414
D.Schranz
Electrophoresis 1991, 12, 414-419
eta!.
5 References [l] Radola, B. J., Electrophoresis 1980, 1, 43-56. [2] Allen, R. C., Electrophoresis 1980, 1, 32-37. 131 Kling, H., Sawatski, G . and Geis, W., Anal. Biochem. 1980,174,589592. [4] Merril, C. R., Goldman, D. and van Keuren, M. L., Electrophoresis 1982, 3, 17-23. [5] Guevara,J., Johnston, D.A. Ramagli,L. S.,Martin B.A., Capitello,S. and Rodriguez, L. V. Electrophoresis 1982, 3, 197-205. [6] Dion, A. S . and Pomenti A. A,, Anal. Biochem. 1983,129, 490-496. 171 Sammons, D. W., Adams, L. D. and Nishizawa, E. E., Electrophoresis 1980,2, 135-140. 181 Slisz, M.L. and van Frank, R. M., Electrophoresis 1980,2, 405-407. [9] Heukeshoven, J. and Dernick, R., Electrophoresis 1985, 6, 103-112. [lo] Merril, C. K. and Goldman, D., in: Celis, J. E. and Bravo, R. (Eds.), Two-dimensional Gel Electrophoresis of Proteins, Academic Press, New York 1984, pp. 93-109. [ I l l Yuksel, K. U. and Gracy, R. W., Electrophoresis 1985, 6 , 361-366.
[I21 Gersten, D. M., Wolf, P. H., Ledley, R. S., Rodriguez, L. V. and Zapolski, E. J., Electrophoresis 1986, 7, 327-332. [13] Merril, C. R. and Pratt M. E., Anal. Biochem. 1986, 156, 96-110. [14] Barker, W. C. (Ed.), Protein Identification Resource, National Biomedical Research Foundation, NBR Press, Washington D C 1990. [lS] Laemmli, U. K., Nature 1970,227, 680-682. [ 161 Johnston, D. A,, Mofftt,M.,Tang, K.,Newton, L. and Covnar, W.,An Interactive General Purpose Image Digitizing System, 5th Edition, Department of Biomathematics, M D Anderson Cancer Center, March 1988. [17] Johnston, D. A., White, R. A,, Barlogie, B., Comput. Biomed. Res. 1978, 11, 393-404. [18] Tal,M.,Silberstein,A.and Nusser,E.,J. 5iol. Chem. 1985,260,99769990. [19] Gersten,D.M.,Wolf,P.H.andZapolski,E.J.,Electrophoresis 1987,8, 545-55 1. [20] Mitchell, J. W., Photogr. Sci. Eng. 1978,22, 249-255. 1211 Miller, M. J., Adv. Electrophoresis 1989, 3, 181-217. [22] Merril, C. R., Methods Enzymol. 1990, 182, 477-488.
Dorota Schranz' Stanislaw Morkowskil Eleonora R. Karamova' Garri I. Abelev'
Counterflow affinity isotachophoresis on cellulose acetate membranes
'Department of Pathology and Immunology of Cancer, Academy of Medicine, Pozna6 'Laboratory of Immunochemistry, All-Union Cancer Research Center, Moscow
Counterflow isotachophoresis on cellulose acetate membranes of human alphafetoprotein (AFP) was performed with concanavalin A, lentil lectin, and castor bean lectin driven by electroendosmotic counterflow. This counterflow caused a uniform stream of lectin to migrate towards the cathode against AFP with carrier ampholytes in steady-state position. Retardation of microheterogeneity forms bound to lectins was observed, giving results comparable to standard crossed affinity immunoelectrophoresis.Smal1er amounts of lectins and more diluted samples of AFP could be used in the described method.
1 Introduction Several variants of isotachophoresis (ITP) on porous membranes have been described recently [l, 21. One of them is carried out on cellulose acetate membranes (ITP-CAM). It offers a nondenaturing system suitable for biological macromolecules, in addition to high resolving power. ITPCAM techniques are easy to combine with various immunochemical methods of specific protein detection: immunofixation, immunoelectrophoresis, immunoblotting etc. [3, 41. Since ITP-CAM is not affected by the molecular weight of the separated molecules it may be applied for studies of diverse sets of interacting biological macromolecules. This includes several important systems, e.g. antiCorrespondence: Dr. Dorota Schranz, Department of Pathology and Immunology of Cancer, Medical Academy, ul. takowa 112, PL-61-787 Poznan. Poland Abbreviations: AFF-ITP, counterflow affinity isotachophoresis; AFP, alpha-fetoprotein; BPB, Bromophenol Blue; CAIE, crossed affinity immunoelectrophoresis; CAM, cellulose acetate membranes; Con A, concanavalin A; LCA, lentil lectin; RCA, castor bean lectin
0VCH Vcrlagsgeseilschaft mbH, D-6940 Weinheim, 1991
genlantibody and glycoproten/lectin. The diversity of the carbohydrate structure of glycoproteins (theirmicroheterogeneity) has been studied widely as a model of specific recognition and regulation processes. It can also serve as an additional tool in cancer diagnosis IS]. Lectins, sugar-recognizing proteins present in various organisms, are often used as specific tools for these studies [6]. The most widely applied techniques used for this purpose are affinity chromatography [7] and affinity electrophoresis [8]. Despite their wide use, they suffer from several disadvantages. Affinity chromatography needs lectins immobilized on a chromatography matrix as well as high sample loading (glycoprotein to be separated). Affinity electrophoresis requires abundant quantities of lectin preparations. Moreover, the determination of lectin concentration in terms of activity is sometimes difficult, since lectins are quite often unstable in solutions [9]. One of the most frequently studied glycoproteins is alphafetoprotein (AFP) [lo]. This oncofetal protein, present in normal human fetuses as well as in other mammalian species, is also widely applied as a tumor marker, specific for hepatocellular carcinoma and germ line tumors having com0173-@835/Y110606-0414 $3.5@+.25/0
Counterflow affinity isotachophoresis
Electruphoresis 1991, 12, 414-419
