EIPcrrophoresir 1991, 12, 367-372
Lurninography
367
Reinhard Schneppenheim' Ulrich Budde' Nicolaus Dahlrnand Peter Rautenberg4
Luminography - a new, highly sensitive visualization method for electrophoresis
'Abteilung Allgemeine Paediatrie, Klinikum der Christian-AlbrechtsUniversitat, Kiel 'Allgemeines Krankenhaus Harburg, Hamburg 'Institut fur Klinische Biochemie, Bonn 4Abteilung Klinische Mikrobiologie, Klinikum der Christian-AlbrechtsUniversitat, Kiel
A highly sensitive method for protein visualization following electrophoresis and protein blotting was developed. The method is based on the light-emitting reaction of luminol and hydrogen peroxide catalyzed by horseradish peroxidase. The luminescent assay can be applied either to the native gel or after protein blotting, and it has a sensitivity two orders of magnitude higher than that achieved with chromogenic detection systems. Analogous to autoradiography the luminescent signal is recorded on an X-ray film with similar sensitivity. We present several examples of application emphasizing the general versatility of this innovative method.
1 Introduction Bio- and chemiluminescent assays have found wide acceptance in research and applied clinical chemistry, biochemistry, and microbiology [I]. One of the first examples was the measurement of ATP, with the luciferase/luciferin system, from firefly lanterns [2]. The potential value of light-emitting detection systems for immunoassays was shown by Schroeder et al. [3], who developed a chemiluminescent immunoassay for biotin. The chemiluminescent system with the widest applicability seems to be the reaction of luminol and hydrogen peroxide catalyzed by horseradish peroxidase. The mechanism of luminol chemiluminescence in aqueous solutions involves the oxidation of luminol by an oxidant (H202),catalyzed by peroxidase, producing a lumino1 radical. After further reactions, an endoperoxide is formed that decomposes, leaving an electronically excited 3-aminophthalate dianion. Light is emitted on its return to the ground state [4]. Horseradish peroxidase is also known as a suitable label ofantibodies,used in enzyme immunoassays and immunohistochemistry, forming a colored complex from a colorless chromogenic substrate. Using lumino1 as a substrate, light will be emitted, which can then be measured by use of a luminometer. Although the high sensitivity of luminescent immunoassays was well known, their applicability as electrophoretic visualization systems was not reported until the end of 1986, when Laing [ 5 ] described a luminescent method for detecting erythrocyte membrane antigens on Western blots. The assay, however, was found to be only four times more sensitive than the chromogenic peroxidase reaction. Concurrently we adapted an enhanced luminescent test tube assay for the von Willebrand factor antigen, described by Wang eta/. [6] for the detection of antibodies to the human immunodeficiency virus (HIV) antigens after sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Correspondence: Dr. R. Schneppenheim, Department of Pediatrics, Klinikum der Christian-Albrechts-Universitat Kid , Schwanenweg 20, W-2300 Kiel 1, Germany Abbreviations: ELISA, enzyme-linked immunosorbent assay; HIV, human immunodeficiency virus; IEF, isoelectric focusing; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; vWd, von Willebrand disease; vWf, von Willebrand factor
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
(SDS-PAGE) and Western blotting [7]. In this article we summarize our experience with the luminescent visualization system, giving some examples of its application to the diagnosis of diseases, diagnosis of bleeding disorders, detection of drug- or food-induced antibodies, and to forensic medicine.
2 Materials and methods 2.1 Electrophoresis and blotting techniques 2.1.1 Detection of horseradish peroxidase isozymes To test the applicability of a luminescent assay to electrophoresis perse, we attempted a preliminary electrophoretic study on peroxidase isozymes. About 100 mg horseradish roots (Amoracia rusticana) were minced in about an equal amount (w/v) of distilled water in an Eppendorf vial, and centrifuged for 5 min in an Eppendorf centrifuge. Two pL supernatant were applied by filter paper onto the surface of a polyacrylamide isoelectric focusing (IEF) gel (pH 3.59.5), cast and run as recommended by Pharmacia (Uppsala, Sweden). The gel was then equilibrated in Tris-buffered saline (TBS: 0.02 M Tris, 0.5 M NaCl, pH 8) for30 min prior to visualization.
