ANALYTICAL
BIOCHEMISTRY
79, 234-239 (1977)
A Rapid, Quantitative, and Highly Specific Assay for Carbohydrate-Binding Protein9
Department
of Biochemistry, University of California, Riverside, California 92502
Received June 10, 1976; accepted December 3, 1976 A rapid, quantitative, and highly specific assay capable of measuring 0.1 pg of soybean agglutinin is described. The method is applicable to determining soybean agglutinin in crude extracts as well as in purified preparations. The highly specific nature of the assay is the product of the chemical specificity between the Sepharose-GalNAc conjugate and soybean agglutinin and the antigenic specificity between soybean agglutinin and r2Wabeled antibody raised against soybean agglutinin. The assay can, in principle, be used to measure any carbohydratebinding protein. It is necessary only to link the appropriate hapten to the Sepharose beads and to raise antibody against the binding protein. While maximum specificity is attained using i2Wabeled antibody raised against a highly purified protein, the inclusion of two distinct specificity requirements in the assay may allow use of antibodies raised against proteins only partially purified.
The most widely used assay to measure lectins involves the agglutination of red blood cells (1). While the agglutination assay is rapid, it is, at best, only semiquantitative and is limited to divalent carbohydrate-binding proteins. Furthermore, due to the heterogenepus nature of surface carbohydrate determinants on red blood cells and since crude plant extracts may contain more than one lectin, the agglutination assay provides minimal information concerning specificity. The ideal assay for carbohydrate-binding proteins should be rapid, quantitative, highly specific, and capable of measuring monovalent carbohydrate-binding proteins in crude extracts as well as in purified preparations. Utilizing affinity chromatography, together with a combination of chemical and antigenic specificity, the assay to be described possesses all of these features. In addition, the assay is sensitive to O.lpg of binding protein. This assay was developed using soybean agglutinin (SBA) which is known to bindN-acetyl-D-galactosamine (GalNac). In principle, the assay can be used for any carbohydrate-binding protein. 1 Supported, in part, by the National Science Foundation * To whom inquiries should be addressed.
(PCM 74-09884 AOl)
234 Copyright 0 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISSN OCW-2697
CARBOHYDRATE-BINDING
MATERIALS
PROTEIN
ASSAY
235
AND METHODS
Preparation of SBA and antibody against SBA. Soybean agglutinin was purified according to Lis and Sharon (2) with the exception that a Bio-Gel P-100 (Bio-Rad, Richmond, Calif. 94804) column replaced a DEAE column in the final purification step. Purified SBA was emulsified with an equal volume of Freund’s complete adjuvant. Two New Zealand white rabbits received 1.0 mg each of SBA, one-half near each popliteal node. This procedure was repeated on 3 successive days. After 4 weeks, each rabbit was boosted with a total of 3 mg of soybean agglutinin in Freund’s incomplete adjuvant as before. Sera from the fourth boost were treated with (NH&SO, to 37% saturation, and the immunoglobulin in the precipitate was purified by passage through DEAE (3). The antibody was concentrated by (NH&SO, precipitation and stored frozen in 50% saturated (NH&SO,. Immediately before use, the antibody was dialyzed ‘against phosphate-buffered saline (PBS), and the protein content was measured spectrophotometrically, (ODzso 1 .O = 0.66 mg of protein/ml). lz5ZZodination of antibody. Lactoperoxidase (50 ~1, 1 mg/ml) and 250 &i of 1251(specific activity: 17 Ci/mg; ICN, Irvine, Calif.) were added to 2 ml of antibody (5 mg/ml). The reaction was initiated by the addition of 25 ~1 of 0.1 M H,O,. After 30 and 60 min, an additional 25 ~1 of H,O, was added. After 90 min, the mixture was applied to a Bio-Gel P-100 column (2.5 x 27 cm). The antibody in the effluent fractions was monitored using the double diffusion Ouchterlony test. The specific activity of the combined fractions was 1.1 x lo6 cpm/mg of protein. SBA Assay. GalNAc linked to Sepharose 4B was obtained from Vector Laboratories (Ignacio, CA 94947). The Sepharose-GalNAc conjugate was suspended in phosphate-buffered saline (1:2, v/v), and 50 ~1 was transferred to a microfuge tube. Normal serum (25 ~1) was added to reduce nonspecific binding of antibody. Soybean agglutinin was added, and the constituents were mixed within the microfuge tube. The SepharoseGalNAc-soybean agglutinin conjugate was pelleted in a Beckman 152 microfuge (5 set), and the supernatant solution was discarded. 1251-Labeled antibody against soybean agglutinin was added and mixed with the pellet. The Sepharose-GalNAc-soybean agglutinin-1251-labeled antibody conjugate was pelleted by centrifugation, and the pellet was washed three times with phosphate-buffered saline (0.2 ml/wash). The microfuge tube containing the Sepharose-GalNAc-soybean agglutinin-1251-labeled antibody conjugate was placed in a counting vial, and radioactivity was measured in a Beckman gamma counter at approximately 70% efficiency.