ponents originating from yolk sac. AFP is an ideal model for microheterogeneity studies due to its relative structural simplicity. AFP contains a single biantennary sugar chain, consisting of mannose, N-acetylglucosamine, galactose, fucose, and sialic acid. Variability of the AFP molecule inciudes differences in sialic acid residues on the ends of the antennas as well as in the presence of side chains of fucose and “bisecting” N-acetylglucosamine moieties [ll]. Different contents of glycosylation variants, reacting and nonreacting with iectins, are characteristic for AFP, arising from different fetal and tumor tissues [12, 131. The principle of counterflow isotachophoresis on cellulose acetate membranes was described earlier [l].The separation of molecules is carried out in a discontinuous buffer system. Two buffers share a common cation, whereas they differ in anions. As anions, chloride (as leading electrolyte) and p-alanine (as terminating electrolyte) are used. Both anions differ in electrophoretic mobilities, and the mobilities of separated macromolecules are in the range between them. Carrier ampholytes, mixed with the sample, serve as spacers, interposed between zones of separated macromolecules. Despite the high electroendosmosis in CAM, its peculiar properties enable a constant flow of liquid through the membrane. Thus the cathodic counterflow, formed in the opposite direction to the electrophoretic movement, provides stable separation conditions. Paradoxically, it creates for isotachophoresis conditions as in an electroendosmosis-free medium. The effective counterflow can also serve as a “conveyer belt”, moving immunoreagents through antigens or antibodies immobilized on the membrane, allowing for semiautomatic immunoblot development. In such experiments all the moving components migrate with the counterflow of the same velocity. In consequence several immunoreagents can be applied in a single run. The antibody directed against the antigen on the membrane and the subsequent immunoreagents (as second antibody, enzyme-labeled antibody, etc.) migrate as isolated zones, separated by free
alettrophoresh 6
-
sample + amphdytes
-
buffer zones (acting as “washing”). Consequently, all the steps (incubations with the immunoreagents and washings) proceed automatically. This method was described by Abelev et a/. [3, 41. This paper contains a modification of affinity electrophoresis, combining the advantages of isotachophoretic separation of proteins with the specificity of lectin/glycoprotein interactions.
2 Materials and methods 2.1 Material and equipment
Cellulose acetate strips (Cellogel) were obtained from M.A.L.T.A. (Milano, Italy) or Serva (Heidelberg, Germany) in 350 pm thick (“normal thickness”) sheets, 4 X 17 cm. Agarose HSA was from Litex (Glostrup, Denmark). Concanavalin A (Con A) was obtained from Pharmacia (Uppsala, Sweden), Ricinus communis lectin (RCA) from Sigma (St. Louis, MO, USA). Lentil lectin (LCA) was extracted with saline from Lens culinaris seeds and purified by affinity chromatography on a Sephadex G-50 column [9]. Carrier ampholytes Servalyt 4-5 and Servalyt 5-7 were from Serva. They were used blended 1:1, diluted 1:lO with anode buffer. D(+)galactose, D(+)glucose and methylalpha-D-glucopyranoside were from Sigma. All other reagents were from Serva. Pooled human amniotic fluid was obtained from early pregnancy, spent culture medium was from human hepatoma cell line HUH6, ascitic fluid from a hitologically proven hepatoma patient, and a serum sample from a patient with testis tumor (yolk sac tumor). All samples contained from 10 to 50 pg AFP/mL. Horse antihuman AFP-antibodies were obtained by courtesy of Dr. Hirai,Tokyo. The electrophoretic equipment was either commercial (supplied by BiophizPribor, Moscow, USSR) or laboratory (all-glass, glued with silicone rubber). .
/-
separated protein bands
counterflow
anodk pocket
I
transfer onto an qarose qel (second dlmenslonl
I
c lecth
+ r
415
bromophand bluc ZOM
(dvhq prdectrophoreslsl
d
h a g a r o s e qel with antibodies
b
Qirst dimension1
Figure 1. AFF-ITP on cellulose acetate membranes. (a) Principle o f counterflow
affinity isotachophoresis. The arrows show direction of migration of the components. (b) Experimental set-up o f the first dimension (isotachophoresis). (c) CAM strip after completing the first-dimensional run. (d) Second dimension (crossed immunoelectrophoresis). The width and length of the CAM strips and the agarose plate have changed proportions in the figure.
41 6
D. Schranz eta/
Elertrophorrrirs 1991, 12. 414-419
2.2.1 Counterflow affinity isotachophoresis (AFF-ITP)
CAM strips, cut 0.5 crn wide, were folded to form two 0.5 cm “pockets” 6.5 cm apart (see Fig. l),soaked with leading buffer (containing traces of Bromophenol Blue, BPB), and placed on the supporting bars of the apparatus. The electrolyte chambers were filled with buffers (50 mL each).The an-
control (no lectin)
Con A
odic chamber contained the leading buffer (0.06 M TrisHCI, pH 6.7).The cathodic chamber contained the terminating buffer (0.012 M Tris-B-alanine, pH 8.6). Two layers of Whatman No. 1filter paper soaked with appropriate electrolyte were used as electrode wicks. Preelectrophoresis was carried out at 150 V constant voltage, until clearly visible, sharp blue zones of BPB were seen on the cathodic sides of
LCA
RCA
amniotic fluid
amniotic fluid
hepatoma ascites
hepatoma ascites
hepatoma medium
hepatoma medium
yolk sac tumor
yolk sac tumor
control (no lectin)
Con A
LCA
RCA
Figure2. Separation of human AFPfrom different samples by AFF-ITP on cellulose acetate membranes. Anode on the right; anode of the second dimension on the top. Samples: top row: amniotic fluid, 10 pL; second row: ascitic fluid from hepatoma patient, 3 pL; third row: spent culture medium from human hepatoma cell line H U H 6 , 2 WL;bottom row: serum from testis tumor patient (yolk sac tumor), 10 pL; left column: control (no lectin); second column: ConA,0.1 mgpersample; third column: LCA0.02 mgpersample; right column: RCA,0.1 mgpersample; Experiment with amnioticfluid with RCA is lacking. Carrier ampholytes added to the sample: 7.5 pL of 1:l mixture of Servalyt 4-5 and Servalyt 5-7 prediluted 1:lO with anode buffer. 0.7 pL/cm2 of horse anti-human AFP antibodies in the second dimensional gel.