2.1.2 Multimeric structure of von Willebrand factor To establish the high sensitivity of luminogaphy, we compared the luminescent assay with '251-autoradiography. Plasma samples from von Willebrand disease (vWd) patients and from normal controls were applied to two identical SDS agarose gels each, and run with a discontinuous buffer system, according to Ruggeri and Zimmermann [S]. After electrophoresis the gels were either fixed, washed, and dried for autoradiography, or equilibrated in transfer buffer for 1 h, with two changes of buffer, prior to electrophoretic transfer onto nitrocellulose for detection by luminography. Transfer buffer was 0.05 M phosphate, pH 7.4, with 0.04% SDS, without methanol [9]. Transfer was carried out using a Hoefer Transphor (Hoefer Scientific Instruments, San Francisco, CA) at a temperature of 16"C, constant voltage of 33 V and 2.5 Amps. Only one gel at a time was transferred in one tank. The gel was placed near the cathode. Transfer was completed in 2 h. 0173-0835/91/0505-0367 $3.50+.25/0
368
Elecrrophoresis 1991, 12, 361-312
R. Schneppenheim e t a / .
2.1.3 Western blot for antibodies to HIV The presence of antibodies to HlV antigens in human sera was tested by means of a commercially available HlV Western blot test kit (DuPont/Biotech, Bad Nauheim, Germany).This test system employs biotin-tagged, anti-human antibodies and peroxidase-labeled avidin. Peroxidase activity was made visible by the chromogenic assay, using 4-chloro-1-naphthol as substrate, and by the luminescent assay to compare their sensitivities. 2.1.4 Heterophilic antibodies A protein extract from bovine pancreatic tissue (Wobenzym, Mucos Pharma, Geretsried, Germany), used as “alternative anti-cancer drug”, was separated by means of SDSPAGE in gels of the Midget-system (Pharmacia-LKB, Uppsala, Sweden) using a linear pore gradient (4-22.5 %T, 4%C) and the Laemmli buffer system [lo] under nonreducing conditions. Twenty pg protein was applied to each slot. Electrophoresis was followed by electrophoretic transfer onto nitrocellulose in a semi-dry blotting device (CTI, Idstein, Germany) according to the manufacturer’s recommendations. 2.1.5 Human IgG antibodies against gliadin SDS-PAGE of gliadin (Sigma, Taufiirchen, Germany) followed by electroblotting was carried out according to Laemmli [lo] and Towbin [ll], respectively. Antibodies against gliadin in a child with coeliac disease were visualized by incubation of this Western blot with the patient’s sera, diluted 1:lOO. Intermediate washing was followed by incubation with a peroxidase labeled mouse antibody against human IgG (Sigma) [12], and subsequently by the detection of the peroxidase label using luminography. 2.1.6 Factor XIIIA Plasma samples (n=218), drawn from nonrelated individuals of a northern German population were investigated by IEF in an agarose gel for polymorphism at the FXIIIA locus. The gel was cast and run as recommended by Pharmacia, using a 1:l mixture of carrier ampholytes, Pharmalyte pH 3.5-9.5 and pH 5-8, respectively (Pharmacia). Plasma samples were diluted 1:20 with distilled water, and 2 pL each was applied to the gel by means of filter papers. After the run, proteins were transferred to nitrocellulose by simple capillary blotting for 30 min.
