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An improved haptoglobin phenotyping procedure using immunofixation after starch gel electrophoresis
C/in& Chimica Acta, 137 (1984) 233-237 Elsevier
233
CCA 02770
Brief technical
note
An improved haptoglobin phenotyping procedure using immunofixation after starch gel electrophoresis Kaarina
Oj ala * and Theodor
Aurora Hospital, Clinical Laboratory, SF-00250 (Received
May 30th; revision October
Key words: Haptoglobin phenotyping;
Immunofixation;
H. Weber Helsinki (Finland) 4th, 198$ Starch gel electrophoresis
Introduction In 1955, by use of starch gel electrophoresis, Smithies [l] discovered the three major types of haptoglobin now designated Hp l-l, Hp 2-l and Hp 2-2. In the starch gel the haptoglobin types are separated on the basis of their molecular size as well as by charge differences. Polyacrylamide gel is often employed instead of starch gel because of better reproducibility and the wide range of molecular sieving effects. Many modifications of these two electrophoretic methods have been developed which were summarised and compared critically in the monograph of Pintera [2]. The common haptoglobin types can also be differentiated by immunoelectrophoresis in agar gel [3]. However, in all hitherto published methods, the hemoglobin-binding capacity of haptoglobin has been used for the detection of phenotypes. The immunofixation procedure presented here is much more sensitive and uses no carcinogenic reagents unlike methods which measure the peroxidase activity of the haptoglobin-hemoglobin complex. No specially designed equipment is needed. The immunofixation technique is compared to a method based on hemoglobin staining. By these two techxiiques the immunological and hemoglobin-binding properties of different haptoglobins can be compared. Materials and methods Apparatus For electrophoresis a commercial system, manufactured by Helena Laboratories (Beaumont, TX, USA), was used. The parts needed are the Zip Zone chamber, the sponge wicks and the gel trays (76 X 102 mm). The gels are sliced by sufficiently * To whom correspondence 0009-8981/84/$03.00
should be addressed.
0 1984 Elsevier Science Publishers
B.V.
234
large tweezers to which a slicing wire (76 mm long) has been attached. The slits for sample insertion are produced by a cutter (about 8 mm wide) made out of a razor blade. Reagents Gel buffer: 0.076 mol/l Tris and 0.005 mol/l citric acid, pH 8.6. Electrophoresis buffer: 0.3 mol/l boric acid and 0.05 mol/l NaOH, pH 8.2. Hemoglobin solution: 10% Hemoglobin Al (Helena Laboratories, Cat. No. 5019). Hemoglobin staining solution: 100 mg of o-dianisidine (Helena Laboratories, Cat. No. 5036) is dissolved in 5 ml of 95% ethanol, 5 ml of 3% hydrogen peroxidase and after that 0.5 ml glacial acetic acid is added. The solution is made just before use. Protein staining solution 2.5 g of Brilliant Blue R (Sigma, St. Louis, MO, USA, Cat. No. B-0630) in a solution containing 500 ml methanol, 400 ml water and 100 ml acetic acid. The same solution without the dye is used for decolorisation. Haptoglobin antiserum solution The haptoglobin antiserum used (Orion Diagnostica, Helsinki, Finland) has a titre of 2 g/l as determined by radial immunodiffusion. One part of haptoglobin antiserum is diluted with four parts of phosphate-buffered saline, pH 5.3, containing 4% of polyethylene glycol (PEG) 6000 (Fluka AG. Switzerland). The solution is made just before use. Preparation of gel Starch (Sigma, Cat. No. S-4501), 12 g, is mixed with heated with continuous swirling on a hot plate until viscosity. After removal from heat, 40 ml of starch solution The tray is then covered with another tray, which is lightly to the gel. After putting a weight on the tray, it is temperature, and after that about 30 min in a refrigerator. tray, it is ready for sample insertion.