Counterflow affinity isotachophoresis
Elertriiphoresis 1991, 12, 414-419
the CAM strips (Fig. 1b). Then the anodic pockets were filled with lectin solutions (previously dialyzed against leading buffer, containing 2 mM CaCl,), 100 pL each. ConA was used in a 1 mg/mL concentration; LCA, 0.2 mg/mL; and RCA, 1 mg/mL. Thus the amount of lectins per sample was 0.1 mg for Con A and RCA and 0.02 mg for LCA. A droplet of vitamin B,, solution was placed on the CAM strip,nearthe anodic pocket,as an eleactrophoreticallyneutral marker of the counterflow [3].When the red colored B,, spots had migrated about 2 cm from the anodic pockets,carrier ampholytes (7.5 pL of the 1 : l O solution in leading buffer) were added to the cathodic pockets. Sample solutions were added to the same pockets. The samples contained approximately 300 ng of AFP each. The current was subsequently increased to 300 V and isotachophoresis was performed until the B,, spot crossed the blue BPB zone and moved an additional 3 cm. Then the individual strips were cut with scissors (Fig. lc) and immediately transferred onto previously prepared agarose gel plates, as described below. They were placed membrane-face down. Before contact with the CAM strip, the agarose surface was blotted for a short time with a strip of Whatman No. 1 filter paper. Location of the blue zones on the CAM strips (albumin-bound BPB and free BPB) were marked on the agarose gel surface by punching the gel. They served as reference points for comparison of the plate with controls (the small puncture on the gel surface stays visible after staining of the gel).The
control (no lectin)
Con A
417
agarose gel plates were cast on 6.5 X 9 cm glass plates. One third of the gel (from the cathode) contained the appropriate inhibitory sugar (2.5 O/o). The anodic two-third part contained horse anti-AFP antibodies 0.7 hL/cm2. Seconddimensional immunoelectrophoresis was carried out according to Grubb [14] with 2 V/cm constant voltage. After 18 h of electrophoresis the gel plates were washed in saline, pressed, dried, and stained in the standard way 1151,
2.2.2 Reference method and controls Agarose gel crossed affinity immunoelectrophoresis (CAIE) was suitable as reference method.It was carried out according to Bog-Hansen [ 161. The first-dimensional gel contained 1 mg Con A or 0.2 mg LCA per mL agarose. For crossed affinity electrophoresis with RCA, affinity chromatography-purified preparations of RCA with a concentration corresponding to 1 mg/mL gel was used. The amount of lectins per sample was 2.25 mg for Con A and RCA, and 0.45 mg for LCA. Samples and antibody for the seconddimensional gel were used as in isotachophoresis. In AFFITP, the anodic (lectin) pockets, filled either with both lectin solutions together, with an excess of inhibitory sugar ( 5 O/o concentration: D(+)galactose for RCA or methylalpha-D-glucopyranoside for Con A and LCA) or with leading buffer alone, were employed as controls.
LCA
RCA
amniotic fluid
amniotic fluid
hepatoma ascites
hepatoma asc ites
hepatoma medium
hepatoma medium
yolk sac tumor
yolk sac tumor
control (no lectin)
Con A
LCA
RCA
Figure 3. CAIE. Anode on the right; anode ofthe second dimension on the top. Samples and antibodies in the second-dimensional gels as in Fig. 2. Left column: control (no lectin); second column: Con A 1 mg/mL agarose gel; third column: LCA,0.2 mglmLge1; right column: RCA,concentration corresponding to 1 mg/mL gel. Experiment with amniotic fluid with RCA is lacking.
418
D. Schranz eta/.
3 Results Experiments with Con A, performed by both AFF-ITP and CAIE have shown resolution of AFP into two variants: Con A-reactive and Con A-nonreactive (Figs. 2 and 3, second column). The areas covered under immunoprecipitates corresponding to either variant were comparable in both techniques.The majority ofAFP in amniotic fluid (top row) and hepatoma patient ascitic fluid (second row) consisted of the reactive variant (73 Yo for amniotic fluid and 92% for hepatoma ascites), whereas the majority ofAFP in hepatoma cell culture medium (65%; third row) and yolk sac tumor patients serum (72%; bottom row) was Con Anonreactive. Both variants were better separated in AFFITP. Experiments with LCA by CAIE (Fig. 3, third column) have shown three AFP variants, i.e. LCA-nonreactive, weakly reactive, and strongly reactive for the amniotic fluid sample (top row). The latter two were incompletely separated. In the AFF-ITP experiments (Fig. 2) results were similar,with slightly better separation of the reactive variants. The hepatoma ascites AFP sample was separated into two variants in CAIE (Fig. 3; second row), whereas the smaller nonreactive variant was split into two (weakly reactive and nonreactive) in AFF-ITP (Fig. 2; second row). Separation of hepatoma cell culture AFP (third row) was similar in both CAIE and AFF-ITP (two variants, the nonreactive one smaller). The yolk sac tumorAFP behaved similarly in both methods, forming a small LCA-nonreactive variant and a reactive, major one. Experiments with RCA were not comparable in these two techniques because the reaction of AFP with RCA is lectin concentration-dependent and does not reach saturation (Breborowicz and Gryska, personal communication). As a result, splitting of AFP into 3-4 peaks in CAIE was observed (Fig. 3, right-hand column). In AFF-ITP up to 5 variants can be seen (Fig. 2, right-hand column). Control experiments (either in runs lacking lectins, or with lectins to-
Elrcfrophoreeiis 1991. 12, 414-419
gether with an inhibitory sugar) showed single AFP peaks in AFF-ITP for three lectins. With increasing amounts of carrier ampholytes, separation of AFP into subfractions differing in electrophoretic mobility was achieved (Fig. 4) without lectins. The same effect was observed earlier in isoelectric focusing [17].