Table 1. Antisera and antibodies used for detection of the antigen described. Antigen First antibody Second antibody Goat anti-rabbit Rabbit FVIIIR: Ag antiserum antibody, peroxidase-labeled Behring, 1:500 Bio-Rad, 1:2000 Anti-human IgG Human serum HIV Western blot biotin-tagged and 1:lOO commercial test peroxidase-labeled (Dupont) avidin as described by Dupont Rabbit anti-human Human serum Wobenzym IgG antibody peroxi1.75 Immunostimulant dase-labeled (Mucos Pharma) Sigma, 1500 Mouse anti-human IgG Human serum Gliadin (Sigma) 1:lOO antibody peroxidaselabeled, Sigma, 1:200O Goat anti-rabbit Rabbit anti-FXIIIA Factor XlIIA antibody peroxidasefrom human plasma Behring, 1:500 labeled, Bio-Rad, 1:2OOO
vWf from human plasma
milk powder) for 10 min, then incubated with the first antibody and, after two washing steps inTTBS,with the second antibody. Antisera and antibodies used in these assays are given in Table 1. After two further washing steps the filters were transferred to 0.02M Tris buffer, pH 7.5, for the chromogenic assay or to 0.02M Tris buffer, pH 8.0, for the luminescent assay. 2.3 Visualization 2.3.1 Autoradiography Autoradiographs were obtained by exposing Kodak X-OMAT AR film to the dried gel in combination with two intensifying screens. Exposure time was 72 h. 2.3.2 Chromogenic assay 4-Chloro-1-naphthol was used as the chromogenic substrate recommended by Bio-Rad (Miinchen, Germany) for use in their immunoblot assay. The staining recipe was according to the Bio-Rad immunoblot manual (60 mg 4-chloro-1-naphthol in 20 mL methanol, 100 mLTBS buffer and 60 pL H,O, (30%). Incubation time was 30-45 min. 2.3.3 Luminescent assay
2.2 Immunoassays
Either the gels or immunoblots were both covered for 1-2 min by an evenly distributed solution of about 5 mL of 2.2.1 Autoradiography 0.02 M Tris, pH 8, containing 0.4 mg/mL of luminol (Sigma), 0.1 mg/mL of 4-iodophenol (Aldrich, Steinheim, GerAutoradiography was carried out essentially as described many), and 2.5 pL/mL of 30 O/o H,O,. Stock solutions of 10 X [8] using 12’I-labeledrabbit anti-human vWf antibody. luminol and 10 X 4-iodophenol can be prepared with dimethy1 sulfoxide (DMSO) and stored in a freezer for an unlimited time. The reaction mixture, however, was prepared 2.2.2 Immunoblot immediately prior to use. In a darkroom either the luminating gel or the Western blot is covered by a thin translucent After electrophoretic or capillary transfer to nitrocellulose, the filters were blocked in antibody buffer ( 0 . 0 2 ~ Tris, 0 . 5 ~ plastic film to protect the X-ray film from getting wet. VariNaCI, 0.05% Tween20, pH 7.5 = TTBS + 5% non-fat dry ous brands of X-ray films are suitable, since all of them are
Ele 10 000 kDa, is visualized by means of 'Z51-labeled antibodies [8].Visualization has also been attempted by use of peroxidase-labeled antibodies and a chromogenic substrate [9, 14-17]. However, these assays are less sensitive than the radioactive one, and, in cases of low antigen concentration, do not allow the reliable analysis of the multimeric structure. To establish the high sensitivity of luminographywe compared the luminescent assay with '251-a~toradiography. Figure 2 compares autoradiography versus luminography in the visualization of the multimeric pattern of vWf. Even at a plasma dilution of 1500, vWf multimers can still be evaluated by either method (Fig. 2a). Assuming the normal plasma concentration of vWf to be 10 pg/mL [18], this dilution would correspond to a quantity of 400 pg vWf in 20 pL
Figure 3. HIV Western blots of a patient who seroconverted after transfusion of a retrospectively contaminated blood unit. (a) Luminography detected seroconversion considerably earlier (day 28 post transfusion) than either the chrornogenic assay or the ELISA screening test (day 57 post transfusion). (b) Comparison of sensitivity between luminography, chromogenic assay, and ELISA screening test. Luminography detected antibodies against HIVat by the chromogenic assay or by ELISA. Exposure time forluminoconcentrations two orders of magnitude lower, at a serum dilution of lo-*versus graphy was 3 min. 3*= same blot as -1g of serum dilution = 3, but exposure time was reduced to 3 s to optimize luminography. Results of the ELISA screening test are stated as '+' or '-'.
E I ~ c t r o p h o r e s i1991, ~
12, 367-372
of sample to be applied to the gel. This quantity is then separated by electrophoresis into a set of up to 20 multimers, each of them resolved as a triplet by our electrophoretic system. By slot blot analysis of different plasma concentrations of vWf, we could estimate the sensitivity of luminography to be down to 10 pg of protein (results not shown). Furthermore, the different types and subtypes of vWd can be reliably discriminated by luminography. There was virtually no difference in the two methods with respect to the banding patterns observed in patients with vWd types IIA, IIB, IIC, IID, IIE, and IIF (Fig. 2b). The multimeric patterns can also be evaluated by densitometry, as shown in Fig. 2c.
