Our screening methods have always included tests for both HBsAg and anti-HBs and have included complement fixation tests, immuno-electroosmophoresis (IEOP), Passive haemagglutination (PHA) and Passive haemagglutination inhibition (PHAI). These anti-HBs

test systems are all sufficiently

specific immunoglobulin production, antibody such as are found in patients

sensitive for finding

high-titre

sera for

but are not capable of detecting low levels of recovering from hepatitis B or in the early stages

of a course of active immunisation. As a result many of our routine specimens require further testing by commercial radioimmunoassay (RIA) kits. The advent of vaccine has brought with it a need for an inexpensive, sensitive anti-HBs screening test to check vaccine response. These factors have prompted the development of a screening test for HBsAg and anti-HBs by RIA, which will provide greater sensitivity than our current PHA/PHAI system. The test system is a modification of the Blood Product Laboratory (BPL) RIA kit for HBsAg detection (Lane, 1981) which is a ‘solid-phase sandwich’ test. The modification which we describe allows the simultaneous assay of HBsAg and anti-

01660934/86/$03.50

0 1986 Elsevier Science Publishers

B.V. (Biomedical

Division)

246

HBs. The test is based in principle

on the RIAQuick

(Immuno

Diagnostika)

method,

but using the BPL reagents. Our modification Laboratories

of the BPL test (M-BPL)

and Regional

Transfusion

could be very useful in Public Health

Centres,

since it employs

reagents

currently

used by them. In this report we describe this modified BPL method and compare its sensitivity, ease of application and cost with those of other commercially available kits. MATERIALS

AND

METHODS

Samples 2670 samples

from our hepatitis

screening

programme

were used.

Standards For sensitivity

comparisons

we used: (a) The British

HBsAg

Reference for Biological Standards) equivalent to 100 BSU/ml The anti-HBs standard equal to 250 mIU/ml (Colindale-subtype were stored at 4°C.

standard

(subtype ad) and (b) ad). Both standards

Normal human serum Normal human sera were selected on the basis of the absence anti-HBs by RIA. They were stored at -20°C. Blocking HBsAg Human serum containing was stored at -20°C.

HBsAg,

subtype

ad, from a healthy

PHA/PHAI for HBsAg and anti-HBs Our Hepatitis Reference laboratory tests specimens

(National

of HBsAg

carrier

and

was used. It

daily for HBsAg and anti-HBs

using Hepatest (Wellcome). Dilutions of sera are made in microtitre plates from l/2 to l/8. 25 pl of blocking HBsAg diluted in Hepatest buffer to give 4 haemagglutinating units (HU), are added 45°C. 25 ~1 of turkey added to the anti-HBs (test settle at room 30 min before

to the l/2 dilution.

The plate is then incubated

red blood cells coated with normal

for 30 min at

horse IgG (control

cells) are

l/4 dilution, and 25 ~1 of turkey red blood cells coated with horse cells) are added to the l/2 and l/8 dilution. The cells are allowed to temperature for 20 min before reading the results of the HBsAg tests and reading the anti-HBs results.

RIA for anti-HBs The RIA used for anti-HBs

detection

was the AUSAB

test (Abbott

Laboratories).

247

RIA for HBsAg The RIAs used for HBsAg tory, Elstree)

detection

and the AUSRIA

were the BPL test (Blood Products

test (Abbott

Laboratories,

N. Chicago,

Labora-

IL).

Equipment An Innotron gamma-counter with a built in microprocessor was used. This enabled us to obtain counts per minute in each sample and to have this value expressed as a ratio with respect to the negative control. M-BPL test for HBsAg and anti-HBs This method detects HBsAg and anti-HBs simultaneously was based essentially on the principle that a positive reaction

in one assay. The method of HBsAg in the BPL test

can be inhibited by the presence of anti-HBs (Fig. 1). Three procedures (A, B, and C) were investigated (Table 1). The concentration of blocking HBsAg was critical. For a high HBsAg sensitivity, the concentration should be low. On the other hand the concentration should be high enough to give an adequate number of counts for good reproducibility of the anti-HBs test result. In all three systems we aimed at the same level of the blocking HBsAg. This level, expressed in counts per minute (cpm) - background, was allowed to range from 400-900. These values correspond with an amount of blocking HBsAg of0.4-0.5 BSU/well depending on the procedure. Positive and negative controls, and the calculation of results were the same in all procedures. The test performance of procedure A for example is described as follows. (1) Add 25 ul of blocking HBsAg (20 BSU/ml) diluted in phosphate buffer pH 7.4, to each well coated with anti-HBs;

