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Detection and characterization of antibodies to polymerized human serum albumin (AAA) by ELISA
Journal oflmmunologicalMethods, 62 (1983) 257-263 Elsevier
257
JIM02733
Detection and Characterization of Antibodies to Polymerized Human Serum Albumin (AAA) by ELISA G. Triolo l, M.G. Filippazzo *, G. Pinzello *, E. Giardina and A. Rinaldi Istituto di Clinica Medica H L University of Palermo, and * Divisione di Medicina Interna, Ospedale V. Cervello, Palermo, Italy
(Received 18 October 1982, accepted 21 March 1983)
Immunodiffusion and passive hemoagglutination with h u m a n or sheep red blood cells for the detection of antibodies to polymerized h u m a n serum albumin (AAA) in sera of patients with various liver diseases have some deficiencies. A sensitive ELISA method for detection and characterization of A A A is described. Key words: anti- albumin antibody - - liver disease - - EL1SA
Introduction
Several reports have demonstrated that sera of patients with liver diseases react with polymerized human albumin (p-HSA) (Imai et al., 1979; Lenkei et al., 1974, 1977, 1979, 1981; O'Neil, 1979; Thung and Gerber, 1981). As well as demonstrating the presence of autoantibodies directed against p-HSA (AAA) some workers have shown that the presence of p-HSA binding sites is associated with Dane and HBsAg particles (Hansson and Purcell, 1979; Neurath and Strick, 1979; Thung and Gerber, 1981). AAA are considered to be a good indicator of the severity of liver damage (Lenkei et al. 1981) and it has been suggested that HBsAg-associated albumin sites are related with HBV infectivity (Stevens et al., 1978). Immunological methods have been developed to detect AAA : Immunodiffusion (Lenkei and Ghetie, 1977), passive hemoagglutination with human or sheep erythrocytes coated with antigen (Lenkei and Ghetie, 1977; Imai et al., 1979; Thung and Gerber, 1981), and radioimmunoassay (RIA) (Hansson and Purcell, 1979;
i Request for reprints should be sent to: Dr. Triolo Giovanni, Istituto di Clinica Medica Ili, Policlinico, Via Del Vespro, 90127 Palermo, Italy. 0022-1759/83/$03.00 © 1983 Elsevier Science Publishers B.V.
258 Neurath and Strick, 1979; Shih et al., 1980). Passive hemoagglutination, however, cannot distinguish AAA from the binding activity of the HBsAg and RIA and was developed only to detect p-HSA receptors on Dane and HBsAg particles. More recently immunofluorescence was used with hemoagglutination to determine the nature of the p-HSA binding activity (Thung and Gerber, 1981). This paper describes an ELISA sensitive method that detects p-HSA binding capacity due to autoantibodies and characterizes their immunoglobulin class.
Material and Methods
Sera Sera from 29 patients with various liver diseases who fulfilled the diagnostic criteria of an International Committee (Fogarty International Center, 1976) were studied. These included sera from 25 patients with chronic active liver disease and inactive cirrhosis (CLD) and 4 patients with chronic persistent hepatitis (CPH). Fourteen health controls were also studied. Five patients had detectable HBsAg by RIA (AUSRIA II Abbott Diagnostic, North Chicago, IL). Additional sera were obtained from a patient with primary biliary cirrhosis (PBC), 2 patients with systemic lupus erythematosus (SLE) and scleroderma respectively, and 8 patients with diabetes mellitus. Antigen Polymerized HSA was prepared according to the method of Lemieux et al. (1974) with minor modifications. Briefly, 500 mg HSA (Behringwerke AG, Marburg) were dissolved in 45 ml of 0.05 M phosphate buffered saline (PBS), pH 6.8, and 5 ml of 25% aqueous solution of glutaraldehyde