273
Clinica Chimica Acta, 61 (1975) 273-278 @ Elsevier Scientific Publishing Company,
Amsterdam
- Printed in The Netherlands
CCA 6985
A NEW RADIOIMMUNOASSAY FOR DEMONSTRATING ANTIBODY TO HEPATITIS-B-SURFACE-ANTIGEN (H&-Ag)
THE
G. THAMER and B. KOMMERELL Department of Gastroenterology, Beigheimerstr. 58 (G. F. R.) (Received
December
Heidelberg
University Hospital, 69 Heidelberg,
16, 1974)
Summary A new radioimmunological system for demonstrating the antibody to hepatitis-B-surface-antigen (HB,-Ag) based on the immunoadsorption principle is described. The optimal conditions for the test are given. False-negative and false-positive results could be excluded by specificity tests on sera from 100 voluntary blood donors. The sensitivity of the system is comparable to that of other more expensive radioimmunological methods.
Introduction Determination of the hepatitis-B-surface-antigen (HB ,-Ag, Australia-Antigen, HAA) in human serum has today attained a secure place in the diagnosis of liver diseases and in prophylaxis of transfusion hepatitis, since specific and sensitive test systems have been developed. In contrast, routine determination of the corresponding antibody (antibody to HB,Ag, anti-HB& still meets with methodological difficulties. Determination of anti-HB, could give information on the humoral immune response of the organism after contact with material positive for hepatitis-B-surface-antigen. Simple, sensitive test systems have not been available up to now. This paper describes a new radioimmunological test system for demonstrating the antibody to hepatitis-B-surface-antigen which can readily be carried out in any immunologically oriented laboratory. All reagents necessary for the test are commercially available. Principleof the test The new test system utilizing the immunoadsorption principle is represented diagrammatically in Fig. 1. The test serum is first incubated with agarose
214
0
Ii
sepha=
0 n
8 cpm
J125-anti-HBs
Gxmting
t-m,-Ag
anti-HB,
II
CPm
Fig. 1. Principle of the test system for demonstrating the antibody to hepatitis-B-surface-antigen. Key, HBs-Ag (hepatitis-B-surface-antigen); anti-HBs, antibody to hepatitis-B-surface-antigen: ’ 2 s I-anti-HBs, * 251-lahelled antibody to HE$Ag; Sepharose/HBs-Ag. Sepharose particles to which HB,-Ag is chemically coupled.
particles to which hepatitis-B-surface-antigen is chemically coupled. When antiHB, are present in the test serum (Fig. 1, left), the binding sites on the agarosefHB,-Ag complex (Seph~ose~HBs-Ag) are occupied by the antibody. These binding sites are hence no longer available to bind the radioactively labelled antibody which is added in a second incubation step. On the other hand, the binding sites on the agarose/HB,Ag complex remain free on incubation with antibody-negative serum. They are occupied by the radioactively labelled antibody in the second incubation step (Fig. 1, right). Since labelled and unlabelled antibody compete for free binding sites, the presence of an antibody to the hepatitis-B-surface-antigen in the test serum can be inferred from the amount of activity bound to the agarosefHBs-Ag complex. Materials and Methods 2 ml of a high-titer HB,-Ag-containing human serum (titer in the complement fixation test 1 : 512, subtype ay) are coupled to 10 g of Sepharose 4-B (Pharmacia, Uppsala) by the cyanogen bromide method according to Porath et al. [I]. After elution of the proteins which have not been bound, the Sepha-
