CLiNICAL
IMMUNOLOGY
AND
iMMUNOPATHOLOCY
7, 176-186 (1977)
Experimental Studies on the Mechanism of Induction of Anti-Nuclear Antibodies by Procainamide’ EUGENE F. GOLD,~ SHLOMO BEN-EFRAIM,~ A. FAIVISEWITZ,~ Z. STEINER,~ AND ABRAHAM KLAJMAN~ Wepartment of Human Microbiology, Sackler School of Medicine, Tel-Aviv University. Tel-Aviv, Israel, and 3Medical Department B and Immunological Laboratory, Meir Hospital, K&r-Saba. lsrael Received Apt4 7, 1976 The occurrence of anti-nuclear factor (ANF) following immunization with procainamide (PrA) conjugates, reactions in vitro between PrA and nuclear components, and the effect of PrA on the immune response to soluble nucleoprotein (SNP) and DNA were examined. Conjugates of PrA with protein elicited formation of antibodies towards PrA in rabbits and guinea pigs without the appearance of ANF even after prolonged immunization. PrA reacted with SNP by forming a precipitate containing PrA and nondissociated SNP. DNA alone or histone was not precipitated by PrA. PrA also bound to isolated mouse liver nuclei. Aggregates of PrA with SNP and a conjugate of PrA with DNA elicited an immune response to both PrA and DNA in rabbits and guinea pigs. A conjugate of azobenzenearsouate prepared in a similar way induced an immune response against the hapten only, without development of anti-DNA antibodies.
INTRODUCTION
A variety of drugs can induce the formation of anti-nuclear antibodies in patients treated for prolonged periods. Among these drugs are isoniazid (I), hydralazine (2), hydantoin (3), chlorpromazine (4) and procainamide (5-9). In our experience, the administration of procainamide (R-A) induced production of antinuclear antibodies in approximately 85% of the treated patients (9). Several hypotheses have been offered to explain the etiology of the induction of anti-nuclear antibodies by drugs treatment: (a) activation of an inherited lupus diathesis by the drug (10); (b) activiation of a latent viral infection (11); (c) the immune reaction to the drug itself is accompanied by the induction of antibodies to DNA (12, 13); and (d) interaction of drugs with nuclear components (H-17) leading to an increase in the ~munogenicity of nuclear antigens. The hypothesis of an immune response to the drugs as responsible for elicitation of anti-DNA antibodies has been put forward in the case of hydralazine for treated human patients (12) and for rabbits immunized with hydralazine-human serum albumin (13). It was claimed in the latter case that antibodies to DNA were cross-reactive with hydralazine, thus suggesting the presence of a determinant common to the drug and DNA (13). Interaction between drugs and nuclei components were reported for hydralazine, and this could alter the viscosity of either nucleoprotein (14) or DNA (17): chlorpromazine reacts with DNA in the presence
1 This study was supported by a grant from the Chief Scientist Offtee, Ministry of Health, Israel. 176 Copyright All rights
Q 1977 by Academic Press. Inc. of reproduction in any form reserved.
