Immunology 1976 30 107
Induction and yield of reaginic antibodies in mouse serum and ascitic fluid* S. B. LEHRER, T. M. ROSE & J. H. VAUGHAN Division of Allergy and Immunology, Department of Clinical Research, Scripps Clinic and Research Foundation, La Jolla, California, U.S.A.
Received 25 June 1975; acceptedfor publication 17 July 1975
Summary. Reaginic antibody in ascitic fluid was isolated from mice immunized with ovalbumin and Al(OH)3 or with ovalbumin and pertussis adjuvant. Partial purification of this reagin by ammonium sulphate precipitation, gel filtration, and ionexchange column chromatography yielded a gammaglobulin fraction highly active in the passive cutaneous anaphylaxis reaction. This was identical in purity and specific activity to a fraction of partially purified reagin isolated from mouse antiserum. The recovery of reaginic activity after purification from ascitic fluid was 19 per cent compared to the 7 8 per cent recovery from anti-serum. These studies demonstrate that ascitic fluid in mice immunized with methods known to produce reaginic antibody provides an excellent source of mouse IgE.
Levine and Vaz, 1970; Vaz, Vaz and Levine, 1971; Lehrer, Vaughan and Tan, 1975ab). Through such studies, reagin has been shown to belong to a separate class of mouse immunoglobulins similar to IgE found in man (Provoust-Danon, Binaghi, Rochas and Bousac-Aron, 1972; Schwartz and Levine, 1973). However, further investigation of mouse reagin has been curtailed in general by the inadequate amount of mouse antisera available containingreaginic antibody. Antibodies are known to be present in ascites isolated from mice treated with Freund's adjuvant (Munoz, 1957) or other inducers (Lieberman, Douglas and Mantel, 1960; Brandt, Buescher and Hetrick, 1967; Dieg, 1974). Furthermore hosts that produce reagin in their sera also have it in their ascitic fluid (Clausen, Munoz and Bergman, 1970). Therefore we investigated the yield of partially purified IgE obtainable from mouse ascitic fluid compared to that from their sera. In experiments to be presented it was demonstrated that reasonable quantities of reagin could be detected in mouse ascitic fluid and that partial purification yielded a preparation of IgE identical in purity and specific activity to that obtained from antiserum. These studies demonstrate that the induction of ascitic fluid in mice producing reaginic antibody provides an excellent source of mouse IgE.
INTRODUCTION Demonstration and isolation of murine reaginic antibodies have been difficult due to the transient nature of their production and low concentration in mouse serum. Nevertheless several experimental immunization regimens have been developed that yield high titres of persistent and boosterable reaginic antibodies (Clausen, Munoz, and Bergman, 1969; * This is publication number 961 from the Division of Allergy and Immunology, Scripps Clinic and Research Foundation. Correspondence: Dr S. B. Lehrer, Department of Medicine, Tulane University School of Medicine, New Orleans, La. 70112, U.S.A.
MATERIALS AND METHODS Immunization of mice Thirty-one 10-week-old female C57B1/6J mice 107
108
S. B. Lehrer, T. M. Rose & J. H. Vaughan
(Jackson Laboratories, Bar Harbour, Maine) were immunized on day 0 with an intraperitoneal (i.p.) injection of 100 pg of ovalbumin (OA) (Sigma Chemical Company, St Louis, Missouri) in 0 5 ml phosphate (001 M) buffered (pH 74) saline (015 NaCI) (PBS) and an intravenous (i.v.) injection of 2 ug of an extract of the histamine-sensitizing factor (HSF) of Bordetella pertussis prepared as described (Lehrer, Tan and Vaughan, 1974). A secondary injection of 10 pg OA in PBS was given i.p. 68 days post-immunization. In a second experiment, thirty-eight female 15-week-old CBA/J mice (Jackson Laboratories, Bar Harbour, Maine) were immunized with 100 ,ug of OA incorporated in 20 mg of Al(OH)3. It had been demonstrated previously with these mice that the latter immunization procedure yielded high titres of reaginic antibodies (Lehrer et al., 1975b).