4 Discussion We have demonstrated that counterflow isotachophoresis may be applied for different immunochemical systems. Its simplicity, lack of any special equipment, together with high sensitivity and easy combination with immunodetection, favors this method for studies of biologically important macromolecules. In this paper we report a new modification of CAM-ITP: afinity isotachophoresis with free lectins driven by electroendosmotic counterflow. This modification can be used alternatively to the standard CAIE method. In the method described above, separated proteins are in a steady state. The electrophoretic movement of separated proteins towards the anode is balanced by the cathodic counterflow. As a consequence, they exhibit no migration after achieving equilibrium. The majority of plant lectins exhibit slow cathodic migration at a pH above 8.0 [8,18]. In the described system they are passively driven towards the cathode due to their low mobility. The uniform stream of lectin, migrating from the anodic pocket, meets the protein in a steady position. Glycoproteins with sugar residues reacting with lectin move together with lectin. Carrier ampholytes, acting as spacers, improve the separation of reacting protein from its nonreacting part. The advantage of the modification described is the simplicity of the system. In comparison to other isotachophoresis systems (e.g. capillary isotachophoresis) it does not need any special equipment. The resolution of the method is high. The amount of lectins to be used in AFF-ITP is small, namely about 20 times, less than required on CAIE. These features qualify the method as the alternative to the stand-
Figure 4. Isotachophoresis of AFP samples
on CAM with increasing amount of carrier ampholytes. Sample: ascitic fluid from hepatoma patient, 3 pL. Carrier ampholytes (diluted 1:lO in leading buffer). Top row: Servalyt 4-5 and Servalyt 5-7 (l:l), from left: 5 pL, 10 pL, 15 pL. Second row: Servalyt 3-10, from left: 5 pL, 10 pL, 15 pL, 20 pL, 25pL. Third row: Servalyt 5-7, from left: 5 pL, 10 pL, 15 pL, 20 pL, 25 pL. Bottom row: Servalyt 4-5,from left: 5 pL. 10 pL, 15 pL; 0.7/cmZ pL of horse antihuman AFP antibodies in the seconddimensional gel.
Counterflow affinity isotachophoresis
E l m m p h o r e s i s 1991. 12, 414-419
ard CAIE. There are also some disadvantages common to AFF-ITP and CAIE, the main one being that each new lectin batch has to be tested because its variability can influence microheterogeneity patterns [ 191. Self-concentrating properties of ITP enable the use of highly diluted biological samples, as in other counterflow-ITP modifications. Previously, interaction of Con A with human serum glycoproteins during isotachophoresis was studied by Bog-Hansen etal. [20]. They applied preparative ITP in a polyacrylamide gel column. The method enabled purification of some serum proteins differing in reactivity with Con A and electrophoretic mobility. For analysis of the isotachophoretic separation in CAM with lectins we have applied crossed immunoelectrophoresis as the second dimension. It allowed us easy comparison with the standard method (CAIE). However, it is also possible to combine AFF-ITP with immunoblotting, as described earlier for ITP alone [3]. Such an approach would be comparable to the lectin affinity electrophoresis followed by the antibody-affinity blotting technique of Taketa etal. [21,22]. Other possibilities are the use of lectins (or other interacting molecules), immobilized on nitrocellulose membrane instead of passing them freely in a counterflow stream. Such experiments are in progress.The presented new modification may find several applications due to its simplicity and versatility. Received December 4, 1990
419
5 References [ l ] Abelev,G.I.andKaramova,E.R.,Anal.Biochem. 1984,142,437-444, 121 Abelev, G. I. and Karamova, E. R.,Sov. Med. Rev. Oncol. 1987, I, 85118. [3] Abelev, G. I. and Karamova, E. R., Molec. Immunol. 1989,26,41-47. [4] Abelev, G. I., Karamova, E. R.,Yazova,A. K . , Molec. Immzrnol. 1989, 2649-52. [5] Brqborowicz, J . and Mackiewicz, A , , Electrophoresis 1989, 10, 568573. [6] Lis, H. and Sharon, N., Annu. Rev. Biochem. 1986, 55,35-67. [7] Smith, C. J. and Kelleher. P. C., Biochim. Biophys. Acta 1973,231. [8] Bog-Hansen,T. C., Bjerrum, 0.J. and Ramlau, J.,Scand. J. Immunol. 1975, 4. Supplement 2, 141. [9] Franz, H., Advances in Lecfin Research, VEB Verlag Volk und Gesundheit, Berlin 1988. [lo] Abelev, G. I., Perova, S. D., Khramkova, N. I . , Postnikova, Z . A. and Irlin, I. S., 7?unsplant. Bull. 1963 I , 174-179. [I I ] Tsuchida,Y.,Yamashita, K., Kobata, A.,Nishi, S . , Endo, S . and Saito, S., Tumor Biol. 1984, 5, 23-34. [12] Rrqborowicz, J., TumorBiol. 1988, 9, 3-14. [13] Taketa, K., Ichikawa, E., Sato, J.,Taga, H. and Hirai, H., ElertrophorpS ~ S1989, 10, 825-829. [14] Grubb, A. O., Scand. J. Immunol. 1983,17, Supplement 10,113-124. [IS] Svendsen, P. J., Weeke, B. and Johanson, B.-G., Scand. J. Immunol. 1983, 17, Supplement 10,3-10. [16] Beg-Hansen,T.C.,Scand.J.Immunol. 1983,17,Supplement 10,243253. Biophys. Acra 1975, [17] Lester,E.P.,Miller,J.B.andYachnin,S.,Biochim. 536, 165-171. [I81 Bog-Hansen, T. C. and Takeo K., Electrophoresis 1980, I , 67-71. [19] Hansen, J.-E. S., Bog-Hansen, T. C., Pedersen, B. and Neland. K., Electrophoresis 1989, 10, 574-578. [20] Beg-Hansen,T. C., Svendsen, P. J . and Bjerrum, 0.J., in: Righetti, P. G., (Ed.), Progress i n Isoelectric Focusing and Isotachophoresis. Elsevier, Amsterdam 1975. [21] Taketa, K., Ichikawa, E., Taga H. and Hirai, H., Electrophoresis 1985, 6, 492-497. [22] Taketa, K. and Hirai, H., Electrophoresis 1989. 10, 562-567.
