COMPARISON OF ANTIHUMAN IMMUNOGLOBULIN AND ANTIHUMAN FAB CONJUGATES FOR THE DETECTION OF TISSUE AUTOANTIBODIES BY IMMUNOFLUORESCENCE Shireen Chantler
Wellcome Research Laboratories Beckenham, Kent, England G . Kolarz, G. D. Johnson, and E. J . Holborow
MRC Rheumatism Unit Canadian Red Cross Memorial Hospital Taplow, Maidenhead, Berkshire, England Screening tests for tissue-reactive autoantibodies by the indirect immunofluorescent procedure are usually performed with fluorescent antihuman immunoglobulin conjugate prepared from the globulin fraction of hyperimmune antiserum obtained by immunizing animals with purified human IgC. Such antisera contain antibodies that cross react with other major immunoglobulin classes by virtue of common antigenic sites and are therefore considered suitable for the detection of IgM and IgA as well as IgG antibodies in screening procedures. Determination of the immunoglobulin class of specific antibody initially detected by this procedure does, however, require the use of immunospecific reagents directed against the Fc fragments of the different immunoglobulin classes. It might be expected that antiserum raised against Fab fragments of human IgG, in which antibody activity is directed against antigenic sites common t o the major classes of immunoglobulin, would provide a satisfactory alternative screening reagent. The finding that a test serum sample was reported to contain tissue-reactive antibodies when examined with the former reagent but was negative when tested with anti-Fab conjugate stimulated us to perform some preliminary comparative tests. In this study, sera previously shown to contain autoantibodies in routine screening with antihuman immunoglobulin conjugate were reexamined with both antihuman immunoglobulin and antihuman Fab con; ugat es. MATERIALS AND METHODS Test Sera
Serum samples were obtained from 96 patients, including 16 cases of chronic active hepatitis, 20 cases of glandular fever (taken within three weeks of onset), 12 cases of Still’s disease, and 48 miscellaneous hospital patients, mainly with connective tissue disease.
Conjuga tcs Sheep antihuman immunoglobulin (Wellcome Reagents, Ltd) was prepared from hyperimmune sera raised against purified IgG obtained from a pool of human serum by chromatography on diethylaminoethyl (DEAE) cellulose.
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Chantler et al. : Tissue Autoantibody Detection
Sheep antihuman Fab was prepared from an antiserum raised against Fab fragments prepared by Dr. Frank Hay by papain digestion of purified IgG obtained from a pool of normal human serum. The digestion products were further purified by fractionation on Sephadex G-100. The second peak was fractionated by ion-exchange chromatography on DEAE cellulose. Both antisera gave good reactions in gel diffusion titrations against whole IgG and F(ab')2 fragments. Anti-immunoglobulin serum reacted strongly against Fc fragments of human IgG, whereas trace reactivity was observed with anti-Fab serum.
Immunofluorescence Tests The standard indirect procedure was followed with cryostat sections of a composite tissue block of rat liver, kidney, and stomach. Patients' sera were usually screened at a dilution of 1 : 10 for antibodies against nuclei, gastric parietal cells, mitochondria, smooth muscle, and reticulin. Conjugates were evaluated by chessboard titration against reference sera that contained antinuclear antibodies of known class, and the working dilution of these polyspecific conjugates was based on t h e results of titrations against both specific IgG and IgM antibodies. The performance of the conjugates at the selected working dilutions was assessed in simultaneous titrations of 10 sera reactive against nuclei, smooth muscle, or gastric parietal cells prior to use in the screening tests. Comparative tests with antihuman immunoglobulin and anti-Fab conjugate were performed in parallel. All readings were made o n a Reichert Zetopan microscope equipped with quartz-halogen illumination and toric cardioid condenser. A Balzer FITC-3 exciter filter and Wratten 1 2 gelatin barrier filter were employed. The intensity of staining was assessed by two individuals o n a predetermined scoring scale. RESULTS There was a lack of agreement between the results obtained with the two conjugates (TABLE 1). A total of 109 autoantibodies were detected in the 96 sera with fluorescent antihuman immunoglobulin, but only 68, i.e., 62.4%, of these were demonstrable when antihuman Fab conjugate was used. TABLE 1 SUMMARY OF RESULTS OF IMMUNOFLUORESCENCE TESTS FOR FIVE DIFFERENT AUTOANTIBODIES PERFORMED ON 96 SERA WITH ANTIHUMAN IMMUNOGLOBULIN AND ANTIHUMAN FAB CONJUGATES Intensity with Anti-Fab Antibody Against
Antiimmunoglobulin No. Positive
Nuclei Gastric parietal cells Smooth muscle Reticulin Mitochondria Totals
Similar No. %
33 15 46 10 5
15 5 18 3 2
45.4 23.3 39.1 30 40
109
