Vol. 1, No. 2 Printed in U.S.A.
JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1975, p. 132-135 Copyright © 1975 American Society for Microbiology
Rapid Method to Detect Rubella Immunoglobulin M and Immunoglobulin A Antibodies H. SCHMITZ,* H. SHIMIZU, D. KAMPA, AND H. W. DOERR Hygiene-Institut, D-78 Freiburg, West Germany
Received for publication 18 November 1974
Immunoglobulin (Ig) G was removed from serum specimens by precipitation with gamma chain-specific anti-human IgG of rabbit origin. The remaining rubella virus-specific IgM (and IgA) antibodies were then detected by the rubella hemagglutination-inhibition test. This procedure has proven to be as reliable as estimations carried out with IgM fractions separated on a sucrose density gradient.
The detection of immunoglobulin (Ig) M antibodies to rubella virus has special diagnostic significance in rubella infections of pregnancy. Several methods for the detection and quantitation of these virus-specific antibodies are available (2, 3, 4, 11). In our laboratory we usually look for rubella IgM antibodies in the IgM fractions isolated from a sucrose gradient (11). However, the separation of the IgM antibodies by means of ultracentrifugation or chromatographic methods is too laborious to be used as a routine test. Therefore, a simpler method for the detection of rubella IgM is needed. Serological studies on rubella patients by Biirgin-Wolff et al. (2) and Cradock-Watson et al. (3), as well as our own studies on antibodies to cytomegalovirus and Epstein-Barr virus (7, 8), have shown that the kinetics of virus-specific IgM and IgA production is almost identical: both immunoglobulin titers decline very rapidly after onset of the disease and can no longer be detected a few weeks later. The presence of these antibodies in a serum specimen indicates a recent infection. Only the IgG antibodies persist over years. Therefore, a residual rubella antibody titer after removal of the IgG will be caused by virusspecific IgM and/or IgA. IgE or IgD antibodies probably do not play a role because of the very low serum concentrations. To remove the IgG antibodies rapidly and specifically from sera, we precipitated the IgG with anti-human IgG (gamma chain specific) antibodies of rabbit origin. MATERIALS AND METHODS Antisera. Rabbits were immunized subcutaneously three times with 0.5, 1, and 5 mg of highly purified human IgG at 2-week intervals. The IgG was 132
prepared by chromatography using QAE Sephadex A50 (Pharmacia, Upsala) (5). Purity of the IgG was demonstrated by polyacrylamide gel electrophoresis. After immunization, antibodies to the light chains were removed from the rabbit antiserum by precipitation with human IgM. The IgG fraction of the antiserum, obtained by chromatography using QAE Sephadex A,0, was dialyzed against 0.01 M tris(hydroxymethyl)aminomethane (Tris) plus 0.1 M NaCi (Tris-NaCl, pH 7) and concentrated to 3 mg of total protein per ml which precipitated 0.35 mg of human IgG/ml (solution A). The gamma chain specificity of the absorbed antiserum had been shown by negative results in immunofluorescence using cytomegalovirus IgM antibody fractions from patients and fluorescein-labeled anti-rabbit globulin (7). Isolation of specific anti-gamma chain antibodies. Anti-gamma chain antibodies were isolated from the anti-gamma chain antiserum using gamma globulin (Behring, Marburg) coupled to a BrCN-sepharose as immunoadsorbent (Pharmacia, Upsala) according to the method of Axen et al. (1). The anti-gamma chain antibody fractions were dialyzed against Tris-NaCl and concentrated by ultrafiltration. The final solution (B) contained 1 mg of total protein per ml and precipitated 0.35 mg of human IgG/ml (see 4). Precipitation of the IgG in serum specimens. Since solution B is difficult to prepare and since the high antibody concentration of B is not needed for precipitation, appropriate amounts of solution A were mixed with solution B to obtain a precipitation solution (PS) which contained about 2.3 mg of total protein per ml and precipitated 0.35 mg of human IgG/ml. PS (0.4 ml) was used to precipitate about 0.03 to 0.14 mg of human IgG. This amount of IgG is to be expected in 0.025 ml of serum diluted 1:4 by heparinMnCl2 treatment. The individual steps of the precipitation can be seen in Fig. 1: 0.025 ml of serum (diluted 1:4) was placed in a small plastic tube (microtubes, Eppendorf, Hamburg) using a micropipette (Eppendorf, Hamburg) and 0.4 ml of PS was added. After mixing and incubating at 20 C for 10 min, the tubes were
VOL. 1, 1975
RUBELLA IGM AND IGA ANTIBODIES
0.025 ml serum (diluted 1:4) +
0.40 ml PS I incubation (10 min)
centrifugation (2 min, 7,000 x g) 0.40 ml supernatant +
150 mg (NH4)2SO4 I incubation (5 min)
centrifugation (2 min, 7,000
x
g)
sedimented protein is dissolved in 0.05 ml Tris-NaCl (pH 8)
I
HI test FIG. 1. Precipitation of IgG. The method is carried out in small plastic tubes (microtubes, Eppendorf).