100 ml of gel buffer and well past the maximum is poured onto a gel tray. oiled to prevent adhesion left to cool at ambient After taking off the upper
Sample preparation For the hemoglobin-binding method, 5 ~1 of Hemoglobin Al solution is added to 100 ~1 of serum. For immunofixation no pre-run sample preparation is needed. Sample insertion The cuts for sample insertion are made about 20 mm from the longer end of the tray. After cutting a slit, a piece of filter paper (about 10 X 7 mm) is put in it. When all slits are made (about eight on one tray), the samples are inserted by taking off the piece of paper and pipetting 5 ~1 of sample into the slit. Electrophoresis 100 ~1 of electrophoresis buffer is poured into each of the outer compartments of the electrophoresis chamber. Two sponge wicks are wetted with buffer and placed in
235
each of the outer compartments against the inner ridges. Two gels can be electrophoresed at the same time. The trays are placed gel side down in the chamber such that the gel layer makes good contact with the top surface of the sponges, and with the application nearest the kathode. The cover is placed on the chamber, which is connected to the power supply. The electrophoresis is carried out for 1 h at approximately 20 mA per plate. The migration distance for free hemoglobin will then be approximately 50 mm. Voltage should be about 100 V. Gel slicing
After electrophoresis the outer surface of the gel must be sliced off, because it shows almost no resolution of the fractions. The tweezers with a slicing wire are placed under the tray so that the wire is above it, the tips of the tweezers being on both sides of the tray. By pulling the tweezers along the sides of the tray, the slicing is easily done. The upper slice is discarded and the gel is ready for staining. Hemoglobin
staining
A solution of o-dianisidine is poured over the gel and it is left to stain for about 10 min. The plate is then first rinsed for about 10 min in 5% acid and after that in tap water. Immunofixation
The gel is overlaid with an antiserum solution, 250 ~1 of antiserum in 1 ml of PEG buffer, covered with another tray, and incubated at room temperature for about 1 h. The plate is then washed once in saline for about 1 h. After pouring off the first washing solution, the plate is left overnight in saline and the following morning a final rinse with tap water is made. After that, the gel is immersed with Brilliant Blue solution for 15 min, destained in the diluent for approximately 30 min and finally rinsed with tap water. Results and discussion Alper and Johnson [4] first employed immunofixation to study protein polymorphism with agarose gel electrophoresis; they recommended washing of starch gels for 72 h. Lieberman and Gaidulis 151,in their method for phenotyping of a,-antitrypsin, used an overnight washing in saline, and this was found suitable for our procedure, too. The antiserum used is polyvalent and reacts with all types of haptoglobin. The concentration of antibody used should be varied, depending on the titre of the antibody employed. By enhancing the antigen-antibody reaction with PEG, more diluted antiserum can be used. The incubation time of 30-60 min is sufficient, when 1 : 5 diluted antiserum, titre 2 g/l, is used. The generally used detection method to identify the haptoglobins by their saturated hemoglobin complexes (HpHb) is not very sensitive. The Hp type may be difficult to recognise, if the concentration is 0.2 g/l or below [2,6]. We tested the sensitivity using both detection methods.
2.36
The results can be seen in Fig. 1, where the electropherograms of three sera with different phenotypes are presented. The samples were analysed both undiluted and diluted 1 : 6, when the concentrations were about 1.2 and 0.2 g/l. respectively. The
Fig. 1. Haptoglobin phenotypes demonstrated by (A) immunofixation and (B) hemoglobin staining after starch gel electrophoresis. The samples to the left contain 1.2 g/l haptoglobin and to the right 0.2 g/l. The arrow denotes the sample application point.
237
TABLE
I
Advantages of the method presented, using immunofixation, employing peroxidase-staining of haptoglobin-hemo~obin 1. 2. 3. 4. 5. 6.
compared
to previously
described
techniques
complexes
Increased sensitivity, i.e. 4 0.2 g/l (Fig. 1) Possibility of detecting non- hemoglobin-binding or weakly binding haptoglobin variants Possibility of distinguishing rare phenotypic variants (Fig. 1) Only non-toxic and non-carcinogenic reagents are used The stained electropherograms are stable for storage for prolonged periods, i.e. several years Free hemoglobin does not show up or interfere
2-2 haptoglobin phenotype, which has the weakest hemoglobin-binding capacity 171, cannot be detected by peroxidase staining at a concentration level of 0.2 g/l. The l-1 and 2-I phenotypes are barely detected at this level. As can be’seen, the phenotypes in the diluted samples are also easy to recognise, when immunofixation is used. In addition immunofixation will also detect haptoglobin variants with an altered hemoglobin-binding capacity [8,9]. The advantages of the method presented are summarised in Table I. Acknowledgement This work was supported
by the Sigrid Juselius
Foundation,
Helsinki,
Finland.