43
39.4
lncreased No. % 8 24.2 5 33.3 0 0 3 60
16
14.7
Reduced No. %
5 1 2 1 0 9
15.2 5.6 4.3 10 8.3
Negative No. %
5 4 26 6 0 41
15.2 26.6 56.5 60 37.6
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Annals New York Academy of Sciences
The staining intensity of individual samples was also found t o vary with the two conjugates. Forty three of the 68 positive samples (70.5%) exhibited a similar intensity of fluorescence with both conjugates. Nine (13.2%) showed decreased staining, and 16 (23.5%) showed an increase intensity of staining with anti-Fab conjugate. T h e enhanced staining with anti-Fab conjugate was not related to the titer of antibodies present. Seven of the 16 antibodies that gave increased fluorescence with anti-Fab conjugate were examined with immunospecific conjugates t o determine the immunoglobulin class of antibody; all contained IgM antibodies exclusively. It can be seen from the Table that the distribution of negatives and the variation in staining intensity are not restricted t o a single autoantibody system. DISCUSSION The detection of tissue-reactive antibodies by indirect immunofluorescence procedures that employ conjugates directed against whole IgG or enzyme digestion products of IgG, i.e., Fab or F(ab‘)* fragments, might b e expected to produce similar results, because both react against antigens common to the major classes of immunoglobulin. Our preliminary results show a surprisingly low level of agreement between the results obtained with the two conjugates. These differences may be due t o qualitative and quantitative differences in the spectrum of antibodies present in the conjugates; the selection of the working dilutions of the conjugates by chessboard titrations with antibodies of defined immunoglobulin class should eliminate effects due to variation in the potency of these polyspecific conjugates for IgG o r IgM antibodies. It is conceivable, however, that configuration changes that occur in the region of the antibody-combining site subsequent t o interaction with antigen may make some individual antibodies, particularly of IgG class, less amenable t o detection with anti-Fab than anti-immunoglobulin conjugate, because the latter contain anti-Fc activity also. Configuration changes might be expected to present fewer difficulties in the detection of IgM antibodies because of the increased number of “unaltered” sites available for combination. In view of this unexpectedly low level of agreement between the results obtained with these conjugates, we feel that it is important t o confirm these data and extend this study b y screening additional sera with both conjugates t o establish which conjugate is more suitable for use in these procedures.
Wellcome Research Laboratories Beckenham, Kent, England G . Kolarz, G. D. Johnson, and E. J . Holborow
MRC Rheumatism Unit Canadian Red Cross Memorial Hospital Taplow, Maidenhead, Berkshire, England Screening tests for tissue-reactive autoantibodies by the indirect immunofluorescent procedure are usually performed with fluorescent antihuman immunoglobulin conjugate prepared from the globulin fraction of hyperimmune antiserum obtained by immunizing animals with purified human IgC. Such antisera contain antibodies that cross react with other major immunoglobulin classes by virtue of common antigenic sites and are therefore considered suitable for the detection of IgM and IgA as well as IgG antibodies in screening procedures. Determination of the immunoglobulin class of specific antibody initially detected by this procedure does, however, require the use of immunospecific reagents directed against the Fc fragments of the different immunoglobulin classes. It might be expected that antiserum raised against Fab fragments of human IgG, in which antibody activity is directed against antigenic sites common t o the major classes of immunoglobulin, would provide a satisfactory alternative screening reagent. The finding that a test serum sample was reported to contain tissue-reactive antibodies when examined with the former reagent but was negative when tested with anti-Fab conjugate stimulated us to perform some preliminary comparative tests. In this study, sera previously shown to contain autoantibodies in routine screening with antihuman immunoglobulin conjugate were reexamined with both antihuman immunoglobulin and antihuman Fab con; ugat es. MATERIALS AND METHODS Test Sera
Serum samples were obtained from 96 patients, including 16 cases of chronic active hepatitis, 20 cases of glandular fever (taken within three weeks of onset), 12 cases of Still’s disease, and 48 miscellaneous hospital patients, mainly with connective tissue disease.
Conjuga tcs Sheep antihuman immunoglobulin (Wellcome Reagents, Ltd) was prepared from hyperimmune sera raised against purified IgG obtained from a pool of human serum by chromatography on diethylaminoethyl (DEAE) cellulose.
