Journal of Virological Methods, 2 (1980) 85 -96

@ Elsevier/North-Holland Biomedical Press

85

COMPARISON OF ENZYME-LABELLED F(ab’), AND IgG CONJUGATES IN AN

ENZ~E-~~~OASSAY FOR ~EPATI~S B ‘e’ ANTIGEN

M. VAN DER WAART, A. SNELTING, F. BRUIJNIS, MM. PRINS-BEKIUS, E. BOS, J. KATCHAKI’ and A. SCHUURS

Organon Scientific Development Group, 0~s; and ‘Public Health Laboratory, Municipal Hospital,

Arnhem, the Netherlands

An improved enzyme-immunoassay (EIA) for the detection of hepatitis B ‘e’ antigen @IBeAg) and its corresponding antibody is described. The present test is as sensitive as the previous one but it is more specific as demonstrated by testing donor/recipient sera, donor plasmas and patients sera.

Interference by antibody against hepatitis B surface antigen (HBsAg) did not occur due to the use of HBsAg-free reagents. Interference by rheumatoid factor could be avoided by using enzyme-labelled F(ab’), rather than IgG conjugates. The application of an F(ab’), conjugate, however, introduced other non-specific reactions, particularly in sera from patients with (auto~mune) liver disorder. Further study into the applicability of F(ab’), conjugates is therefore indicated.

INTRODUCTION

Since its introduction in 197 1 enzyme-immunoassay (EIA, ELISA) (Engvall and Perl-

mann, 1971; Van Weemen and Schuurs, 1971) has become a well-establi~ed procedure

for the laboratory diagnosis of a variety of diseases (Schuurs and Van Weemen, 1977).

One of the areas of application is the screening for markers of hepatitis A and B (Duer-

meyer and Van der Veen, 1978; Duermeyer et al., 1979; Wolters et al., 1976,1979).

We have described a sandwich-type EIA for the detection of hepatitis B ‘e’ antigen

(HBeAg) (Van der Waart et al., 1978). Anti-HBe IgG was prepared from human sera,

which were also positive for hepatitis B surface antigen (HBsAg). In a first evaluation, this

EIA appeared to be sensitive and could be applied to the screening of donors and hepatitis

B patients (Katchaki et al., 1979; Heytink et al., 1980; Niermeyer et al., 1980). Occasion-

ally, false positive reactions were observed. In a number of cases this could be attributed

to the presence of antibody directed against HBsAg (anti-HBs) or to rheumatoid factor

(RF). Interference by anti-HBs was presumably caused by the presence of trace amounts

of HBsAg in the conjugate and in the anti-HBe coating of the microtitre plates. Further

purification of these reagents was therefore required.

Interference by rheumatoid factor, an autoantibody frequently occurring in hepatitis,

is due to its reaction with the Fc region of the anti-HBe IgG (Kunkel and Tan, 1964).

86

~e~atoid factor interference can be suppressed by removal of this factor from serum

through addition of heat-aggregated IgG (Meurman and Ziola, 1978). A more practical

approach, particularly in EIA, might be the use of Fab’ or F(ab’)a fragments instead of

whole IgG for the preparation of conjugates (Duermeyer et al., 1979; Kato et al., 1979).

In this paper we describe the results of a comparison between F(ab& and IgG con-

jugates labelled with ho~eradish peroxidase (HRP) in EIA for ~BeAg~anti-H3e, using

anti-HBe-containing reagents that were purified by immune adsorption with anti-HBs

coupled to Sepharose 4B.

MATERIALS AND METHODS

~epara~~~z uf anti-HBe IgG

Two different pools, each composed of five human sera positive for anti-HBe, were

used for the preparation of conjugate and coated microtitre plates. These sera were posi-

tive for anti-HBe by EIA (titre 9 3000) and by ~munodiffusion (ID; at least 2 subtypes),

positive for HBsAg by EIA and negative for RF and anti-HBs (EIA or RIA).

From the pool for coating, HBsAg was removed by batchwise immune adsorption with

sheep anti-HBs coupled to Sepharose 413, in two successive steps at 4’C overnight and at

room temperature for 4 h respectively. The Sepharose beads were spun down, washed

twice with 0.04 mol/l Na2HP04/0.00.5 mol/l KH2P04 buffer pH 6.9, containing 0.06

mol/l EDTA and 0.15 mol/l NaCl and the supernatants were collected. I~unoglobulins

were precipitated with 140 g/l Na2S04 and were dissolved in and dialysed against 0.1

mol/l sodium phosphate buffered saline, pH 7.4, at a final concentration of 10 mg/ml

IgG. This fraction was used for the coating of the microtitre plates. HBsAg was not re-

moved from the other anti-HBe pool, but immunoglobulins were precipitated as des-

cribed above. This pool was used for the preparation of the conjugate.