Induction of ascites C57BI/6J mice were injected i.v. with 2 ug of HSF and i.p. with 1 ml Freund's complete adjuvant (FCA) from Difco Laboratories, Detroit, Michigan 68 days post-immunization. An additional injection of 0 5 ml of Freund's incomplete adjuvant (FIA) was given i.p. 80 days post-immunization. CBA/J mice were injected i.v. with 2 pg of HSF and i.p. with 1 ml of FCA 8 days post-immunization. Two additional injections of 0 5 ml of IFA were given i.p. 13 and 20 days post-immunization. Our method of inducing ascites in mice differs in two respects from that reported by others (Munoz, 1957). First, in addition to FCA, HSF was used, since preliminary experiments suggested that a greater percentage of mice had ascites when injected with both (Lehrer, unpublished observations). Secondly, mice were immunized with antigen prior to induction of ascites by Freund's adjuvant. This was done in order to favour maximal reagin production, since incorporation of antigen in FCA or FIA does not yield optimal titres of reagin (Clausen et al., 1970).
removed, spun at 2000 g, the supernate removed and stored at -20°. Mice containing ascites (determined by observations of distended abdomens) were anaesthetized with ether, an 18-gauge needle was inserted into the peritoneal cavity, and the ascites slowly removed with an attached 10-ml syringe. After exsanguination, a short incision was made through the peritoneal wall, and the ascites removed with a Pasteur pipette. The amount of ascites obtained varied from 1 to 10 ml per mouse. Ascites samples per group from each strain were pooled, kept at 24° for 2 h and at 40 overnight. During this period, a small clot formed. Usually the ascitic fluid contained large amounts of lipid which were removed after centrifugation at 20,000 g by aspiration of the upper lipid layer. The remaining supernatant was removed and stored at -20°. Passive cutaneous anaphylaxis (PCA) Reaginic antibody was assayed by the PCA reaction in 6-week-old female CFW mice obtained from Carworth, Farms, Portage, Michigan. This was performed according to the procedure of Ovary (1964) and detected IgE antibodies (Provoust-Danon et al., 1972; Schwartz et al., 1973; Lehrer and Vaughan, 1975). 0 5 ml of diluted serum or ascitic fluid (diluent-PBS) was injected intradermally into the CFW recipients. Each animal was challenged 48 h later with an i.v. injection of 0 2 ml of PBS containing 1 mg of OA in 0 5 per cent Evans blue and killed 30 min later. A positive response was blueing on the subcutaneous surface of the skin of at least 5 mm in size. All samples were tested in duplicate and results expressed as a geometric mean. All antibodies detected by this assay will be referred to as IgE or reaginic antibodies. During subsequent fractionation procedures to be described below, it was necessary to quantitate reagin. This was done by calculation of the number of PCA units = [sample volume . volume injected in PCA] x sample titre.
Ammonium sulphate precipitation and column chro-
Isolation of serum and ascitic fluid containing reagin All mice were bled from the orbital plexus postimmunization (yield approximately 0 5 ml per mouse). After anaesthetizing the animal with ether, the final bleeding was performed by cutting the auxillary artery (yield 1 to 14 ml per mouse). All blood samples from each strain were pooled, clotted at 240 for 2 h and at 40 overnight. Serum was
matography All steps in the ammonium sulphate precipitation were carried out at 4°. Ten to 15 ml serum or ascitic fluid were 30 per cent saturated by the addition of 100 per cent saturated ammonium sulphate solution in PBS, added slowly and stirred continually. After being mixed for 2 hr the solution was centrifuged (10,OCO g) and the precipitate was resuspended in
109
Murine reaginic antibodies PBS and dialysed exhaustively against PBS. The supernatant was 40 per cent saturated with ammonium sulphate as described and the resulting precipitate was dissolved in and dialysed against PBS. The ammonium sulphate precipitation was repeated at 50, 60 and 80 per cent saturation, and all pellets resuspended in and dialysed against PBS as described above. Chromatographic separations followed in part the methods reported for isolation of rat IgE by Isersky, Kulczycki and Metzger (1974). Proteins precipitating between 40 and 50 per cent ammonium sulphate saturation were equilibrated with 0 2 M borate buffer (pH 8), containing 0-9 per cent NaCI and then applied to a 2-5 x 100 cm Bio Gel A-5 m column (Bio Rad Laboratories, Richmond, California). Reagin-rich fractions were concentrated by negative pressure, dialysed against 0 005 M phosphate buffer, pH 8 0. Approximately 50-70 ml of 0 005 M sodium phosphate buffer, pH 8-0, was passed through the column and then a linear phosphate gradient from 0-005 M phosphate, pH 8 0, to 01 M phosphate buffer, pH 8 0, begun. Column fractions were assayed for reagin by the PCA reaction and the reagin rich fraction concentrated by negative pressure and stored at -20°.