66
67
Chantler et al. : Tissue Autoantibody Detection
Sheep antihuman Fab was prepared from an antiserum raised against Fab fragments prepared by Dr. Frank Hay by papain digestion of purified IgG obtained from a pool of normal human serum. The digestion products were further purified by fractionation on Sephadex G-100. The second peak was fractionated by ion-exchange chromatography on DEAE cellulose. Both antisera gave good reactions in gel diffusion titrations against whole IgG and F(ab')2 fragments. Anti-immunoglobulin serum reacted strongly against Fc fragments of human IgG, whereas trace reactivity was observed with anti-Fab serum.
Immunofluorescence Tests The standard indirect procedure was followed with cryostat sections of a composite tissue block of rat liver, kidney, and stomach. Patients' sera were usually screened at a dilution of 1 : 10 for antibodies against nuclei, gastric parietal cells, mitochondria, smooth muscle, and reticulin. Conjugates were evaluated by chessboard titration against reference sera that contained antinuclear antibodies of known class, and the working dilution of these polyspecific conjugates was based on t h e results of titrations against both specific IgG and IgM antibodies. The performance of the conjugates at the selected working dilutions was assessed in simultaneous titrations of 10 sera reactive against nuclei, smooth muscle, or gastric parietal cells prior to use in the screening tests. Comparative tests with antihuman immunoglobulin and anti-Fab conjugate were performed in parallel. All readings were made o n a Reichert Zetopan microscope equipped with quartz-halogen illumination and toric cardioid condenser. A Balzer FITC-3 exciter filter and Wratten 1 2 gelatin barrier filter were employed. The intensity of staining was assessed by two individuals o n a predetermined scoring scale. RESULTS There was a lack of agreement between the results obtained with the two conjugates (TABLE 1). A total of 109 autoantibodies were detected in the 96 sera with fluorescent antihuman immunoglobulin, but only 68, i.e., 62.4%, of these were demonstrable when antihuman Fab conjugate was used. TABLE 1 SUMMARY OF RESULTS OF IMMUNOFLUORESCENCE TESTS FOR FIVE DIFFERENT AUTOANTIBODIES PERFORMED ON 96 SERA WITH ANTIHUMAN IMMUNOGLOBULIN AND ANTIHUMAN FAB CONJUGATES Intensity with Anti-Fab Antibody Against
Antiimmunoglobulin No. Positive
Nuclei Gastric parietal cells Smooth muscle Reticulin Mitochondria Totals
Similar No. %
33 15 46 10 5
15 5 18 3 2
45.4 23.3 39.1 30 40
109
43
39.4
lncreased No. % 8 24.2 5 33.3 0 0 3 60
16
14.7
Reduced No. %
5 1 2 1 0 9
15.2 5.6 4.3 10 8.3
Negative No. %
5 4 26 6 0 41
15.2 26.6 56.5 60 37.6
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Annals New York Academy of Sciences
The staining intensity of individual samples was also found t o vary with the two conjugates. Forty three of the 68 positive samples (70.5%) exhibited a similar intensity of fluorescence with both conjugates. Nine (13.2%) showed decreased staining, and 16 (23.5%) showed an increase intensity of staining with anti-Fab conjugate. T h e enhanced staining with anti-Fab conjugate was not related to the titer of antibodies present. Seven of the 16 antibodies that gave increased fluorescence with anti-Fab conjugate were examined with immunospecific conjugates t o determine the immunoglobulin class of antibody; all contained IgM antibodies exclusively. It can be seen from the Table that the distribution of negatives and the variation in staining intensity are not restricted t o a single autoantibody system. DISCUSSION The detection of tissue-reactive antibodies by indirect immunofluorescence procedures that employ conjugates directed against whole IgG or enzyme digestion products of IgG, i.e., Fab or F(ab‘)* fragments, might b e expected to produce similar results, because both react against antigens common to the major classes of immunoglobulin. Our preliminary results show a surprisingly low level of agreement between the results obtained with the two conjugates. These differences may be due t o qualitative and quantitative differences in the spectrum of antibodies present in the conjugates; the selection of the working dilutions of the conjugates by chessboard titrations with antibodies of defined immunoglobulin class should eliminate effects due to variation in the potency of these polyspecific conjugates for IgG o r IgM antibodies. It is conceivable, however, that configuration changes that occur in the region of the antibody-combining site subsequent t o interaction with antigen may make some individual antibodies, particularly of IgG class, less amenable t o detection with anti-Fab than anti-immunoglobulin conjugate, because the latter contain anti-Fc activity also. Configuration changes might be expected to present fewer difficulties in the detection of IgM antibodies because of the increased number of “unaltered” sites available for combination. In view of this unexpectedly low level of agreement between the results obtained with these conjugates, we feel that it is important t o confirm these data and extend this study b y screening additional sera with both conjugates t o establish which conjugate is more suitable for use in these procedures.
