Comparison of four newly developed immunoblot assays with RIBA II for detection of HCV antibodies

Serodiagnosis & lmmunotherapy IN INFECTIOUS DISEASE

ELSEVIER

Serodiagnosis and Immunotherapy in InfectiousDisease8 (1996) 79-83

Comparison of four newly developed immunoblot assays with RIBA II for detection of HCV antibodies Hanns Hofmann Department of Clinical Virology, GeneralHospital V'wma, W'ahringerGf~rtel 18-20. A-f090 V'wra-4.Austria

Received 10 September 1995; accepted 30 October 1995

Abstract ELISA, the usual screening test for HCV infection, may yield nonspecilic results. Most laboratories, therefore, for corroboration perform PCR. However, a negative H C V - P C R does not prove nonspeciiity of the initial ELISA test and therefore an immuaobiot has to be performed. RIBA-II was used for that purpose for several years, but suffers from a high number ofindetermlnate results. We therefore compared RIBA II results of 75 sera with those of RIBA III, Matrix, Western Blot (Mure~) and INNO-LIA tests. Of 34 sera that were positive in RIBA II, all were also positive in the four other immunoblots. Similarly, these 4 tests showed concordantly positive results in 13 of 27 RIBA I1 indeterminate sera. In the remaining 14 (RIBA II indeterminate) sera the four immunoblots displayed no uniform results but various combinations of positive, indeterminate and even negative results. Simihr results were found with 13 RIBA II negative (ELISA positive) sera. These data may indicate less sensitivity as well as some nonspecificity ofsome of the immtmoblots. In general, however, the four newly developed immunoblots proved to be more sensitive than RIBA If. This obviously is not caused by the inclusion of the NSs-antigen but by improvement of the conventional antigens. Only one serum was found in which the NSs-band was crucial for its positivity. In conclusion, for corroboration of some HCVELISA positive, PCR negative sera, more than one immunoblot may be ~ r y . Key~ords: Hepatitis C virus; lmmtmoblot a~ay; RIBA; ELISA; Screening test

1. Introduction The ELISA is used worldwide as the screening test for diagnosis of hepatitis C virus (I-ICV) infection [I-3]. Similarly to other ELISAs in diagnostic virology, a positive HCV test may be nonspecitic, i.e. false positive. Such results therefore have to be corroborated by another test based on a different principle. Initially for that purpose immunoblots were developed. At present, however, most diagnostic laboratories prefer to perform a reverse transcription-polymerase chain reaction (RTPCR) to detect HCV-RNA, which provides information about viral activity in addition to the specificity o f the positive ELISA test [1-3]. However, a negative HCVPCR does not always indicate that the initial HCVELISA was unspeofic. The HCV infection might resolve and the viremia might cease, while antibodies are still detectable, or a chronic infection might improve tem0g~078@9@$15.00 © 1996 Elses~ ScienceB.V. All ri..thtsn~en~l SSD! 0888-0786(95)01054-C

poratily, resulting in negative P C R results for only a short period [1,3]. F o r these cases corroboration by immtmoblot is still needed. The best known test o f thi~ type is the RIBA, which in its second generation version (RIBA ED has been widely employed in recent years [1,3]. However, it ha~ suffered from a relatively high number o f indeterminate results [4-10]. F o r example, in our ~ o f 3022 RIBA H tests 1895 (62.7%) were positive, 344 (II.4%) were aegative and 783 (25.9%) w e ~ i a & s ~ a i a a ~ In the meantime the R I B A test has been im!woved by replacing both core and Ns4 recombinant antigeas by synthetic peptides and an antigen o f the 1 ~ n~gion has been included, resulting in the dfird generation ( R I B A l i d test [3,81. However, other m a n u f a a m e s s offer a comparable product for corroborating HC'3/-ELISApositive results. We t l m ~ o r e compared four _emmnercially available h~munoblots: M a l i m (Abbott), WeMern

80

H. Hofmann / Serodiagnosis and lmmunotherapy in Infectious Disease 8 (1996) 79-83

Blot (Murex) RIBA III (Chiton) and INNO-LIPA (Immunogenetics) with RIBA II, especially with respect to inconclusive results. 2. M a t e r i a l s and m e t h o d s

