Automated enzyme-linked fluorescence immunoassays for antiviral antibodies: A test for anti-HBs

Journal of VirologicalMethods, Elsevier Biomedical

AUTOMATED ANTIVIRAL

ENZYME-LINKED ANTIBODIES:

NEURATH’

A. ROBERT

4 (1982)

303

291-

291

Press

FLUORESCENCE

IMMUNOASSAYS

FOR

A TEST FOR ANTI-HBs

, NATHAN

STRICK’

and SETH GOODMAN*

’ The Lindsley F. Kimball Research Institute of the New York Blood Center, New York, NY 10021; and ’ Gilson MedicaI Electronics, (Accepted

18 February

A solid-phase antigen

linked

The

test samples antibodies

fluorescence

subsequently

was automated.

of a commercial

U.S.A.

immunoassay

coli p-galactosidase

is described.

were adsorbed were

fluorescence

to Escherichia

umbelliferyl-p-D-galactopyranoside) from

WI 53562,

1982)

enzyme-linked

covalently

Inc., Middleton,

Antibodies

to wells of 96-well detected

The sensitivity

using

(ELFA)

for

antiviral

and a fluorogenic to hepatitis

aminopolystyrene enzyme-conjugated

antibodies

substrate

B surface

antigen

plates precoated HBsAg.

of the test was approximately

using

(4-methyl(HBsAg)

with HBsAg.

The measurement

50 times higher

of

than that

radioimmunoassay.

enzyme-linked

fluorescence

immunoassay

hepatitis

B surface

antigen

surface

antibody

INTRODUCTION

Enzyme-linked fluorescence immunoassays (ELFA) combine the advantages of enzyme-labeled immunoassays (ELISA), i.e. stability of reagents and lack of potential health

hazards,

and of radioimmunoassays,

i.e. high sensitivity

(Ishikawa

and Kato,

1978; Yolken and Stopa, 1979; Neurath and Strick, 1981). Enzyme-labeled antibodies were utilized in these ELFA tests. The usefulness of ELFA for diagnostic purposes would be further extended by the availability of enzyme-labeled antigens and by the automation of fluorescence measurements. In accordance with such aims, we developed an automated ELFA for antibodies to hepatitis B surface antigen (HBsAg) using HBsAg conjugated with /I-galactosidase. MATERIALS

AND METHODS

Covalent linking of HBsAg to polystyrene

microtiter plates

Wells of 96-well polystyrene tissue culture plates (cat. No. 3042, Falcon Products, Oxnard, CA) were nitrated and subsequently reduced with dithionite as described before (Neurath and Strick, 1981). Wells of the resulting aminopolystyrene plates were filled 0166-0934/82/0000-0000/$02.75

@ 1982 Elsevier Biomedical

Press

with 200 ~1 of a solution containing HBsAg subtype ad (100 pg/ml), oxidized as described below, and incubated overnight at 4°C. The unattached HBsAg was removed by aspiration. The wells were filled with a solution of bovine hemoglobin (10 mg/ml in 0.14 M NaCl, 0.01 M Tris, 0.02% NaNa, pH 7.2 (TS)) and the plates were incubated for 4 h at 4°C. After removal of hemoglobin

the plates were washed with TS and stored wet at

4°C. HBsAg for coating of the plates was pretreated with ditions: HBsAg (500 I.cg/ml in 0.2 M sodium acetate, pH concentration 100 mM) for 30 min at 0°C dialyzed for phate, pH 6, and finally diluted 5-fold in 0.05 M sodium

NaI04 under the following con6) was treated with NaI04 (final 2 h against 0.01 M sodium phosphosphate, pH 8.5.