Preparation and purificatiort of anti-HBe F(ab’)2

We used slightly modified procedures of Kato et al. (1976a) and Wilcsek and Hambur-

ger (1978). Briefly, the IgG fraction was dialysed against 0.1 mol/l sodium acetate buffer

pH 4.5 overnight, diluted to 5 mg/ml IgG and digested with pepsin for 16 h at 37°C

using an IgG/pepsin weight ratio of SO/l. The reaction was stopped by adjusting the pH

to 8.0. The concentrated pepsin digest was chromatographed at room temperature on

Sephacryl S200 equilibrated with 0.1 mol/l boric acid pH 8.0. The fractions containing

F(ab’)s were pooled and concentrated to a final protein concentration of 60 mg/ml.

Preparation ~fHRP-~abelled anti-HBe Ffab’),

The F(ab’)a concentrate was coupled to HRP according to a slightly modified ‘two-

step’ procedure of Avrameas and Ternynck (1971). We used an F(ab’):!/enzyme weight

ratio of l/4 and a glutar~dehyde concentration of 40 g/l in a 1 mol/l NaHC03 buffer

pH 9.0. The HRP-IabelIed anti-HBe F(ab’), conjugate was recovered by precipitation

with 140 g/l Na2S04. The precipitate was dissolved in 9 g/l NaCl and stored at -20°C.

Prior to use, the conjugates were dissolved in an 0.2 mol/l Tris buffer, pH 7.4, containing

250 ml/l of a mixture of five different normal human sera (NHS). These sera were nega-

tive for HBsAg, anti-HBs, HBeAg, anti-HBe and RF.

Preparation of HRP-labelled anti-HBe IgG

Part of the IgG-containing dialysate (see above) was concentrated to a concentration

of 60 mg/ml IgG. It was conjugated with HRP as described above, but for the precipitation

of the conjugate 200 g/i Na2S04 was used.

Coating of the microtitre plates

Polystyrene Microtiter@ flat-bottom plates (Cooke) were coated with anti-HBe as was

described before (Van der Waart et al., 1978), but 0.1 instead of 0.2 mg/ml IgG was used.

Coupling of sheep an ti-HBs to Sepharose 4B

We followed the instructions of the manufacturer (Pharmacia), slightly modified by

Kuijpers and Wohers (personal communization).

Briefly, a 140 g/l Na2S04 precipitate of sheep anti-HBs serum was added to CNBr-

activated Sepharose 4B, prewashed with 1 mmol/l HCl. Coupling was carried out in 0.1

mol/l NaHCOa, pH 8.3, overnight at 4°C. Free cyanide groups were blocked with

1 moljl monoethanolamine, pH 9.0, for 2 h at room temperature. The gel was washed

with acetic acid pH 2.8, and 0.1 mol/l NaHCOs pH 8.3, alternately and finally with a

0.014 molff NazHP04 buffer pH 6.9, containing 0.06 mol/l EDTA and 0.15 mol/I NaCI.

Anti-NHS active sites were blocked by incubation with NHS overnight at 4°C.

Polyacrylamide gel electrophoresis (PAGE)

Protein samples were analysed by sodium dode~ylsulphate-PAGE according to the

procedure of Laemmli (1970). In the experiments described, gel electrophoresis was per-

formed on gradient gels with a linearly increasing monomer concentration of 30-260 g/l

and completed by a 30 g/l spacer gel.

Gels were stained with 5 g/l Coomassie brilliant blue R250 in 400 ml/l methanol, 100

ml/I acetic acid and were destained in 100 ml/l acetic acid.

Other assays and reagents

HBsAg was tested by EIA (Hepanostika @, Organon Teknika, Oss, The Netherlands) or

88

RIA (Ausria, Abbott, IL, U.S.A.) according to the instructions of the manufacturer.

Anti-HBs was assayed with Ausab@ (Abbott, IL, U.S.A.) or with EIA (Wolters et al.,

1979). Results were expressed as I.U./ml (based on the W.H.O. standard).

Antibody directed against hepatitis B ‘core’ antigen (anti-HBc) was tested with Corab@

(Abbott, IL, U.S.A.).