were added dropwise with stirring. The mixture was incubated for 3 h at room temperature with continuous gentle stirring and then dialysed overnight against 0.05 M PBS, pH 7.2. The glutaraldehyde treated HSA was concentrated with a Minicon B 15 concentrator (Amicon) to a volume of 10 ml and run through a Sephadex G-200 column (50 × 2.5) equilibrated with the same buffer. The fractions of the first peak containing p-HSA were pooled and concentrated to a protein content of 10 mg/ml. Conjugates Rabbit anti-human IgA, IgG, IgM and albumin (Behringwerke) were coupled to alkaline phosphatase type VII S (Sigma) by the method of Voller et al. (1976). Substrate p-nitrophenyl disodium phosphate (Sigma) was dissolved in 1 M diethanolamine HCI buffer, pH 9.8, at a concentration of 1 mg/ml. A ssay Wells of microtiter plates (Falcon) were coated as follows: 200/al of a solution of p-HSA (100 #g/ml) in sodium carbonate buffer, pH 9.6 (sodium carbonate 0.015 M,
259
sodium bicarbonate 0.035 M, sodium azide 0.02%) were added to each well. The plates were incubated for 24-48 h at 4°C, and then washed 3 times (allowing at least 2 min standing between each filling) with PBS containing 0.05% Tween 20 and 0.02% sodium azide, and stored empty at 4°C. No change in activity was noted on storage for up 2 weeks. Separate plates were used to detect IgA, I g G and lgM AAA. Sera were diluted in 0.05 M PBS containing 4% BSA. Two hundred microliters of serial 2-fold dilutions of test sera were added to each well and the plates incubated for 2 h at 37°C with shaking. After washing, 200 ~1 of the alkaline phosphatase conjugates diluted 1 : 500 with 0.05 M PBS containing 1% BSA were added to each well, and the plates incubated again at room temperature for 18 h, washed again, and 200/~1 of substrate solution then added. After 1 h incubation the reaction was stopped with 50 /~1 of 3 M N a O H and absorptions measured in a Titertek-Multiscan.
10
O0 O0O 000
00
0000
L 7 ©
000
00
• •
O0 =OI
• •
•
000
O0
HB~jAg +
• • • Oi
0000
0000
H BsAg-
CAH OR CIRRHOSIS
CPH
MISCELLANY
CONTROLS
Fig. l. lgG antibodies to polymerized human serum albumin in sera of patients with various liver diseases and normal controls. The miscellaneous group included the patient with primary biliary cirrhosis, 2 patients with SLE and scleroderma respectively, and 8 diabetic patients. The dotted line indicates the normal cut-off value.
260 Sera were considered to contain A A A when a b s o r b a n c e values (A405) were at least 3 S.D. ab o v e the mean a b s o r b a n c e of a control set of 14 n o r m a l sera.
Results
IgG AAA (Fig. 1) IgG A A A were detected in 70% of patients with HBsAg-negative C L D (mean geometric titer 6.5 + 1.1) and in 40% of HBsAg-positive C L D (mean titer 3.4 + 0.9). IgG A A A were also detected in 1 CPH patient and in 1 of 14 controls, at low titer (1:8). In the miscellaneous group, IgG A A A was detected in the sera of the PBC patient and in 1 of 8 diabetics up to a dilution of 1 : 128.
lO •
I g A - AAA
•
IgM
AAA
8
6-
E Z ©
4
&AA
2
Itt AAAAA AAAAA
• • •
lOOm
o
&&
mmmmo
H Bs A g P BC
CONTROLS
C A H OR C I R R H O S I S
Fig. 2. IgA and lgM antibodies to polymerized human albumin in sera of patients with various liver diseases and controls.
261
IgA and IgM AAA (Fig. 2) IgA AAA and IgM AAA were tested for in the sera of HBsAg-negative CLD, in the PBC serum and in control sera. IgA AAA and IgM AAA were detected in 20% (mean geometric titer 4.25 _+ 0.5) and 40% (mean geometric titer 5.25 + 0.88) of HBsAg-negative CLD sera respectively. None of the control group, nor the PBC serum was positive.
Experimental variables Binding of p-HSA to the solid phase.