rose/HB,-Ag complex which is formed is resuspended in 300 ml Tris/HCl buffer (100 mM, pH 7.4 with added sodium aside). The antibody to hepatitis-B-surface-antigen which is labelled with ’ * ‘1 is obtained from the commercially available test kits for the detection of HB,-Ag (AUSRIA-125: Abbott Laboratories). According to the manufacturers’ data [2], this heterologous guinea pig antibody reacts against the antigenic determinants a, d and y. In the test, 500 ~1 test serum and 500 ~.tl Sepharose/HB,-Ag complex suspension were incubated for 24 hours at 24°C in disposable test tubes. They were subsequently reincubated for 24 hours at 24°C with ’ * ‘I-labelled antibody. The amount of labelled antibody which is added is so adjusted that about 8000 to 10 000 cpm (as a rule about 20 ~1) are bound in antibody-negative serum. After the second incubation step, the incubate is quantitatively transferred to small columns. The proteins which are not bound to the Sepharose/HB,-Ag complex are removed by washing four times with 2 ml Tris/HCl buffer (100 mM, pH 7.4). We use as columns 2-ml disposable syringes (Co. Braun, Melsungen) of which the outlets are plugged with cellulose after removal of the plungers. The activity bound to the Sepharose/HB,-Ag complex in the columns after washing is determined in the gamma counter. A serum is designated positive for antibody to hepatitis-B-surface-antigen if the bound activity is more than 3 standard deviations less than the average of 10 certain antibodynegative sera. Experimental
Results
To ascertain the optimal test conditions, a few parameters of the experimental preparation are varied and the effect on the binding of the labelled antibody studied. The dependence of antibody binding on the amount of the Sepharose/HB,-Ag complex in the test mixture is shown in Fig. 2. Decreasing amounts of a Sepharose/HB,-Ag complex suspension (10 g/20 ml) are incubated with labelled antibody for 24 hours at 24°C and the activity bound to the complex determined. With large amounts of Sepharose/HBs-Ag complex, labelled and unlabelled antibody are bound almost quantitatively. When small quantities of Sepharose/HB,-Ag complex are added, the binding sites are no longer sufficient to bind the labelled antibody, even in antibody-negative serum. The region which is optimal for the test lies at the maximum of the difference curve. It is determined by the amount of HB,-Ag coupled to the Sepharose and must hence be determined anew for each Sepharose/HB,-Ag complex preparation. If the titer of the coupled serum is sufficiently high, the complex formed from 2 ml serum and 10 g Sepharose 4-B is sufficient for about 600 determinations. The Sepharose/HB,-Ag complex keeps for several months at -20°C. The effect of the length of incubation on the binding of labelled antibody to the Sepharose/HB,-Ag complex is shown in Fig. 3. If labelled anti-HB, is incubated with Sepharose to which HB,-Ag-negative serum has been coupled, antibody is attached unspecifically to the complex with increasing time of incubation. On incubation of labelled antibody with Sepharose to which HB,Ag-positive serum is bound, a maximal value is not attained even with an
Fig. 2. Dependence of binding of labelled anti-HBs (ordinate, cpm) on the amount of Sepharose/HBs-Ag complex in the incubate (abscissa, ml SepharosefHq-Ag-suspension, 10 g/Z0 ml). O------O, preparation preparation with any-HBs-poetize serum; X -X, difference with anti-HBgnegative serum; e----*, cunte.
. 7..
1.. 48
b time Ihours
Fig. 3. Dependence of antibody binding (ordinate, cpm) on the duration of incubation (abscissa, hours). incubation -. incubation with Sepharose to which HBs-Ag-positive serum is coupled: o----O, with Sepharose to which HBs-Ag-negative serum is coupled.