RF was assayed with Rheumanosticon@ (Organon Teknika, Oss, The Netherlands).

For HBeAg/anti-HBe detection and for the confirmation of presumptive HBeAg-

positive results we used an EIA as described elsewhere (Van der Waart et al., 1978).

Results for HBeAg were expressed as positive~negative (P/N) ratio, being the absorbance

of a test sample divided by that of the average of five negative human sera, both corrected

for the enzyme substrate blank. Dilution series of HBeAg and anti-HBe-positive sera were

made in 9 g/l NaCl, 100 g/l NHS.

Subtyping of sera positive for anti-HBe was performed as described before (Van der

Waart et al., 1978).

IgG concentrations were calculated by measuring E280 and EzhO in a Gilford model

250 and applying the formula: IgG (mglml) = (1.45 X Ezse) - (0.75 X Eaee) X dil. factor.

Fractions were concentrated by ultrafiltration on PM-10 filters, using Amicon equip-

ment.

HRP activity was determined with o~hophenylenediamine/urea peroxide as the sub-

strate (Wolters et al., 1976).

Pepsin was purchased from Worthington (Freehold, U.S.A.) (2700 U/mg). Sephacryl

S200 and CNBr-activated Sepharose 4B were obtained from Pharmacia (Uppsala, Sweden).

All other reagents were of analytical grade and distilled water was used throughout for

the preparation of solutions.

Sera

Donor sera, positive for HBsAg/anti-HBe and HBsAg/HBeAg, were obtained from Dr.

Babes (Bucharest, Rumania), via Dr. Bulgini (Milano, Italy), via Dr. Orlowska (Warsaw,

Poland) and from Dr. Stute (Dusseldorf, G.F.R.). HBsAg-negative plasmas were kindly

provided by Dr. Stute (Institute for Blood Coagulation and Blood Transfusion, University

of Dusseldorf, G.F.R.). RF-positive sera were obtained from Dr. Banffer (Municipal

Health Service, Rotterdam, The Netherlands) and Dr. Aay (Central Laboratory of the

Blood Transfusion Service of the Netherlands Red Cross, CLB, Amsterdam, The Nether-

lands). Anti-HBs-positive sera were supplied by Drs. Lafeber-Schut and Reerink (CLB,

Amsterdam). Sera from various patients suffering from (auto~mune) liver disorders

other than hepatitis B were provided by one of us (J.N.K.).

RESULTS

Preparation of HBsAg-free anti-HBe

The pools of sera positive for both HBsAg and anti-HBe, as well as the anti-HBe pre-

89

paration, obtained after immune adsorption with anti-HBs-coupled Sepharose 4B, were

titrated in the EIAs for HBsAg and anti-HBe. On average, the recovery for anti-HBe was

at least 90% and was less than 0.1% for HBsAg. After Na2S04 precipitation HBsAg was

no longer detectable in the precipitate.

Preparation of HRP-labelled anti-HBe F(ab’J2

After pepsin digestion of the Naz SO4 precipitate, the elution profile of the Sephacryl

S200 chromatography showed four peaks (Fig. 1). As was revealed with PAGE these

peaks presumably consisted of aggregated molecules and some undigested IgG, F(ab’)z

without any detectable IgG, Fc fragments and small degradation products, respectively

(Fig. 2a). The fractions from the second peak containing F(ab’), were used for the pre-

paration of the anti-HBe F(ab’)s conjugate.

The F(ab’), and the IgG conjugates were analysed with PAGE. As demonstrated in

Fig. 2b, both conjugates contained free HRP and they were highly heterogeneous. The

molecular weight of the F(ab’)* conjugate ranged from 150,000 to at least 600,000 and

that of IgG from 190,000 to at least 600,000. In comparison with anti-HBe IgG a five-

fold lower yield was obtained with the preparation of the anti-HBe F(ab’)a conjugate.

F(ab’)s conjugates were as stable as IgG conjugates when stored frozen at -20°C, or

lyophilised at 4*C.

% transmission

Peak I Peak II Peak 111 Peak IV

P I I- -r- * t

40- / t

I I

I I I I I I I I

60- I I

I I I I I I I I

80-

0 200 400 600 800 1000 1200

Elution volume (ml)

Fig. 1. Sephacryl S200 chromatography of anti-HBe pepsin digest. The second peak contained F(ab’), fragments (see Fig. 2a).