Absorption of p-HSA to the polystyrene surface of wells was investigated by incubation with enzyme-labeled anti-human albumin. The most efficient binding of AAA was obtained if wells were coated with 100 ~ g / m l of p-HSA. The effect of different coating concentrations is shown in Fig. 3. Selective binding of AAA to the solid-phase p-HSA. AAA bound to the solid-phase p-HSA but non-specific binding of immunoglobulins was almost completely prevented by aspiration and washing. Fig. 4 shows results with positive sera incubated in uncoated wells or wells coated with native HSA. When sera of AAA positive
A(4051
.400 \
\
.300
.200
.100
;-5 Fig. 3. Effect of coating concentration on absorption values obtained with a positive (o) and a negative (a,) reference serum, p-HSA concentration: - - , 100 # g / m l ; - - - - - - , 50 ~ g / m l ; . . . . . , 10 ~tg / m l .
262
AI405I .500
,\ %, .400
.300
\
.200
.100
SERUM DILUTION WELLS
"'-
2'-3 21 UNCOATED
2'-5
2'43 2[4 2[5 COATED WITH NATIVE H S A
2;3 2-'4 2'-5 COATED WITH P-HSA
Fig. 4. Selective binding of A A A to the solid-phase: dilutions of 4 positive reference sera were incubated in uncoated wells, and wells coated with native HSA and p-HSA.
patients were treated with native and p-HSA as inhibiting antigens and then tested again in the assay, inhibition was seen with p-HSA only.
Discussion It is known that p-HSA produced by ageing or giutaraldehyde treatment of native albumin induces anti-p-HSA antibodies (AAA). A A A determinations have been shown to be of diagnostic value in assessment of liver cell dysfunction (Lenkei et al., 1981). It has also been suggested that A A A in HBsAg-positive patients may interfere with the attachement of HBV to hepatocytes via a p-HSA receptor and may thus be protective by decreasing infectivity (Imai et al., 1979; Thung and Gerber, 1981). Immunodiffusion and passive hemoagglutination have been used to detect AAA, but the ELISA described here detects all classes of anti-albumin autoantibodies and is thought to be more sensitive. The results obtained with ELISA among patients with severe, mild or no liver lesions confirm previous data. A A A titers between different assays were observed to vary only slightly, due to small temperature variations during incubation or technical inaccuracies.
263 T h e f i n d i n g o f high titer A A A in a d i a b e t i c p a t i e n t does n o t c o n f l i c t with the p o s t u l a t e d close r e l a t i o n s h i p b e t w e e n A A A a n d liver cell d a m a g e . Liver e n l a r g e m e n t a n d a p o s i t i v e test for liver specific p r o t e i n - c o n t a i n i n g i m m u n e c o m p l e x e s were p r e s e n t , a r g u i n g s u b c l i n i c a l liver disease.
References Engvall, E. and P. Perlmann, 1971, Immunochemistry 8, 871. Hansson, B.G. and R.H. Purcell, 1979, Infect. lmmun. 26, 125. Imai, N., Y. Yanase, T. Nojiri, Y. Miyakawa and M. Mayumi, 1979, Gastroenterology 26, 125. Lemieux, S., S. Avrameas, A.E. Bussard, 1974, Immunochemistry 11,261. Lenkei, R. and V. Ghetie, 1977, J. Immunol. Methods 16, 23. Lenkei, R., G. Mota, M.E. Dan, M. Laky, 1974, Rev. Roum. Biochim. 11,271. Lenkei, R., V.T. Babej, M.E. Dan, A. Musten and I. Dobre, 1977, J. Med. Virol. I, 29. Lenkei, R., V.T. Babej, A. Mustea and I. Dobre, 1979, J. Med. Virol. 4, 137. Lenkei, R., L. Buligescu, I. Belascu, D. Pospai and I. Dobre, 1981, Clin. Exp. Immunol. 43, 381. Neurath, A.R. and N. Strick, 1979, Intervirology 11, 128. O'Neil, S.P., 1979, J. Med. Virol. 4, 177. Shih, J.W.R., P.L. Tan and J.L. Gerin, 1980, Infect. Immun. 28, 459. Stevens, C.E., A.R. Neurath, W. Szmuness, R.F. Beasley and H. Ikram, 1978, in: Viral Hepatitis, eds. G.N. Vyas, S.N. Cohen and R. Schimd (The Franklin Institute Press, Philadelphia) p. 211. Thung, S. and M.A. Gerber, 1981, Gastroenterology 80, 260. Voller, A., D.E. Bidwell, and A. Bartlett, 1976, Bull. W.H.O. 53, 55.