(2) cover the plate and incubate

for 20 h at room temperature

(23°C); (3) wash the wells 8 times in distilled water using a plate washer and dry well; (4) continue with the normal procedure for the BPL test, i.e. add 100 ul of test sample to HBsAg positive

Negative

Anti-HBs positive

for

HBsAg and anti-HBs

sample CR >= 1.3

0.5 < CR < 1.3

sample CR <= 0.5

Solid phase (removawell)

Fig. 1. Modified

BPL method.

ratio with respect to negative

h, Anti-HBs; control.

., HBsAg;

6, blocking

HBsAg;

2, “‘1 anti-HBs;

CR, count

248

TABLE

1

Summary

of procedures

Procedure

A

Reaction

Volumes

Blocking

steps

(111)

HBsAg/BSU/ml)

Wash

time (h)

temp. (“C)

yes/no

20

23

yes

SP-Ab+ Ag+

B

25

20

Sample+

100

1.5

50

yes

Ab-I,,5

100

1.5

50

yes

Sp-Ab+

C

20

37

Yes

Sample+

100

1.5

50

yes

Ab-I,,,

100

1.5

50

yes

Agf

25

20

Sp-Ab+ 20

37

no

Sample+

100

2

50

Yes

Ab-I,,,

100

1

50

yes

Ag+

Ag-t,

Incubation

blocking

25

antigen;

each well. Incubate

Ab-I,,,,

iodinated

15

anti-HBs;

SP-AbS,

solid phase coated

with anti-HBs.

for 1.5 h at 50°C; (5) wash the wells as in step 3; (6) add 100 ul of

goat anti-HBs labeled with ‘*‘I. Incubate Controls included in each test are: 8 negative (i.e. free from HBsAg 1 positive HBsAg ( 20 ng/ml),

for 1.5 h at 50°C; (7) wash the wells as in step 3.

and anti-HBs),

2 positive

HBsAg

(

4 ng/ml),

2 positive 2 positive 1 positive

HBsAg anti-HBs anti-HBs

( 1 ng/ml), (250 mIU/ml), (500 mIU/ml).

Count specimen

each well for 60 set in a gamma-counter. was calculated thus:

and The count

ratio

(CR) of each

count-background CR = negative

mean-background

Interpretation of the results is as follows. CR > 1.3 - positive for HBsAg, negative for anti-HBs; CR < 0.5 - positive for anti-HBs, negative for HBsAg; 0.5 < CR < 1.3 - negative for HBsAg and anti-HBs. The sample is considered anti-HBs positive if the response of the blocking HBsAg is reduced by more than 50% by the sample.

249

RESULTS

Performance of procedures A, B and C (1) Sensitivity of anti-HBs detection Dilutions of the Colindale anti-HBs standard were tested with procedures A, B and C. A typical result is shown in Fig. 2. The graph illustrates a similar response for all 3 procedures. Procedure B is the most sensitive, due to the improved binding of blocking HBsAg achieved by incubation at 37°C thus allowing a higher level of antigen in which to observe

inhibition

by any anti-HBs

in the test sample.

(2) Sensitivity of HBsAg detection Dilutions of the British HBsAg standard were also tested. A typical result is shown in Fig. 3. Again a similar response is observed. Procedure A is marginally more sensitive. Although procedure A was the most sensitive for HBsAg, and B most sensitive for anti-HBs they both had the disadvantages of giving ‘false positive’ anti-HBs results with fresh sera. When repeated the following day these proved negative for anti-HBs. Procedure C did not have this serious disadvantage and was chosen as a compromise in sensitivity, it being intermediate between method A and B in detection of both HBsAg and anti-HBs. Procedure C was chosen for further evaluation and comparative l,l-

(+)

A B

(*I

c

(0)

0.9-

2

0.7_

2 s

0.5_

1;

Ii0

Concentration antl-HB5 miu/ml (log stole)

Fig. 2. Comparison

of sensitivity

for anti-HBs

between

procedures.