Antisera Goat antisera to mouse gamma-i globulin, gamma-2 globulin, gamma A globulin, gamma M globulin, gamma globulins and whole mouse sera were obtained from Meloy Laboratories, Springfield, Virginia. Rabbit antiserum to the reagin-rich pool isolated from reaginic antisera was prepared by injecting 40 ,ug into the lymph nodes of a rabbit and 100 pg subcutaneously and into the footpads 27 days later as described (Goudie, Horne and Wilkinson, 1966). The rabbit was bled 10 days after the last injection; the serum was isolated after clotting and stored at -200.
Detroit, Michigan) in 005 M barbital buffer, pH 8.6, at 240. RESULTS
Yield of reaginic serum and ascitic fluid from immunized mice
C57B1/6J mice immunized with OA and HSF adjuvant and CBA/J mice immunized with OA and AI(OH)3 adjuvant were later injected with HSF, FCA and FIA in order to induce ascites. Abdomens with swollen ascites were observed in mice 12 days after the first injection of FCA (corresponding to 80 days post-immunization for C57Bl/6J mice and 20 days post-immunization for CBA/J mice). The mice were then bled and their peritoneal cavity tapped to obtain serum and ascites samples respectively. The reaginic activity in each sample was determined by the PCA reaction and the number of PCA units calculated. Approximately 1-10 ml ascites was isolated per mouse. It should be noted that during the course of this experiment some of the animals died as indicated by the reduced number of mice on subsequent bleeding. Their deaths were thought to result from the stresses of repeated anaesthetizing, bleeding and tapping of ascites. Reagin activity was detected in sera and ascitic fluid of both strains of mice (Table 1). Ascitic fluid from the C57B1/6J mice consistently had lower reagin titres as compared to those of their sera. However, ascitic fluid and sera from CBA/J animals always had identical reagin titres. When the total reagin activity was calculated as PCA units (a calculation that takes into account both activity and volume), ascitic fluid was comparable or superior to serum as a source of reaginic antibodies in both strains of mice. Recovery and yield of reagin from serum
Immunodiffusion Gel diffusion was performed in 0 4 per cent agarose (Marine Colloids, Rockland, Maine) in PBS. Approximately 0-05 ml antiserum and antigen were added to the wells as indicated and the immunodiffusion plate kept at 24° overnight. Immunoelectrophoresis (IEP) was performed according to the procedure of Grabar and Williams (1953) in 1-0 per cent agar (Special Agar-Noble, Difco Laboratories,
Reaginic antisera, isolated from C57BI/6J female mice treated similarly to those described in the previous section were fractionated by ammonium sulphate precipitation. The results summarized in Table 2 demonstrate that no detectable reagin precipitated at ammonium sulphate concentrations less than 40 per cent or greater than 60 per cent saturation. Most of the reagin precipitated between
110
S. B. Lehrer, T. M. Rose & J. H. Vaughan Table 1. Yield and reaginic activity of serum and ascitic fluid in different strains of mice
Serum
Ascitic fluid Mouse strain
Days postimmunization
C57B1/6J C57B1/6J C57BI/6J CBA/J CBA/J CBA/J
73
801 84
20t 27 31
Mice
Volume (ml)
48-h PCA
ascites/ total
Range Total
Titre*
Units
0/31 23/31 19/22 30/38 11/29 11/21
None None 1-10 83 42 1-5 42 1-5 25 1-6 20 1-4
None 50 40 80 201 160
None 83,000 33,600 67,200 100,500 64,000
*
Volume (ml) 10 10 14 11 5 10 21
48-h PCA
Titre*
Units
160 160 80 80 201 160
32,000 32,000 22,400 18,400 40,200 67,200
Reciprocal antibody dilution.
t Corresponding to 12 days after injection of FCA. Table 2. Recovery of reaginic antibody in serum or ascitic fluid after ammonium sulphate precipitation
12r
PCA units Percentage ammonium sulphate
Serum
Ascitic fluid
-i
E 0
0-30 30-40 40-50 50-60 60-80
Induction and yield of reaginic antibodies in mouse serum and ascitic fluid* S. B. LEHRER, T. M. ROSE & J. H. VAUGHAN Division of Allergy and Immunology, Department of Clinical Research, Scripps Clinic and Research Foundation, La Jolla, California, U.S.A.