257
JIM02733
Detection and Characterization of Antibodies to Polymerized Human Serum Albumin (AAA) by ELISA G. Triolo l, M.G. Filippazzo *, G. Pinzello *, E. Giardina and A. Rinaldi Istituto di Clinica Medica H L University of Palermo, and * Divisione di Medicina Interna, Ospedale V. Cervello, Palermo, Italy
(Received 18 October 1982, accepted 21 March 1983)
Immunodiffusion and passive hemoagglutination with h u m a n or sheep red blood cells for the detection of antibodies to polymerized h u m a n serum albumin (AAA) in sera of patients with various liver diseases have some deficiencies. A sensitive ELISA method for detection and characterization of A A A is described. Key words: anti- albumin antibody - - liver disease - - EL1SA
Introduction
Several reports have demonstrated that sera of patients with liver diseases react with polymerized human albumin (p-HSA) (Imai et al., 1979; Lenkei et al., 1974, 1977, 1979, 1981; O'Neil, 1979; Thung and Gerber, 1981). As well as demonstrating the presence of autoantibodies directed against p-HSA (AAA) some workers have shown that the presence of p-HSA binding sites is associated with Dane and HBsAg particles (Hansson and Purcell, 1979; Neurath and Strick, 1979; Thung and Gerber, 1981). AAA are considered to be a good indicator of the severity of liver damage (Lenkei et al. 1981) and it has been suggested that HBsAg-associated albumin sites are related with HBV infectivity (Stevens et al., 1978). Immunological methods have been developed to detect AAA : Immunodiffusion (Lenkei and Ghetie, 1977), passive hemoagglutination with human or sheep erythrocytes coated with antigen (Lenkei and Ghetie, 1977; Imai et al., 1979; Thung and Gerber, 1981), and radioimmunoassay (RIA) (Hansson and Purcell, 1979;
i Request for reprints should be sent to: Dr. Triolo Giovanni, Istituto di Clinica Medica Ili, Policlinico, Via Del Vespro, 90127 Palermo, Italy. 0022-1759/83/$03.00 © 1983 Elsevier Science Publishers B.V.
258 Neurath and Strick, 1979; Shih et al., 1980). Passive hemoagglutination, however, cannot distinguish AAA from the binding activity of the HBsAg and RIA and was developed only to detect p-HSA receptors on Dane and HBsAg particles. More recently immunofluorescence was used with hemoagglutination to determine the nature of the p-HSA binding activity (Thung and Gerber, 1981). This paper describes an ELISA sensitive method that detects p-HSA binding capacity due to autoantibodies and characterizes their immunoglobulin class.
Material and Methods
Sera Sera from 29 patients with various liver diseases who fulfilled the diagnostic criteria of an International Committee (Fogarty International Center, 1976) were studied. These included sera from 25 patients with chronic active liver disease and inactive cirrhosis (CLD) and 4 patients with chronic persistent hepatitis (CPH). Fourteen health controls were also studied. Five patients had detectable HBsAg by RIA (AUSRIA II Abbott Diagnostic, North Chicago, IL). Additional sera were obtained from a patient with primary biliary cirrhosis (PBC), 2 patients with systemic lupus erythematosus (SLE) and scleroderma respectively, and 8 patients with diabetes mellitus. Antigen Polymerized HSA was prepared according to the method of Lemieux et al. (1974) with minor modifications. Briefly, 500 mg HSA (Behringwerke AG, Marburg) were dissolved in 45 ml of 0.05 M phosphate buffered saline (PBS), pH 6.8, and 5 ml of 25% aqueous solution of glutaraldehyde were added dropwise with stirring. The mixture was incubated for 3 h at room temperature with continuous gentle stirring and then dialysed overnight against 0.05 M PBS, pH 7.2. The glutaraldehyde treated HSA was concentrated with a Minicon B 15 concentrator (Amicon) to a volume of 10 ml and run through a Sephadex G-200 column (50 × 2.5) equilibrated with the same buffer. The fractions of the first peak containing p-HSA were pooled and concentrated to a protein content of 10 mg/ml. Conjugates Rabbit anti-human IgA, IgG, IgM and albumin (Behringwerke) were coupled to alkaline phosphatase type VII S (Sigma) by the method of Voller et al. (1976). Substrate p-nitrophenyl disodium phosphate (Sigma) was dissolved in 1 M diethanolamine HCI buffer, pH 9.8, at a concentration of 1 mg/ml. A ssay Wells of microtiter plates (Falcon) were coated as follows: 200/al of a solution of p-HSA (100 #g/ml) in sodium carbonate buffer, pH 9.6 (sodium carbonate 0.015 M,