3.3 HIV Western blot Figure 3 shows the results of testing for HIV antibodies in human sera. By luminography the detection of antibodies to HIV in one patient was made possible 28 days after the assumed infection by a retrospectively contaminated blood transfusion. Neither the chromogenic Western blot nor the enzyme-linked immunosorbent assay (ELISA) screening test were reactive prior to day 57 after transfusion. Sensitivity with respect to the detection of antibodies was shown to be 100 times higher in luminography than in the conventional chromogenic assay (Fig. 3b).
3.4 Heterophilic antibodies Immunoassays of the “sandwich type, performed directly on serum, can yield aberrant results due to heterophilic antibodies in the specimen, which may bind to reagent mouse antibodies, causing false positive results. These interferences are potentially serious pitfalls. Acceptance of spuriously elevated results leads to a great deal of unnecessary followup testing or, even worse, of superfluous therapy. Pre-
Figure 4. Detection of heterophilic antibodies reactive to the immunostimulant Wobenzym, with crossreactivity to mouse immunoglobulins. Comparison between luminography and chromogenic visualization subsequent to SDS-PAGE. (A) molecular weight markers; (B)-(E) Wobenzym, (B) unspecific silver stain of Wobenzym, (C) chromogenic and (D) luminographic detection after incubation with patient’s serum, (E) negative luminographic control incubated only with second antibody without prior incubation with patient’s serum.
Luminography
37 1
viously, we reported on a patient who underwent adjuvant chemotherapy because of a spuriously elevated tumor marker-alphafetoprotein (AFP) [19].The factor which was responsible for this false increase could be purified and was identified as an IgG molecule 1201. In addition to the patient’s recommended cancer therapy, he used 18 different preparations of alternative anti-cancer drugs. To obtain clues pertaining to the etiology of the heterophilic antibody, we analyzed these preparations. All of them were immune-stimulating substances of partly plant, partly animal origin. Of all 18 preparations tested, only “Wobenzym”, a protein extract from bovine pancreatic tissue (Mucos Pharma, Geretsried, Germany), was reactive with the IgG fraction of the patient’s serum. As shown in Fig. 4, detection of heterophilic antibodies was questionable, using the chromogenic assay, although the sandwich assay was clearly positive. However, using luminography, a strong signal can be obtained, which is even more intense than the unspecific silver stain, indicating clearly the existence of IgG antibodies to Wobenzym.
3.5 Human IgG antibodies against gliadin Elevated titers of IgG and IgA antibodies against gliadin are of diagnostic value in coeliac disease of childhood [21]. Figure 5 shows the detection of antigliadin IgG antibodies in a child with coeliac disease by means of a gliadin Western blot subsequent to SDS-PAGE. The diagnosis was confirmed by small bowel biopsy and an ELISA, specific for antigliadin antibodies (results not shown). Incubation of the Western blot with sera of healthy control persons failed to visualize any protein bands.
Figure 5. Luminographic detection of human antigliadin antibodies by means of immunoblotting subsequent to SDS-PAGE of gliadin. MW, molecular weight markers, P, visualization after incubation with serum from a patient with coeliac disease, C, incubation with serum from a healthy control. Exposure time for luminography, P = 3 s, C = 3 min.
372
R Schneppenliciiii
E/cctrophurrsis 1991. 12, 367-372
cio/
3.6 Factor XIIla polymorphim Factor XIIIA, a transglutaminase, catalyzes the last step in the blood coagulation cascade, the crosslinking of the fibrin clot at y-glutamylle-lysine sites to a stable product. Polymorphism at the FXIIIAlocus is responsible for the existence oftwo common alleles (FXIIIA-I and FXIIIA-2) with reported allele frequencies of 0.8 and 0.2, respectively [22]. This polymorphism makes FXIIIA a useful marker in studies of human population genetics, linkage analysis, and forensic medicine. A typical presentation of FXIIIA electromorphs is shown in Fig. 6. FXIIIA, a dimeric enzyme, displays a 3-banded pattern in the heterozygous state and one band in the homozygous state. Among 218 individuals, only the two common alleles (FXIIIA-1, FXIIIA-2) were detected. Allele frequencies were 0.773 for FXIIIA-1 and 0.227 for FXIIIA-2.The distribution ofphenotypes was consistent with random mating expectations according to the Hardy-Weinberg principle [23] k:=O.128;p=O.74). Studies on 3 families confirmed autosomal codominant inheritance of these alleles.