277
incubation of long duration. The upper curve in Fig. 3 thus represents a summation of specific and non-specific binding. Both specific and non-specific antibody binding increases with a raised temperature of incubation in the range 0-45°C. A 24-hour incubation at 24°C was chosen for the test. Alterations in the molarity of the incubation buffer in a range from 10 to 1000 mM and changes of pH in the range pH 5 to 9 do not show any significant effect on antibody binding. Optimal conditions for qualitative demonstration of the antibody to hepatitis-B-surface-antigen are as follows: (1) test serum, 500 ~1. (2) Sepharose/HB,-Ag complex, 500 1.11of a Sepharose/HB,-Ag complex suspension in Tris/HCl buffer (100 mM, pH 7.4). Dilution of the complex depends on the height of the titer of the coupled serum and the effectiveness of the cyanogen bromide coupling. (3) time of incubation, 24 hours. (4) temperature of incubation, 24°C. (5) labelled antibody, charge dependent, adjusted so that about 8000 to 10 000 cpm are bound in anti-HB,-negative serum. After optimization of the test conditions sera from 100 voluntary blood donors from the Heidelberg area were tested. Sera were designated anti-HB,negative when the test results lay within 2 standard deviations from the mean of the normally distributed results. When the bound activity was less than 3 standard deviations from the mean, the serum is termed anti-HB,-positive. Such an antibody to hepatitis-B-surface-antigen was demonstrated in 6 of 100 voluntary blood donors. To exclude false-negative results, the test was repeated with Sepharose to which HB,-Ag-negative serum had been coupled. In no case was the bound activity as high as in the preparation with Sepharose/HB,-Ag complex. Falsewm
-.aJa 1 -1 10
.
I
Id2
1
o-3
lo-’
l
. I
0-o
ml anti-I-W,
co
Fig. 4. Dependence of the amount of the lebelled anti-H&-Ag (ordinate. CPd HB,-containing eenun in the essay (absciesn.ml).
on the axxxoud of enti-
2’i8
positive results were excluded by absorbing the six sera which were found to be anti-HB, positive with serum containing a high titer of HB,-Ag. With our test system, antibody was no longer demonstrable in the supernatant of any serum after ultracentrifugation. The test system enabled the antibody to hepatitis-B-surface-antigen in the test sera to be qu~tified. Fig. 4 shows the dependence of binding of labelled antibody on the amount of an anti-HB,-containing serum in the incubate. Unknown antibody titers can be quantified on the basis of this calibration curve. Discussion The radioimmunological test system for demonstrating antibody against hepatitis-B-surface-antigen which is described and which utilizes the immunoadsorption principle is distinguished by technical simplicity. Since all reagents, including the labelled ~tibody, are commercially available, the tesjt can be carried out in any immunologically orientated laboratory without substantial methodological difficulties. False positive and false negative results could be excluded by tests of specificity which were carried out on 100 HB,-Ag-negative sera from voluntary blood donors. Of course, antibody cannot be demonstrated in HB,-Ag-positive sera owing to the principle of the test. The free antigen in the test preparation leads to reduced binding of the labelled antibody since the free HB,-Ag and the agarose-bound HB,-Ag compete for the labelled antibody. This competition for the labelled antibody can be utilized for a qu~titative dete~ination of hepatitis-B-surface-antigen, as will be reported in a later paper. Antibodies against HB,-Ag of differing subtype specificity can be detected with this test system. The subtype specificity depends on the subtype specificity of the labelled antibody and the subtype of the coupled HB,-Ag. The labelled heterologous guinea pig antibody (Abbott Laboratories) which is used contains antibodies against the antigenic determinants a, d and y (according to the manufacturers’ specifications [ 21). Depending on which agarose-coupled HB,Ag is chosen, antibodies of the specificity anti-a, anti-d and anti-y can be detected. The sensitivity of the system appears to be comparable with that of other, methodologically more expensive, radioimmunological test systems. Comparably high antibody frequencies in voluntary blood donors were found by several authors [ 3,4 ] (Schober, personal communication) using other methods. Its technical simplicity, high sensitivity and specificity appear to make this new radioimmunological test system suitable for investigating the humoral immune response after contact with hepatitis-B-surface-antigen-positive material even in routine clinical procedures. References 1 Porath, J.. Axen, R. and Emback, S. (1967) Nature 215, 1491 2 Overby, LR.. Decker. R.H. and Ling, C.M. (1973) Science 182, 1368 3 Lander, J.. Holland, P.V., Alter, H.J., Chanock. R.M. and Purcell, R.H. (1972) J. Am. Med. Assoc. 220, 1079 4 Szmunes. W., Prince, A.M., I3xotmau. B. and Hirsch, R.L. (1973) J. Infect. Dis. 127-17
Clinica Chimica Acta, 61 (1975) 273-278 @ Elsevier Scientific Publishing Company,
Amsterdam
- Printed in The Netherlands
CCA 6985
A NEW RADIOIMMUNOASSAY FOR DEMONSTRATING ANTIBODY TO HEPATITIS-B-SURFACE-ANTIGEN (H&-Ag)
THE
G. THAMER and B. KOMMERELL Department of Gastroenterology, Beigheimerstr. 58 (G. F. R.) (Received
December
Heidelberg
University Hospital, 69 Heidelberg,
16, 1974)
Summary A new radioimmunological system for demonstrating the antibody to hepatitis-B-surface-antigen (HB,-Ag) based on the immunoadsorption principle is described. The optimal conditions for the test are given. False-negative and false-positive results could be excluded by specificity tests on sera from 100 voluntary blood donors. The sensitivity of the system is comparable to that of other more expensive radioimmunological methods.