D.Schranz
Electrophoresis 1991, 12, 414-419
eta!.
5 References [l] Radola, B. J., Electrophoresis 1980, 1, 43-56. [2] Allen, R. C., Electrophoresis 1980, 1, 32-37. 131 Kling, H., Sawatski, G . and Geis, W., Anal. Biochem. 1980,174,589592. [4] Merril, C. R., Goldman, D. and van Keuren, M. L., Electrophoresis 1982, 3, 17-23. [5] Guevara,J., Johnston, D.A. Ramagli,L. S.,Martin B.A., Capitello,S. and Rodriguez, L. V. Electrophoresis 1982, 3, 197-205. [6] Dion, A. S . and Pomenti A. A,, Anal. Biochem. 1983,129, 490-496. 171 Sammons, D. W., Adams, L. D. and Nishizawa, E. E., Electrophoresis 1980,2, 135-140. 181 Slisz, M.L. and van Frank, R. M., Electrophoresis 1980,2, 405-407. [9] Heukeshoven, J. and Dernick, R., Electrophoresis 1985, 6, 103-112. [lo] Merril, C. K. and Goldman, D., in: Celis, J. E. and Bravo, R. (Eds.), Two-dimensional Gel Electrophoresis of Proteins, Academic Press, New York 1984, pp. 93-109. [ I l l Yuksel, K. U. and Gracy, R. W., Electrophoresis 1985, 6 , 361-366.
[I21 Gersten, D. M., Wolf, P. H., Ledley, R. S., Rodriguez, L. V. and Zapolski, E. J., Electrophoresis 1986, 7, 327-332. [13] Merril, C. R. and Pratt M. E., Anal. Biochem. 1986, 156, 96-110. [14] Barker, W. C. (Ed.), Protein Identification Resource, National Biomedical Research Foundation, NBR Press, Washington D C 1990. [lS] Laemmli, U. K., Nature 1970,227, 680-682. [ 161 Johnston, D. A,, Mofftt,M.,Tang, K.,Newton, L. and Covnar, W.,An Interactive General Purpose Image Digitizing System, 5th Edition, Department of Biomathematics, M D Anderson Cancer Center, March 1988. [17] Johnston, D. A., White, R. A,, Barlogie, B., Comput. Biomed. Res. 1978, 11, 393-404. [18] Tal,M.,Silberstein,A.and Nusser,E.,J. 5iol. Chem. 1985,260,99769990. [19] Gersten,D.M.,Wolf,P.H.andZapolski,E.J.,Electrophoresis 1987,8, 545-55 1. [20] Mitchell, J. W., Photogr. Sci. Eng. 1978,22, 249-255. 1211 Miller, M. J., Adv. Electrophoresis 1989, 3, 181-217. [22] Merril, C. R., Methods Enzymol. 1990, 182, 477-488.
Dorota Schranz' Stanislaw Morkowskil Eleonora R. Karamova' Garri I. Abelev'
Counterflow affinity isotachophoresis on cellulose acetate membranes
'Department of Pathology and Immunology of Cancer, Academy of Medicine, Pozna6 'Laboratory of Immunochemistry, All-Union Cancer Research Center, Moscow
Counterflow isotachophoresis on cellulose acetate membranes of human alphafetoprotein (AFP) was performed with concanavalin A, lentil lectin, and castor bean lectin driven by electroendosmotic counterflow. This counterflow caused a uniform stream of lectin to migrate towards the cathode against AFP with carrier ampholytes in steady-state position. Retardation of microheterogeneity forms bound to lectins was observed, giving results comparable to standard crossed affinity immunoelectrophoresis.Smal1er amounts of lectins and more diluted samples of AFP could be used in the described method.
1 Introduction Several variants of isotachophoresis (ITP) on porous membranes have been described recently [l, 21. One of them is carried out on cellulose acetate membranes (ITP-CAM). It offers a nondenaturing system suitable for biological macromolecules, in addition to high resolving power. ITPCAM techniques are easy to combine with various immunochemical methods of specific protein detection: immunofixation, immunoelectrophoresis, immunoblotting etc. [3, 41. Since ITP-CAM is not affected by the molecular weight of the separated molecules it may be applied for studies of diverse sets of interacting biological macromolecules. This includes several important systems, e.g. antiCorrespondence: Dr. Dorota Schranz, Department of Pathology and Immunology of Cancer, Medical Academy, ul. takowa 112, PL-61-787 Poznan. Poland Abbreviations: AFF-ITP, counterflow affinity isotachophoresis; AFP, alpha-fetoprotein; BPB, Bromophenol Blue; CAIE, crossed affinity immunoelectrophoresis; CAM, cellulose acetate membranes; Con A, concanavalin A; LCA, lentil lectin; RCA, castor bean lectin
0VCH Vcrlagsgeseilschaft mbH, D-6940 Weinheim, 1991
genlantibody and glycoproten/lectin. The diversity of the carbohydrate structure of glycoproteins (theirmicroheterogeneity) has been studied widely as a model of specific recognition and regulation processes. It can also serve as an additional tool in cancer diagnosis IS]. Lectins, sugar-recognizing proteins present in various organisms, are often used as specific tools for these studies [6]. The most widely applied techniques used for this purpose are affinity chromatography [7] and affinity electrophoresis [8]. Despite their wide use, they suffer from several disadvantages. Affinity chromatography needs lectins immobilized on a chromatography matrix as well as high sample loading (glycoprotein to be separated). Affinity electrophoresis requires abundant quantities of lectin preparations. Moreover, the determination of lectin concentration in terms of activity is sometimes difficult, since lectins are quite often unstable in solutions [9]. One of the most frequently studied glycoproteins is alphafetoprotein (AFP) [lo]. This oncofetal protein, present in normal human fetuses as well as in other mammalian species, is also widely applied as a tumor marker, specific for hepatocellular carcinoma and germ line tumors having com0173-@835/Y110606-0414 $3.5@+.25/0
Counterflow affinity isotachophoresis
Electruphoresis 1991, 12, 414-419