exposed “hard copies” as permanent records can be obtained from one gel to optimize the result. (v) After elution of the antibodies, even multiple probing for the same or different antigens is possible. (vi) Using non-fat milk as blocking agent for the nitrocellulose membranes, avery low background results, even after prolonged exposure. (vii) Reagents are extremely cheap, which makes the method economical. These advantages make luminography a superior tool for the evaluation of protein electrophoresis. It combines ideally the high sensitivity of autoradiography with the relatively nonhazardous nature of chromogenic assays. It is tempting to also apply this method to the analysis of DNA; however, several experiments with different nonradioactive labeling techniques (e.g. digoxigenin labeling of DNA probes in combination with peroxidase-labeled antidigoxigenin antibodies) failed to show higher sensitivity of DNA luminography when compared with chromogenic assays employing peroxidase or alkaline phosphatase labels.
We thank Mrs. H. El Abd Miiller and Mrs. R . Rosenberg f o r their excellent technical assistance and Mrs. Reimers f o r the photographs. We greatly appreciate suggestions made by J. Ingerslev on electrotransfer methods j o r vW$ This work was supported in part by the Deutsche Forschungsgemeinschaft (grant No. Bu 608/1-1 and grant No. Schn 325/1-1). Received November 7, 1990
5 References
figure 6. Luminographic identification offactorXIII.4 polymorphism following agarose IEF and capillary transfer to nitrocellulose. 1-1, homozygous phenotype ofthe common allele 1; 2-2, homozygous phenotype of allele 2; 1-2, heterozygous phenotype. The three-banded pattern displayed by heterozygotes are consistent with the dimeric structure of FXIIIA.
4 Concluding remarks The potential value of luminography as a highly sensitive visualization method based on peroxidase-mediated light emission has been proven by its successful application to a variety of electrophoretic systems. Peroxidase itself was the subject of electrophoresis in our isozyme study. However, the widest applicability of this enzyme is seen in analyzing antigens and antibodies. Thanks to the availability of specific antibodies to many relevant proteins, adequate visualization assays after electrophoresis of these antigens can easily be designed. Furthermore, membrane-immobilized particular antigens can be used to detect specific antibodies in a sample. The method has many advantages: (i) Luminography is as sensitive as ‘*’I-autoradiographyin the analysis of multimers of vWf without the potential hazards and disadvantages of radioactive isotopes. (ii) The sensitivity is two orders of magnitude higher than the conventional chromogenic peroxidase assay in the detection of specific antibodies to HIV, which is of great clinical relevance. (iii) The method is reliable and reproducible in analyzing particular electrophoretic patterns of proteins at low concentrations, e.g. in vWd and in forensic medicine, as shown by the study of FXIIIA polymorphism. (iv) The method is fast. Expocure times vary between ceconds and minutes, compared with days in the case of autoradiography. Many differently
Kricka,L. J., Stanley,P. E..Thorpe,G.and Whitehead,T.,(Eds.),Anulytiral Applications qf Bioluminescence and Chemiluminescence, 3rd International Symposion on Analytical Applications of Bioluminescence and Chemiluminescence, Academic Press, New York 1984. Strehler, B. L. and Totter, J. R., Arch. Biochem. Biophys. 1952,40,28. Schroeder, H. R.,Vogelhut,P. O., Carrico,R. J.,Boguslaski,R. C.and Buckler, R. T., Anal. Chem. 1976, 48, 1933-1937. Misra, H. P. and Squatrito. P. M., Arch. Biochnn. Biophys. 1982,215, 59-65. Laing, P., J. i m m u n o l . Me.thods 1986, 92, 161-165. Wang, H.X., George, J.,Thorpe, G. H., Stott,R.A., Kricka,L. J. and Whitehead,T. P., J. Clin. Pathol. 1985, 38, 317-319. Schneppenheim, R. and Rautenberg, P., Eur. J. Clin.Microbiol. 1987, 6, 49-5 1. Ruggeri, Z . M. and Zimmermann, T. S., Blood 1981, 57, 1140-1143. Bukh, A., Ingerslev, J., Stenbjerg, S. and Moller, N. P., Thromb. Res. 1986,43,579-84. Laemmli, U. K., Nature 1970,227, 680-685. Towbin. H., Staehelin, T. and Gordon, J., Proc. Narl. Arud. Sci. USA 1979, 76,4350-4354. Schneppenheim, R., Plcndl, H. and Budde, U., Thromb. Haemost. 1988, 60. 