RESULTS AND DISCUSSION The effect of increasing amounts of 1251-labeled antibody on the radioactivity bound to a fixed amount of Sepharose- GalNAc- soybean agglutinin
236
HOWARD
AND SHANNON
01 0 12?
Ab
100 fug)
I 200
FIG. 1. Influence of anti-soybean agglutinin 1*51-labeled antibody concentration on the amount of antibody bound to Sepharose-GalNAc conjugate. Soybean agglutinin (3 pg) was added to 50 ~1 of Sepharose-GalNAc conjugate followed by anti-soybean agglutinin lz510) labeled antibody (Specific activity: 1.1 x lo8 cpm/mg of protein). The solid circles (0 represent counts/minute bound in excess of control (Sepharose-GalNAc conjugate without soybean agglutinin). The open circles (0 0) represent counts/minute as a percentage of control and was calculated from the ratio of counts/minute in Sepharose-GalNAc treated with 3 fig of soybean agglutinin to the control (Sepharose-GalNAc without soybean agglutinin).
conjugate is noted in Fig. 1. It can be seen (solid circles) that, up to a point, the amount of bound 1251-labeled antibody is dependent on the quantity of antibody added, and then it levels off. The plateau results from saturation of the antigenic determinants on soybean agglutinin present in the conjugate. It is also noted that, at the highest antibody concentration, the ratio of [conjugate with soybean agglutinin (cpm)/conjugate without soybean agglutinin (cpm)] decreased (open circles). The decreased ratio is the consequence of a large increase in nonspecific binding of antibody at the high antibody concentration. These observations indicate that, while the antibody concentration must be sufficient to saturate the antigenic determinants on soybean agglutinin, a large excess of antibody may decrease the sensitivity of the assay. The data in Fig. 2 indicate that the amount of radioactivity bound to the Sepharose conjugate increased linearly with soybean agglutinin additions from 0.1 to 4 pg. Upon increasing soybean agglutinin additions to 50 pg, the curve representing the amount of radioactivity bound to the Sepharose conjugate appeared as a rectangular hyperbola (Fig. 2; inset). A double reciprocal plot (data not shown) gave a correlation coefficient of 0.998. The following experiments were performed to determine if the assay was able to measure accurately the soybean agglutinin in crude extracts. Aliquots of a crude extract from soybean seed were added to microfuge
CARBOHYDRATE-BINDING
SBA
PROTEIN
237
ASSAY
(ug)
FIG. 2. Influence of soybean agglutinin concentration on the amount of antibody bound to Sepharose-GalNAc conjugate. Varying amounts of soybean agglutinin were added to 50 ~1 of Sepharose-GalNAc conjugate followed by 90 pg of anti-soybean agglutinin **Wabeled antibody, and the bound antibody was measured as described in Materials and Methods. The open circles (0 0) represent the linear portion of the complete curve (inset).