ponents originating from yolk sac. AFP is an ideal model for microheterogeneity studies due to its relative structural simplicity. AFP contains a single biantennary sugar chain, consisting of mannose, N-acetylglucosamine, galactose, fucose, and sialic acid. Variability of the AFP molecule inciudes differences in sialic acid residues on the ends of the antennas as well as in the presence of side chains of fucose and “bisecting” N-acetylglucosamine moieties [ll]. Different contents of glycosylation variants, reacting and nonreacting with iectins, are characteristic for AFP, arising from different fetal and tumor tissues [12, 131. The principle of counterflow isotachophoresis on cellulose acetate membranes was described earlier [l].The separation of molecules is carried out in a discontinuous buffer system. Two buffers share a common cation, whereas they differ in anions. As anions, chloride (as leading electrolyte) and p-alanine (as terminating electrolyte) are used. Both anions differ in electrophoretic mobilities, and the mobilities of separated macromolecules are in the range between them. Carrier ampholytes, mixed with the sample, serve as spacers, interposed between zones of separated macromolecules. Despite the high electroendosmosis in CAM, its peculiar properties enable a constant flow of liquid through the membrane. Thus the cathodic counterflow, formed in the opposite direction to the electrophoretic movement, provides stable separation conditions. Paradoxically, it creates for isotachophoresis conditions as in an electroendosmosis-free medium. The effective counterflow can also serve as a “conveyer belt”, moving immunoreagents through antigens or antibodies immobilized on the membrane, allowing for semiautomatic immunoblot development. In such experiments all the moving components migrate with the counterflow of the same velocity. In consequence several immunoreagents can be applied in a single run. The antibody directed against the antigen on the membrane and the subsequent immunoreagents (as second antibody, enzyme-labeled antibody, etc.) migrate as isolated zones, separated by free
alettrophoresh 6
-
sample + amphdytes
-
buffer zones (acting as “washing”). Consequently, all the steps (incubations with the immunoreagents and washings) proceed automatically. This method was described by Abelev et a/. [3, 41. This paper contains a modification of affinity electrophoresis, combining the advantages of isotachophoretic separation of proteins with the specificity of lectin/glycoprotein interactions.
2 Materials and methods 2.1 Material and equipment
Cellulose acetate strips (Cellogel) were obtained from M.A.L.T.A. (Milano, Italy) or Serva (Heidelberg, Germany) in 350 pm thick (“normal thickness”) sheets, 4 X 17 cm. Agarose HSA was from Litex (Glostrup, Denmark). Concanavalin A (Con A) was obtained from Pharmacia (Uppsala, Sweden), Ricinus communis lectin (RCA) from Sigma (St. Louis, MO, USA). Lentil lectin (LCA) was extracted with saline from Lens culinaris seeds and purified by affinity chromatography on a Sephadex G-50 column [9]. Carrier ampholytes Servalyt 4-5 and Servalyt 5-7 were from Serva. They were used blended 1:1, diluted 1:lO with anode buffer. D(+)galactose, D(+)glucose and methylalpha-D-glucopyranoside were from Sigma. All other reagents were from Serva. Pooled human amniotic fluid was obtained from early pregnancy, spent culture medium was from human hepatoma cell line HUH6, ascitic fluid from a hitologically proven hepatoma patient, and a serum sample from a patient with testis tumor (yolk sac tumor). All samples contained from 10 to 50 pg AFP/mL. Horse antihuman AFP-antibodies were obtained by courtesy of Dr. Hirai,Tokyo. The electrophoretic equipment was either commercial (supplied by BiophizPribor, Moscow, USSR) or laboratory (all-glass, glued with silicone rubber). .
/-
separated protein bands
counterflow
anodk pocket
I
transfer onto an qarose qel (second dlmenslonl
I
c lecth
+ r
415
bromophand bluc ZOM
(dvhq prdectrophoreslsl
d
h a g a r o s e qel with antibodies
b
Qirst dimension1
Figure 1. AFF-ITP on cellulose acetate membranes. (a) Principle o f counterflow
affinity isotachophoresis. The arrows show direction of migration of the components. (b) Experimental set-up o f the first dimension (isotachophoresis). (c) CAM strip after completing the first-dimensional run. (d) Second dimension (crossed immunoelectrophoresis). The width and length of the CAM strips and the agarose plate have changed proportions in the figure.
41 6
D. Schranz eta/
Elertrophorrrirs 1991, 12. 414-419
2.2.1 Counterflow affinity isotachophoresis (AFF-ITP)
CAM strips, cut 0.5 crn wide, were folded to form two 0.5 cm “pockets” 6.5 cm apart (see Fig. l),soaked with leading buffer (containing traces of Bromophenol Blue, BPB), and placed on the supporting bars of the apparatus. The electrolyte chambers were filled with buffers (50 mL each).The an-
control (no lectin)
Con A
odic chamber contained the leading buffer (0.06 M TrisHCI, pH 6.7).The cathodic chamber contained the terminating buffer (0.012 M Tris-B-alanine, pH 8.6). Two layers of Whatman No. 1filter paper soaked with appropriate electrolyte were used as electrode wicks. Preelectrophoresis was carried out at 150 V constant voltage, until clearly visible, sharp blue zones of BPB were seen on the cathodic sides of
LCA
RCA
amniotic fluid
amniotic fluid
hepatoma ascites
hepatoma ascites
hepatoma medium
hepatoma medium
yolk sac tumor
yolk sac tumor
control (no lectin)
Con A
LCA
RCA
Figure2. Separation of human AFPfrom different samples by AFF-ITP on cellulose acetate membranes. Anode on the right; anode of the second dimension on the top. Samples: top row: amniotic fluid, 10 pL; second row: ascitic fluid from hepatoma patient, 3 pL; third row: spent culture medium from human hepatoma cell line H U H 6 , 2 WL;bottom row: serum from testis tumor patient (yolk sac tumor), 10 pL; left column: control (no lectin); second column: ConA,0.1 mgpersample; third column: LCA0.02 mgpersample; right column: RCA,0.1 mgpersample; Experiment with amnioticfluid with RCA is lacking. Carrier ampholytes added to the sample: 7.5 pL of 1:l mixture of Servalyt 4-5 and Servalyt 5-7 prediluted 1:lO with anode buffer. 0.7 pL/cm2 of horse anti-human AFP antibodies in the second dimensional gel.
