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A chimera antibody erythroimmunoassay for detecting HBsAg in human sera
© INSTITUTPASTEUR/ELSEVIER Paris 1990
Res. Virol. 1990, !4!, 33%342
TECHNICAL NOTE
A CHIMERA ANTIBODY ERYTHROIMMUNOASSAY FOR D E T E C T I N G H B s A g in H U M A N SERA
Y. Chen (*), S.C. Yang and Q.H. Luo Department of Immunology, Institute of Infecttbus Diseases, Feng-Tai Road 26, 100039 Be(ling (PRC)
SUMMARY A highly efficient chimera antibody, a monoclonal anti-hepatitis-B-surface (anti-HBs) antibody coupled with polyclonal anti-sheep-red-blood-ceU (anti-SRBC) antibody was prepared using a heterobifunctional reagent, N-succinimidyl-3-(2-pyridyldithiopropionate) (SPDP). Using SRBC as a marker, we established a sensitive solid-phase chimera antibody erythroimmunoassay (CAEIA) according to Guesdon's method. The sensitivity of this assay was 2-20 times higher than the reverse passive haemagglutination assay (RPHA) for detecting HBsAg in serially diluted sera from 10 hepatitis B patients. The weakest quantity of HBsAg detected by this assay was 4.5 ng/ml, while RPHA was unable to detect less than 75 ng/ml of HBsAg. The assay was as sensitive as the enzyme-linked immunosorbent assay (ELISA) and gave more accurate, reproducible and stable results than ELISA; specificity was also satisfactory. KEY-WORDS: Chimera antibody, Erythroimmunoassay, HBsAg, Hepatitis B; Diagnosis. INTRODUCTION Various methods exist for detecting hepatitis B surface antigens (HBsAg) in human sera. Enzyme-linked immunosorbent assay (ELISA) and reverse passive haemagglutination assay (RPHA) are among those in widespread use and are sensitive and relatively easy to perform. However, they have some disadvantages during application: the instability of ELISA and its substrates may interfere with its application, and RPHA has low sensitivity and low specificity. A chimera antibody erythroimmunoassay (CAEIA) based on both ELISA and haemagglutination assay was established by Guesdon et al. (1983). Sensitivity and specificity were satisfactory for detecting some antigens and antibodies (Guesdon et al., 1980, 1983; Gupta et r~i., 1985 ; Germani et al., 1987).
Submitted February 2, 1989, accepted January 8, 1990. (*) Correspondingauthor.
Y. C H E N E T AL.
338
In the preseat study, a highly efficient chimera antibody was prepared with monoclonai anti-HBs antibodies and polyclonal anti-SRBC (sheep red blood cells) antibodies using SPDP, a heterobifunctional reagent. CAEIA was established using this chimera antibody and SRBC instead of enzyme antibody-conjugates and substrates in ELISA according to Guesdon's description. Satisfactory results were achieved for detecting HBsAg in human sera.
MATERIALS AND METHODS
Reagents. All reagents were of analytical grade. SPDP was obtained from the Fourth Military Medical University (China); 1-4-dithiothreitol (DTT) was obtained from Feinbiochemica (West Germany); horseradish peroxidase (HRP) grade VI from Sigma; commercial HBsAg from an R P H A kit (National Institute of Vaccine and Serum, Beijing). HBsAg vaccine (30 t~g/ml) purified from human sera; ascites containing monoclonal a n t i - H B s " a " determinant and rabbit anti-SRBC antisera were obtained from the National Institute of Vaccine and Serum (Beijing); SRBC were stored at 4°C in sterile Elsever's solution until use; normal and pathological human sera with hepatitis B were obtained from our laboratory.
Preparation of the enzyme-antibody conjugate. Monoclonal a n t i - H B s " a " IgG were coupled with H R P by a modified Wilson's method (Wilson, 1978). The conjugate was stored at - 2 0 ° C with an added equal volume of glycerol until used.