centrifuged (Eppendorf-Centrifuge, Hamburg; 7,000 x g) and the supernatant fluid was pipetted into another tube which contained 150 mg of (NH4)2SO4, giving an overall saturation of 50%. The sealed tubes were thoroughly shaken, and after 5 min incubation and centrifugation (7,000 x g, 5 min), the supernatant fluid was carefully removed. The sediment consisted mainly of rabbit IgG (about 80%). The rest was human albumin IgM and IgA. The final amount of protein (and the solubility of the protein in a small volume of buffer) therefore depends upon the amount of nonspecific rabbit IgG, which remains in the sediment after (NH4)2SO4 precipitation. The amount of nonspecific rabbit IgG should be kept low, i.e., the total rabbit IgG of the PS should not exceed by more than five to seven times the amount of the precipitated human IgG. If this is the case, the protein sedimented by (NH4)2SO4 precipitation can be dissolved in 0.05 ml Tris-NaCl (pH 8). This corresponds to a final dilution of the IgM and IgA antibodies of 1:8
133
the IgG, IgA, and IgM concentrations in a serum specimen, the appropriate Tri-, S-, and LC-Partigen immunodiffusion plates (Behring, Marburg) were used. The LC-plates detect minimum concentrations of at least 0.01 mg of IgG per ml. Hemagglutination inhibition (HI) test. The HI test was carried out according to the Center for Disease Control standard procedure (10). Bovine serum albumin was added in a concentration of 2 mg/ml to the 0.25% chicken erythrocyte suspension to improve the agglutination. Separation of the IgM antibodies. The separation of IgM antibodies by sucrose gradient centrifugation has been described elsewhere (7, 11). Sera examined. Seven sera were obtained from adults with clinical rubella. In three of these cases, pregnancy was additionally diagnosed. One serum specimen was taken from a case of congenital rubella. Two sera showing high titers in the HI test (2,048) were obtained from individuals who had had a rubella infection 6 to 12 months previously. Additionally, 20 sera from blood donors with a rubella antibody titer in the HI test of > 256 were included.
RESULTS The question of what concentrations of IgM, IgA, and IgG antibodies could still be detected in the serum samples after precipitation with PS was of particular significance. One problem was co-precipitation. IgA and IgM antibodies are co-precipitated, particularly in small reaction volumes, if the concentration of the IgGanti-IgG precipitates becomes so high that in the subsequent centrifugation a considerable cosedimentation of IgM and IgA molecules can occur. On the other hand, with excessive volumes (under our conditions more than 0.7 ml total volume) IgG was not completely precipito 1:10. Quantitation of the anti-IgG titer. The amount of tated. To find the optimal conditions for precipIgG (milligrams) which was precipitated during 10 itation, we varied the total volume while the min at 20 C by 1 ml of anti-human IgG globulin amount of anti-gamma chain antibody and (anti-IgG titer) was determined according to the human serum remained constant. The greatest method of Sewell (9). To simplify the estimations of possible degree of precipitation of IgG antithe anti-IgG titer, we additionally performed a single bodies without too great loss of IgM and IgA radial immunodiffusion test (6): gamma globulin was found at a total volume of 0.4 ml. The im(Behring, Marburg) was mixed with melted agarose at munoglobulin concentrations before and after 56 C (final concentration 0.01 mg/ml, 2% agarose). serum (diFifteen milliliters of the mixture was poured into a precipitation of 0.025 ml of human petri dish (10-cm diameter). Two-millimeter holes luted 1:4) with 0.4 ml of PS are shown in Table 1. After precipitation, the IgG content in the were made in the agarose and 0.01 ml of an unknown anti-IgG, or anti-gamma chain antiserum, was placed serum was reduced to less than 0.3% of the in each hole. The diameters of the precipitation rings initial concentration. On the other hand, 64% of were measured 48 h later. The anti-human IgG titer the IgM and 76% of the IgA were still present. (T) of different dilutions (T = 0.04, 0.08 ... 0.8 mg of Assuming an equal reduction of all IgM and IgA precipitated IgG/ml of a standard antibody solution) antibodies, a rubella HI titer based on IgG determined by the method of Sewell was plotted antibodies must be reduced from 1,024 to
JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1975, p. 132-135 Copyright © 1975 American Society for Microbiology
Rapid Method to Detect Rubella Immunoglobulin M and Immunoglobulin A Antibodies H. SCHMITZ,* H. SHIMIZU, D. KAMPA, AND H. W. DOERR Hygiene-Institut, D-78 Freiburg, West Germany
Received for publication 18 November 1974
Immunoglobulin (Ig) G was removed from serum specimens by precipitation with gamma chain-specific anti-human IgG of rabbit origin. The remaining rubella virus-specific IgM (and IgA) antibodies were then detected by the rubella hemagglutination-inhibition test. This procedure has proven to be as reliable as estimations carried out with IgM fractions separated on a sucrose density gradient.