90

Cl

A B

275K-

28K- -

15K-

C D b

E F GIH 1 .I I m

I I

/ I - 250K

I - lSOK

t - 7fOK

I n ,I - 80K

I I I

- 25K -

Fig. 2. a) Polyacrylamide gel electrophoretic analyses (PAGE) of pooled human anti-HBe sera (A), of a 140 g/l Na,SO, precipitate of this serum pool (B), of a pepsin digest of the precipitate (C) and of 4

peaks obtained after chormatographic separation of the pepsin digest (see Fig. l), cf. peak I (D), peak

11 (E), peak III (F) and peak IV (G). b) PAGE of F(ab’), (H) and of IgG conjugate (I) labelled with

HRP. Both conjugates contained free HRP (MW 40,000), HRP dimers (MW 80,000), free F&b’), and

IgG, respectively, and a variety of different conjugate molecules.

Chu~~cter~stie~ of HBeAg EIA with both eonj~g~tes

Sensitivity

HBeAg-positive sera were titrated and tested in EIA with F(ab’)z and IgG conjugates.

A typical result is given in Fig. 3, showing that both conjugates were comp~able with

respect to sensitivity. Interpolated titres of 5000 and 6400 were found with the F(ab’),

and the IgG conjugate, respectively. In ID this serum had a titre of 8.

Specificity

The reactions in EIA for HBeAg of 128 donor sera positive for anti-HBs but negative

for HBsAg and HBeAg were investigated. In the first instance, they were tested by EIA,

prepared from reagents which were not treated with anti-HBs coupled Sepharose. With

the F(ab’)z and the IgG conjugate five and two sera respectively were found positive. The

one serum that gave the highest P/N ratio was retested in the EIA prepared from HBsAg-

free reagents. In addition, seven other sera moderately or strongly positive for anti-HBs

but negative for HBsAg and HBeAg were tested as well. None of these sera containing up

to 28 I.U./ml anti-HBs reacted with either of the two conjugates in EIA for HBeAg.

The specificity with respect to interference by RF was investigated with a panel of

HBsAg-negative~RF-positive sera. They either consisted of pools of different sera with

the same Waaler-Rose titre or they were derived from individuals. As summarised in

Table 1, seven out of 14 sera reacted positively in the EIA for HBeAg with the IgG con-

91

P/N ratio Titre --._

EIA ID

I9G W’12

l/6400 l/5cmO 8

Dilution of HBeAg containing serum

Fig. 3. Dose-response curves of HBeAg in EIA using two different conjugates. Pos/neg (P/N) ratio 20

corresponded with A,,, = 1.847. For NHS, values ofA,,, = 0.106 were found. The reagent blank was

A 492 = 0.015.

TABLE 1

Specificity of EIA for HBeAg with respect to interference by RF using two different conjugates

Serum code Type RF titre HBeAg-EIA (P/N ratio)

Waaler-Rose __ IgG conjugate F(ab’), conjugate

B 17575 - 2 - 5 - 6 - 7 - 8

B6217 - I B 17559 - 15

3185 3665 3546 3161 3487 3565 3359

pool

individual

16 128

256 512

1024 512 256

NT NT NT NT NT NT NT

3.0

2.3 2.7 2.3 2.4 6.0

2.2

NT = not tested -means P/N ratio < 2.1

92

TABLE 2

Precision of EIA for HBeAg using different conjugates

Assay variation Conjugate No. tests A 492 cv (%)

Within Ffab’>, 90 1.52 5.1 Within 1gG 90 1.55 6.7 Total kG 24 3.63 8.5

jugate, but none was found positive with the F(ab’), conjugate. None.of the seven

positives found with the IgG conjugate could be confirmed in the confirmation test.

Precision The withy-assay variation for the HBeAg EIA was established for the two conjugates.

Using two microtitre plates, we tested a pool of eight different sera and distributed the

two conjugates randomly over both plates. Coefficients of variation (CV) of 5.1% and 6.7% were found for the F(ab’)z and the IgG conjugates respectively (Table 2). For the

TABLE 3

First results of a retrospective study into the role of anti-HBc-positive serum as a possible cause of are compiled where the recipients became positive for either HBeAg or anti-HBe. 86 donors and 86

Donor serum

Donor

Test

Recipient

HBsAg HBeAg’

code

Anti-HBe Anti-HBc Anti-HBs a b c d e f g

9 - + +(&gy _ _ _ _ _ _ _

25 - + + +cc-g) - - +3>5 + + + +

(8) (31) (26) (26) (24) 29 + + + --_- _ -_

49 + + + ___- _ --

62 - + + + + + + +

(4) (4) (6) (5) (5) (5) (4) 122 - f t t f + + +

(2) (4) (4) (3) (31 (2) (2)

133 - + _ NT4_ _ _ _ _

’ Sample a: before transfusion, sample b--g: after transfusion. * Between brackets the period of positivity (see note 1). 3 Between brackets the P/N ratio. 4 NT = not tested. B Recipient of donor 25 was true positive for HBeAg, recipients of donors 62 and 122 appeared to be false positive for HBeAg with the IgG conjugate and were negative with the F(ab’), conjugate.