Preparation of chimera antibody. • . e I~ were precipitated nrst by 50 °7/o (NH4)2SO4 then by 33 o70 (NH4)2SO 4 before use. The monoclonal anti-HBs (Abl) 5 mg/ml and rabbit polyclonal anti-SRBC (Ab2) 5 mg/ml were dialysed overnight at 4°C against 0.1 M phosphate buffer pH 7.5 containing 0.1 M NaCI. To this, 50 ~1 of SPDP dissolved in ethanol (1 mg/ml) were added drop by drop with stirring and allowed to react for 30 min at 25°C with stirring. Abl and Ab2 were dialysed overnight at 4°C against 0.1 M phosphate buffer pH 7.5 c o n t t ~ i n g 0.1 M NaC1 and against 0. l M acetate buffer pH 4.5 containing 0.1 M NaCl; the buffers were changed five times. To Ab2, 50 mM of DTT were added with gentle stirring and allowed to react for 30 min at 25°C; then, Abl and Ab2 were mixed rapidly and allowed to incubate at 25°C for 30 min. The mixture was dialysed against 0.1 M phosphate buffer at 4°C overnight, centrifuged 8,000 rpm for 10 min, and the supernatant was added to an equal volume of glycerol and stored at - 2 0 ° C until use.
BSA CAEIA DTT EIA ELISA HBsAg
= = = = = =
bovine serum albumin. chimera antibody EIA. dithiothreitol, erythroimmunoassay. enzyme-linkedimmunosorbentassay. hepatitis B surface antigen.
] ! [ !
HPR = horseradish peroxidase. RPHA = reverse passive haemagglutination assay. SPDC = N-succinimidyl-3-(2-pyridyldithio(propionate)). SRBC = sileep red blood cell.
A CHIMERA A b EIA FOR HBsAg I N H U M A N SERA
339
EIA procedure. Polyvinyl chloride plates with U-shaped wells (Tian Jin, China) were coated with 100 pd of monoclonal anti-HBs antibody at a concentration of 5 ~g/ml in 0.05 M pH 9.5 carbonate buffer. Coating was performed for 2 h at 37°C, then for 16 h at 4°C; then plates were washed with PBS + 0.1 070 Tween-20. Next, 100 Izl of sera samples, undiluted or serially diluted from 1/10 to 1/64,000 in PBS + 0.1 070Tween-20 were added. The plates were incubated for 1 h at 37°C, washed with PBS + 0.1 070 Tween-20. The chimera antibodies (100 Fd, about 0.5 ptg/ml) were added to the wells. After further incubation (1 h at 37°C) and washing, each well was filled with 100 I~l of 0.05 O7osuspension of SRBC in PBS, previously washed 5 times with PBS. The plates were left undisturbed and incubated for 1-2 h at 37°C. Positive HBsAg or negative HBsAg were determined according to the degree of SRBC adsorbed as seen by the naked eye.
Enzyme-linked immunosorbent assay (ELISA). Polystyrene plates with flat-bottomed wells were coated, washed and the samples were added as described for CAEIA; then the HRP-monoclonal anti-HBs IgG (about 1 ptg/ml) conjugate was added to the wells (100 ~l) and incubated for 1 h at 37°C. After washing the wells, 100 bd of substrate solution composed of 0.05 M citrate buffer containing 0.4 mg o-phenylenediamine/ml and 0.06 070 H202 were added to the wells. The enzyme reaction was stopped by adding 25 izl of 2 M H2SO4 a r d absorbance was read at 492 nm.
Reverse passive haemagglutination assay (RPHA). Plexiglass plates with U-shaped wells were added to 25 ~ of human serum samples and 25 I~l of polyclonal horse anti-HBs-antibody-sensitized SRBC suspension (commercial kit). The plates were incubated for 2 h at 37°C; results were assessed by the naked eye. RESULTS Characterization of the chimera antibody was based on the description of Pain and Surolia (198 ~). The number of 2-pyridyl disulphide groups introduced into both IgG proteins wv:~ calculated by detecting the amount of pyridine-2-thione released on treatment of the IgG proteins with 100 mM DTT at 280 nm (before adding DTT) and at 343 nm (after adding DTT). When the molar concentration ratios of SPDP/IgG proteins were about 5-8, satisfactory results were obtained. The titration of chimera antibody was determined by CAEIA (table I). When the chimera antibody was diluted 1/4,800-1/9,600 in PBS + 1 07o BSA (approximately
TABLE I. Human serum samples
1/30
HBsAg + HBsAg-
4+ -
-
-
Titration of chimera antibody in EIA.