References Smithies 0. Zone electrophoresis in starch gels; group variations in serum proteins of normal human adults. Biochem J 1955; 61: 629-641. Pintera J. The biochemical, genetic and clinicopathological aspects of haptoglobin. Ser Haematol 1971; 4: l-183. Hirschfield J. A simple method of determining haptoglobin group in human sera by means of agar-gel electrophoresis. Acta Path01 Microbial Stand 1959; 47: 160-169. Alper CA, Johnson AM. Immunofixation electrophoresis: a technique for the study of protein polymorphism. VOX Sang 1969; 17: 44-452. Lieberman J, Gaidulis L. Simplified a,-antitrypsin phenotyping by immunofixation of acid-starch gels. J Lab Ciin Med 1976; 87: 710-716. Laurel1 CB, Gr6nvalI C. Hapto~obins. In: Sabotka H, Stewart CP, eds. Advances of clinical chemistry, Vol. 5. New York, London: Academic Press, 1962: 152. Javid J. The effect of haptoglobin poiymer size on hemoglobin binding capacity. VOX Sang 1965; 10: 320-325. De Castro I, Fumier F, Waks M. Haptogiobin Porto Alegre. In: Peeters H, ed. Protides, biological fluids, Vol. 18. New York: Pergamon Press, 1970: 373-376. Engler R, Degrelle H, Jayle MF. Evidence of a plasmatic factor inhibiting the haptogIobin-hemoglobin binding at the onset of an inflammatory reaction. In: Peeters H, ed. Protides, biological fluids, Vol. 18, New York: Pergamon Press, 1970: 377-379.
233
CCA 02770
Brief technical
note
An improved haptoglobin phenotyping procedure using immunofixation after starch gel electrophoresis Kaarina
Oj ala * and Theodor
Aurora Hospital, Clinical Laboratory, SF-00250 (Received
May 30th; revision October
Key words: Haptoglobin phenotyping;
Immunofixation;
H. Weber Helsinki (Finland) 4th, 198$ Starch gel electrophoresis
Introduction In 1955, by use of starch gel electrophoresis, Smithies [l] discovered the three major types of haptoglobin now designated Hp l-l, Hp 2-l and Hp 2-2. In the starch gel the haptoglobin types are separated on the basis of their molecular size as well as by charge differences. Polyacrylamide gel is often employed instead of starch gel because of better reproducibility and the wide range of molecular sieving effects. Many modifications of these two electrophoretic methods have been developed which were summarised and compared critically in the monograph of Pintera [2]. The common haptoglobin types can also be differentiated by immunoelectrophoresis in agar gel [3]. However, in all hitherto published methods, the hemoglobin-binding capacity of haptoglobin has been used for the detection of phenotypes. The immunofixation procedure presented here is much more sensitive and uses no carcinogenic reagents unlike methods which measure the peroxidase activity of the haptoglobin-hemoglobin complex. No specially designed equipment is needed. The immunofixation technique is compared to a method based on hemoglobin staining. By these two techxiiques the immunological and hemoglobin-binding properties of different haptoglobins can be compared. Materials and methods Apparatus For electrophoresis a commercial system, manufactured by Helena Laboratories (Beaumont, TX, USA), was used. The parts needed are the Zip Zone chamber, the sponge wicks and the gel trays (76 X 102 mm). The gels are sliced by sufficiently * To whom correspondence 0009-8981/84/$03.00
should be addressed.
0 1984 Elsevier Science Publishers
B.V.