Conjugation of HBsAg with @-galactosidase

Two mg of HBsAg subtype ad in ~1 ml of 0.1 M sodium phosphate-O.1 M NaCl, pH 7.5 (PBS) were mixed with 10 ~1 of a solution ofN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (10 mg/ml in ethanol; obtained either from Pharmacia Fine Chemicals AB, Uppsala, Sweden, or from Pierce Chemical Co ., Rockford, IL). After 30 min at room temperature, the mixture was chromatographed on a 0.7 X 20 cm column of Sephadex G-25 in 0.1 M acetate-O.1 M NaCl, pH 4.5. Fractions containing protein were pooled and solid dithiothreitol was added (7.7 mg/ml). After 20 min at room temperature, the mixture was chromatographed on a 0.7 X 20 cm column of Sephadex G-25 in PBS. Fractions containing the major portion of protein were pooled and mixed with 1 mg of E. coli fl-galactosidase (600 units/mg; cat. No. 567779; Boehringer Mannheim Biochemicals, Indianapolis, IN) for 2.5 h at 20°C followed by 16 h at 4°C. The volume of the mixture was reduced to l-2 ml and submitted to rate zonal centrifugation (16 h at 25,000 r.p.m., Spinco rotor SW27) in a 10-35s (v/v) glycerol gradient in TS. Fractions of 2 ml were collected from the bottom of the gradients. Appropriate aliquots of the fractions were tested for HBsAg (1 ~1) by radioimmunoassay (RIA) and for P-galactosidase activity (0.075 ~1). The percentage of HBsAg recovered per fraction was calculated from a calibration curve relating radioactive counts measured in RIA to serial 5-fold dilutions of HBsAg-enzyme conjugate prior to centrifugation. Fractions containing the highest concentration of HBsAg (fractions 5-10, Fig. 1) were pooled and stored at 4°C. One ~1 aliquots of this pool were used per ELFA test for anti-HBs. Performance

of ELFA

Samples (200 ~1) containing appropriate dilutions of anti-HBs positive serum in a solution containing bovine serum albumin (50 mg/ml) in TS (BSA-TS) were added to wells of microtiter plates precoated with HBsAg. BSA-TS was used for control blanks which were processed in the same way as serum specimens. For routine screening of human sera, specimens including negative control sera were diluted lo-fold in BSA-TS. After standing overnight at room temperature, the samples were removed by suction and

299

the plates were washed with TS. 200 ~1 of the HBsAg-/3-galactosidase conjugate diluted in BSA-TS were added per well. After 4 h at room temperature, the plates were washed with TS, 300 ~1 of the substrate solution (5 X lo-’ M 4-methylumbelliferyl-~-D-galactopyranoside in 0.01 M phosphate, pH 7.0; Sigma Chemical Co., St. Louis, MO) were added and 4-methylumbelliferone released after 30 min at 37°C was determined as described before (Neurath and Strick, 1981).

fluorometrically

Fluorescence measurements Fluorescence was measured against control blanks (= BSA-TS) in the Spectra/glo fluorometer (Gilson Medical Electronics, Inc., Middleton, WI, cat. No. 041001) equipped with filters used for determination of o-phtalaldehyde. The fluorescence was determined from the following formula: fluorescence

= 100 X

fluorometer fluorometer

printout sensitivity

range

Specimens were considered anti-HBs positive if the fluorescence than that corresponding to normal human sera. Automated

was > 2.1-fold

higher

jluorescence measurements

The system for automated fluorescence measurements from 96-well microtiter plates consists of an automatic sampler (Model 212 liquid handler, Gilson Medical Electronics, cat. No. 23101 l), a fluorometer, a peristaltic transfer pump (Gilson Model 311, cat. No. 24101 l), and a data logger (Gilson prototype). Liquids flow in the system from the probe on the sampler, through the fluorometer, peristaltic pump, and out to waste. The sampler aspirates sample from the appropriate well into the Teflon line. It introduces air bubbles before and after the sample to prevent mixing with the rinse liquid. Next, the sampler aspirates sufficient

rinse to move the sample into the fluorometer

logger then prints out sample number the next well and the whole procedure

flow cell. The data

and fluorescence. The sampler then advances to is repeated until samples from all wells are with-

drawn. The volumes of sample, air bubbles, and rinse are all programmable by controlling the amount of time the pump is on during each phase of the cycle. The pump is a special design that runs at a single constant speed and starts and stops very rapidly so that it may be turned on for very short times reproducibly. About 75 ~1 of sample is used to fill the 45 1.11fluorometer flowcell (cat. No. 04047001) so there is sufficient sample left in the well to read it several times if desired. With a continuous flow system such as this, a major concern is sample dilution and carryover. Several steps are taken to minimize these problems. 1. All tubing leading to the flowcell is Teflon which is non-wetting and reduces mixing of flowing liquids. 2. All tubing joints are of the zero dead volume type to avoid mixing. 3. The sample is separated