93

IgG conjugate the total-assay variation was investigated by testing during 6 consecutive

weeks a pool of four different HBeAg-positive sera. The CV was 8.5%.

Screening of blood donors and their recipients (Table 3)

As part of a retrospective study of the role of anti-HBc-positive donor blood as a

possible cause of post-transfusion hepatitis B (PTHB) we tested 86 donors and their 86

recipients. The donors were negative for HBsAg and either positive or negative for anti-

HBc. They were tested once for HBeAg and anti-HBe. None of the donors was found

positive for HBeAg with the IgG conjugate but 24/86 were positive for anti-HBe, always

running in parallel with anti-HBc. The recipients were not selected and received serum for

a variety of reasons. They were tested for HBeAg/anti-HBe prior to transfusion and at

monthly intervals up to 7 months after transfusion. One out of 86 recipients developed

post-transfusion hepatitis B and was found positive for HBeAg 12 weeks after transfusion

(from donor No. 29, two others were weakly (false) positive for the antigen (from donor

Nos. 62 and 122). This result was obtained with the IgG conjugate. When applying the

F(ab’), conjugate, the one true HBeAg-positive result could be confirmed (from donor

No. 2.5) but the false positive sera were now negative for HBeAg.

post-transfusion hepatitis B. All donors were negative for HBsAg and HBeAg. In the Table those cases recipients were investigated.

Anti-HBe’ Anti-HBc Anti-HBs

Remarks

a b C def g

_ _ - -++ + + (d-g) + (f-g) Clinical hep. _ - _ --- _ +(d-g) +(b) Clinical hep.

t t + + t t + + (a-s) + (a-g) - + + tt- - + (b-d) +(b-d) _ _ - -__ _

t NT t t t t t + (a-g) + (a-g)

94

One recipient, who developed post-transfusion hepatitis B, was found positive for

anti-HBe (and HBsAg) 20 weeks after transfusion (from donor No. 9). Three other reci-

pients who did not develop post-transfusion hepatitis B were permanently or only tran-

siently positive for anti-HBe, anti-HBc and anti-HBs, but in their case we did not observe

real seroconversion (from donor Nos. 29,49 and 133).

Screening of donor plasmas

A group of 76 donor plasmas, either positive or negative for HBsAg, was tested in EIA

for HBeAg~anti-HBe with the F(ab’)z and IgG conjugate. We obtained identical results

with both conjugates. None of the sera gave a false positive reaction for HBeAg, as could

be demonstrated with the confirmation test.

Screening of patients sera

A group of 30 sera from patients suffering from (autoimmune) liver disorders other

than hepatitis B were tested for HBeAg/anti-HBe with the F(ab’), and the IgG conjugate.

Two samples were false positive with the IgG conjugate (P/N ratio 3.0; 9.9) and four

reacted false positive with the F(ab’)s conjuagte (P/N ratio 3; 14; 15; 23).

DISCUSSION

Our previously published method for the detection of HBeAg and anti-HBe could be

applied to the screening of blood donors and for testing hepatitis B patients (Van der

Waart et al., 1978). However, we observed that anti-HBs and rheumatoid factor occasion-

ally gave rise to false positive reactions. Therefore, we investigated whether interference

by anti-HBs could be removed by the treatment of reagents containing both HBsAg and

anti-HBe with anti-HBs-coupled Sepharose 4B. After adsorption and subsequent salt

fractionation anti-HBe was recovered, but HBsAg was no longer detectable. With these

reagents false positive reactions were no longer found in the EIA for HBeAg when applied

to a group of donors negative for HBsAg but positive for anti-HBs. For the elimination

of interference by rheumatoid factor in the EIA for HBeAg we relied on the favourable

results of Kato et al. (1979) and Duermeyer et al. (1979). They used Fab’ and F(ab’)z

conjugates in EIAs for insulin and hepatitis A antibody, respectively, and found no

rheumatoid factor interference.