KG-4 owe-I??9
PRO~AINAMIDE
AND
ANTI-DNA
RESPONSE
177
of ultraviolet light (18) and procainamide can bind to native and single-stranded DNA (17) or form a photochemical complex with DNA (15). The aim of the present work was to investigate two possibilities for explaining the induction of anti-nuclear antibodies by procdnamide: (a) induction of antinuclear antibodies as a result of the immune response to procainamide, and (b) increase in the immunogenicity of nuclear components resulting from an interaction between procainamide and nuclear components. The results we obtained indicate that the second possibility is more likely to occur. MATERIALS
AND METHODS
A~~~uZ~.Rabbits, 2-2.5 kg, ~ndo~y bred Hartley guinea pigs, 300-350 g, and female BALBlc mice, 8-12 weeks old, were obtained from a local breeding colony. Compounds and preparations. Procainamide-HCl (PrA) was purchased from Squibb Laboratories. Tritium-labeled F’rA (PrA*, 163 mCi/mmol) and tritiumlabeled aniline (An*, 22.5 Ci/mmol) were obtained from the Nuclear Research Centre, Beer-Sheba, Israel. Calf thymus native DNA (nDNA) was obtained from Worthington Biochemical Corporation and calf thymus histone (HIS) from N.B.C. Denatured DNA (dDNA) was prepared by heating nDNA in a water bath at 100°C for 10 min and subsequent rapid cooling in an ice bath. Human serum albumin fraction V @ISA) and bovine serum albumin fraction V (BSA) were obtained from Si8ma. Rabbit serum ~bumin (RSA) was purchased from Marm Laboratories. p-Arsanilic acid (Fluka, AG) was used in addition to PrA for prep aration of conjugates. Conjugates of PrA with proteins (PrA-BSA, PrA-RSA, PrA-HSA, PrA-HIS) and of azobenzenearsonate(ARS-HSA and ARS-BSA) were prepared by diazotation (19). The same method was also used for preparation of either PrA- or ARS-DNA conjugates. Diazotized ARS or PrA (0.37 M) was added to 100 mg of dDNA in N/25 NaOH. The mixtures were placed in the cold overnight. Afterwards the pH was adjusted to 7-7.5, and the product was dialyzed against water prior to lyophilization. Spectral readings of the samplesusing dDNA as a blank had an absorbance peak at 460 nm, indicating the presence of the diazo bond. This is compatible with electrophilic substitution of the diazo bond with the p~midine cytosine at position 5 (20). For the preparation of lightly labeled ARS-DNA, one-tenth the mount of arsanilic acid was used. The ARS-DNA and PrA-DNA conjugates were shown to be protected from enzymatic hydrolysis by DNase. The method used was based on change in OD at 260 nm. Coupling of either PrA or ARS with sheep red blood cells (RrA-SRBC or ARS-SRBC) was also performed by diazotization (19). Soluble nucleoprotein (SNP) was prepared in the cold from fi-esh calf thymus gland as described (21). Briefly, this consists of the homogenization of calfthymus gland in saline-Versene buffer, pH 8.6, containing a small amount of octanol to prevent foaming, followed by a series of six cycles of resuspension of the homogenized centrifuged sediment. The final sediment (chromatin material) was homogenized in water, stirred for one hour and dialyzed extensively against 0.7 m&f potassium phosphate buffer, pH 6.8. Fo~owi~ dialysis, any nucleoprotein in the form of gel particles was removed by ultracen~gation for 30 min at 10,000g.
178
GOLD
ET AL