Dilution of chimera antibody 1/300 1/600 1/1,200 1/2,400 1/4,800 1/9,600 1/19,200 4+ .
4+ .
4+ .
.
4+ .
4+ .
3+ .
-+
Y. C H E N E T A L .
340
0.5-1 Is.g/ml) for detecting HBsAg in human sera, satisfactory results could be read by the naked eye. Sera from 10 HBsAg-positive human sera and normal control human sera were titrated by CAEIA and RPHA. The highest titres of the serially diluted sera by CAEIA were 2-20 times higher than by R P H A ; results are given in table II. HBsAg was serially diluted from 300 to 1.125 ng/ml; the quantit? of HBsAg was determined by CAEIA and R P H A ; as little as 4.5 ng of HBsAg/ml could be titrated by CAEIA; 75 ng of HBsAg/ml were detected by RPHA. The results were given in table III. The specificity of CAEIA was assessed by adding different antigens instead of HBsAg. These antigens were HBeAg, HBcAg and viral-cultured supernatants from HAV, ADV, CMV and parainfluenza virus type I, HA-2 strain. Most of these antigens did not react with the chimera antibody, excepl for HBeAg solution containing some HBsAg (see table IV).
TABLE II. - - Qualitative comparison of HBsAg detection in human sera using CAEIA or RPHA. Titres of HBsAg-positive sera from 10 patients CAEIA RPHA
1/1,000 1/2,000 1/2,000 1/8,000 1/1,000 1/32,000 1/4,000 1/16,000 1/4,000 1/16,000 1/100 1/100 1/1,000 1/2,000 1/100 1/8,000 1/2,000 1/8,000 1/1,000 1/2,000
TABLE I I I . - - Quantitative comparison of HBsAg detection in vaccine solution using CAEIA or RPHA.
CAEIA RPHA
300
150
HBsAg concentration (ng/mi) 75 37.3 18 9 4.5
4+ 4+
4+ 3+
4+ 2+
4+ .
4+ .
.
2+ .
+
2.3
1
m
D
m
w
TABLE IV. - - Specificity control of CAEIA by adding different antigens and antibodies. Monoclonal anti-HBs"a" HBsAg+ HBsAg- HBeAg(*)HBcAg(**)HAV ADV HA-2 CMV (5 t~g/ml) serum serum (5 ~tg/ml) (supernatant) Chimera antibody (1 ~g/ml)
4+
-
3+
(*) HBeAg solution containing some IIBsAg. (**) HBcAg tom E. coil transinfected by plasmid containing HBcAg DNA. HAV = hepatitis A virus; ADV = adenovirus; HA-2= parainfluenza virus type 1, HA-2 strain; CMV = cytomegalovirus.
A CHIMERA
Ab EIA FOR HBsAg IN HUMAN
SERA
34!
Sera from 432 patients with or without hepatitis B were detected by CAEIA and ELISA results are given in table V.
TABLE V.
-
-
Comparison of HBsAg detection in sera from 432 patients with hepatitis B using C A E | A and ELISA.
CAEIA ELISA
+
-
Total
+ -
259 8
2 163
261 171
Total
267
165
432
CAEIA: ELISA, X2=2.50, P >0.05.
DISCUSSION
In the present work, we have prepared a kind of chimera antibody by coupling monoclonal anti-HBs to polyclonal rabbit anti-SRBC; coupling was done using a heterobifunctional reagent-SPDP. CAEIA was established according to Guesdon's description. From the results obtained, it was found that the sensitivity of CAEIA was better than in RPHA, and the stability and reproducibility in CAEIA were better than in ELISA. Many types of coupling reagents exist for preparing various protein-protein conjugates. Glutaraldehyde is a simple and effective reagent and has been successfully used for preparing various conjugates, including the chimera antibodies (Guesdon et al., 1980, 1983; Gupta et al., 1985; Germani et al., 1987). As a homob~'unctional coupling reagent, glutaraldehyde has some disadvantages. It is possible to form a cross-link between the two different proteins or two identical proteins, e.g. P1-PI and P2-P2 as well as the desired PI-P2 conjugates (Wilson et al., 1978). To avoid this disadvantage, the heterobifunctional coupling reagents can be used for preparing conjugates such as chimera antibodies. SPDP is such a heterobifunctional reagent. An efficient chimera antibody was prepared using SPDP when the ratio between SPDP and IgG was near 1/1 in our laboratory. Many methods exist for detecting HBsAg in human serum. ELISA and RPHA have been used conveniently by many laboratories and the results have been satisfactory. There are also some disadvantages in these methods. ELISA is unstable and does not always give accurate and reliable results in primitive environments. RIA may be unsafe for operators, and the sensitivity and specificity of R P H A are not entirely satisfactory. CAEIA has been used for detecting some antigens and antibodies. Results have been satisfactory in terms of sensitivity, specificity and reproducibility. It is believe that CAEIA may be conveniently suitable for poorly equipped clinical laboratories. ACKNOWLEDGEMENTS
We thank Dr. J.-L. Guesdon for his kind comments and for revision of this manuscript.