Introduction Determination of the hepatitis-B-surface-antigen (HB ,-Ag, Australia-Antigen, HAA) in human serum has today attained a secure place in the diagnosis of liver diseases and in prophylaxis of transfusion hepatitis, since specific and sensitive test systems have been developed. In contrast, routine determination of the corresponding antibody (antibody to HB,Ag, anti-HB& still meets with methodological difficulties. Determination of anti-HB, could give information on the humoral immune response of the organism after contact with material positive for hepatitis-B-surface-antigen. Simple, sensitive test systems have not been available up to now. This paper describes a new radioimmunological test system for demonstrating the antibody to hepatitis-B-surface-antigen which can readily be carried out in any immunologically oriented laboratory. All reagents necessary for the test are commercially available. Principleof the test The new test system utilizing the immunoadsorption principle is represented diagrammatically in Fig. 1. The test serum is first incubated with agarose
214
0
Ii
sepha=
0 n
8 cpm
J125-anti-HBs
Gxmting
t-m,-Ag
anti-HB,
II
CPm
Fig. 1. Principle of the test system for demonstrating the antibody to hepatitis-B-surface-antigen. Key, HBs-Ag (hepatitis-B-surface-antigen); anti-HBs, antibody to hepatitis-B-surface-antigen: ’ 2 s I-anti-HBs, * 251-lahelled antibody to HE$Ag; Sepharose/HBs-Ag. Sepharose particles to which HB,-Ag is chemically coupled.
particles to which hepatitis-B-surface-antigen is chemically coupled. When antiHB, are present in the test serum (Fig. 1, left), the binding sites on the agarosefHB,-Ag complex (Seph~ose~HBs-Ag) are occupied by the antibody. These binding sites are hence no longer available to bind the radioactively labelled antibody which is added in a second incubation step. On the other hand, the binding sites on the agarose/HB,Ag complex remain free on incubation with antibody-negative serum. They are occupied by the radioactively labelled antibody in the second incubation step (Fig. 1, right). Since labelled and unlabelled antibody compete for free binding sites, the presence of an antibody to the hepatitis-B-surface-antigen in the test serum can be inferred from the amount of activity bound to the agarosefHBs-Ag complex. Materials and Methods 2 ml of a high-titer HB,-Ag-containing human serum (titer in the complement fixation test 1 : 512, subtype ay) are coupled to 10 g of Sepharose 4-B (Pharmacia, Uppsala) by the cyanogen bromide method according to Porath et al. [I]. After elution of the proteins which have not been bound, the Sepha-
rose/HB,-Ag complex which is formed is resuspended in 300 ml Tris/HCl buffer (100 mM, pH 7.4 with added sodium aside). The antibody to hepatitis-B-surface-antigen which is labelled with ’ * ‘1 is obtained from the commercially available test kits for the detection of HB,-Ag (AUSRIA-125: Abbott Laboratories). According to the manufacturers’ data [2], this heterologous guinea pig antibody reacts against the antigenic determinants a, d and y. In the test, 500 ~1 test serum and 500 ~.tl Sepharose/HB,-Ag complex suspension were incubated for 24 hours at 24°C in disposable