SNP was precipitated by 0.15 M NaCl. The precipitate was dissolved in buffer, pH 6.8, and dialyzed versus the same buffer prior to lyophilization. The lyophilized preparation was stored in the cold. The average yield was 40 mg of SNP from 20 g of calf thymus gland. The lyophilized material was soluble at 1.O mg/ml in low ionic strength, 0.7 mM buffer (21). The DNA content of the SNP was determined by absorption at 260 nm and also by the diphenylamine method (22). The A26,jA28,sratio of SNP was 1.6 to 1.8. The spectral curve of SNP showed elevated adsorption at 230 nm, a peak at 260 nm, and little or no adsorption at 320 nm. No peak at 280 nm was discernible. The protein content of SNP was also determined by the method of Lowry et al. (23) and found to be approximately 40-60s dry weight. Immunizations. Rabbits were immunized with either PrA-RSA, PrA-BSA, ARS-HSA, PrA-DNA, PrA-HIS, PI-A-SNP aggregate (for preparation, see Results). or SNP alone. At least two animals were taken for each compound. Groups of 10 guinea pigs each, were immunized with either PrA-RSA, ARS-HSA, PrA-DNA, or ARS-DNA. In all cases the immunizing compound was emulsified at a ratio of 1: 1 in Freund’s complete adjuvant (FCA, Difco). The schedule of immunizations is given in Results. Skin tests. Concentrations of 10 and 20 pug of the conjugate used for immunization were injected intradermally in guinea pigs at day 13 after immunization for detection of delayed-type reactions. The readings of the skin reactions were made at 4 and 24 hr after the intradermal injections. Reactions at 24 hr with erythema having a diameter of more than 10 mm and with marked induration were considered positive. Anti-nuclearfactor. Anti-nuclear factor was determined by the immunofluorescence technique reported previously (7). Hemagglutination tests. SRBC were sensitized for hemagglutination (HA) in different ways according to the compound used for sensitization: (a) tannic acid treatment with protein conjugates (24); (b) direct coupling of SRBC with either diazotized PrA or diazotized ARS (19); and (c) coupling of either SNP or dDNA to formalin-treated SRBC by chromium chloride (25). Quantities of 100 pg of SNP or dDNA/ml of packed erythrocytes were used. Because the stability and the solubility of SNP is ionic strength dependent (21), the coupling of SNP to SRBC was performed in 0.7 mM phosphate buffer, pH 6.8. The specificity of HA titers was shown by inhibition with the adequate compound as reported in results. Serial 0.5-ml dilutions of sera were preineubated for 1 hr at room temperature with O.l-ml quantities of the inhibitor. The effect of adding 0.1 M 2-mercaptoethanol (Sigma) on the HA titer was determined (26) as an indicator for the IgM nature of the antibodies. Quantitative precipitin test. The quantity of anti-PrA antibodies (milligrams per mililiter of serum) was determined by precipitation of a constant amount of nondiluted serum (0.5 ml) with various amounts of antigen (27). Radioimmunoassay for antibodies to DNA (28). The sera tested were heated at 56°C for 0.5 hr. Volumes of 0.3 ml of dDN.$ (1 mg/ml in PBS, pH 7.2) were placed in wells of plastic trays and kept at 4°C for 24 hr. The wells were wash.ed six times with PBS. Volumes of 0.5 ml of serial two-fold dilutions of sera (1:2 to 1:16) in PBS were placed in the wells. The trays were kept for 24 hr in the cold and
PRO~AI~AMID~
AND
ANTI-DNA
RESPONSE
179
afterwards washed six times with PBS. Native or denatured tritium-labeled DNA (DNA*, 100,000 cpm) was added in the amount of 0.3 ml per well and the contents placed for, another 24 hr in the cold. The trays were washed eight times with PBS. After drying, the plastic wells were cut out with scissors and placed into glass scintillation bottles. Ten milliliters of scintillation fluid [PPO, 4 g/liter, and POPOP, 0.2 g/liter (Packard) dissolved in toluene] were added and the samples counted for radioactivity (Tri-Carb, Packard). Sera taken from guinea pigs immunized with either PrA-DNA or ARS-DNA and from rabbits immunized, respectively, with HSA, ARS-HSA, PrA-DNA, PrA-SNP aggregate, and SNP alone were tested. Three kinds of controls were included in each experiment: without serum, with sera taken from rabbits or guinea pigs before immunization (normal sera), and with cold DNA only. Binding ofPrA fo ~~~~~~e~ ptuclei. Isolated nuclei (29) were prepared from livers of adult Balblc mice. The mice were bled before killing, and the livers