300

from the preceding and following rinse liquid by air bubles to avoid sample dilution. Because of the flow cell and detector design, bubbles pass through easily and it is not necessary to remove them as is often done. 4. The probe dips into a trough to aspirate rinse liquid during the rinse phase of the cycle. Provision is made for a flowing rinse to prevent accumulation of sample components in the rinse liquid. The complete cycle takes about 23 s per well, or 37 min for the 96-well plate. Other methods HBsAg was purified as described previously {Neurath et al., 1978). Commercial test kits (AUSRIA II and AUSAB, Abbott Laboratories, North Chicago, 11)were used for RIA of HBsAg and anti-HBs, respectively. RESULTS

AND DIS~~JSSION

Covalent linking of HBsAg to aminopolystyrene plates The advantages for antigen immunoassays of covalently linking antibodies to the solid phase have been described (Herrmann et al., 1979; Neurath and Strick, 1981). We used ~utaraldehyde-activated a~nopolystyrene to bind immunoglobu~s to the solid phase. Preliminary experiments (own unpublished data) revealed that the antibody binding capacity of HBsAg is diminshed as a result of reaction with glutaraldehyde. Therefore, other methods of linking HBsAg to polystyrene microtiter plates had to be considered. We chose to introduce aldehyde groups, expected to react directly with polyaminostyrene, into the saccharide moiety of HBsAg by periodate oxidation of the terminal sialic acid residues (Van Lenten and Ashwell, 1971; Neurath et al., 1975). AUSRKA tests on serial dilutions (in normal human serum) of HBsAg before and after treatment with NaI04 revealed similar titration endpoints, although the calibration curve relating counts per minute to antigen concentration was steeper for intact HBsAg (data not shown). Approximately 80% (= 16 &well) of added oxidized HBsAg was attached to the plastic and was not eluted when the coated plates were exposed for 30 min to an alkaline (pH 10.9) solution containing 10 mg/ml of sodium dodecyl sulfate. This suggests that HRsAg was covalently linked to the solid support. Conj~~ti~n ofIi&& with ~-galaetosidase The heterobifunctional

reagent N-succinimidyl-3-(2-pyridyldithio)propionate

(SPDP)

is the cross-linking agent of choice for conjugation of antibodies with /3-galactosidase (Neurath and Strick, 1981). In considering the use of SPDP for linking of this enzyme with HBsAg, we were concerned with possible alterations of the immunological specificity of HBsAg after exposure to the reducing agent dithiothreitol (Neurath and Strick, 1980) used in the course of the preparation of the conjugate. Of additional concern

301

was the possible sterical hindrance Comparative

AUSRIA

of HBsAg antigen&

tests on serial dilutions

sites by the attached

enzyme.

of HBsAg before and after conjugation

indicated that the derivatization resulted in an acceptable 60% decrease of HBsAg titer. The HBsAg-/I-galactosidase conjugate was separated from the bulk of free enzyme by rate zonal centrifugation (Fig. 1). The activity of the conjugate did not decrease during storage at 4°C for 4 months (longer periods were not tested). The successful conjugation of HBsAg with fl-galactosidase suggests that other virus antigens, including those with disulfide bonds essential for their antigenicity, may be linked with enzymes using SPDP and that the resulting conjugates may become useful for development of ELFA or ELISA tests. EL FA tests of an ti-HBs Preliminary

tests revealed the presence

in normal human sera of a component(s)

re-

IO-

oI

I I I I I I I I 3 5 7 3 II 13 I5 17 FRACTION NUMBER

Fig. 1. Comparative in lo-

cross-linking in which ment.

results

35% (v/v) glycerol of HBsAg maximum

of rate zonal gradients

with

quantities

centrifugations

of p-galactosidase

p-galactosidase

(bottom).

