Rabbit-derived anti-HD antibodies for HDAg immunoblotting

JournalofHepatology, 1991; 13(Suppl.4): S130-S133 @ 1991Elsevier Science Publishers B.V. Ail rights reserved. 0168-8278/91/$03.50

s130 HEPAT 01077

Rabbit-derived Floriano

Rosina’,

the

wxitivity

antibodies for

Antonina Fabiano’, Antonietta Garripoli’, Antonina Smedile’, Albert0 Michael R. Eckart’, Michael Houghton’ and Ferruccio Bonino’

‘Dil$&,le& Ch~roe~rreroiogia,

\$‘e studied

anti-

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Molirleue.

Toho.

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and specificity of two rabbit-derived

Co.

I Enwr~dle.

CA, lIded

Mattalia’,

States of America

antibodies (W2 and W3) raised against two synthetic

peptides of the HDAg: the C-terminus of the p24 protein (W2) and the C-terminus of the p27 protein (W3) (Chiron

Co., Emeryville. CA. U.S.A.), The results were compared with those obtained with a human polyclonal anti-HD (Wl). We have tested W2 and W3 against blotted serum samples from 25 patients (20 HBsAg positive anti-HD positive, three HBsAg positive anti-KD negative and two HBsAg negative anti-HD negative) and liver extracts from five KBsAg seropositive patients, two anti-HD positive and three anti-HD negative. In serum samples W3 sporadically reacted with a p31 protein present in both anti-HD positive and negative sera; W2 identified the p24 protein in 7/10 Wl positive samples but in none of 10 Wl negative; the p27 protein was captured by W2 only in one highly viremic serum. In every sample of liver extract W3 recognized a 4%kDa band. Wl and W2 stained 5 bands at 45, 27. 24, 16 and 12 kDa. The ~45, p27 and p24 proteins were peculiar of HDV-infected livers: p16 and p12 were also detected in HDV free livers. W2 identified an additional 54-kDa protein in 4/5 liver extracts. For diagnostic purposes human polyclonal anti-HD represents the most sensitive probe for HDAg immunoblotting, with a specificity similar to that of rabbit-derived antibodies. The affinity of W2 and W3 antibodies for serum and liver HDAg appears to be lower than for HDAg recombinant. The structural organization of HDAg. possibly related to nucleic acids binding. could account for this in vivo low affinity.

T’he hepatitis delta virus (HDV) is a small RNA virus which is dependent on helper functions provided by (HBV) (I).

Virological and molecular studies have shown that the HDV particle contains an approximately 1680-base circular RNA (HDV-RNA) and the hepatitis delta antigen (HDAg) surrounded by the surface antigen of HBV (HBsAg) (2-4). Although both genomic and antigenomic RNA contain multiple open reading frames, available evidence suggests that only one is actually expressed (ORF-5) (5). This open reading frame encodes two HDAg species of I95 and 214 amino acids, depending on the variable

presence of an amber stop codon at position 196 (6). HDAg in HDV particles is comprised of the two molecular species with different migration rates in SDS-

Correspondence:

E

PAGE of 24 kDa and 27 kDa (7) or 27 kDa and 29 kDa (8). Until now immunoblot detection has been made using anti-HD positive human sera or immunoglobulin fractions (Wl) (7.8). To better characterize the HDV-specific proteins, rabbit antibodies have been raised against peptides derived from the C-terminus of the p24 (W2) and p27 (W3) HDAg proteins. Immunoblots with lysates of Escherichiu coli expressing p24 and p27 had been performed using W2 and W3 antisera. W2 antiserum reacted with both p24 and ~27, W3 did not react with ~24 but did react with the p27 protein. We tested the reactivity of these two rabbit-derived antibodies on sera and liver extracts from HDV-infected patients and results have been compared with those obtained using the immunoglobulin fraction from an anti-HD-positive human serum.

Rosina. Department of Gastroenterology, Molinette Hospital. Corso 13ramante88, I-10126Turin, Itzly.