were freed of red blood cells. The sample of isolated nuclei (120 mg dry weight of lyophilized material) was divided into three equal portions of 10 ml each of 0.25 M sucrose solution containing either PrA* (6 &i in 0.5 ml), An* (6 &i in 0.5 ml), or plain buffer. The samples were incubated for 30 min at room temperature. The treated nuclei samples were washed four times in buffer (supernatants S-I, S-II, S-III and S-IV), followed by exposure to 5% trichloroacetic acid (TCA-I and TCA-II). The sediment of treated nuclei was dissolved in 3.5 ml of a solution composed of 0.5 ml of concentrated formic acid + 3 ml of absolute ethanol. Radioactivity counts were performed in triplicate from the whole sediment (3.5 ml of dissolved sediment + 7 ml of scintillation fluid) and from the aqueous and TCA washings (0.2 ml of supernatant + 10 ml of scintillation fluid). RESULTS Immune Response to Hapten-Protein Conjugates As shown in Table 1, injection of either PrA- or ARS-protein conjugates induced a specific immune response towards the haptenic determinant. The occurrence of an immune response against PrA was also shown in quantitative precipitin tests (Fig. 1). Immuni~tion of guinea pigs with either PrA-RSA or ARS-HSA also induced skin reactions of the delayed type (Table 1). PrA alone or PrA-poly (Tyr) in FCA were nonimmunogenic. The specificity of the immune response towards PrA was proven by the occurrence of titers with heterologous PrA conjugates and by inhibition tests with,PrA alone. Thus, preincubation of sera with 20 pg of PrAIO.5 ml of serum dilution prevented hemagglutination almost completely: The titer against PrA-RSA of anti-PrA-RSA serum was reduced to 1: 160 instead of 1:5120, and the titer against PrA-RSA of anti-PrA-BSA serum was 1:80 instead of 1: 10,240. Injection of either PrA-RSA or PrA-BSA in rabbits and of PrA in guinea pigs did not elicit formation of anti-nuclear antibodies even after prolonged treatment. The immunofluorescence tests for ANF were performed before immunization and on various days after the beginning of the immuni~tion, after 1,2,3, and 4 im~u~zing injections, and were invariably negative.
180
GOLD
ET AL.
TABLE THE
IMMUNE
1
REsrnr-isE TO FROCAINAMIDE @A)PROTEIN CONJUGATES IN RABBITS
AND AND
AZOBENZENEARSONE GUINEA Fks
HA Species Rabbit
Immunizing antigen PrA-RSA”
PrA-BSAb ARS-HSAC Guinea pig
PRA-RSAd ARS-HSA* pr At RQ&GyrF
Test antigen PrA-BSA PrA-RSA BSA RSA PrA-SRBC FrA-RSA RSA ARS-BSA BSA PrA-SRBC ARS-SRBC PrA-RSA PrA-RSA
(ARS)-
.Titer
ANF
5,120
Negative (0, 17. 40, 47y
40 640
IMMUNOLOGY
AND
iMMUNOPATHOLOCY
7, 176-186 (1977)
Experimental Studies on the Mechanism of Induction of Anti-Nuclear Antibodies by Procainamide’ EUGENE F. GOLD,~ SHLOMO BEN-EFRAIM,~ A. FAIVISEWITZ,~ Z. STEINER,~ AND ABRAHAM KLAJMAN~ Wepartment of Human Microbiology, Sackler School of Medicine, Tel-Aviv University. Tel-Aviv, Israel, and 3Medical Department B and Immunological Laboratory, Meir Hospital, K&r-Saba. lsrael Received Apt4 7, 1976 The occurrence of anti-nuclear factor (ANF) following immunization with procainamide (PrA) conjugates, reactions in vitro between PrA and nuclear components, and the effect of PrA on the immune response to soluble nucleoprotein (SNP) and DNA were examined. Conjugates of PrA with protein elicited formation of antibodies towards PrA in rabbits and guinea pigs without the appearance of ANF even after prolonged immunization. PrA reacted with SNP by forming a precipitate containing PrA and nondissociated SNP. DNA alone or histone was not precipitated by PrA. PrA also bound to isolated mouse liver nuclei. Aggregates of PrA with SNP and a conjugate of PrA with DNA elicited an immune response to both PrA and DNA in rabbits and guinea pigs. A conjugate of azobenzenearsouate prepared in a similar way induced an immune response against the hapten only, without development of anti-DNA antibodies.