133-136. Ruggeri, Z. M. and Zimmerman, T. S., Blood 1987, 70, 895-904. Schneppenheim, R., Plendl, H. and Grote,W., in: Neuhoff,V., (Ed.), Electrophoresis ’84,Verlag Chemie, Weinheim 1984, 481-483. Miller, M. A . , Palascak, J. E., Thompson, M. R. and Martelo, 0. J . , Thromb. Res. 1985, 39, 777-780. Aihara,M., Sawada,Y., Ueno,K., Morimoto, S.,Yoshida,Y., de Serres, M., Cooper, H . A. and Wagner,R. H., Thromb. Haemost. 1986, jS, 263-267. Lombard;, R., Gelfi, C., Righetti, P. G., Lattuada, A . and Mannucci, P. M., Thromb. Haemnst. 1986, 55. 246-249. Abildgaard, C. F., Suzuki, Z., Harrison, J., Jefcoat, K. and Zimmerman,T. S., Blood 1980, 56,712-716. Dahlmann, N. and Hartlapp, J. H., Lancer 1988. 1, 1172-1173. Dahlmann, N. and Hartlapp, J. H., k’lin. Wochenschr. 1989, 67,408412. Lebenthal, E. and Heitlinger, L. A, J. fediatr. 1983, 102, 711-712. Board, P. G., Am. J. Hum. Genet. 1979, 31, 116-124. Hardy, G . H., Science 1980. 28, 49-50.
Lurninography
367
Reinhard Schneppenheim' Ulrich Budde' Nicolaus Dahlrnand Peter Rautenberg4
Luminography - a new, highly sensitive visualization method for electrophoresis
'Abteilung Allgemeine Paediatrie, Klinikum der Christian-AlbrechtsUniversitat, Kiel 'Allgemeines Krankenhaus Harburg, Hamburg 'Institut fur Klinische Biochemie, Bonn 4Abteilung Klinische Mikrobiologie, Klinikum der Christian-AlbrechtsUniversitat, Kiel
A highly sensitive method for protein visualization following electrophoresis and protein blotting was developed. The method is based on the light-emitting reaction of luminol and hydrogen peroxide catalyzed by horseradish peroxidase. The luminescent assay can be applied either to the native gel or after protein blotting, and it has a sensitivity two orders of magnitude higher than that achieved with chromogenic detection systems. Analogous to autoradiography the luminescent signal is recorded on an X-ray film with similar sensitivity. We present several examples of application emphasizing the general versatility of this innovative method.
1 Introduction Bio- and chemiluminescent assays have found wide acceptance in research and applied clinical chemistry, biochemistry, and microbiology [I]. One of the first examples was the measurement of ATP, with the luciferase/luciferin system, from firefly lanterns [2]. The potential value of light-emitting detection systems for immunoassays was shown by Schroeder et al. [3], who developed a chemiluminescent immunoassay for biotin. The chemiluminescent system with the widest applicability seems to be the reaction of luminol and hydrogen peroxide catalyzed by horseradish peroxidase. The mechanism of luminol chemiluminescence in aqueous solutions involves the oxidation of luminol by an oxidant (H202),catalyzed by peroxidase, producing a lumino1 radical. After further reactions, an endoperoxide is formed that decomposes, leaving an electronically excited 3-aminophthalate dianion. Light is emitted on its return to the ground state [4]. Horseradish peroxidase is also known as a suitable label ofantibodies,used in enzyme immunoassays and immunohistochemistry, forming a colored complex from a colorless chromogenic substrate. Using lumino1 as a substrate, light will be emitted, which can then be measured by use of a luminometer. Although the high sensitivity of luminescent immunoassays was well known, their applicability as electrophoretic visualization systems was not reported until the end of 1986, when Laing [ 5 ] described a luminescent method for detecting erythrocyte membrane antigens on Western blots. The assay, however, was found to be only four times more sensitive than the chromogenic peroxidase reaction. Concurrently we adapted an enhanced luminescent test tube assay for the von Willebrand factor antigen, described by Wang eta/. [6] for the detection of antibodies to the human immunodeficiency virus (HIV) antigens after sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Correspondence: Dr. R. Schneppenheim, Department of Pediatrics, Klinikum der Christian-Albrechts-Universitat Kid , Schwanenweg 20, W-2300 Kiel 1, Germany Abbreviations: ELISA, enzyme-linked immunosorbent assay; HIV, human immunodeficiency virus; IEF, isoelectric focusing; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; vWd, von Willebrand disease; vWf, von Willebrand factor
0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
(SDS-PAGE) and Western blotting [7]. In this article we summarize our experience with the luminescent visualization system, giving some examples of its application to the diagnosis of diseases, diagnosis of bleeding disorders, detection of drug- or food-induced antibodies, and to forensic medicine.