2.1. Sera

All sera tested were sent to the Department Of Clinical Virology for routine diagnosis from the different clinical departments of the General Hospital Vienna. Seventeen patients suffered from chronic hepatitis, 21 were on hemodyalysis treatment, 10 were haemophiliacs, 5 HIV positive, 2 drug users, and in 20 persons the HCV-antibodies were detected by chance either in connection with a voluntary blood donation or preoperatively. Initially the Abbott HCV-ELISA (second generation test) was performed. For routine diagnosis a positive ELISA result was corroborated by recombinant immunoblot assay (RIBA II, Chiron Corporation, Emeryville, CA) and by RT-PCR as described previously [11,12]. For the study 75 sera were selected (all HCV-ELISA positive): 35 RIBA II positive, 13 negative and 27 indeterminate. The only selection criterion was that enough serum was left to perform all the four immunoblots. 2.2. Immunoblots

Sera were tested initially by RIBA II (R 2) and thereafter retested by Matrix (Ma) from Abbott Laboratories, Chicago, IL; by RIBA III (R 3) from Chiron Corporation, Emeryville, CA; by INNO-LIA (LI) from Innogenetics, Zwijnaarde, Belgium; and by Wellcocyme HCV Western Blot (WB) from Murex, Dartford, UK. All immunoblot assays were performed according to the manufacturer's instructions. 2.2.1. R I B A I I (R2)

This assay provides four virus-specific recombinant viral antigens, 5-1-1, c100-3, c33c, c22-3, fixed on nitrocellulose membrane strips. Antigen 5-1-1 (from the NS 4 region) and c33c (from the NS3 region) are produced by recombinant techniques in E. cord, the antigens c100-3 (from the NS4 region) and c 22-3 (from the core region) in yeast. Additionally, three control bands are included: one SOD band (which should be negative) and two control bands (levels 1 and 2), which measure intensity of a virus-specific band. Intensity of a band equal to level 1 is quoted as I+, positive intensity between level 1 and 2 as 2+ positive, intensity equal to level 2 as 3+ positive and stronger than level 2 as 4+ positive. For a serum to be scored positive, at least 2 bands have to be 1+ positive.

2.2.2. RIBA I l l (R 3)

The test works by the same principle as R 2, but uses synthetic peptides for the c-22 (core) and the el00 (NS4) regions, and the concentrations have been increased to improve sensitivity. C-33c antigen is produced in E. coli and NSs-antigen in yeast. The control bands and criteria for interpretation of results are the same as in R 2. 2.2.3. Matrix Assay ( M a )

The following recombinant antigens are fixed as spots on a nitrocellulose layer: HC-34 (core - - E. coli), HC 29 (NS3 - - E. coli), HC 23 (NS4 - - E. coli), and c100-3 (NS 4 - - yeast). The test procedure is performed automatically and read by a special Abbott analyzer. A serum is considered as positive when antibodies against at least two antigens are detectable. Since the NS4 region is represented twice (E. coil- and yeastproduced antigens), a serum is considered NS4-positive only when both reactions yield positive results. 2.2.4. Wellcozyme HCV-Western Blot ( W B )

The recombinant antigens used in this assay are separated on a polyacrylamide gel and then transferred to a nitrocellulose layer. The recombinant antigens represent the core, NS3, NS4 and NS5 regions, respectively. In each separate run a low and high positive control have to be included. A band with an intensity similar to the low positive control is quoted as I+ and similar to the high positive control as 3+. Thus the intensity score is from 1+ to 4+, similar to RIBA (see above). A band clearly visible but weaker than the low positive serum is ±. A serum reads positive if at least two bands are I+ or if one band is at least 1+ and two others are 4-. 2.2.5. INNO-LIA (LI) Synthetic antigens representing core, E2/NSb NS3, NS4 and NSs proteins have been coated as discrete lines on nylon strips with plastic backing. Four control lines -- three for intensity comparison, one for streptavidin control -- are included. In contrast to the other immunoblots, I N N O - L I A is positive ifat leastone band is

Table I Immunoblot-resultsof 75 HCV-ELISA-positivesera Test

Positive

Negative Indeterminate

RIBA I1 RIBA III Matrix Western Blot INNO-LIA INNO-LIA(R)a

35 53 55 55 59 50

13 15 6 7 13 16

27 7 14 13 3 9

alNNO-LIA assessed by RIBA criteria (positive= at least two 1+ bands).