INTRODUCTION
A variety of drugs can induce the formation of anti-nuclear antibodies in patients treated for prolonged periods. Among these drugs are isoniazid (I), hydralazine (2), hydantoin (3), chlorpromazine (4) and procainamide (5-9). In our experience, the administration of procainamide (R-A) induced production of antinuclear antibodies in approximately 85% of the treated patients (9). Several hypotheses have been offered to explain the etiology of the induction of anti-nuclear antibodies by drugs treatment: (a) activation of an inherited lupus diathesis by the drug (10); (b) activiation of a latent viral infection (11); (c) the immune reaction to the drug itself is accompanied by the induction of antibodies to DNA (12, 13); and (d) interaction of drugs with nuclear components (H-17) leading to an increase in the ~munogenicity of nuclear antigens. The hypothesis of an immune response to the drugs as responsible for elicitation of anti-DNA antibodies has been put forward in the case of hydralazine for treated human patients (12) and for rabbits immunized with hydralazine-human serum albumin (13). It was claimed in the latter case that antibodies to DNA were cross-reactive with hydralazine, thus suggesting the presence of a determinant common to the drug and DNA (13). Interaction between drugs and nuclei components were reported for hydralazine, and this could alter the viscosity of either nucleoprotein (14) or DNA (17): chlorpromazine reacts with DNA in the presence
1 This study was supported by a grant from the Chief Scientist Offtee, Ministry of Health, Israel. 176 Copyright All rights
Q 1977 by Academic Press. Inc. of reproduction in any form reserved.
KG-4 owe-I??9
PRO~AINAMIDE
AND
ANTI-DNA
RESPONSE
177
of ultraviolet light (18) and procainamide can bind to native and single-stranded DNA (17) or form a photochemical complex with DNA (15). The aim of the present work was to investigate two possibilities for explaining the induction of anti-nuclear antibodies by procdnamide: (a) induction of antinuclear antibodies as a result of the immune response to procainamide, and (b) increase in the immunogenicity of nuclear components resulting from an interaction between procainamide and nuclear components. The results we obtained indicate that the second possibility is more likely to occur. MATERIALS
AND METHODS
A~~~uZ~.Rabbits, 2-2.5 kg, ~ndo~y bred Hartley guinea pigs, 300-350 g, and female BALBlc mice, 8-12 weeks old, were obtained from a local breeding colony. Compounds and preparations. Procainamide-HCl (PrA) was purchased from Squibb Laboratories. Tritium-labeled F’rA (PrA*, 163 mCi/mmol) and tritiumlabeled aniline (An*, 22.5 Ci/mmol) were obtained from the Nuclear Research Centre, Beer-Sheba, Israel. Calf thymus native DNA (nDNA) was obtained from Worthington Biochemical Corporation and calf thymus histone (HIS) from N.B.C. Denatured DNA (dDNA) was prepared by heating nDNA in a water bath at 100°C for 10 min and subsequent rapid cooling in an ice bath. Human serum albumin fraction V @ISA) and bovine serum albumin fraction V (BSA) were obtained from Si8ma. Rabbit serum ~bumin (RSA) was purchased from Marm Laboratories. p-Arsanilic acid (Fluka, AG) was used in addition to PrA for prep aration of conjugates. Conjugates of PrA with proteins (PrA-BSA, PrA-RSA, PrA-HSA, PrA-HIS) and of azobenzenearsonate(ARS-HSA and ARS-BSA) were prepared by diazotation (19). The same method was also used for preparation of either PrA- or ARS-DNA conjugates. Diazotized ARS or PrA (0.37 M) was added to 100 mg of dDNA in N/25 NaOH. The mixtures were placed in the cold overnight. Afterwards the pH was adjusted to 7-7.5, and the product was dialyzed against water prior to lyophilization. Spectral readings of the samplesusing dDNA as a blank had an absorbance peak at 460 nm, indicating the presence of the diazo bond. This is compatible with electrophilic substitution of the diazo bond with the p~midine cytosine at position 5 (20). For the preparation of lightly labeled ARS-DNA, one-tenth the mount of arsanilic acid was used. The ARS-DNA and PrA-DNA conjugates were shown to be protected from enzymatic hydrolysis by DNase. The method used was based on change in OD at 260 nm. Coupling of either PrA or ARS with sheep red blood cells (RrA-SRBC or ARS-SRBC) was also performed by diazotization (19). Soluble nucleoprotein (SNP) was prepared in the cold from fi-esh calf thymus gland as described (21). Briefly, this consists of the homogenization of calfthymus gland in saline-Versene buffer, pH 8.6, containing a small amount of octanol to prevent foaming, followed by a series of six cycles of resuspension of the homogenized centrifuged sediment. The final sediment (chromatin material) was homogenized in water, stirred for one hour and dialyzed extensively against 0.7 m&f potassium phosphate buffer, pH 6.8. Fo~owi~ dialysis, any nucleoprotein in the form of gel particles was removed by ultracen~gation for 30 min at 10,000g.