tubes containing Sepharose-GalNAc conjugate. This was followed by 1251-labeled antibody raised against purified soybean agglutinin. The amount of radioactivity bound to the conjugate increased linearly with additions of the crude extract, Fig. 3 (0 0). In a separate experiment, 0.4 pg of purified soybean agglutinin was added to microfuge tubes containing, respectively, OS-, l.O-, and 2.0-p] aliquots of crude extract from soybean seed. Sepharose-GalNAc conjuTABLE INFLUENCEOF
1
VARIOUSADDITIONSTOSEPHAROSE-GaINAC ON ANTI-SBA
L251-L~~~~~~
Treatment Sepharose-GalNAc conjugate (control) + 3pgofSBA + 3 pg of SBA and 40 pg of GalNAc +50 pg of PHA or lima bean lectin
ANTIBODY
CONJUGATE BINDING”
Counts/minute in excess of control 2700
0 loo
n Anti-SBA 1251-labeled antibody (90 pg; specific activity: 1.1 x lo@cpm/mg of protein) was added to Sepharose-GalNAc conjugate in the presence of the additions listed, and the bound antibody was measured as described in Materials and Methods.
238
HOWARD
AND SHANNON
8-
8-
4-
OO
I CRUDE SEED EXTRACT (~1)
2
FIG. 3. Assay of soybean agglutinin in crude extracts of soybean seed. Soybean seeds were soaked overnight and then homogenized in 4 vol of water using a mortar and pestle. The debris were removed by centrifugation, and aliquots of the crude seed extract were added to 50 ~1 of Sepharose-GalNAc conjugate, followed by 90 pg of anti-soybean agglutinin rzsI antibody. The solid circles (0 0) represent counts/minute of rz51-labeled antibody bound from crude extract in excess of control (1251-labeled antibody added to Sepharose-GalNAc conjugate in absence of crude extract). The open triangles (A A) represent the measured counts/minute bound to Sepharose conjugate when 0.4 pg of purified soybean agglutinin was added to 0.5, 1.0, and 2.0 ~1 of crude extract. The open circles (0 0) represent the calculated summation of radioactivity from 0.5, 1.0, and 2.0 ~1 of crude extract (0 0) and from 0.4 pg of purified soybean agglutinin (Fig. 2).
gate and 1251-labeled antibody were then added. The measured radioactivity bound to the Sepharose conjugate (A A) and the calculated summation of radioactivities (0 0) are also presented in Fig. 3. The observations that the measured and calculated values are nearly superimposed and are nearly parallel with the curve representing increased aliquots of crude extract indicate that the assay accurately measures soybean agglutinin in crude extracts. Table 1 illustrates the highly specific nature of the assay. Each treatment received 90 pg of anti-soybean agglutinin 1251-labeled antibody. It may be noted that, in the presence of 3 pg of soybean agglutinin, the amount of
CARBOHYDRATE-BINDING
PROTEIN
ASSAY
239
1251-labeled antibody which bound the Sepharose conjugate exceeded the control by 2700 cpm. When the carbohydrate binding sites of soybean agglutinin were saturated with free GalNAc, the lectin failed to bind the Sepharose conjugate, and the amount of bound 1251-labeled antibody was the same as the control. These observations illustrate the chemical specificity between the Sepharose-GalNAc conjugate and soybean agglutinin. In the presence of 50 pg of phytohemaglutinin (PHA) or lima bean lectin, both of which bind GalNAc (l), the amount of 1251-labeled antibody which bound the Sepharose-GalNAc conjugate was also the same as the control. This observation indicates that, even though lectins other than soybean agglutinin bind the carbohydrate moiety of the Sepharose conjugate, antigenic specificity to soybean agglutinin is required for a positive assay. The above experiments provide convincing evidence for the highly specific nature of this assay. It is important to note that the assay is the product of two types of specificity: (i) chemical specificity between soybean agglutinin and the Sepharose-GalNAc conjugate; and (ii) antigenic specificity between soybean agglutinin and 1251-labeled antibody raised against soybean agglutinin. Only those compounds which possess both types of specificity yield a positive assay. REFERENCES 1. Lis, H., and Sharon, N. (1973) Ann. Rev. Biochem. 42,541-574. 2. Lis, H., and Sharon, N. (1972) in Methods in Enzymology (Ginsburg, V.. ed.), Vol. 28. Part B, p. 360, Academic Press, New York. 3. Shannon, L. M., and Mills, S. E. (1976) Eur. J. Biochem. 63, 563-568.