H. Hofmann/ Serodiagnasis and Immunotherapy in Infectious Disease 8 (1996) 79-83 Table 2 Results from 27 RIBA ll-indeterminate sera Pattern

Number of sera

All 4 immunoblots positive 3 immunoblots positive, I indeterminate Ma indeterminate R 3 indeterminate WB indeterminate 2 immunoblots positive, 2 indeterminate WB, R 3 indeterminate Ma, WB indeterminate 3 immunoblots indeterminate, 1 positive (LI) 3 immunoblots indeterminate, I negative (R 3) 2 immunoblots indeterminate, 2 negative (R3/LI) ! Immunoblot indeterminate (Ma), 3 negative

13 5 2 (1 x PCR+, 1 x PCR-) 2 (2 x PCR-) ! (PCR-) 4 2 (2 x PCR-) 2 (2 x PCR+) ! (PCR+) 1 (PCR-) 2 (2 × PCR-) 1 (PCR-)

+2 or stronger. Additionally, two bands at least 1+ also read positive. 3. Results and discussion Our study clearly shows that the four newly developed immunoblots yielded more positive results than RIBA II (Table I). The number of indeterminate results, however, was substantially lower. The highest number of positives was found by INNO-LIA. This test, however, applies different criteria for interpretation to the other assays. While in RIBA a serum is positive if two bands are at least 1+, one 2+ band is sufficient for INNO-LIA. However, applying the RIBA criteria for INNO-LIA, the number of positive (and indeterminate) results are in

the same range as with the other immunoblots (see Table 1). Of the 34 sera that were positive in RIBA II, all were also positive in the four other immunoblots. However, one additional RIBA II positive (and also PCR positive) serum was positive only in three immunoblots but clearly negative in INNO-LIA. Unfortunately, there was no serum left to repeat the INNO-LIA test, thus a mistake or a serum mix-up cannot be excluded. The results of 27 RIBA II indeterminate sera are shown in Table 2. Thirteen of these sera were positive in all four immunoblots, but the remaining 14 displayed various combinations of positive, indeterminate and negative results. Thus five sera were positive in three tests but indeterminate in only one. Four more sera were positive in two, but indeterminate in the remaining two tests. In two cases three immunoblots were found in= determinate, and the fourth positive or negative. In two sera two immunoblots were found indeterminate and two negative, and one serum was negative in three tests and indeterminate in the fourth. No correlation between tests was found. However, taking only the four PCR positive sera with discordant immunoblot results, we found the Matrix test to be indeterminate in all four, the Western Blot in three and RIBA III in only one. This may indicate a lesser sensitivity of those tests. Of those four sera, however, with negative PCR and at least one negative immunoblot, which therefore of all RIBA II indeterminate sera (Table 2)are most likely to be true negative, all four were indeterminate in Matrix, three in Western Blot and only one in LIA. This may indicate nonspecifity of those tests. Of 13 sera negative in RIBA II, only three were also negative in the other four immunoblots. The remaining 10 sera displayed different patterns, as shown in Table 3. Comparable discordant results were also found by other authors [7,13-19] comparing immunoblots. This

Table 3 Results from 13 RIBA II-negative sera Pattern

Number of sera

All 4 immunoblots negative 2 immunoblots positive, 1 indeterminate (LI), 1 negative (R 3) 1 immunoblot positive (WB), 2 indeterminate (Ma, LI), i negative (R 3) 1 immunoblot positive (WB), l indeterminate (Ma), 2 negative 1 immunoblot positive, 3 negative Ma positive LI positive 3 immunoblots indeterminate, 1 negative (LI) 2 immunoblots indeterminate, 2 negative WB, LI negative R 3, LI negative 1 immunoblot indeterminate (WB), 3 negative

3 I (PCR-) I (PCR nd) 1 (PCR-) 2 I (PCR nd)

nd, not done.