342
Y. C H E N
ET AL.
RI~SUMI~ ERYTHRO-IMMUNODOSAGE
AVEC DES ANTICORPS CHIM~RES
DE L'ANTIGi~.NE H l s
POUR LA Di~TECTION
D A N S L E S SI~RUMS H U M A I N S
Un anticorps chim6re fortement actif, form6 par couplage entre un anticorps monoclonal anti-HBs et un anticorps polyclonal anti-~rythrocyte de mouton, a 6t6 pr6par6/t l'aide d'un r6actif h6t~ro-bifonctioanel, le N-succinimydyl-3-(2-pyridyldithio)-proponiate. Nous avons mis au point an ~rythro-immunodosage en phase solide, utilisant ces anticorps chim6res et des ~rythrocytes de mouton comme marqueurs, en accord avec les m6thodes de Guesdon. Nous avons appliqu~ cette technique ~ la recherche de l'antig6ne HBs dans des s6rums, dilu6s en s6rie, de 10 patients atteints d'h~patite B. Ce dosage est 2/t 20 fois plus sensible que le dosage par h6magglutination passive inverse. La plus faible quantit6 d'antig~ne HBs d~tect~e par ce dosage est de 4,5 ng/ml, alors que l'h6magglutination passive inverse ne permet pas de d6tecter moins de 75 ng/ml du m~me antigone. Cet 6rythro-immunodosage est aussi sensible que I'ELISA et donne des r6sultats plus precis, reproductibles et fiables que I'ELISA. Sa sp6cificit6 est aussi satisfaisante. MOTS-CLI~S: H6patite B, HBsAg, Erythro-immunodosage, Anticorps chim6re; Diagnostic.
REFERENCES
GERMAN1,Y., GUESDON,J.-L., Bi~GAUD,E. & MOREAU,J.-P. (1987), Antibody chimera technique applied to Escherichia coli heat-labile enterotoxin. J. Immunol. Methods, 98, 83-89. t". . . . . . . . I I ~ A ......... S . t l t a o ~ x ~ _ _ _ : . : . . . . : . . . . : A - _ ~ __~-" !_ _ ..! -'__ Uuiza~a~ J.-It.J. / ' ~ ¥ Itu~MgA~*, [17OOJ, Ol~llSItlVl~ tltli~l.tlO|l U[ dlltlOOUlt~i r d l l d it.ntlg•ns---'-" . . . . . . . uslng-'-erythro-immunoassay. Ann. Immunol. (Inst. Pasteur), 131C, 389-396. GuEst~P,, J.-L., NAQUIRAVELARDE, F. & AVRAMEAS, S. (1983), Solid-phase immunoassays using chimera antibodies prepared with monoclonal or polyclonal anti-enzyme and antierythrocyte antibodies. Ann. Immunol. (Inst. Pasteur), 134C, 265-274. GUPTA,S.K., GUESDON,J.-L., AVRAM-~S, S. & TALWAR,G.P. (1985), Solid-phase competitive and sandwich-type erythro-immunoassays for human chorionic gonadotropin. J. Imraunol. Methods, 80, 177-187. PAIN, D. & SUROUA,A. (1981), Preparation of protein A-peroxidase monoconjugate using a heterobifunctional reagent, and its use in enzyme immunoassays. J. Immunol. Methods, 40, 219-230. W I L S O N , N.B. et al. (1978), Recent developments in the periodates method of conjugation HRPO to antibodies, in "Immunofluorescence and related staining techniques" (Knapp, W. et al.) (pp. 215-224). Elsevier, North-Holland. Biochemical Press, Amsterdam.