259
sodium bicarbonate 0.035 M, sodium azide 0.02%) were added to each well. The plates were incubated for 24-48 h at 4°C, and then washed 3 times (allowing at least 2 min standing between each filling) with PBS containing 0.05% Tween 20 and 0.02% sodium azide, and stored empty at 4°C. No change in activity was noted on storage for up 2 weeks. Separate plates were used to detect IgA, I g G and lgM AAA. Sera were diluted in 0.05 M PBS containing 4% BSA. Two hundred microliters of serial 2-fold dilutions of test sera were added to each well and the plates incubated for 2 h at 37°C with shaking. After washing, 200 ~1 of the alkaline phosphatase conjugates diluted 1 : 500 with 0.05 M PBS containing 1% BSA were added to each well, and the plates incubated again at room temperature for 18 h, washed again, and 200/~1 of substrate solution then added. After 1 h incubation the reaction was stopped with 50 /~1 of 3 M N a O H and absorptions measured in a Titertek-Multiscan.
10
O0 O0O 000
00
0000
L 7 ©
000
00
• •
O0 =OI
• •
•
000
O0
HB~jAg +
• • • Oi
0000
0000
H BsAg-
CAH OR CIRRHOSIS
CPH
MISCELLANY
CONTROLS
Fig. l. lgG antibodies to polymerized human serum albumin in sera of patients with various liver diseases and normal controls. The miscellaneous group included the patient with primary biliary cirrhosis, 2 patients with SLE and scleroderma respectively, and 8 diabetic patients. The dotted line indicates the normal cut-off value.
260 Sera were considered to contain A A A when a b s o r b a n c e values (A405) were at least 3 S.D. ab o v e the mean a b s o r b a n c e of a control set of 14 n o r m a l sera.
Results
IgG AAA (Fig. 1) IgG A A A were detected in 70% of patients with HBsAg-negative C L D (mean geometric titer 6.5 + 1.1) and in 40% of HBsAg-positive C L D (mean titer 3.4 + 0.9). IgG A A A were also detected in 1 CPH patient and in 1 of 14 controls, at low titer (1:8). In the miscellaneous group, IgG A A A was detected in the sera of the PBC patient and in 1 of 8 diabetics up to a dilution of 1 : 128.
lO •
I g A - AAA
•
IgM
AAA
8
6-
E Z ©
4
&AA
2
Itt AAAAA AAAAA
• • •
lOOm
o
&&
mmmmo
H Bs A g P BC
CONTROLS
C A H OR C I R R H O S I S
Fig. 2. IgA and lgM antibodies to polymerized human albumin in sera of patients with various liver diseases and controls.
261
IgA and IgM AAA (Fig. 2) IgA AAA and IgM AAA were tested for in the sera of HBsAg-negative CLD, in the PBC serum and in control sera. IgA AAA and IgM AAA were detected in 20% (mean geometric titer 4.25 _+ 0.5) and 40% (mean geometric titer 5.25 + 0.88) of HBsAg-negative CLD sera respectively. None of the control group, nor the PBC serum was positive.
Experimental variables Binding of p-HSA to the solid phase.
Absorption of p-HSA to the polystyrene surface of wells was investigated by incubation with enzyme-labeled anti-human albumin. The most efficient binding of AAA was obtained if wells were coated with 100 ~ g / m l of p-HSA. The effect of different coating concentrations is shown in Fig. 3. Selective binding of AAA to the solid-phase p-HSA. AAA bound to the solid-phase p-HSA but non-specific binding of immunoglobulins was almost completely prevented by aspiration and washing. Fig. 4 shows results with positive sera incubated in uncoated wells or wells coated with native HSA. When sera of AAA positive
A(4051
.400 \
\
.300
.200
.100
;-5 Fig. 3. Effect of coating concentration on absorption values obtained with a positive (o) and a negative (a,) reference serum, p-HSA concentration: - - , 100 # g / m l ; - - - - - - , 50 ~ g / m l ; . . . . . , 10 ~tg / m l .