81

l (PCR-) l (PCR-) 2 1 (PCR nd) I (PCR-) 2 (l x PCR-, I x P C R nd)

82

H. Hofmann/ Serodiagnosis and Immunotherapy in Infectious Disease 8 (1996) 79-83

Table 4 Immunoblot results of 47 sera with respect to PCR PCR positive

RIBA I1 RIBA III Matrix Western Blot INNO-LIA

PCR negative

Pos.

lndet.

Neg.

Pos.

Indet.

12 15 13 14 16

4

1 3 2

0

0

13

9

9

0 0

15 18

5 8

11 5

0

19

7

5

0

21

2

8

makes it difficult to establish a reliable diagnosis based on immunoblot results only. However, in all cases PCR was negative (given that sera remained for PCR). In the six discordant cases with negative PCR the following immunoblots were positive/indeterminate: Western Blot 2/2, Matrix 1/3, LIA l/1 and RIBA III O/l. Again these results are suspected of being nonspecific and correspond well to those mentioned above from PCR negative and RIBA II indeterminate sera. A general overview on immunoblot results with respect to PCR is given in Table 4. The overall larger number of positives indicates higher sensitivity of the four newly developed immunoblots compared with RIBA II. Three ( R I I l , WB, LI) of them additionally to the conventional antigens include the NSs protein and the INNO-LIA, and moreover an antigen from the E2/NSt region. However, the higher sensitivity obviously is not caused by the inclusion of new antigens, namely from the NS5 region, but by the higher concentration of the conventional antigens. This is indicated by the results from the Matrix assay, which lacks the NS 5 antigen but displayed about the same number of positives as the three other tests, and by the detailed analysis of antibodies against the NS 5 antigen assessed by RIBA III, Western Blot and INNO-LIA. Accordingly, improvement of the third generation ELISA was achieved not so much by inclusion of the NS 5 antigen as by higher concentrations of the conventional antigens [20-22]. Table 5 summarizes the results from all the sera that

Table 5 Sera with one band only in immunoblot Test

RIBA III Matrix

Western Blot INNO.LIA ha, not applicable.

N

8 9

12 5

Neg.

displayed only one band. Matrix and RIBA III predominantly had single core bands, while Western Blot most often showed single NS4 reactivity. Single bands in I N N O - L I A were rare - - five cases only. Only one serum was found that had a single NS5-band (in Western Blot) and therefore was interpreted as indeterminate. The same results were found in Matrix and RIBA III, but from a single core-band, while I N N O LIA and RIBA II were hegative. An additional serum (also in WB) yielded two bands, of which one was the NSs-band. By the other three immunoblots, however, this serum was also positive or indeterminate, based on positivity of antibodies against different antigens: in RIBA Ill we found antibodies against core and NS3, in INNO-LIA against core and NS4 and in Matrix against core only - - PCR was negative. No serum was found in the INNO-LIA test that had a single E2/NSi-band. These findings indicate that different immunoblots are not equally sensitive for the various antibodies. How often antibodies against the different antigens were detected by the various immunoblots is shown in Table 6. Of all the sera for which at least one ± band was detected, RIBA II and III, Matrix and I N N O - L I A most often displayed core antibodies, namely in 89%, 92%, 91% and 77%, respectively. Western Blot, however, detected antibodies most often against NS4 (88%), but against core in only 60%. Our study clearly shows that the four newly developed immunoblots are superior to RIBA II. However, its results are not always identical, therefore for corrobora-

Table 6 Antibodiesagainst variousantigens assessedby differentimmunoblots

Antigen region C

NS3

NS4

NS5

6 7 I

2 0 I

0 2 9

0 na I

1

3

I

0

RIBA II RIBA II1 Matrix

Western Blot INNO-LIA

N

Core

E2/NSI

NS3

NS4

NSs

63 62 68 68 62

89% 92% 91% 60% 77%

na na na na 320

67% 90% 75% 78% 74%

56% 61% 63% 88% 58%

na 27% na 440 21%

N, number of sera with at least one -~-band; ha, not applicable.

H. Hofmann / Serodiagnosis and lmmunotherapy in Infectious Disease 8 (1996) 79-83

tion o f a positive H C V - E L I S A we therefore r e c o m m e n d P C R . O n l y P C R negative sera s h o u l d be tested by imm u n o b l o t . I n critical cases two p r o d u c t s , therefore, might sometimes be necessary.

[11] [12]

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