Res. Virol. 1990, !4!, 33%342
TECHNICAL NOTE
A CHIMERA ANTIBODY ERYTHROIMMUNOASSAY FOR D E T E C T I N G H B s A g in H U M A N SERA
Y. Chen (*), S.C. Yang and Q.H. Luo Department of Immunology, Institute of Infecttbus Diseases, Feng-Tai Road 26, 100039 Be(ling (PRC)
SUMMARY A highly efficient chimera antibody, a monoclonal anti-hepatitis-B-surface (anti-HBs) antibody coupled with polyclonal anti-sheep-red-blood-ceU (anti-SRBC) antibody was prepared using a heterobifunctional reagent, N-succinimidyl-3-(2-pyridyldithiopropionate) (SPDP). Using SRBC as a marker, we established a sensitive solid-phase chimera antibody erythroimmunoassay (CAEIA) according to Guesdon's method. The sensitivity of this assay was 2-20 times higher than the reverse passive haemagglutination assay (RPHA) for detecting HBsAg in serially diluted sera from 10 hepatitis B patients. The weakest quantity of HBsAg detected by this assay was 4.5 ng/ml, while RPHA was unable to detect less than 75 ng/ml of HBsAg. The assay was as sensitive as the enzyme-linked immunosorbent assay (ELISA) and gave more accurate, reproducible and stable results than ELISA; specificity was also satisfactory. KEY-WORDS: Chimera antibody, Erythroimmunoassay, HBsAg, Hepatitis B; Diagnosis. INTRODUCTION Various methods exist for detecting hepatitis B surface antigens (HBsAg) in human sera. Enzyme-linked immunosorbent assay (ELISA) and reverse passive haemagglutination assay (RPHA) are among those in widespread use and are sensitive and relatively easy to perform. However, they have some disadvantages during application: the instability of ELISA and its substrates may interfere with its application, and RPHA has low sensitivity and low specificity. A chimera antibody erythroimmunoassay (CAEIA) based on both ELISA and haemagglutination assay was established by Guesdon et al. (1983). Sensitivity and specificity were satisfactory for detecting some antigens and antibodies (Guesdon et al., 1980, 1983; Gupta et r~i., 1985 ; Germani et al., 1987).
Submitted February 2, 1989, accepted January 8, 1990. (*) Correspondingauthor.
Y. C H E N E T AL.
338
In the preseat study, a highly efficient chimera antibody was prepared with monoclonai anti-HBs antibodies and polyclonal anti-SRBC (sheep red blood cells) antibodies using SPDP, a heterobifunctional reagent. CAEIA was established using this chimera antibody and SRBC instead of enzyme antibody-conjugates and substrates in ELISA according to Guesdon's description. Satisfactory results were achieved for detecting HBsAg in human sera.
MATERIALS AND METHODS
Reagents. All reagents were of analytical grade. SPDP was obtained from the Fourth Military Medical University (China); 1-4-dithiothreitol (DTT) was obtained from Feinbiochemica (West Germany); horseradish peroxidase (HRP) grade VI from Sigma; commercial HBsAg from an R P H A kit (National Institute of Vaccine and Serum, Beijing). HBsAg vaccine (30 t~g/ml) purified from human sera; ascites containing monoclonal a n t i - H B s " a " determinant and rabbit anti-SRBC antisera were obtained from the National Institute of Vaccine and Serum (Beijing); SRBC were stored at 4°C in sterile Elsever's solution until use; normal and pathological human sera with hepatitis B were obtained from our laboratory.
Preparation of the enzyme-antibody conjugate. Monoclonal a n t i - H B s " a " IgG were coupled with H R P by a modified Wilson's method (Wilson, 1978). The conjugate was stored at - 2 0 ° C with an added equal volume of glycerol until used.