Counterflow affinity isotachophoresis
Elertriiphoresis 1991, 12, 414-419
the CAM strips (Fig. 1b). Then the anodic pockets were filled with lectin solutions (previously dialyzed against leading buffer, containing 2 mM CaCl,), 100 pL each. ConA was used in a 1 mg/mL concentration; LCA, 0.2 mg/mL; and RCA, 1 mg/mL. Thus the amount of lectins per sample was 0.1 mg for Con A and RCA and 0.02 mg for LCA. A droplet of vitamin B,, solution was placed on the CAM strip,nearthe anodic pocket,as an eleactrophoreticallyneutral marker of the counterflow [3].When the red colored B,, spots had migrated about 2 cm from the anodic pockets,carrier ampholytes (7.5 pL of the 1 : l O solution in leading buffer) were added to the cathodic pockets. Sample solutions were added to the same pockets. The samples contained approximately 300 ng of AFP each. The current was subsequently increased to 300 V and isotachophoresis was performed until the B,, spot crossed the blue BPB zone and moved an additional 3 cm. Then the individual strips were cut with scissors (Fig. lc) and immediately transferred onto previously prepared agarose gel plates, as described below. They were placed membrane-face down. Before contact with the CAM strip, the agarose surface was blotted for a short time with a strip of Whatman No. 1 filter paper. Location of the blue zones on the CAM strips (albumin-bound BPB and free BPB) were marked on the agarose gel surface by punching the gel. They served as reference points for comparison of the plate with controls (the small puncture on the gel surface stays visible after staining of the gel).The
control (no lectin)
Con A
417
agarose gel plates were cast on 6.5 X 9 cm glass plates. One third of the gel (from the cathode) contained the appropriate inhibitory sugar (2.5 O/o). The anodic two-third part contained horse anti-AFP antibodies 0.7 hL/cm2. Seconddimensional immunoelectrophoresis was carried out according to Grubb [14] with 2 V/cm constant voltage. After 18 h of electrophoresis the gel plates were washed in saline, pressed, dried, and stained in the standard way 1151,
2.2.2 Reference method and controls Agarose gel crossed affinity immunoelectrophoresis (CAIE) was suitable as reference method.It was carried out according to Bog-Hansen [ 161. The first-dimensional gel contained 1 mg Con A or 0.2 mg LCA per mL agarose. For crossed affinity electrophoresis with RCA, affinity chromatography-purified preparations of RCA with a concentration corresponding to 1 mg/mL gel was used. The amount of lectins per sample was 2.25 mg for Con A and RCA, and 0.45 mg for LCA. Samples and antibody for the seconddimensional gel were used as in isotachophoresis. In AFFITP, the anodic (lectin) pockets, filled either with both lectin solutions together, with an excess of inhibitory sugar ( 5 O/o concentration: D(+)galactose for RCA or methylalpha-D-glucopyranoside for Con A and LCA) or with leading buffer alone, were employed as controls.
LCA
RCA
amniotic fluid
amniotic fluid
hepatoma ascites
hepatoma asc ites
hepatoma medium
hepatoma medium
yolk sac tumor
yolk sac tumor
control (no lectin)
Con A
LCA
RCA
Figure 3. CAIE. Anode on the right; anode ofthe second dimension on the top. Samples and antibodies in the second-dimensional gels as in Fig. 2. Left column: control (no lectin); second column: Con A 1 mg/mL agarose gel; third column: LCA,0.2 mglmLge1; right column: RCA,concentration corresponding to 1 mg/mL gel. Experiment with amniotic fluid with RCA is lacking.
418
D. Schranz eta/.
3 Results Experiments with Con A, performed by both AFF-ITP and CAIE have shown resolution of AFP into two variants: Con A-reactive and Con A-nonreactive (Figs. 2 and 3, second column). The areas covered under immunoprecipitates corresponding to either variant were comparable in both techniques.The majority ofAFP in amniotic fluid (top row) and hepatoma patient ascitic fluid (second row) consisted of the reactive variant (73 Yo for amniotic fluid and 92% for hepatoma ascites), whereas the majority ofAFP in hepatoma cell culture medium (65%; third row) and yolk sac tumor patients serum (72%; bottom row) was Con Anonreactive. Both variants were better separated in AFFITP. Experiments with LCA by CAIE (Fig. 3, third column) have shown three AFP variants, i.e. LCA-nonreactive, weakly reactive, and strongly reactive for the amniotic fluid sample (top row). The latter two were incompletely separated. In the AFF-ITP experiments (Fig. 2) results were similar,with slightly better separation of the reactive variants. The hepatoma ascites AFP sample was separated into two variants in CAIE (Fig. 3; second row), whereas the smaller nonreactive variant was split into two (weakly reactive and nonreactive) in AFF-ITP (Fig. 2; second row). Separation of hepatoma cell culture AFP (third row) was similar in both CAIE and AFF-ITP (two variants, the nonreactive one smaller). The yolk sac tumorAFP behaved similarly in both methods, forming a small LCA-nonreactive variant and a reactive, major one. Experiments with RCA were not comparable in these two techniques because the reaction of AFP with RCA is lectin concentration-dependent and does not reach saturation (Breborowicz and Gryska, personal communication). As a result, splitting of AFP into 3-4 peaks in CAIE was observed (Fig. 3, right-hand column). In AFF-ITP up to 5 variants can be seen (Fig. 2, right-hand column). Control experiments (either in runs lacking lectins, or with lectins to-
Elrcfrophoreeiis 1991. 12, 414-419
gether with an inhibitory sugar) showed single AFP peaks in AFF-ITP for three lectins. With increasing amounts of carrier ampholytes, separation of AFP into subfractions differing in electrophoretic mobility was achieved (Fig. 4) without lectins. The same effect was observed earlier in isoelectric focusing [17].