(16 h at 25,000

r.p.m.,

Spinco

($0 pg; top) and of the product Arrow

of free HBsAg were recovered

indicates

the fraction

when centrifuged

rotor resulting

SW 27) from

of the gradient

in a separate

experi-

302

acting with the HBsAg-fl-galactosidase conjugate. This resulted in high levels of background fluorescence for anti-HBs-negative sera. Moreover, this fluorescence varied for different human sera. Therefore, all sera screened for anti-HBs were diluted at least lofold in BSA-TS. This resulted in a substantial decrease of fluorescence corresponding to anti-HBs-negative blanks and in increased sensitivity of anti-HBs detection (Fig. 2). Comparative ELFA and AUSAB tests on serial dilutions of the same sera indicated that the sensitivity of ELFA is about 50 times higher than that of the AUSAB test. To assess the comparative efficiency of the AUSAB and ELFA tests in screening of sera for anti-HBs, serum specimens from 100 randomly selected blood donors were analyzed by both tests. Anti-HBs was detected in five sera by the AUSAB test. The same sera (diluted lo-fold in BSA-TS) were also positive as determined by ELFA. Additional two sera were positive using the latter test. (The mean fluorescence corresponding to negative specimens was 1.5 with a variance of 1 .l; positive specimens had a fluorescence > 6.3). Addition of 50 pg of each of HBsAg subtypes ad and ay to sera which appeared anti-HBs positive by ELFA resulted in decreased fluorescence readings, thus confirming that all seven sera contained anti-HBs. As mentioned before, ELFA tests combine the advantages of ELISA and RIA tests. However, previously described ELFA tests (Ishikawa and Kato, 1978; Yolken and Stopa,

l60-

kj 120-

:

z &oo-

1

z g

ao-

3 k

60-

ot,, 10-7

10-6

DILUTION Fig. 2. Calibration

anti-HBs-positive line indicates

lo-5

curve human

background

the substrate

solution

still positive

by

normal

human

and decreased

IO-~ IO-~ IO-’

IO-~

OF ANTI-HBs

the

serum further

POSITIVE

for anti-HBs. serum

Serial 2-fold

in a solution

fluorescence

which AUSAB

SERUM

corresponding

was not incubated test

was

dilutions

reaching

to blanks.

2 X 10-5-fold. background

with a lo-fold

in TS) were tested. Fluorescence

at 37°C. The highest

was the same as that corresponding by dilution

(starting

of BSA (50 mg/ml

Fluorescence

dilution

levels at a 10e4 dilution.

of an

horizontal

was measured at which

corresponding

to 6 x 10e5 diluted

dilution)

Broken

against

this serum was to 10-l

diluted

anti-HBs-positive

serum

303

1979; Yolken fluorescence mits

rapid

et al., 1980; Neurath readings.

and Strick,

Such automation,

fluorescence

described

measurements

1981) did not utilize automation

of

for the first time in this report, per-

(?J 23 s/sample)

and eliminates

the need for

manual operations in the course of reading 96 samples corresponding to one microtiter plate. Work now in progress promises to reduce this time substantially. Our results show that ELFA tests can be automated and applied for screening of large numbers of specimens in a clinical laboratory setting. ACKNOWLEDGEMENTS

This study was supported in part by Grant C 172100 from the New York State Health Research Council. We thank Dr. L. Baker for sera containing HBsAg and anti-HBs, respectively. REFERENCES

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J.E., R.M. Hendry E. and K. Kato,

and M.F. Collins,

1978, Stand.

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1981, J. Virol. Methods

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A.R., N. Hashimoto

and A.M. Prince,

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1980, J. Med. Virol. 6,309.

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YoIken,

R.H., V.M. Torsch,

3,155.

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34,414.

1971, J. Biol. Chem. 246, 1889.

1979, J. Clin. Microbial. R. Berg, B.R. Murphy

10,317.

and Y.C. Lee, 1980, J. Infect.

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