BIOCHEMISTRY
79, 234-239 (1977)
A Rapid, Quantitative, and Highly Specific Assay for Carbohydrate-Binding Protein9
Department
of Biochemistry, University of California, Riverside, California 92502
Received June 10, 1976; accepted December 3, 1976 A rapid, quantitative, and highly specific assay capable of measuring 0.1 pg of soybean agglutinin is described. The method is applicable to determining soybean agglutinin in crude extracts as well as in purified preparations. The highly specific nature of the assay is the product of the chemical specificity between the Sepharose-GalNAc conjugate and soybean agglutinin and the antigenic specificity between soybean agglutinin and r2Wabeled antibody raised against soybean agglutinin. The assay can, in principle, be used to measure any carbohydratebinding protein. It is necessary only to link the appropriate hapten to the Sepharose beads and to raise antibody against the binding protein. While maximum specificity is attained using i2Wabeled antibody raised against a highly purified protein, the inclusion of two distinct specificity requirements in the assay may allow use of antibodies raised against proteins only partially purified.
The most widely used assay to measure lectins involves the agglutination of red blood cells (1). While the agglutination assay is rapid, it is, at best, only semiquantitative and is limited to divalent carbohydrate-binding proteins. Furthermore, due to the heterogenepus nature of surface carbohydrate determinants on red blood cells and since crude plant extracts may contain more than one lectin, the agglutination assay provides minimal information concerning specificity. The ideal assay for carbohydrate-binding proteins should be rapid, quantitative, highly specific, and capable of measuring monovalent carbohydrate-binding proteins in crude extracts as well as in purified preparations. Utilizing affinity chromatography, together with a combination of chemical and antigenic specificity, the assay to be described possesses all of these features. In addition, the assay is sensitive to O.lpg of binding protein. This assay was developed using soybean agglutinin (SBA) which is known to bindN-acetyl-D-galactosamine (GalNac). In principle, the assay can be used for any carbohydrate-binding protein. 1 Supported, in part, by the National Science Foundation * To whom inquiries should be addressed.
(PCM 74-09884 AOl)
234 Copyright 0 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.
ISSN OCW-2697
CARBOHYDRATE-BINDING
MATERIALS
PROTEIN
ASSAY
235
AND METHODS
Preparation of SBA and antibody against SBA. Soybean agglutinin was purified according to Lis and Sharon (2) with the exception that a Bio-Gel P-100 (Bio-Rad, Richmond, Calif. 94804) column replaced a DEAE column in the final purification step. Purified SBA was emulsified with an equal volume of Freund’s complete adjuvant. Two New Zealand white rabbits received 1.0 mg each of SBA, one-half near each popliteal node. This procedure was repeated on 3 successive days. After 4 weeks, each rabbit was boosted with a total of 3 mg of soybean agglutinin in Freund’s incomplete adjuvant as before. Sera from the fourth boost were treated with (NH&SO, to 37% saturation, and the immunoglobulin in the precipitate was purified by passage through DEAE (3). The antibody was concentrated by (NH&SO, precipitation and stored frozen in 50% saturated (NH&SO,. Immediately before use, the antibody was dialyzed ‘against phosphate-buffered saline (PBS), and the protein content was measured spectrophotometrically, (ODzso 1 .O = 0.66 mg of protein/ml). lz5ZZodination of antibody. Lactoperoxidase (50 ~1, 1 mg/ml) and 250 &i of 1251(specific activity: 17 Ci/mg; ICN, Irvine, Calif.) were added to 2 ml of antibody (5 mg/ml). The reaction was initiated by the addition of 25 ~1 of 0.1 M H,O,. After 30 and 60 min, an additional 25 ~1 of H,O, was added. After 90 min, the mixture was applied to a Bio-Gel P-100 column (2.5 x 27 cm). The antibody in the effluent fractions was monitored using the double diffusion Ouchterlony test. The specific activity of the combined fractions was 1.1 x lo6 cpm/mg of protein. SBA Assay. GalNAc linked to Sepharose 4B was obtained from Vector Laboratories (Ignacio, CA 94947). The Sepharose-GalNAc conjugate was suspended in phosphate-buffered saline (1:2, v/v), and 50 ~1 was transferred to a microfuge tube. Normal serum (25 ~1) was added to reduce nonspecific binding of antibody. Soybean agglutinin was added, and the constituents were mixed within the microfuge tube. The SepharoseGalNAc-soybean agglutinin conjugate was pelleted in a Beckman 152 microfuge (5 set), and the supernatant solution was discarded. 1251-Labeled antibody against soybean agglutinin was added and mixed with the pellet. The Sepharose-GalNAc-soybean agglutinin-1251-labeled antibody conjugate was pelleted by centrifugation, and the pellet was washed three times with phosphate-buffered saline (0.2 ml/wash). The microfuge tube containing the Sepharose-GalNAc-soybean agglutinin-1251-labeled antibody conjugate was placed in a counting vial, and radioactivity was measured in a Beckman gamma counter at approximately 70% efficiency.