ANTI-HD

ANTIBODICS

FOR HDAg l~MUN~5~D~~N~

S131

-l-ABLE I Reactivity of human (WI )- and rabbit (W2 and W3)-derived anti-HD antibodies against blotted serum samples Protein sizes (kDa)

Serum samples

Wl

w2

w3

WI

w2

w3

45 31 27 24

20120 O/20 10120 10/20

20120 O/20 1120 7120

20120 2120 O/20 0120

515 015 015 o/5

s/s

515

015 01.5 015

115 015

anti-ND positive

anti-l-ID negative

o/5

WI: immunoglobulin fractions of an anti-HD-positive human serum. W2: rabbit-derived anti-ND antibody against the C-terminus of the p24 HDAg protein. W3: rabbit-derived anti-HD antibody against the C-terminus of the p27 HDAg protein.

Patients Sera from 23 I-IBsAg-positive patients with chronic liver disease (20 anti-HD positive and three anti-MD negative) and two healthy subjects were tested. Liver extracts were obtained from five HBsAg-positive cirrhotic patients (two HDAg positive and three DAg negative by immunohistochemistry) who received a liver graft in Italy. Methods Serum samples (250 ~1) were layered over 11 ml of 20% sucrose, 0.02 M Hepes (PI-I 7.4), 0.01 M CaCl,, 0.01 M MgCl, and 0.1% bovine serum albumin (BSA) and were centrifuged in an SW41 rotor (Beckman Instruments) for 5 h at 150 000 X g. Liver samples (about 1 g of tissue) were minced with scissors. Samples were homogenized with a PotterElvejhem apparatus in 2 ml of TKM and centrifwged at 1000 x g for 10 min. The pellet was resuspended in 60% sucrose-TKM until the sucrose concentration reached 3.: ~.~:kx! on 60% sucrose-TKM and centrifuged at 120 000 X g for 30 min in a swing-out rotor. Sera and liver pellets were boiled in 0.05 M Tris (pH 6.8), 2% SDS and 1% 2-mercaptoethanol and run on 12% polyacrylamide gels. Proteins were electrically transferred to nitrocellulose filters and detected using immunoglobulin fractions of a serum from a patient with chronic HDV hepatitis (Wl) and two rabbit-derived anti-HD antibodies raised against the C-terminus of p24 (W2) and p27 (W3). An tz51-labeled protein A was used to detect bound antibodies.

Rg. I. Immunohlots of human serum samples. Lanes a-c represent an HBsAg-positive. anti-HD-positive serum sample; lanes d, e and fan HBsAg-negative, anti-HD-negative serum sample. Lanes a and d are probed with immunoglobuling fractions of a human anti-HDpositive serum (Wl); lanes b and e with a rabbit-derived anti-HD antibody agaiust the C-terminus of the p24 HDAg protein (W2); lanes c and f with a rabbit-derived anti-HD antibody against the C-terminus of the p27 HDAg protein (W3).

Sera Wl, W2 and W3 reacted with a 45kDa protein common to anti-HD-positive and -negative sera (Table 1 and Fig. 1, lanes a-f). Wl identified both p27 and p24 in half (10/20) of the HBsAg-positive, anti-HD-positive samples, but in none of the anti-HD-negative (Table 1 and Fig. 1, lanes a and d). The p27 protein was usually less reactive than the ~24. W2 identified the p24 protein in 7/20 anti-HD-positive samples (all reactive with Wl), but in none of the antiHD-negative samples (Table 1 and Fig. 1, lanes b and e). The p27 protein was detected by W2 only in one highly viremic serum. W3 never reacted with the p27 protein. Reactivity with a ~31 protein was shared by anti- D-positive and -negative samples (Table 1 and Fig. 1, lanes c and f). Liver extracts Wl and W2 identified three proteins of 45, 27 and 24

E RBSINA et al.