Preparation of chimera antibody. • . e I~ were precipitated nrst by 50 °7/o (NH4)2SO4 then by 33 o70 (NH4)2SO 4 before use. The monoclonal anti-HBs (Abl) 5 mg/ml and rabbit polyclonal anti-SRBC (Ab2) 5 mg/ml were dialysed overnight at 4°C against 0.1 M phosphate buffer pH 7.5 containing 0.1 M NaCI. To this, 50 ~1 of SPDP dissolved in ethanol (1 mg/ml) were added drop by drop with stirring and allowed to react for 30 min at 25°C with stirring. Abl and Ab2 were dialysed overnight at 4°C against 0.1 M phosphate buffer pH 7.5 c o n t t ~ i n g 0.1 M NaC1 and against 0. l M acetate buffer pH 4.5 containing 0.1 M NaCl; the buffers were changed five times. To Ab2, 50 mM of DTT were added with gentle stirring and allowed to react for 30 min at 25°C; then, Abl and Ab2 were mixed rapidly and allowed to incubate at 25°C for 30 min. The mixture was dialysed against 0.1 M phosphate buffer at 4°C overnight, centrifuged 8,000 rpm for 10 min, and the supernatant was added to an equal volume of glycerol and stored at - 2 0 ° C until use.
BSA CAEIA DTT EIA ELISA HBsAg
= = = = = =
bovine serum albumin. chimera antibody EIA. dithiothreitol, erythroimmunoassay. enzyme-linkedimmunosorbentassay. hepatitis B surface antigen.
] ! [ !
HPR = horseradish peroxidase. RPHA = reverse passive haemagglutination assay. SPDC = N-succinimidyl-3-(2-pyridyldithio(propionate)). SRBC = sileep red blood cell.
A CHIMERA A b EIA FOR HBsAg I N H U M A N SERA
339
EIA procedure. Polyvinyl chloride plates with U-shaped wells (Tian Jin, China) were coated with 100 pd of monoclonal anti-HBs antibody at a concentration of 5 ~g/ml in 0.05 M pH 9.5 carbonate buffer. Coating was performed for 2 h at 37°C, then for 16 h at 4°C; then plates were washed with PBS + 0.1 070 Tween-20. Next, 100 Izl of sera samples, undiluted or serially diluted from 1/10 to 1/64,000 in PBS + 0.1 070Tween-20 were added. The plates were incubated for 1 h at 37°C, washed with PBS + 0.1 070 Tween-20. The chimera antibodies (100 Fd, about 0.5 ptg/ml) were added to the wells. After further incubation (1 h at 37°C) and washing, each well was filled with 100 I~l of 0.05 O7osuspension of SRBC in PBS, previously washed 5 times with PBS. The plates were left undisturbed and incubated for 1-2 h at 37°C. Positive HBsAg or negative HBsAg were determined according to the degree of SRBC adsorbed as seen by the naked eye.
Enzyme-linked immunosorbent assay (ELISA). Polystyrene plates with flat-bottomed wells were coated, washed and the samples were added as described for CAEIA; then the HRP-monoclonal anti-HBs IgG (about 1 ptg/ml) conjugate was added to the wells (100 ~l) and incubated for 1 h at 37°C. After washing the wells, 100 bd of substrate solution composed of 0.05 M citrate buffer containing 0.4 mg o-phenylenediamine/ml and 0.06 070 H202 were added to the wells. The enzyme reaction was stopped by adding 25 izl of 2 M H2SO4 a r d absorbance was read at 492 nm.
Reverse passive haemagglutination assay (RPHA). Plexiglass plates with U-shaped wells were added to 25 ~ of human serum samples and 25 I~l of polyclonal horse anti-HBs-antibody-sensitized SRBC suspension (commercial kit). The plates were incubated for 2 h at 37°C; results were assessed by the naked eye. RESULTS Characterization of the chimera antibody was based on the description of Pain and Surolia (198 ~). The number of 2-pyridyl disulphide groups introduced into both IgG proteins wv:~ calculated by detecting the amount of pyridine-2-thione released on treatment of the IgG proteins with 100 mM DTT at 280 nm (before adding DTT) and at 343 nm (after adding DTT). When the molar concentration ratios of SPDP/IgG proteins were about 5-8, satisfactory results were obtained. The titration of chimera antibody was determined by CAEIA (table I). When the chimera antibody was diluted 1/4,800-1/9,600 in PBS + 1 07o BSA (approximately
TABLE I. Human serum samples
1/30
HBsAg + HBsAg-
4+ -
-
-
Titration of chimera antibody in EIA.
Dilution of chimera antibody 1/300 1/600 1/1,200 1/2,400 1/4,800 1/9,600 1/19,200 4+ .