178
GOLD
ET AL
SNP was precipitated by 0.15 M NaCl. The precipitate was dissolved in buffer, pH 6.8, and dialyzed versus the same buffer prior to lyophilization. The lyophilized preparation was stored in the cold. The average yield was 40 mg of SNP from 20 g of calf thymus gland. The lyophilized material was soluble at 1.O mg/ml in low ionic strength, 0.7 mM buffer (21). The DNA content of the SNP was determined by absorption at 260 nm and also by the diphenylamine method (22). The A26,jA28,sratio of SNP was 1.6 to 1.8. The spectral curve of SNP showed elevated adsorption at 230 nm, a peak at 260 nm, and little or no adsorption at 320 nm. No peak at 280 nm was discernible. The protein content of SNP was also determined by the method of Lowry et al. (23) and found to be approximately 40-60s dry weight. Immunizations. Rabbits were immunized with either PrA-RSA, PrA-BSA, ARS-HSA, PrA-DNA, PrA-HIS, PI-A-SNP aggregate (for preparation, see Results). or SNP alone. At least two animals were taken for each compound. Groups of 10 guinea pigs each, were immunized with either PrA-RSA, ARS-HSA, PrA-DNA, or ARS-DNA. In all cases the immunizing compound was emulsified at a ratio of 1: 1 in Freund’s complete adjuvant (FCA, Difco). The schedule of immunizations is given in Results. Skin tests. Concentrations of 10 and 20 pug of the conjugate used for immunization were injected intradermally in guinea pigs at day 13 after immunization for detection of delayed-type reactions. The readings of the skin reactions were made at 4 and 24 hr after the intradermal injections. Reactions at 24 hr with erythema having a diameter of more than 10 mm and with marked induration were considered positive. Anti-nuclearfactor. Anti-nuclear factor was determined by the immunofluorescence technique reported previously (7). Hemagglutination tests. SRBC were sensitized for hemagglutination (HA) in different ways according to the compound used for sensitization: (a) tannic acid treatment with protein conjugates (24); (b) direct coupling of SRBC with either diazotized PrA or diazotized ARS (19); and (c) coupling of either SNP or dDNA to formalin-treated SRBC by chromium chloride (25). Quantities of 100 pg of SNP or dDNA/ml of packed erythrocytes were used. Because the stability and the solubility of SNP is ionic strength dependent (21), the coupling of SNP to SRBC was performed in 0.7 mM phosphate buffer, pH 6.8. The specificity of HA titers was shown by inhibition with the adequate compound as reported in results. Serial 0.5-ml dilutions of sera were preineubated for 1 hr at room temperature with O.l-ml quantities of the inhibitor. The effect of adding 0.1 M 2-mercaptoethanol (Sigma) on the HA titer was determined (26) as an indicator for the IgM nature of the antibodies. Quantitative precipitin test. The quantity of anti-PrA antibodies (milligrams per mililiter of serum) was determined by precipitation of a constant amount of nondiluted serum (0.5 ml) with various amounts of antigen (27). Radioimmunoassay for antibodies to DNA (28). The sera tested were heated at 56°C for 0.5 hr. Volumes of 0.3 ml of dDN.$ (1 mg/ml in PBS, pH 7.2) were placed in wells of plastic trays and kept at 4°C for 24 hr. The wells were wash.ed six times with PBS. Volumes of 0.5 ml of serial two-fold dilutions of sera (1:2 to 1:16) in PBS were placed in the wells. The trays were kept for 24 hr in the cold and
PRO~AI~AMID~
AND
ANTI-DNA
RESPONSE
179