262
AI405I .500
,\ %, .400
.300
\
.200
.100
SERUM DILUTION WELLS
"'-
2'-3 21 UNCOATED
2'-5
2'43 2[4 2[5 COATED WITH NATIVE H S A
2;3 2-'4 2'-5 COATED WITH P-HSA
Fig. 4. Selective binding of A A A to the solid-phase: dilutions of 4 positive reference sera were incubated in uncoated wells, and wells coated with native HSA and p-HSA.
patients were treated with native and p-HSA as inhibiting antigens and then tested again in the assay, inhibition was seen with p-HSA only.
Discussion It is known that p-HSA produced by ageing or giutaraldehyde treatment of native albumin induces anti-p-HSA antibodies (AAA). A A A determinations have been shown to be of diagnostic value in assessment of liver cell dysfunction (Lenkei et al., 1981). It has also been suggested that A A A in HBsAg-positive patients may interfere with the attachement of HBV to hepatocytes via a p-HSA receptor and may thus be protective by decreasing infectivity (Imai et al., 1979; Thung and Gerber, 1981). Immunodiffusion and passive hemoagglutination have been used to detect AAA, but the ELISA described here detects all classes of anti-albumin autoantibodies and is thought to be more sensitive. The results obtained with ELISA among patients with severe, mild or no liver lesions confirm previous data. A A A titers between different assays were observed to vary only slightly, due to small temperature variations during incubation or technical inaccuracies.
263 T h e f i n d i n g o f high titer A A A in a d i a b e t i c p a t i e n t does n o t c o n f l i c t with the p o s t u l a t e d close r e l a t i o n s h i p b e t w e e n A A A a n d liver cell d a m a g e . Liver e n l a r g e m e n t a n d a p o s i t i v e test for liver specific p r o t e i n - c o n t a i n i n g i m m u n e c o m p l e x e s were p r e s e n t , a r g u i n g s u b c l i n i c a l liver disease.
References Engvall, E. and P. Perlmann, 1971, Immunochemistry 8, 871. Hansson, B.G. and R.H. Purcell, 1979, Infect. lmmun. 26, 125. Imai, N., Y. Yanase, T. Nojiri, Y. Miyakawa and M. Mayumi, 1979, Gastroenterology 26, 125. Lemieux, S., S. Avrameas, A.E. Bussard, 1974, Immunochemistry 11,261. Lenkei, R. and V. Ghetie, 1977, J. Immunol. Methods 16, 23. Lenkei, R., G. Mota, M.E. Dan, M. Laky, 1974, Rev. Roum. Biochim. 11,271. Lenkei, R., V.T. Babej, M.E. Dan, A. Musten and I. Dobre, 1977, J. Med. Virol. I, 29. Lenkei, R., V.T. Babej, A. Mustea and I. Dobre, 1979, J. Med. Virol. 4, 137. Lenkei, R., L. Buligescu, I. Belascu, D. Pospai and I. Dobre, 1981, Clin. Exp. Immunol. 43, 381. Neurath, A.R. and N. Strick, 1979, Intervirology 11, 128. O'Neil, S.P., 1979, J. Med. Virol. 4, 177. Shih, J.W.R., P.L. Tan and J.L. Gerin, 1980, Infect. Immun. 28, 459. Stevens, C.E., A.R. Neurath, W. Szmuness, R.F. Beasley and H. Ikram, 1978, in: Viral Hepatitis, eds. G.N. Vyas, S.N. Cohen and R. Schimd (The Franklin Institute Press, Philadelphia) p. 211. Thung, S. and M.A. Gerber, 1981, Gastroenterology 80, 260. Voller, A., D.E. Bidwell, and A. Bartlett, 1976, Bull. W.H.O. 53, 55.