4+ .
4+ .
.
4+ .
4+ .
3+ .
-+
Y. C H E N E T A L .
340
0.5-1 Is.g/ml) for detecting HBsAg in human sera, satisfactory results could be read by the naked eye. Sera from 10 HBsAg-positive human sera and normal control human sera were titrated by CAEIA and RPHA. The highest titres of the serially diluted sera by CAEIA were 2-20 times higher than by R P H A ; results are given in table II. HBsAg was serially diluted from 300 to 1.125 ng/ml; the quantit? of HBsAg was determined by CAEIA and R P H A ; as little as 4.5 ng of HBsAg/ml could be titrated by CAEIA; 75 ng of HBsAg/ml were detected by RPHA. The results were given in table III. The specificity of CAEIA was assessed by adding different antigens instead of HBsAg. These antigens were HBeAg, HBcAg and viral-cultured supernatants from HAV, ADV, CMV and parainfluenza virus type I, HA-2 strain. Most of these antigens did not react with the chimera antibody, excepl for HBeAg solution containing some HBsAg (see table IV).
TABLE II. - - Qualitative comparison of HBsAg detection in human sera using CAEIA or RPHA. Titres of HBsAg-positive sera from 10 patients CAEIA RPHA
1/1,000 1/2,000 1/2,000 1/8,000 1/1,000 1/32,000 1/4,000 1/16,000 1/4,000 1/16,000 1/100 1/100 1/1,000 1/2,000 1/100 1/8,000 1/2,000 1/8,000 1/1,000 1/2,000
TABLE I I I . - - Quantitative comparison of HBsAg detection in vaccine solution using CAEIA or RPHA.
CAEIA RPHA
300
150
HBsAg concentration (ng/mi) 75 37.3 18 9 4.5
4+ 4+
4+ 3+
4+ 2+
4+ .
4+ .
.
2+ .
+
2.3
1
m
D
m
w
TABLE IV. - - Specificity control of CAEIA by adding different antigens and antibodies. Monoclonal anti-HBs"a" HBsAg+ HBsAg- HBeAg(*)HBcAg(**)HAV ADV HA-2 CMV (5 t~g/ml) serum serum (5 ~tg/ml) (supernatant) Chimera antibody (1 ~g/ml)
4+
-
3+
(*) HBeAg solution containing some IIBsAg. (**) HBcAg tom E. coil transinfected by plasmid containing HBcAg DNA. HAV = hepatitis A virus; ADV = adenovirus; HA-2= parainfluenza virus type 1, HA-2 strain; CMV = cytomegalovirus.
A CHIMERA
Ab EIA FOR HBsAg IN HUMAN
SERA
34!
Sera from 432 patients with or without hepatitis B were detected by CAEIA and ELISA results are given in table V.
TABLE V.
-
-
Comparison of HBsAg detection in sera from 432 patients with hepatitis B using C A E | A and ELISA.
CAEIA ELISA
+
-
Total
+ -
259 8
2 163
261 171
Total
267
165
432
CAEIA: ELISA, X2=2.50, P >0.05.
DISCUSSION
In the present work, we have prepared a kind of chimera antibody by coupling monoclonal anti-HBs to polyclonal rabbit anti-SRBC; coupling was done using a heterobifunctional reagent-SPDP. CAEIA was established according to Guesdon's description. From the results obtained, it was found that the sensitivity of CAEIA was better than in RPHA, and the stability and reproducibility in CAEIA were better than in ELISA. Many types of coupling reagents exist for preparing various protein-protein conjugates. Glutaraldehyde is a simple and effective reagent and has been successfully used for preparing various conjugates, including the chimera antibodies (Guesdon et al., 1980, 1983; Gupta et al., 1985; Germani et al., 1987). As a homob~'unctional coupling reagent, glutaraldehyde has some disadvantages. It is possible to form a cross-link between the two different proteins or two identical proteins, e.g. P1-PI and P2-P2 as well as the desired PI-P2 conjugates (Wilson et al., 1978). To avoid this disadvantage, the heterobifunctional coupling reagents can be used for preparing conjugates such as chimera antibodies. SPDP is such a heterobifunctional reagent. An efficient chimera antibody was prepared using SPDP when the ratio between SPDP and IgG was near 1/1 in our laboratory. Many methods exist for detecting HBsAg in human serum. ELISA and RPHA have been used conveniently by many laboratories and the results have been satisfactory. There are also some disadvantages in these methods. ELISA is unstable and does not always give accurate and reliable results in primitive environments. RIA may be unsafe for operators, and the sensitivity and specificity of R P H A are not entirely satisfactory. CAEIA has been used for detecting some antigens and antibodies. Results have been satisfactory in terms of sensitivity, specificity and reproducibility. It is believe that CAEIA may be conveniently suitable for poorly equipped clinical laboratories. ACKNOWLEDGEMENTS
We thank Dr. J.-L. Guesdon for his kind comments and for revision of this manuscript.