4 Discussion We have demonstrated that counterflow isotachophoresis may be applied for different immunochemical systems. Its simplicity, lack of any special equipment, together with high sensitivity and easy combination with immunodetection, favors this method for studies of biologically important macromolecules. In this paper we report a new modification of CAM-ITP: afinity isotachophoresis with free lectins driven by electroendosmotic counterflow. This modification can be used alternatively to the standard CAIE method. In the method described above, separated proteins are in a steady state. The electrophoretic movement of separated proteins towards the anode is balanced by the cathodic counterflow. As a consequence, they exhibit no migration after achieving equilibrium. The majority of plant lectins exhibit slow cathodic migration at a pH above 8.0 [8,18]. In the described system they are passively driven towards the cathode due to their low mobility. The uniform stream of lectin, migrating from the anodic pocket, meets the protein in a steady position. Glycoproteins with sugar residues reacting with lectin move together with lectin. Carrier ampholytes, acting as spacers, improve the separation of reacting protein from its nonreacting part. The advantage of the modification described is the simplicity of the system. In comparison to other isotachophoresis systems (e.g. capillary isotachophoresis) it does not need any special equipment. The resolution of the method is high. The amount of lectins to be used in AFF-ITP is small, namely about 20 times, less than required on CAIE. These features qualify the method as the alternative to the stand-
Figure 4. Isotachophoresis of AFP samples
on CAM with increasing amount of carrier ampholytes. Sample: ascitic fluid from hepatoma patient, 3 pL. Carrier ampholytes (diluted 1:lO in leading buffer). Top row: Servalyt 4-5 and Servalyt 5-7 (l:l), from left: 5 pL, 10 pL, 15 pL. Second row: Servalyt 3-10, from left: 5 pL, 10 pL, 15 pL, 20 pL, 25pL. Third row: Servalyt 5-7, from left: 5 pL, 10 pL, 15 pL, 20 pL, 25 pL. Bottom row: Servalyt 4-5,from left: 5 pL. 10 pL, 15 pL; 0.7/cmZ pL of horse antihuman AFP antibodies in the seconddimensional gel.
Counterflow affinity isotachophoresis
E l m m p h o r e s i s 1991. 12, 414-419
ard CAIE. There are also some disadvantages common to AFF-ITP and CAIE, the main one being that each new lectin batch has to be tested because its variability can influence microheterogeneity patterns [ 191. Self-concentrating properties of ITP enable the use of highly diluted biological samples, as in other counterflow-ITP modifications. Previously, interaction of Con A with human serum glycoproteins during isotachophoresis was studied by Bog-Hansen etal. [20]. They applied preparative ITP in a polyacrylamide gel column. The method enabled purification of some serum proteins differing in reactivity with Con A and electrophoretic mobility. For analysis of the isotachophoretic separation in CAM with lectins we have applied crossed immunoelectrophoresis as the second dimension. It allowed us easy comparison with the standard method (CAIE). However, it is also possible to combine AFF-ITP with immunoblotting, as described earlier for ITP alone [3]. Such an approach would be comparable to the lectin affinity electrophoresis followed by the antibody-affinity blotting technique of Taketa etal. [21,22]. Other possibilities are the use of lectins (or other interacting molecules), immobilized on nitrocellulose membrane instead of passing them freely in a counterflow stream. Such experiments are in progress.The presented new modification may find several applications due to its simplicity and versatility. Received December 4, 1990
419
5 References [ l ] Abelev,G.I.andKaramova,E.R.,Anal.Biochem. 1984,142,437-444, 121 Abelev, G. I. and Karamova, E. R.,Sov. Med. Rev. Oncol. 1987, I, 85118. [3] Abelev, G. I. and Karamova, E. R., Molec. Immunol. 1989,26,41-47. [4] Abelev, G. I., Karamova, E. R.,Yazova,A. K . , Molec. Immzrnol. 1989, 2649-52. [5] Brqborowicz, J . and Mackiewicz, A , , Electrophoresis 1989, 10, 568573. [6] Lis, H. and Sharon, N., Annu. Rev. Biochem. 1986, 55,35-67. [7] Smith, C. J. and Kelleher. P. C., Biochim. Biophys. Acta 1973,231. [8] Bog-Hansen,T. C., Bjerrum, 0.J. and Ramlau, J.,Scand. J. Immunol. 1975, 4. Supplement 2, 141. [9] Franz, H., Advances in Lecfin Research, VEB Verlag Volk und Gesundheit, Berlin 1988. [lo] Abelev, G. I., Perova, S. D., Khramkova, N. I . , Postnikova, Z . A. and Irlin, I. S., 7?unsplant. Bull. 1963 I , 174-179. [I I ] Tsuchida,Y.,Yamashita, K., Kobata, A.,Nishi, S . , Endo, S . and Saito, S., Tumor Biol. 1984, 5, 23-34. [12] Rrqborowicz, J., TumorBiol. 1988, 9, 3-14. [13] Taketa, K., Ichikawa, E., Sato, J.,Taga, H. and Hirai, H., ElertrophorpS ~ S1989, 10, 825-829. [14] Grubb, A. O., Scand. J. Immunol. 1983,17, Supplement 10,113-124. [IS] Svendsen, P. J., Weeke, B. and Johanson, B.-G., Scand. J. Immunol. 1983, 17, Supplement 10,3-10. [16] Beg-Hansen,T.C.,Scand.J.Immunol. 1983,17,Supplement 10,243253. Biophys. Acra 1975, [17] Lester,E.P.,Miller,J.B.andYachnin,S.,Biochim. 536, 165-171. [I81 Bog-Hansen, T. C. and Takeo K., Electrophoresis 1980, I , 67-71. [19] Hansen, J.-E. S., Bog-Hansen, T. C., Pedersen, B. and Neland. K., Electrophoresis 1989, 10, 574-578. [20] Beg-Hansen,T. C., Svendsen, P. J . and Bjerrum, 0.J., in: Righetti, P. G., (Ed.), Progress i n Isoelectric Focusing and Isotachophoresis. Elsevier, Amsterdam 1975. [21] Taketa, K., Ichikawa, E., Taga H. and Hirai, H., Electrophoresis 1985, 6, 492-497. [22] Taketa, K. and Hirai, H., Electrophoresis 1989. 10, 562-567.