RESULTS AND DISCUSSION The effect of increasing amounts of 1251-labeled antibody on the radioactivity bound to a fixed amount of Sepharose- GalNAc- soybean agglutinin
236
HOWARD
AND SHANNON
01 0 12?
Ab
100 fug)
I 200
FIG. 1. Influence of anti-soybean agglutinin 1*51-labeled antibody concentration on the amount of antibody bound to Sepharose-GalNAc conjugate. Soybean agglutinin (3 pg) was added to 50 ~1 of Sepharose-GalNAc conjugate followed by anti-soybean agglutinin lz510) labeled antibody (Specific activity: 1.1 x lo8 cpm/mg of protein). The solid circles (0 represent counts/minute bound in excess of control (Sepharose-GalNAc conjugate without soybean agglutinin). The open circles (0 0) represent counts/minute as a percentage of control and was calculated from the ratio of counts/minute in Sepharose-GalNAc treated with 3 fig of soybean agglutinin to the control (Sepharose-GalNAc without soybean agglutinin).
conjugate is noted in Fig. 1. It can be seen (solid circles) that, up to a point, the amount of bound 1251-labeled antibody is dependent on the quantity of antibody added, and then it levels off. The plateau results from saturation of the antigenic determinants on soybean agglutinin present in the conjugate. It is also noted that, at the highest antibody concentration, the ratio of [conjugate with soybean agglutinin (cpm)/conjugate without soybean agglutinin (cpm)] decreased (open circles). The decreased ratio is the consequence of a large increase in nonspecific binding of antibody at the high antibody concentration. These observations indicate that, while the antibody concentration must be sufficient to saturate the antigenic determinants on soybean agglutinin, a large excess of antibody may decrease the sensitivity of the assay. The data in Fig. 2 indicate that the amount of radioactivity bound to the Sepharose conjugate increased linearly with soybean agglutinin additions from 0.1 to 4 pg. Upon increasing soybean agglutinin additions to 50 pg, the curve representing the amount of radioactivity bound to the Sepharose conjugate appeared as a rectangular hyperbola (Fig. 2; inset). A double reciprocal plot (data not shown) gave a correlation coefficient of 0.998. The following experiments were performed to determine if the assay was able to measure accurately the soybean agglutinin in crude extracts. Aliquots of a crude extract from soybean seed were added to microfuge
CARBOHYDRATE-BINDING
SBA
PROTEIN
237
ASSAY
(ug)
FIG. 2. Influence of soybean agglutinin concentration on the amount of antibody bound to Sepharose-GalNAc conjugate. Varying amounts of soybean agglutinin were added to 50 ~1 of Sepharose-GalNAc conjugate followed by 90 pg of anti-soybean agglutinin **Wabeled antibody, and the bound antibody was measured as described in Materials and Methods. The open circles (0 0) represent the linear portion of the complete curve (inset).