234
large tweezers to which a slicing wire (76 mm long) has been attached. The slits for sample insertion are produced by a cutter (about 8 mm wide) made out of a razor blade. Reagents Gel buffer: 0.076 mol/l Tris and 0.005 mol/l citric acid, pH 8.6. Electrophoresis buffer: 0.3 mol/l boric acid and 0.05 mol/l NaOH, pH 8.2. Hemoglobin solution: 10% Hemoglobin Al (Helena Laboratories, Cat. No. 5019). Hemoglobin staining solution: 100 mg of o-dianisidine (Helena Laboratories, Cat. No. 5036) is dissolved in 5 ml of 95% ethanol, 5 ml of 3% hydrogen peroxidase and after that 0.5 ml glacial acetic acid is added. The solution is made just before use. Protein staining solution 2.5 g of Brilliant Blue R (Sigma, St. Louis, MO, USA, Cat. No. B-0630) in a solution containing 500 ml methanol, 400 ml water and 100 ml acetic acid. The same solution without the dye is used for decolorisation. Haptoglobin antiserum solution The haptoglobin antiserum used (Orion Diagnostica, Helsinki, Finland) has a titre of 2 g/l as determined by radial immunodiffusion. One part of haptoglobin antiserum is diluted with four parts of phosphate-buffered saline, pH 5.3, containing 4% of polyethylene glycol (PEG) 6000 (Fluka AG. Switzerland). The solution is made just before use. Preparation of gel Starch (Sigma, Cat. No. S-4501), 12 g, is mixed with heated with continuous swirling on a hot plate until viscosity. After removal from heat, 40 ml of starch solution The tray is then covered with another tray, which is lightly to the gel. After putting a weight on the tray, it is temperature, and after that about 30 min in a refrigerator. tray, it is ready for sample insertion.
100 ml of gel buffer and well past the maximum is poured onto a gel tray. oiled to prevent adhesion left to cool at ambient After taking off the upper
Sample preparation For the hemoglobin-binding method, 5 ~1 of Hemoglobin Al solution is added to 100 ~1 of serum. For immunofixation no pre-run sample preparation is needed. Sample insertion The cuts for sample insertion are made about 20 mm from the longer end of the tray. After cutting a slit, a piece of filter paper (about 10 X 7 mm) is put in it. When all slits are made (about eight on one tray), the samples are inserted by taking off the piece of paper and pipetting 5 ~1 of sample into the slit. Electrophoresis 100 ~1 of electrophoresis buffer is poured into each of the outer compartments of the electrophoresis chamber. Two sponge wicks are wetted with buffer and placed in
235
each of the outer compartments against the inner ridges. Two gels can be electrophoresed at the same time. The trays are placed gel side down in the chamber such that the gel layer makes good contact with the top surface of the sponges, and with the application nearest the kathode. The cover is placed on the chamber, which is connected to the power supply. The electrophoresis is carried out for 1 h at approximately 20 mA per plate. The migration distance for free hemoglobin will then be approximately 50 mm. Voltage should be about 100 V. Gel slicing
After electrophoresis the outer surface of the gel must be sliced off, because it shows almost no resolution of the fractions. The tweezers with a slicing wire are placed under the tray so that the wire is above it, the tips of the tweezers being on both sides of the tray. By pulling the tweezers along the sides of the tray, the slicing is easily done. The upper slice is discarded and the gel is ready for staining. Hemoglobin
staining
A solution of o-dianisidine is poured over the gel and it is left to stain for about 10 min. The plate is then first rinsed for about 10 min in 5% acid and after that in tap water. Immunofixation
The gel is overlaid with an antiserum solution, 250 ~1 of antiserum in 1 ml of PEG buffer, covered with another tray, and incubated at room temperature for about 1 h. The plate is then washed once in saline for about 1 h. After pouring off the first washing solution, the plate is left overnight in saline and the following morning a final rinse with tap water is made. After that, the gel is immersed with Brilliant Blue solution for 15 min, destained in the diluent for approximately 30 min and finally rinsed with tap water. Results and discussion Alper and Johnson [4] first employed immunofixation to study protein polymorphism with agarose gel electrophoresis; they recommended washing of starch gels for 72 h. Lieberman and Gaidulis 151,in their method for phenotyping of a,-antitrypsin, used an overnight washing in saline, and this was found suitable for our procedure, too. The antiserum used is polyvalent and reacts with all types of haptoglobin. The concentration of antibody used should be varied, depending on the titre of the antibody employed. By enhancing the antigen-antibody reaction with PEG, more diluted antiserum can be used. The incubation time of 30-60 min is sufficient, when 1 : 5 diluted antiserum, titre 2 g/l, is used. The generally used detection method to identify the haptoglobins by their saturated hemoglobin complexes (HpHb) is not very sensitive. The Hp type may be difficult to recognise, if the concentration is 0.2 g/l or below [2,6]. We tested the sensitivity using both detection methods.