Received 25 June 1975; acceptedfor publication 17 July 1975
Summary. Reaginic antibody in ascitic fluid was isolated from mice immunized with ovalbumin and Al(OH)3 or with ovalbumin and pertussis adjuvant. Partial purification of this reagin by ammonium sulphate precipitation, gel filtration, and ionexchange column chromatography yielded a gammaglobulin fraction highly active in the passive cutaneous anaphylaxis reaction. This was identical in purity and specific activity to a fraction of partially purified reagin isolated from mouse antiserum. The recovery of reaginic activity after purification from ascitic fluid was 19 per cent compared to the 7 8 per cent recovery from anti-serum. These studies demonstrate that ascitic fluid in mice immunized with methods known to produce reaginic antibody provides an excellent source of mouse IgE.
Levine and Vaz, 1970; Vaz, Vaz and Levine, 1971; Lehrer, Vaughan and Tan, 1975ab). Through such studies, reagin has been shown to belong to a separate class of mouse immunoglobulins similar to IgE found in man (Provoust-Danon, Binaghi, Rochas and Bousac-Aron, 1972; Schwartz and Levine, 1973). However, further investigation of mouse reagin has been curtailed in general by the inadequate amount of mouse antisera available containingreaginic antibody. Antibodies are known to be present in ascites isolated from mice treated with Freund's adjuvant (Munoz, 1957) or other inducers (Lieberman, Douglas and Mantel, 1960; Brandt, Buescher and Hetrick, 1967; Dieg, 1974). Furthermore hosts that produce reagin in their sera also have it in their ascitic fluid (Clausen, Munoz and Bergman, 1970). Therefore we investigated the yield of partially purified IgE obtainable from mouse ascitic fluid compared to that from their sera. In experiments to be presented it was demonstrated that reasonable quantities of reagin could be detected in mouse ascitic fluid and that partial purification yielded a preparation of IgE identical in purity and specific activity to that obtained from antiserum. These studies demonstrate that the induction of ascitic fluid in mice producing reaginic antibody provides an excellent source of mouse IgE.
INTRODUCTION Demonstration and isolation of murine reaginic antibodies have been difficult due to the transient nature of their production and low concentration in mouse serum. Nevertheless several experimental immunization regimens have been developed that yield high titres of persistent and boosterable reaginic antibodies (Clausen, Munoz, and Bergman, 1969; * This is publication number 961 from the Division of Allergy and Immunology, Scripps Clinic and Research Foundation. Correspondence: Dr S. B. Lehrer, Department of Medicine, Tulane University School of Medicine, New Orleans, La. 70112, U.S.A.
MATERIALS AND METHODS Immunization of mice Thirty-one 10-week-old female C57B1/6J mice 107
108
S. B. Lehrer, T. M. Rose & J. H. Vaughan
(Jackson Laboratories, Bar Harbour, Maine) were immunized on day 0 with an intraperitoneal (i.p.) injection of 100 pg of ovalbumin (OA) (Sigma Chemical Company, St Louis, Missouri) in 0 5 ml phosphate (001 M) buffered (pH 74) saline (015 NaCI) (PBS) and an intravenous (i.v.) injection of 2 ug of an extract of the histamine-sensitizing factor (HSF) of Bordetella pertussis prepared as described (Lehrer, Tan and Vaughan, 1974). A secondary injection of 10 pg OA in PBS was given i.p. 68 days post-immunization. In a second experiment, thirty-eight female 15-week-old CBA/J mice (Jackson Laboratories, Bar Harbour, Maine) were immunized with 100 ,ug of OA incorporated in 20 mg of Al(OH)3. It had been demonstrated previously with these mice that the latter immunization procedure yielded high titres of reaginic antibodies (Lehrer et al., 1975b).