100;

250

5

4

0 3 2

3-

z ::

2_

I 10

~OnCentr~tfon HBsAg Bsum Fig. 3. Comparison

of sensitivity

for HBsAg

between

(109

I

I

100

1000

scofe)

procedures.

studies. It was the most attractive system with respect to the routine working laboratory as it required a low dilution of blocking HBsAg, one washing step is omitted and it can be completed within 3.5 h. The most important anti-HBs results with fresh human sera.

factor was that it gave few false

Comparison of the M-BPL with other methods for HBsAg and anti-HI& detection End point dilutions of the standard test systems used in our laboratory.

anti-HBs and HBsAg were determined The results are given in Table 2.

for all the

To ensure the sensitivity of each screening test we included positive controls close to the end point dilution for HBsAg and anti-HBs. Thus in a M-BPL test the 4 and 20 ng/ml positive.

HBsAg Failure

control

and the 250 and 500 mIU/ml

in this wouId indicate

Comparison of M-BPL and PHA/PHAi

an unacceptable

anti-HBs

control

were always

test.

in the routine laboratory

Initially procedure B was used to screen routine specimens. However, many fresh specimens (i.e. they had not been frozen and thawed) gave rise to false anti-HBs results. Heat treatment of the sera (WC for 10 min) removed this effect. The conclusion was that a heat labile protein was inhibiting the binding of label to the blocking HBsAg. Use of procedure C eliminated this problem and therefore did not require the addition-

251

TABLE

2

Sensitivity

titration

of standard

anti-HBs

HBsAg

and anti-HBs.

Yi

End point results

HBsAg

(mIU/ml)

x

(BSWml)

mBPL A

235-240

231.5

1.4-2.5

1.95

mBPL B

135-220

166.25

1.0-4.0

2.23

210-225

217.5

1.9-3.4

2.61

mBPL C PHAI

2,500

AUSAB

NA

1.0

NA

PHA

NA

10.0

BPL

NA

0.5

AUSRIA

NA

0.1

NA, not applicable.

al step of heat inactivation inactivation of specimens Hepatitis A IgM.)

and was chosen was inappropriate

for use in the laboratory.

(n.b. heat

since many are subsequently

tested for

Modified BPL method C was run in parallel with PHA/PHAI over a period of 2.5 mth. 2,670 samples were tested by both methods and the results are shown in Table 3. Twice as many anti-HBs positive samples were detected by M-BPL. positive samples were detected by M-BPL. They included: (A) five cord bloods from carrier mothers; (B) one patient with chronic active hepatitis; (C) three patients

with a recent history

of hepatitis

B;

(D) three antenatal screens from the Blood Transfusion (E) one haemophiliac patient recovering from hepatitis

Service; B;

(F) two patients who had a history of jaundice; (G) two asymptomatic homosexual patients; (H) three pooled (I)

one alcoholic

TABLE

sera from the clinical patient

of procedure

C and PHA/PHAI M-BPL

HBsAg

laboratory;

3

Comparison

anti-HBs

chemistry

with hepatomegaly.

positive positive

(%)

240 (9) 164 (6)

on 2,670 routine PHA/PHAI 219 (8) 94(3.5)

Negative

2,266(85)

2,357 (88.5)

Total

2,670

2,670

(%)

sera

and

21 extra HBsAg

252

Normally further reported

specimens

testing

in categories

by RIA.

negative

The others

positive

would

have been missed

have been selected for by PHA/PHAI

and

for HBsAg and anti-HBs.

In order to check the specificity BPL or AUSRIA,

A, C, D, and E would

of the results all HBsAg positives

and any anti-HBs

for anti-core

positives

confirmed

were confirmed

by AUSAB.

by

They were also

by IEOP or RIA.