The detection of immunoglobulin (Ig) M antibodies to rubella virus has special diagnostic significance in rubella infections of pregnancy. Several methods for the detection and quantitation of these virus-specific antibodies are available (2, 3, 4, 11). In our laboratory we usually look for rubella IgM antibodies in the IgM fractions isolated from a sucrose gradient (11). However, the separation of the IgM antibodies by means of ultracentrifugation or chromatographic methods is too laborious to be used as a routine test. Therefore, a simpler method for the detection of rubella IgM is needed. Serological studies on rubella patients by Biirgin-Wolff et al. (2) and Cradock-Watson et al. (3), as well as our own studies on antibodies to cytomegalovirus and Epstein-Barr virus (7, 8), have shown that the kinetics of virus-specific IgM and IgA production is almost identical: both immunoglobulin titers decline very rapidly after onset of the disease and can no longer be detected a few weeks later. The presence of these antibodies in a serum specimen indicates a recent infection. Only the IgG antibodies persist over years. Therefore, a residual rubella antibody titer after removal of the IgG will be caused by virusspecific IgM and/or IgA. IgE or IgD antibodies probably do not play a role because of the very low serum concentrations. To remove the IgG antibodies rapidly and specifically from sera, we precipitated the IgG with anti-human IgG (gamma chain specific) antibodies of rabbit origin. MATERIALS AND METHODS Antisera. Rabbits were immunized subcutaneously three times with 0.5, 1, and 5 mg of highly purified human IgG at 2-week intervals. The IgG was 132
prepared by chromatography using QAE Sephadex A50 (Pharmacia, Upsala) (5). Purity of the IgG was demonstrated by polyacrylamide gel electrophoresis. After immunization, antibodies to the light chains were removed from the rabbit antiserum by precipitation with human IgM. The IgG fraction of the antiserum, obtained by chromatography using QAE Sephadex A,0, was dialyzed against 0.01 M tris(hydroxymethyl)aminomethane (Tris) plus 0.1 M NaCi (Tris-NaCl, pH 7) and concentrated to 3 mg of total protein per ml which precipitated 0.35 mg of human IgG/ml (solution A). The gamma chain specificity of the absorbed antiserum had been shown by negative results in immunofluorescence using cytomegalovirus IgM antibody fractions from patients and fluorescein-labeled anti-rabbit globulin (7). Isolation of specific anti-gamma chain antibodies. Anti-gamma chain antibodies were isolated from the anti-gamma chain antiserum using gamma globulin (Behring, Marburg) coupled to a BrCN-sepharose as immunoadsorbent (Pharmacia, Upsala) according to the method of Axen et al. (1). The anti-gamma chain antibody fractions were dialyzed against Tris-NaCl and concentrated by ultrafiltration. The final solution (B) contained 1 mg of total protein per ml and precipitated 0.35 mg of human IgG/ml (see 4). Precipitation of the IgG in serum specimens. Since solution B is difficult to prepare and since the high antibody concentration of B is not needed for precipitation, appropriate amounts of solution A were mixed with solution B to obtain a precipitation solution (PS) which contained about 2.3 mg of total protein per ml and precipitated 0.35 mg of human IgG/ml. PS (0.4 ml) was used to precipitate about 0.03 to 0.14 mg of human IgG. This amount of IgG is to be expected in 0.025 ml of serum diluted 1:4 by heparinMnCl2 treatment. The individual steps of the precipitation can be seen in Fig. 1: 0.025 ml of serum (diluted 1:4) was placed in a small plastic tube (microtubes, Eppendorf, Hamburg) using a micropipette (Eppendorf, Hamburg) and 0.4 ml of PS was added. After mixing and incubating at 20 C for 10 min, the tubes were
VOL. 1, 1975
RUBELLA IGM AND IGA ANTIBODIES
0.025 ml serum (diluted 1:4) +
0.40 ml PS I incubation (10 min)
centrifugation (2 min, 7,000 x g) 0.40 ml supernatant +
150 mg (NH4)2SO4 I incubation (5 min)
centrifugation (2 min, 7,000
x
g)
sedimented protein is dissolved in 0.05 ml Tris-NaCl (pH 8)
I
HI test FIG. 1. Precipitation of IgG. The method is carried out in small plastic tubes (microtubes, Eppendorf).