Induction of ascites C57BI/6J mice were injected i.v. with 2 ug of HSF and i.p. with 1 ml Freund's complete adjuvant (FCA) from Difco Laboratories, Detroit, Michigan 68 days post-immunization. An additional injection of 0 5 ml of Freund's incomplete adjuvant (FIA) was given i.p. 80 days post-immunization. CBA/J mice were injected i.v. with 2 pg of HSF and i.p. with 1 ml of FCA 8 days post-immunization. Two additional injections of 0 5 ml of IFA were given i.p. 13 and 20 days post-immunization. Our method of inducing ascites in mice differs in two respects from that reported by others (Munoz, 1957). First, in addition to FCA, HSF was used, since preliminary experiments suggested that a greater percentage of mice had ascites when injected with both (Lehrer, unpublished observations). Secondly, mice were immunized with antigen prior to induction of ascites by Freund's adjuvant. This was done in order to favour maximal reagin production, since incorporation of antigen in FCA or FIA does not yield optimal titres of reagin (Clausen et al., 1970).
removed, spun at 2000 g, the supernate removed and stored at -20°. Mice containing ascites (determined by observations of distended abdomens) were anaesthetized with ether, an 18-gauge needle was inserted into the peritoneal cavity, and the ascites slowly removed with an attached 10-ml syringe. After exsanguination, a short incision was made through the peritoneal wall, and the ascites removed with a Pasteur pipette. The amount of ascites obtained varied from 1 to 10 ml per mouse. Ascites samples per group from each strain were pooled, kept at 24° for 2 h and at 40 overnight. During this period, a small clot formed. Usually the ascitic fluid contained large amounts of lipid which were removed after centrifugation at 20,000 g by aspiration of the upper lipid layer. The remaining supernatant was removed and stored at -20°. Passive cutaneous anaphylaxis (PCA) Reaginic antibody was assayed by the PCA reaction in 6-week-old female CFW mice obtained from Carworth, Farms, Portage, Michigan. This was performed according to the procedure of Ovary (1964) and detected IgE antibodies (Provoust-Danon et al., 1972; Schwartz et al., 1973; Lehrer and Vaughan, 1975). 0 5 ml of diluted serum or ascitic fluid (diluent-PBS) was injected intradermally into the CFW recipients. Each animal was challenged 48 h later with an i.v. injection of 0 2 ml of PBS containing 1 mg of OA in 0 5 per cent Evans blue and killed 30 min later. A positive response was blueing on the subcutaneous surface of the skin of at least 5 mm in size. All samples were tested in duplicate and results expressed as a geometric mean. All antibodies detected by this assay will be referred to as IgE or reaginic antibodies. During subsequent fractionation procedures to be described below, it was necessary to quantitate reagin. This was done by calculation of the number of PCA units = [sample volume . volume injected in PCA] x sample titre.
Ammonium sulphate precipitation and column chro-
Isolation of serum and ascitic fluid containing reagin All mice were bled from the orbital plexus postimmunization (yield approximately 0 5 ml per mouse). After anaesthetizing the animal with ether, the final bleeding was performed by cutting the auxillary artery (yield 1 to 14 ml per mouse). All blood samples from each strain were pooled, clotted at 240 for 2 h and at 40 overnight. Serum was
matography All steps in the ammonium sulphate precipitation were carried out at 4°. Ten to 15 ml serum or ascitic fluid were 30 per cent saturated by the addition of 100 per cent saturated ammonium sulphate solution in PBS, added slowly and stirred continually. After being mixed for 2 hr the solution was centrifuged (10,OCO g) and the precipitate was resuspended in
109
Murine reaginic antibodies PBS and dialysed exhaustively against PBS. The supernatant was 40 per cent saturated with ammonium sulphate as described and the resulting precipitate was dissolved in and dialysed against PBS. The ammonium sulphate precipitation was repeated at 50, 60 and 80 per cent saturation, and all pellets resuspended in and dialysed against PBS as described above. Chromatographic separations followed in part the methods reported for isolation of rat IgE by Isersky, Kulczycki and Metzger (1974). Proteins precipitating between 40 and 50 per cent ammonium sulphate saturation were equilibrated with 0 2 M borate buffer (pH 8), containing 0-9 per cent NaCI and then applied to a 2-5 x 100 cm Bio Gel A-5 m column (Bio Rad Laboratories, Richmond, California). Reagin-rich fractions were concentrated by negative pressure, dialysed against 0 005 M phosphate buffer, pH 8 0. Approximately 50-70 ml of 0 005 M sodium phosphate buffer, pH 8-0, was passed through the column and then a linear phosphate gradient from 0-005 M phosphate, pH 8 0, to 01 M phosphate buffer, pH 8 0, begun. Column fractions were assayed for reagin by the PCA reaction and the reagin rich fraction concentrated by negative pressure and stored at -20°.