DISCUSSION

The M-BPL in addition to screening for HBsAg, tests for anti-HBs. This latter marker is valuable for epidemiological studies and in monitoring patients, medical, nursing and laboratory staff in hospitals and for investigating high risk groups, such as drug addicts and homosexuals. Detection of anti-HBs is also useful for following the presence of passive immunity against hepatitis B in persons who have received specific immune globulin for prophylaxis. Now that active immunisation is available, tests for anti-HBs will give the first indication that immunisation has been effective. We attempted to find the most sensitive modification (with minimal adaptation) of the original RIA for HBsAg. Anti-HBs is detected on the basis of inhibition of the HBsAg reaction by anti-HBs in the test sample. Our experience showed us that procedure C was the most suitable for routine use. The fewer false anti-HBs positive results may be explained by the fact that any excess blocking HBsAg in solution may mop up the heat labile protein from the the binding of label to the blocking HBsAg. blocking HBsAg was necessary to obtain due to the relatively small volume (0.025

test sample, preventing it from inhibiting For all procedures the accurate addition of reliable results. This was, of course critical ml) of blocking antigen used.

Some of the initial sensitivity of the BPL test had to be compromised in order to develop a convenient screening test which can fit easily into a working day. The test is completed in 3.5 h and could be easily introduced into a laboratory with RIA facilities. The BPL test is an inexpensive RIA for HBsAg detection produced in the U.K. and our modification tests for anti-HBs decreased our useage of commercial confirm

equivocal

results.

tion over our previous sensitivity

provided

at no extra cost. The use of this modification has RIA kits, so that these are now only required to

The improved

anti-HBs

screening

by specific commercial

sensitivity method

of the M-BPL in anti-HBs means

that in practice

detec-

the extra

RIA systems is very rarely required.

The

M-BPL is suitable for dealing with large numbers of specimens and can be mechanised and computerised. The M-BPL has many advantages over PHA. It eliminates the problem of non-specific agglutination and is approximately 10 times more sensitive for anti-HBs and 5 times more sensitive for HBsAg. It can be used to test many types of specimen, e.g. plasma, breast milk, bile, knee aspirates, urine and saliva. Results are obtained faster than by some enzyme immunoassays (EIA) which can take up to 5 h (Wolters, 1979). Only 100 ul of specimen is required for both HBsAg and anti-HBs assay. The control

253

specimens

are of human

Although

the results

easily adapted standard

origin and therefore

at present

to calculate

curve included

react in a similar way to patient

are not expressed

HBsAg

the system can be

in terms of British Standard

Units (BSU) from a

in each assay. In the short term we are continuing

samples found positive for HBsAg by Hepatest monitoring

samples.

quantitatively,

the course of acute hepatitis.

to titrate all

as this has proved useful in the past for

Anti-HBs

could be expressed

in mIU/ml,

allowing assessment of immunity. Extensive screening for anti-HBs will help to assess the level of antibody required to give immunity. Although we may speculate that a direct sandwich RIA for HBsAg will be more sensitive than our M-BPL, we conclude from our results that the M-BPL meets the current requirements for HBsAg sensitivity. It has proved to be more sensitive than PHA, which is widely accepted as a sensitive test. In our hands it is as sensitive for HBsAg as the RIAQuick test which detects 5 BSU/ml, and is as sensitive for anti-HBs as the EIA-L test evaluated by Haas and Hotz (1980), since this test detects 390 mIU/ml of the WHO standard for anti-HBs. The reliability of the BPL test has been proven by its extensive use in the Blood Transfusion Service. As a modification of this test the M-BPL has been equally reliable. It has an additional advantage in the routine laboratory, since screening for HBsAg and anti-HBs can be done with the same reagents at the same time. Since January 1985, we have been using the M-BPL as our routine screening test. To date, approximately 12,000 specimens have been processed by the M-BPL. We feel that this has given an improved service to the clinician and their patients. REFERENCES

Haas,

H. and G. Hotz,

Ionescu-Matiu,

1980, J. Virol. Methods

I., S. Yanuario,

2, 63.

H.A. Fields and G.R. Dreesman,

1983, J. Viral. Methods

Lane, R.S., 1981, Med. Lab. Sci. 38, 323. Walters,

G., L. Kuijpers

and A. Schuurs,

1979, J. Clin. Pathol.

32, 1264.

6, 41.