Slightly modified procedures of Kato et al. (1976b) and Wilcsek and Hamburger

(1978), using pepsin digestion, were applicable for the preparation of F(ab’)z fragments

from human anti-HBe IgG. This could be deduced from the chromatographic separation

of the pepsin digest (Fig. 1) and gel electrophoretic analysis of the fractions (Fig. 2a). A

small fraction of IgG was not digested by pepsin, but it could be separated from F(ab’)z.

Slight differences were observed in the Sephacryl S200 elution profile in the pepsin

digest of different serum pools. This may have been caused by the presence in each

95

serum pool of variable amounts of aggregated IgG which seems not to be susceptible to

pepsin digestion. This aggregated IgG will appear in the first peak of the Sephacryl S200

eluate. Moreover, variable amounts of lipid were also recovered in the first peak.

F(ab’)* as well as IgG could be used for the preparation of enzyme-labelled conjugates,

although the yield of F(ab’)* conjugates was poor. This was mainly due to the low re-

covery of anti-HBe activity in the pooled F(ab’), fractions (peak II, Fig. 1). Pepsin

probably also partially degraded IgG to smaller molecules other than F(ab’), (peak IV,

Fig. 1).

The enzyme-labelled conjugates, prepared from F(ab’)z and IgG, according to the two-

step method of Avrameas and Ternynck (1971), appeared to be highly heterogeneous

as was revealed by PAGE (Fig. 2b). Ford et al. (1978) made similar observations by

analysing conjugates on Sepharose gels and sucrose density gradients. In contrast, con-

jugates prepared by Boorsma and Streefkerk (1979) were mainly of low molecular

weight.

The heterogeneity of our conjugates may point to the presence of aggregated F(ab’)2

fragments and IgG molecules. This might be disadvantageous because it could give rise to

enhanced reactivity with rheumatoid factor (Meurman and Ziola, 1978). However, in

practice, no false positive reactions were observed in rheumatoid factor positive sera when

applying the F(ab’)2 conjugate in the EIA for HBeAg (Table 1). Obviously, the lack of

Fc fragments in the F(ab’), conjugates completely compensated for the possible pre-

sence of aggregates.

The specificity of the present EIA for HBeAg/anti-HBe was established by the screen-

ing of a group of donor plasmas and a group of sera from donors and their recipients

(Table 3). Almost identical results were obtained with F(ab’)* and IgG conjugates.

However, some sera positive for RF reacted false positive for HBeAg with the IgG con-

jugate but were negative with the IgG conjugate. Further insight into the specificity of

EIA, using the two different conjugates, was obtained by testing sera from patients

suffering from (autoimmune) liver disorders other than hepatitis B. In this group of sera

some non-specific reactions for HBeAg were observed with both conjugates. Remarkably,

some sera negative with the IgG conjugate were strongly false positive for HBeAg with

the F(ab’), conjugate. These non-specific reactions could not be attributed to the pre-

sence of rheumatoid factor. One might speculate that they were caused by antibodies

directed against human anti-HBe digested with pepsin.

The sensitivity of our HBeAg EIA was neither impaired nor improved by the use of

F(ab’), conjugates. Almost identical titres were found in dilution series of HBeAg-

positive sera (Fig. 3). Kato et al. (1976a) suggested that the use of Fab’ conjugates

allowed the development of extremely sensitive EIAs. This was more or less confirmed

by Hamaguchi et al. (1979) who found lower non-specific adsorption in EIAs with Fab’

conjugates. However, no direct comparisons between dose-response curves or between

ultimate detection levels of EIAs applying the two types of conjugates were given. We

therefore believe that the use of Fab’ or F(ab’), conjugates, based merely on an expected

higher sensitivity, is premature.

96

In conclusion, the present EIA, using an IgG conjugate, is at least as sensitive and pre-

cise as the method described previously (Van der Waart et al., 1978) but it is more spe-

cific with respect to anti-HBs interference. Non-specific reactions due to the presence of rheumatoid factor can be avoided by using an F(ab’)* instead of an IgG conjugate. The

application of an F(ab’)* conjugate, however, appears to introduce other non-specific

reactions, particularly in sera from patients with (autoimmune) liver disorders. Thus, a

definite decision regarding the usefulness of F(ab’)z conjugates awaits a more extensive

study of sera collected from patients.

ACKNOWLEDGEMENT

The authors would like to thank Cisca Nipvlieg and Ine Wassing for skilful assistance.

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