tubes containing Sepharose-GalNAc conjugate. This was followed by 1251-labeled antibody raised against purified soybean agglutinin. The amount of radioactivity bound to the conjugate increased linearly with additions of the crude extract, Fig. 3 (0 0). In a separate experiment, 0.4 pg of purified soybean agglutinin was added to microfuge tubes containing, respectively, OS-, l.O-, and 2.0-p] aliquots of crude extract from soybean seed. Sepharose-GalNAc conjuTABLE INFLUENCEOF
1
VARIOUSADDITIONSTOSEPHAROSE-GaINAC ON ANTI-SBA
L251-L~~~~~~
Treatment Sepharose-GalNAc conjugate (control) + 3pgofSBA + 3 pg of SBA and 40 pg of GalNAc +50 pg of PHA or lima bean lectin
ANTIBODY
CONJUGATE BINDING”
Counts/minute in excess of control 2700
0 loo
n Anti-SBA 1251-labeled antibody (90 pg; specific activity: 1.1 x lo@cpm/mg of protein) was added to Sepharose-GalNAc conjugate in the presence of the additions listed, and the bound antibody was measured as described in Materials and Methods.
238
HOWARD
AND SHANNON
8-
8-
4-
OO
I CRUDE SEED EXTRACT (~1)
2
FIG. 3. Assay of soybean agglutinin in crude extracts of soybean seed. Soybean seeds were soaked overnight and then homogenized in 4 vol of water using a mortar and pestle. The debris were removed by centrifugation, and aliquots of the crude seed extract were added to 50 ~1 of Sepharose-GalNAc conjugate, followed by 90 pg of anti-soybean agglutinin rzsI antibody. The solid circles (0 0) represent counts/minute of rz51-labeled antibody bound from crude extract in excess of control (1251-labeled antibody added to Sepharose-GalNAc conjugate in absence of crude extract). The open triangles (A A) represent the measured counts/minute bound to Sepharose conjugate when 0.4 pg of purified soybean agglutinin was added to 0.5, 1.0, and 2.0 ~1 of crude extract. The open circles (0 0) represent the calculated summation of radioactivity from 0.5, 1.0, and 2.0 ~1 of crude extract (0 0) and from 0.4 pg of purified soybean agglutinin (Fig. 2).
gate and 1251-labeled antibody were then added. The measured radioactivity bound to the Sepharose conjugate (A A) and the calculated summation of radioactivities (0 0) are also presented in Fig. 3. The observations that the measured and calculated values are nearly superimposed and are nearly parallel with the curve representing increased aliquots of crude extract indicate that the assay accurately measures soybean agglutinin in crude extracts. Table 1 illustrates the highly specific nature of the assay. Each treatment received 90 pg of anti-soybean agglutinin 1251-labeled antibody. It may be noted that, in the presence of 3 pg of soybean agglutinin, the amount of
CARBOHYDRATE-BINDING
PROTEIN
ASSAY
239
1251-labeled antibody which bound the Sepharose conjugate exceeded the control by 2700 cpm. When the carbohydrate binding sites of soybean agglutinin were saturated with free GalNAc, the lectin failed to bind the Sepharose conjugate, and the amount of bound 1251-labeled antibody was the same as the control. These observations illustrate the chemical specificity between the Sepharose-GalNAc conjugate and soybean agglutinin. In the presence of 50 pg of phytohemaglutinin (PHA) or lima bean lectin, both of which bind GalNAc (l), the amount of 1251-labeled antibody which bound the Sepharose-GalNAc conjugate was also the same as the control. This observation indicates that, even though lectins other than soybean agglutinin bind the carbohydrate moiety of the Sepharose conjugate, antigenic specificity to soybean agglutinin is required for a positive assay. The above experiments provide convincing evidence for the highly specific nature of this assay. It is important to note that the assay is the product of two types of specificity: (i) chemical specificity between soybean agglutinin and the Sepharose-GalNAc conjugate; and (ii) antigenic specificity between soybean agglutinin and 1251-labeled antibody raised against soybean agglutinin. Only those compounds which possess both types of specificity yield a positive assay. REFERENCES 1. Lis, H., and Sharon, N. (1973) Ann. Rev. Biochem. 42,541-574. 2. Lis, H., and Sharon, N. (1972) in Methods in Enzymology (Ginsburg, V.. ed.), Vol. 28. Part B, p. 360, Academic Press, New York. 3. Shannon, L. M., and Mills, S. E. (1976) Eur. J. Biochem. 63, 563-568.