2 Materials and methods 2.1 Electrophoresis and blotting techniques 2.1.1 Detection of horseradish peroxidase isozymes To test the applicability of a luminescent assay to electrophoresis perse, we attempted a preliminary electrophoretic study on peroxidase isozymes. About 100 mg horseradish roots (Amoracia rusticana) were minced in about an equal amount (w/v) of distilled water in an Eppendorf vial, and centrifuged for 5 min in an Eppendorf centrifuge. Two pL supernatant were applied by filter paper onto the surface of a polyacrylamide isoelectric focusing (IEF) gel (pH 3.59.5), cast and run as recommended by Pharmacia (Uppsala, Sweden). The gel was then equilibrated in Tris-buffered saline (TBS: 0.02 M Tris, 0.5 M NaCl, pH 8) for30 min prior to visualization.
2.1.2 Multimeric structure of von Willebrand factor To establish the high sensitivity of luminogaphy, we compared the luminescent assay with '251-autoradiography. Plasma samples from von Willebrand disease (vWd) patients and from normal controls were applied to two identical SDS agarose gels each, and run with a discontinuous buffer system, according to Ruggeri and Zimmermann [S]. After electrophoresis the gels were either fixed, washed, and dried for autoradiography, or equilibrated in transfer buffer for 1 h, with two changes of buffer, prior to electrophoretic transfer onto nitrocellulose for detection by luminography. Transfer buffer was 0.05 M phosphate, pH 7.4, with 0.04% SDS, without methanol [9]. Transfer was carried out using a Hoefer Transphor (Hoefer Scientific Instruments, San Francisco, CA) at a temperature of 16"C, constant voltage of 33 V and 2.5 Amps. Only one gel at a time was transferred in one tank. The gel was placed near the cathode. Transfer was completed in 2 h. 0173-0835/91/0505-0367 $3.50+.25/0
368
Elecrrophoresis 1991, 12, 361-312
R. Schneppenheim e t a / .
2.1.3 Western blot for antibodies to HIV The presence of antibodies to HlV antigens in human sera was tested by means of a commercially available HlV Western blot test kit (DuPont/Biotech, Bad Nauheim, Germany).This test system employs biotin-tagged, anti-human antibodies and peroxidase-labeled avidin. Peroxidase activity was made visible by the chromogenic assay, using 4-chloro-1-naphthol as substrate, and by the luminescent assay to compare their sensitivities. 2.1.4 Heterophilic antibodies A protein extract from bovine pancreatic tissue (Wobenzym, Mucos Pharma, Geretsried, Germany), used as “alternative anti-cancer drug”, was separated by means of SDSPAGE in gels of the Midget-system (Pharmacia-LKB, Uppsala, Sweden) using a linear pore gradient (4-22.5 %T, 4%C) and the Laemmli buffer system [lo] under nonreducing conditions. Twenty pg protein was applied to each slot. Electrophoresis was followed by electrophoretic transfer onto nitrocellulose in a semi-dry blotting device (CTI, Idstein, Germany) according to the manufacturer’s recommendations. 2.1.5 Human IgG antibodies against gliadin SDS-PAGE of gliadin (Sigma, Taufiirchen, Germany) followed by electroblotting was carried out according to Laemmli [lo] and Towbin [ll], respectively. Antibodies against gliadin in a child with coeliac disease were visualized by incubation of this Western blot with the patient’s sera, diluted 1:lOO. Intermediate washing was followed by incubation with a peroxidase labeled mouse antibody against human IgG (Sigma) [12], and subsequently by the detection of the peroxidase label using luminography. 2.1.6 Factor XIIIA Plasma samples (n=218), drawn from nonrelated individuals of a northern German population were investigated by IEF in an agarose gel for polymorphism at the FXIIIA locus. The gel was cast and run as recommended by Pharmacia, using a 1:l mixture of carrier ampholytes, Pharmalyte pH 3.5-9.5 and pH 5-8, respectively (Pharmacia). Plasma samples were diluted 1:20 with distilled water, and 2 pL each was applied to the gel by means of filter papers. After the run, proteins were transferred to nitrocellulose by simple capillary blotting for 30 min.