test tubes. They were subsequently reincubated for 24 hours at 24°C with ’ * ‘I-labelled antibody. The amount of labelled antibody which is added is so adjusted that about 8000 to 10 000 cpm (as a rule about 20 ~1) are bound in antibody-negative serum. After the second incubation step, the incubate is quantitatively transferred to small columns. The proteins which are not bound to the Sepharose/HB,-Ag complex are removed by washing four times with 2 ml Tris/HCl buffer (100 mM, pH 7.4). We use as columns 2-ml disposable syringes (Co. Braun, Melsungen) of which the outlets are plugged with cellulose after removal of the plungers. The activity bound to the Sepharose/HB,-Ag complex in the columns after washing is determined in the gamma counter. A serum is designated positive for antibody to hepatitis-B-surface-antigen if the bound activity is more than 3 standard deviations less than the average of 10 certain antibodynegative sera. Experimental
Results
To ascertain the optimal test conditions, a few parameters of the experimental preparation are varied and the effect on the binding of the labelled antibody studied. The dependence of antibody binding on the amount of the Sepharose/HB,-Ag complex in the test mixture is shown in Fig. 2. Decreasing amounts of a Sepharose/HB,-Ag complex suspension (10 g/20 ml) are incubated with labelled antibody for 24 hours at 24°C and the activity bound to the complex determined. With large amounts of Sepharose/HBs-Ag complex, labelled and unlabelled antibody are bound almost quantitatively. When small quantities of Sepharose/HB,-Ag complex are added, the binding sites are no longer sufficient to bind the labelled antibody, even in antibody-negative serum. The region which is optimal for the test lies at the maximum of the difference curve. It is determined by the amount of HB,-Ag coupled to the Sepharose and must hence be determined anew for each Sepharose/HB,-Ag complex preparation. If the titer of the coupled serum is sufficiently high, the complex formed from 2 ml serum and 10 g Sepharose 4-B is sufficient for about 600 determinations. The Sepharose/HB,-Ag complex keeps for several months at -20°C. The effect of the length of incubation on the binding of labelled antibody to the Sepharose/HB,-Ag complex is shown in Fig. 3. If labelled anti-HB, is incubated with Sepharose to which HB,-Ag-negative serum has been coupled, antibody is attached unspecifically to the complex with increasing time of incubation. On incubation of labelled antibody with Sepharose to which HB,Ag-positive serum is bound, a maximal value is not attained even with an
Fig. 2. Dependence of binding of labelled anti-HBs (ordinate, cpm) on the amount of Sepharose/HBs-Ag complex in the incubate (abscissa, ml SepharosefHq-Ag-suspension, 10 g/Z0 ml). O------O, preparation preparation with any-HBs-poetize serum; X -X, difference with anti-HBgnegative serum; e----*, cunte.
. 7..
1.. 48
b time Ihours
Fig. 3. Dependence of antibody binding (ordinate, cpm) on the duration of incubation (abscissa, hours). incubation -. incubation with Sepharose to which HBs-Ag-positive serum is coupled: o----O, with Sepharose to which HBs-Ag-negative serum is coupled.