centrifuged (Eppendorf-Centrifuge, Hamburg; 7,000 x g) and the supernatant fluid was pipetted into another tube which contained 150 mg of (NH4)2SO4, giving an overall saturation of 50%. The sealed tubes were thoroughly shaken, and after 5 min incubation and centrifugation (7,000 x g, 5 min), the supernatant fluid was carefully removed. The sediment consisted mainly of rabbit IgG (about 80%). The rest was human albumin IgM and IgA. The final amount of protein (and the solubility of the protein in a small volume of buffer) therefore depends upon the amount of nonspecific rabbit IgG, which remains in the sediment after (NH4)2SO4 precipitation. The amount of nonspecific rabbit IgG should be kept low, i.e., the total rabbit IgG of the PS should not exceed by more than five to seven times the amount of the precipitated human IgG. If this is the case, the protein sedimented by (NH4)2SO4 precipitation can be dissolved in 0.05 ml Tris-NaCl (pH 8). This corresponds to a final dilution of the IgM and IgA antibodies of 1:8
133
the IgG, IgA, and IgM concentrations in a serum specimen, the appropriate Tri-, S-, and LC-Partigen immunodiffusion plates (Behring, Marburg) were used. The LC-plates detect minimum concentrations of at least 0.01 mg of IgG per ml. Hemagglutination inhibition (HI) test. The HI test was carried out according to the Center for Disease Control standard procedure (10). Bovine serum albumin was added in a concentration of 2 mg/ml to the 0.25% chicken erythrocyte suspension to improve the agglutination. Separation of the IgM antibodies. The separation of IgM antibodies by sucrose gradient centrifugation has been described elsewhere (7, 11). Sera examined. Seven sera were obtained from adults with clinical rubella. In three of these cases, pregnancy was additionally diagnosed. One serum specimen was taken from a case of congenital rubella. Two sera showing high titers in the HI test (2,048) were obtained from individuals who had had a rubella infection 6 to 12 months previously. Additionally, 20 sera from blood donors with a rubella antibody titer in the HI test of > 256 were included.
RESULTS The question of what concentrations of IgM, IgA, and IgG antibodies could still be detected in the serum samples after precipitation with PS was of particular significance. One problem was co-precipitation. IgA and IgM antibodies are co-precipitated, particularly in small reaction volumes, if the concentration of the IgGanti-IgG precipitates becomes so high that in the subsequent centrifugation a considerable cosedimentation of IgM and IgA molecules can occur. On the other hand, with excessive volumes (under our conditions more than 0.7 ml total volume) IgG was not completely precipito 1:10. Quantitation of the anti-IgG titer. The amount of tated. To find the optimal conditions for precipIgG (milligrams) which was precipitated during 10 itation, we varied the total volume while the min at 20 C by 1 ml of anti-human IgG globulin amount of anti-gamma chain antibody and (anti-IgG titer) was determined according to the human serum remained constant. The greatest method of Sewell (9). To simplify the estimations of possible degree of precipitation of IgG antithe anti-IgG titer, we additionally performed a single bodies without too great loss of IgM and IgA radial immunodiffusion test (6): gamma globulin was found at a total volume of 0.4 ml. The im(Behring, Marburg) was mixed with melted agarose at munoglobulin concentrations before and after 56 C (final concentration 0.01 mg/ml, 2% agarose). serum (diFifteen milliliters of the mixture was poured into a precipitation of 0.025 ml of human petri dish (10-cm diameter). Two-millimeter holes luted 1:4) with 0.4 ml of PS are shown in Table 1. After precipitation, the IgG content in the were made in the agarose and 0.01 ml of an unknown anti-IgG, or anti-gamma chain antiserum, was placed serum was reduced to less than 0.3% of the in each hole. The diameters of the precipitation rings initial concentration. On the other hand, 64% of were measured 48 h later. The anti-human IgG titer the IgM and 76% of the IgA were still present. (T) of different dilutions (T = 0.04, 0.08 ... 0.8 mg of Assuming an equal reduction of all IgM and IgA precipitated IgG/ml of a standard antibody solution) antibodies, a rubella HI titer based on IgG determined by the method of Sewell was plotted antibodies must be reduced from 1,024 to