342
Y. C H E N
ET AL.
RI~SUMI~ ERYTHRO-IMMUNODOSAGE
AVEC DES ANTICORPS CHIM~RES
DE L'ANTIGi~.NE H l s
POUR LA Di~TECTION
D A N S L E S SI~RUMS H U M A I N S
Un anticorps chim6re fortement actif, form6 par couplage entre un anticorps monoclonal anti-HBs et un anticorps polyclonal anti-~rythrocyte de mouton, a 6t6 pr6par6/t l'aide d'un r6actif h6t~ro-bifonctioanel, le N-succinimydyl-3-(2-pyridyldithio)-proponiate. Nous avons mis au point an ~rythro-immunodosage en phase solide, utilisant ces anticorps chim6res et des ~rythrocytes de mouton comme marqueurs, en accord avec les m6thodes de Guesdon. Nous avons appliqu~ cette technique ~ la recherche de l'antig6ne HBs dans des s6rums, dilu6s en s6rie, de 10 patients atteints d'h~patite B. Ce dosage est 2/t 20 fois plus sensible que le dosage par h6magglutination passive inverse. La plus faible quantit6 d'antig~ne HBs d~tect~e par ce dosage est de 4,5 ng/ml, alors que l'h6magglutination passive inverse ne permet pas de d6tecter moins de 75 ng/ml du m~me antigone. Cet 6rythro-immunodosage est aussi sensible que I'ELISA et donne des r6sultats plus precis, reproductibles et fiables que I'ELISA. Sa sp6cificit6 est aussi satisfaisante. MOTS-CLI~S: H6patite B, HBsAg, Erythro-immunodosage, Anticorps chim6re; Diagnostic.
REFERENCES
GERMAN1,Y., GUESDON,J.-L., Bi~GAUD,E. & MOREAU,J.-P. (1987), Antibody chimera technique applied to Escherichia coli heat-labile enterotoxin. J. Immunol. Methods, 98, 83-89. t". . . . . . . . I I ~ A ......... S . t l t a o ~ x ~ _ _ _ : . : . . . . : . . . . : A - _ ~ __~-" !_ _ ..! -'__ Uuiza~a~ J.-It.J. / ' ~ ¥ Itu~MgA~*, [17OOJ, Ol~llSItlVl~ tltli~l.tlO|l U[ dlltlOOUlt~i r d l l d it.ntlg•ns---'-" . . . . . . . uslng-'-erythro-immunoassay. Ann. Immunol. (Inst. Pasteur), 131C, 389-396. GuEst~P,, J.-L., NAQUIRAVELARDE, F. & AVRAMEAS, S. (1983), Solid-phase immunoassays using chimera antibodies prepared with monoclonal or polyclonal anti-enzyme and antierythrocyte antibodies. Ann. Immunol. (Inst. Pasteur), 134C, 265-274. GUPTA,S.K., GUESDON,J.-L., AVRAM-~S, S. & TALWAR,G.P. (1985), Solid-phase competitive and sandwich-type erythro-immunoassays for human chorionic gonadotropin. J. Imraunol. Methods, 80, 177-187. PAIN, D. & SUROUA,A. (1981), Preparation of protein A-peroxidase monoconjugate using a heterobifunctional reagent, and its use in enzyme immunoassays. J. Immunol. Methods, 40, 219-230. W I L S O N , N.B. et al. (1978), Recent developments in the periodates method of conjugation HRPO to antibodies, in "Immunofluorescence and related staining techniques" (Knapp, W. et al.) (pp. 215-224). Elsevier, North-Holland. Biochemical Press, Amsterdam.