277
incubation of long duration. The upper curve in Fig. 3 thus represents a summation of specific and non-specific binding. Both specific and non-specific antibody binding increases with a raised temperature of incubation in the range 0-45°C. A 24-hour incubation at 24°C was chosen for the test. Alterations in the molarity of the incubation buffer in a range from 10 to 1000 mM and changes of pH in the range pH 5 to 9 do not show any significant effect on antibody binding. Optimal conditions for qualitative demonstration of the antibody to hepatitis-B-surface-antigen are as follows: (1) test serum, 500 ~1. (2) Sepharose/HB,-Ag complex, 500 1.11of a Sepharose/HB,-Ag complex suspension in Tris/HCl buffer (100 mM, pH 7.4). Dilution of the complex depends on the height of the titer of the coupled serum and the effectiveness of the cyanogen bromide coupling. (3) time of incubation, 24 hours. (4) temperature of incubation, 24°C. (5) labelled antibody, charge dependent, adjusted so that about 8000 to 10 000 cpm are bound in anti-HB,-negative serum. After optimization of the test conditions sera from 100 voluntary blood donors from the Heidelberg area were tested. Sera were designated anti-HB,negative when the test results lay within 2 standard deviations from the mean of the normally distributed results. When the bound activity was less than 3 standard deviations from the mean, the serum is termed anti-HB,-positive. Such an antibody to hepatitis-B-surface-antigen was demonstrated in 6 of 100 voluntary blood donors. To exclude false-negative results, the test was repeated with Sepharose to which HB,-Ag-negative serum had been coupled. In no case was the bound activity as high as in the preparation with Sepharose/HB,-Ag complex. Falsewm
-.aJa 1 -1 10
.
I
Id2
1
o-3
lo-’
l
. I
0-o
ml anti-I-W,
co
Fig. 4. Dependence of the amount of the lebelled anti-H&-Ag (ordinate. CPd HB,-containing eenun in the essay (absciesn.ml).
on the axxxoud of enti-
2’i8
positive results were excluded by absorbing the six sera which were found to be anti-HB, positive with serum containing a high titer of HB,-Ag. With our test system, antibody was no longer demonstrable in the supernatant of any serum after ultracentrifugation. The test system enabled the antibody to hepatitis-B-surface-antigen in the test sera to be qu~tified. Fig. 4 shows the dependence of binding of labelled antibody on the amount of an anti-HB,-containing serum in the incubate. Unknown antibody titers can be quantified on the basis of this calibration curve. Discussion The radioimmunological test system for demonstrating antibody against hepatitis-B-surface-antigen which is described and which utilizes the immunoadsorption principle is distinguished by technical simplicity. Since all reagents, including the labelled ~tibody, are commercially available, the tesjt can be carried out in any immunologically orientated laboratory without substantial methodological difficulties. False positive and false negative results could be excluded by tests of specificity which were carried out on 100 HB,-Ag-negative sera from voluntary blood donors. Of course, antibody cannot be demonstrated in HB,-Ag-positive sera owing to the principle of the test. The free antigen in the test preparation leads to reduced binding of the labelled antibody since the free HB,-Ag and the agarose-bound HB,-Ag compete for the labelled antibody. This competition for the labelled antibody can be utilized for a qu~titative dete~ination of hepatitis-B-surface-antigen, as will be reported in a later paper. Antibodies against HB,-Ag of differing subtype specificity can be detected with this test system. The subtype specificity depends on the subtype specificity of the labelled antibody and the subtype of the coupled HB,-Ag. The labelled heterologous guinea pig antibody (Abbott Laboratories) which is used contains antibodies against the antigenic determinants a, d and y (according to the manufacturers’ specifications [ 21). Depending on which agarose-coupled HB,Ag is chosen, antibodies of the specificity anti-a, anti-d and anti-y can be detected. The sensitivity of the system appears to be comparable with that of other, methodologically more expensive, radioimmunological test systems. Comparably high antibody frequencies in voluntary blood donors were found by several authors [ 3,4 ] (Schober, personal communication) using other methods. Its technical simplicity, high sensitivity and specificity appear to make this new radioimmunological test system suitable for investigating the humoral immune response after contact with hepatitis-B-surface-antigen-positive material even in routine clinical procedures. References 1 Porath, J.. Axen, R. and Emback, S. (1967) Nature 215, 1491 2 Overby, LR.. Decker. R.H. and Ling, C.M. (1973) Science 182, 1368 3 Lander, J.. Holland, P.V., Alter, H.J., Chanock. R.M. and Purcell, R.H. (1972) J. Am. Med. Assoc. 220, 1079 4 Szmunes. W., Prince, A.M., I3xotmau. B. and Hirsch, R.L. (1973) J. Infect. Dis. 127-17