2.36
The results can be seen in Fig. 1, where the electropherograms of three sera with different phenotypes are presented. The samples were analysed both undiluted and diluted 1 : 6, when the concentrations were about 1.2 and 0.2 g/l. respectively. The
Fig. 1. Haptoglobin phenotypes demonstrated by (A) immunofixation and (B) hemoglobin staining after starch gel electrophoresis. The samples to the left contain 1.2 g/l haptoglobin and to the right 0.2 g/l. The arrow denotes the sample application point.
237
TABLE
I
Advantages of the method presented, using immunofixation, employing peroxidase-staining of haptoglobin-hemo~obin 1. 2. 3. 4. 5. 6.
compared
to previously
described
techniques
complexes
Increased sensitivity, i.e. 4 0.2 g/l (Fig. 1) Possibility of detecting non- hemoglobin-binding or weakly binding haptoglobin variants Possibility of distinguishing rare phenotypic variants (Fig. 1) Only non-toxic and non-carcinogenic reagents are used The stained electropherograms are stable for storage for prolonged periods, i.e. several years Free hemoglobin does not show up or interfere
2-2 haptoglobin phenotype, which has the weakest hemoglobin-binding capacity 171, cannot be detected by peroxidase staining at a concentration level of 0.2 g/l. The l-1 and 2-I phenotypes are barely detected at this level. As can be’seen, the phenotypes in the diluted samples are also easy to recognise, when immunofixation is used. In addition immunofixation will also detect haptoglobin variants with an altered hemoglobin-binding capacity [8,9]. The advantages of the method presented are summarised in Table I. Acknowledgement This work was supported
by the Sigrid Juselius
Foundation,
Helsinki,
Finland.
References Smithies 0. Zone electrophoresis in starch gels; group variations in serum proteins of normal human adults. Biochem J 1955; 61: 629-641. Pintera J. The biochemical, genetic and clinicopathological aspects of haptoglobin. Ser Haematol 1971; 4: l-183. Hirschfield J. A simple method of determining haptoglobin group in human sera by means of agar-gel electrophoresis. Acta Path01 Microbial Stand 1959; 47: 160-169. Alper CA, Johnson AM. Immunofixation electrophoresis: a technique for the study of protein polymorphism. VOX Sang 1969; 17: 44-452. Lieberman J, Gaidulis L. Simplified a,-antitrypsin phenotyping by immunofixation of acid-starch gels. J Lab Ciin Med 1976; 87: 710-716. Laurel1 CB, Gr6nvalI C. Hapto~obins. In: Sabotka H, Stewart CP, eds. Advances of clinical chemistry, Vol. 5. New York, London: Academic Press, 1962: 152. Javid J. The effect of haptoglobin poiymer size on hemoglobin binding capacity. VOX Sang 1965; 10: 320-325. De Castro I, Fumier F, Waks M. Haptogiobin Porto Alegre. In: Peeters H, ed. Protides, biological fluids, Vol. 18. New York: Pergamon Press, 1970: 373-376. Engler R, Degrelle H, Jayle MF. Evidence of a plasmatic factor inhibiting the haptogIobin-hemoglobin binding at the onset of an inflammatory reaction. In: Peeters H, ed. Protides, biological fluids, Vol. 18, New York: Pergamon Press, 1970: 377-379.