Antisera Goat antisera to mouse gamma-i globulin, gamma-2 globulin, gamma A globulin, gamma M globulin, gamma globulins and whole mouse sera were obtained from Meloy Laboratories, Springfield, Virginia. Rabbit antiserum to the reagin-rich pool isolated from reaginic antisera was prepared by injecting 40 ,ug into the lymph nodes of a rabbit and 100 pg subcutaneously and into the footpads 27 days later as described (Goudie, Horne and Wilkinson, 1966). The rabbit was bled 10 days after the last injection; the serum was isolated after clotting and stored at -200.
Detroit, Michigan) in 005 M barbital buffer, pH 8.6, at 240. RESULTS
Yield of reaginic serum and ascitic fluid from immunized mice
C57B1/6J mice immunized with OA and HSF adjuvant and CBA/J mice immunized with OA and AI(OH)3 adjuvant were later injected with HSF, FCA and FIA in order to induce ascites. Abdomens with swollen ascites were observed in mice 12 days after the first injection of FCA (corresponding to 80 days post-immunization for C57Bl/6J mice and 20 days post-immunization for CBA/J mice). The mice were then bled and their peritoneal cavity tapped to obtain serum and ascites samples respectively. The reaginic activity in each sample was determined by the PCA reaction and the number of PCA units calculated. Approximately 1-10 ml ascites was isolated per mouse. It should be noted that during the course of this experiment some of the animals died as indicated by the reduced number of mice on subsequent bleeding. Their deaths were thought to result from the stresses of repeated anaesthetizing, bleeding and tapping of ascites. Reagin activity was detected in sera and ascitic fluid of both strains of mice (Table 1). Ascitic fluid from the C57B1/6J mice consistently had lower reagin titres as compared to those of their sera. However, ascitic fluid and sera from CBA/J animals always had identical reagin titres. When the total reagin activity was calculated as PCA units (a calculation that takes into account both activity and volume), ascitic fluid was comparable or superior to serum as a source of reaginic antibodies in both strains of mice. Recovery and yield of reagin from serum
Immunodiffusion Gel diffusion was performed in 0 4 per cent agarose (Marine Colloids, Rockland, Maine) in PBS. Approximately 0-05 ml antiserum and antigen were added to the wells as indicated and the immunodiffusion plate kept at 24° overnight. Immunoelectrophoresis (IEP) was performed according to the procedure of Grabar and Williams (1953) in 1-0 per cent agar (Special Agar-Noble, Difco Laboratories,
Reaginic antisera, isolated from C57BI/6J female mice treated similarly to those described in the previous section were fractionated by ammonium sulphate precipitation. The results summarized in Table 2 demonstrate that no detectable reagin precipitated at ammonium sulphate concentrations less than 40 per cent or greater than 60 per cent saturation. Most of the reagin precipitated between
110
S. B. Lehrer, T. M. Rose & J. H. Vaughan Table 1. Yield and reaginic activity of serum and ascitic fluid in different strains of mice
Serum
Ascitic fluid Mouse strain
Days postimmunization
C57B1/6J C57B1/6J C57BI/6J CBA/J CBA/J CBA/J
73
801 84
20t 27 31
Mice
Volume (ml)
48-h PCA
ascites/ total
Range Total
Titre*
Units
0/31 23/31 19/22 30/38 11/29 11/21
None None 1-10 83 42 1-5 42 1-5 25 1-6 20 1-4
None 50 40 80 201 160
None 83,000 33,600 67,200 100,500 64,000
*
Volume (ml) 10 10 14 11 5 10 21
48-h PCA
Titre*
Units
160 160 80 80 201 160
32,000 32,000 22,400 18,400 40,200 67,200
Reciprocal antibody dilution.
t Corresponding to 12 days after injection of FCA. Table 2. Recovery of reaginic antibody in serum or ascitic fluid after ammonium sulphate precipitation
12r
PCA units Percentage ammonium sulphate
Serum
Ascitic fluid
-i
E 0
0-30 30-40 40-50 50-60 60-80