afterwards washed six times with PBS. Native or denatured tritium-labeled DNA (DNA*, 100,000 cpm) was added in the amount of 0.3 ml per well and the contents placed for, another 24 hr in the cold. The trays were washed eight times with PBS. After drying, the plastic wells were cut out with scissors and placed into glass scintillation bottles. Ten milliliters of scintillation fluid [PPO, 4 g/liter, and POPOP, 0.2 g/liter (Packard) dissolved in toluene] were added and the samples counted for radioactivity (Tri-Carb, Packard). Sera taken from guinea pigs immunized with either PrA-DNA or ARS-DNA and from rabbits immunized, respectively, with HSA, ARS-HSA, PrA-DNA, PrA-SNP aggregate, and SNP alone were tested. Three kinds of controls were included in each experiment: without serum, with sera taken from rabbits or guinea pigs before immunization (normal sera), and with cold DNA only. Binding ofPrA fo ~~~~~~e~ ptuclei. Isolated nuclei (29) were prepared from livers of adult Balblc mice. The mice were bled before killing, and the livers were freed of red blood cells. The sample of isolated nuclei (120 mg dry weight of lyophilized material) was divided into three equal portions of 10 ml each of 0.25 M sucrose solution containing either PrA* (6 &i in 0.5 ml), An* (6 &i in 0.5 ml), or plain buffer. The samples were incubated for 30 min at room temperature. The treated nuclei samples were washed four times in buffer (supernatants S-I, S-II, S-III and S-IV), followed by exposure to 5% trichloroacetic acid (TCA-I and TCA-II). The sediment of treated nuclei was dissolved in 3.5 ml of a solution composed of 0.5 ml of concentrated formic acid + 3 ml of absolute ethanol. Radioactivity counts were performed in triplicate from the whole sediment (3.5 ml of dissolved sediment + 7 ml of scintillation fluid) and from the aqueous and TCA washings (0.2 ml of supernatant + 10 ml of scintillation fluid). RESULTS Immune Response to Hapten-Protein Conjugates As shown in Table 1, injection of either PrA- or ARS-protein conjugates induced a specific immune response towards the haptenic determinant. The occurrence of an immune response against PrA was also shown in quantitative precipitin tests (Fig. 1). Immuni~tion of guinea pigs with either PrA-RSA or ARS-HSA also induced skin reactions of the delayed type (Table 1). PrA alone or PrA-poly (Tyr) in FCA were nonimmunogenic. The specificity of the immune response towards PrA was proven by the occurrence of titers with heterologous PrA conjugates and by inhibition tests with,PrA alone. Thus, preincubation of sera with 20 pg of PrAIO.5 ml of serum dilution prevented hemagglutination almost completely: The titer against PrA-RSA of anti-PrA-RSA serum was reduced to 1: 160 instead of 1:5120, and the titer against PrA-RSA of anti-PrA-BSA serum was 1:80 instead of 1: 10,240. Injection of either PrA-RSA or PrA-BSA in rabbits and of PrA in guinea pigs did not elicit formation of anti-nuclear antibodies even after prolonged treatment. The immunofluorescence tests for ANF were performed before immunization and on various days after the beginning of the immuni~tion, after 1,2,3, and 4 im~u~zing injections, and were invariably negative.
180
GOLD
ET AL.
TABLE THE
IMMUNE
1
REsrnr-isE TO FROCAINAMIDE @A)PROTEIN CONJUGATES IN RABBITS
AND AND
AZOBENZENEARSONE GUINEA Fks
HA Species Rabbit
Immunizing antigen PrA-RSA”
PrA-BSAb ARS-HSAC Guinea pig
PRA-RSAd ARS-HSA* pr At RQ&GyrF
Test antigen PrA-BSA PrA-RSA BSA RSA PrA-SRBC FrA-RSA RSA ARS-BSA BSA PrA-SRBC ARS-SRBC PrA-RSA PrA-RSA
(ARS)-
.Titer
ANF
5,120
Negative (0, 17. 40, 47y
40 640