S132 TABLE 2 Reactivity

an$HD Protein sizes

of human (Wl)and rabbit (W2 and W3)-derived antibodies against blotted liver extracts -

Liver samples HDAg

HDAg negativea

positivea

~___~

@Da)

54 48 45 27 24 16 12

Wl

w2

o/2 012 2i2 2i2 212 212 212

212 o/2 212 212 212 212 2t2

w3 o/2 212 o/2 o/2 o/2 o/2 012 ._._.___.__

Wl

w2

w3

oi3 o/3 013 013 013 313 313 .._._~~~

2/3 o/3 oi3 o/3 013 313 313 ..~

o/3 3i3 o/3 O/3 o/3 o/3 013

~__ ..__ _ _.

WI: immunoglobulin fractions of an anti-HD-positive human serum. W2: rabbit-derived anti-HD antibody against the C-terminus of the p2J HDAg protein. W3: rabbit-derived anti-HD antibody against the C-terminus of the p27 HDAg protein. “HDAg positive or negative at immunohistochemistry.

kDa in HDAg-positive samples (Table 2 and Fig. 2, lanes a, b, d and e) and two further proteins of 16 and 12 kDa in HDAg-positive and -negative samples (Table 2 and def

abc

ghi

WI w2w3 wlw2w ‘3 F@. 2. Immunoblotsof human liver extracts. Lanes a-f represent liver extracts from two HBsAg-positive cirrhotic patients. HDAg positive at immunohistochemistry: lanes g. h and i liver extracts from one HBsAg-positive cirrhotic patient. HDAg-negative at immunohistochemistry.Lanes a. d and g are probed with immunoglobulin fractions of a human anti-HD-positive serum (Wl): lanes b. e and h with a rabbit-derived anti-HD antibody against the C-terminus of the p24 HDAg protein (W2): lanes c. f and i with a rabbit-derived anti-HD antibody against the C-terminus of the p27 HDAg

protein (W3).

Fig. 2, lanes a, b, d. e, g and h). WI stained several proteins between 24 kDa and 16 kDa, which W2 was unable to recognize (Table 2 and Fig. 2, lanes a. b. d and e). W2 reacted with a 54-kDa protein in both HDAgpositive and -negative liver specimens (Table 2 and Fig. 2. lane b). W3 did not bind the p27 protein but a 48-kDa protein shared by HDAg-positive and -negative samples (Table 2 and Fig. 2, lanes c, f and i).

Discllssion The rabbit anti-HD raised against a synthetic peptide derived from the C-terminus of the p24 HDAg protein (W2) recognizes the p24 protein both in serum and liver samples. while it stains the p27 HDAg protein poorly. The p27 protein is usually expressed at a lower degree than ~24, however, the different staining capacity of W2 antibody for the two HDAg proteins does not correlate with their relative amounts as detectable with a human derived anti-HD antibody (Wl). Other factors, such as changes in the molecular structure of the C-terminus of ~24, induced by the addition of amino acids from 196 to 214. could explain this discrepancy. W3. the rabbit-derived anti-HD raised against the C-terminus of p27 HDAg protein, is capable of binding to the p27 protein expressed in E. co/i. but does not identify the p27 protein of human origin. This poor affinity can again be explained by the lower level of the p27 protein in human samples, but other mechanisms could also be involved. Expression of recombinant HDAg gene in E. coli could lead to a structural organization of the p27 protein that significantly differs from that of the protein expressed in eucaryotic cells. Alternatively the epitopes of the p27 protein could be masked in vivo. possibly by nucleic acids: the HDAg-associated proteins were shown to possess an RNA-binding activity (9,lO). The ~45 protein recognized by both Wl and W2 could represent a multimer of p24 and p27 or of small molecular weight HDAg species: HDAg dimers or multimers have been demonstrated in vitro (11,12). This protein could otherwise represent complexes of HDAg with intracellular proteins or nucleic acids. Since W2, raised against the C-terminus of the p24 protein, does not recognize the lower molecular weight HDAg species detected by Wl (ranging between p24 and p16), these species probably represent terminally truncated translation products of ORF-5. For diagnostic purposes, immunoglobulin fractions of anti-HD-positive sera represent the most sensitive probe

ANTI-MD ANTIBODIES

PQR HDAg IMMUNQBLQTTING

for HDAg immunoblotting. Their specificity is co ble with that of rabbit W2 antibody. W2 and W3 antibodies appear to be suitable tools to

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of

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