Table 1. Antisera and antibodies used for detection of the antigen described. Antigen First antibody Second antibody Goat anti-rabbit Rabbit FVIIIR: Ag antiserum antibody, peroxidase-labeled Behring, 1:500 Bio-Rad, 1:2000 Anti-human IgG Human serum HIV Western blot biotin-tagged and 1:lOO commercial test peroxidase-labeled (Dupont) avidin as described by Dupont Rabbit anti-human Human serum Wobenzym IgG antibody peroxi1.75 Immunostimulant dase-labeled (Mucos Pharma) Sigma, 1500 Mouse anti-human IgG Human serum Gliadin (Sigma) 1:lOO antibody peroxidaselabeled, Sigma, 1:200O Goat anti-rabbit Rabbit anti-FXIIIA Factor XlIIA antibody peroxidasefrom human plasma Behring, 1:500 labeled, Bio-Rad, 1:2OOO
vWf from human plasma
milk powder) for 10 min, then incubated with the first antibody and, after two washing steps inTTBS,with the second antibody. Antisera and antibodies used in these assays are given in Table 1. After two further washing steps the filters were transferred to 0.02M Tris buffer, pH 7.5, for the chromogenic assay or to 0.02M Tris buffer, pH 8.0, for the luminescent assay. 2.3 Visualization 2.3.1 Autoradiography Autoradiographs were obtained by exposing Kodak X-OMAT AR film to the dried gel in combination with two intensifying screens. Exposure time was 72 h. 2.3.2 Chromogenic assay 4-Chloro-1-naphthol was used as the chromogenic substrate recommended by Bio-Rad (Miinchen, Germany) for use in their immunoblot assay. The staining recipe was according to the Bio-Rad immunoblot manual (60 mg 4-chloro-1-naphthol in 20 mL methanol, 100 mLTBS buffer and 60 pL H,O, (30%). Incubation time was 30-45 min. 2.3.3 Luminescent assay
2.2 Immunoassays
Either the gels or immunoblots were both covered for 1-2 min by an evenly distributed solution of about 5 mL of 2.2.1 Autoradiography 0.02 M Tris, pH 8, containing 0.4 mg/mL of luminol (Sigma), 0.1 mg/mL of 4-iodophenol (Aldrich, Steinheim, GerAutoradiography was carried out essentially as described many), and 2.5 pL/mL of 30 O/o H,O,. Stock solutions of 10 X [8] using 12’I-labeledrabbit anti-human vWf antibody. luminol and 10 X 4-iodophenol can be prepared with dimethy1 sulfoxide (DMSO) and stored in a freezer for an unlimited time. The reaction mixture, however, was prepared 2.2.2 Immunoblot immediately prior to use. In a darkroom either the luminating gel or the Western blot is covered by a thin translucent After electrophoretic or capillary transfer to nitrocellulose, the filters were blocked in antibody buffer ( 0 . 0 2 ~ Tris, 0 . 5 ~ plastic film to protect the X-ray film from getting wet. VariNaCI, 0.05% Tween20, pH 7.5 = TTBS + 5% non-fat dry ous brands of X-ray films are suitable, since all of them are
Ele 10 000 kDa, is visualized by means of 'Z51-labeled antibodies [8].Visualization has also been attempted by use of peroxidase-labeled antibodies and a chromogenic substrate [9, 14-17]. However, these assays are less sensitive than the radioactive one, and, in cases of low antigen concentration, do not allow the reliable analysis of the multimeric structure. To establish the high sensitivity of luminographywe compared the luminescent assay with '251-a~toradiography. Figure 2 compares autoradiography versus luminography in the visualization of the multimeric pattern of vWf. Even at a plasma dilution of 1500, vWf multimers can still be evaluated by either method (Fig. 2a). Assuming the normal plasma concentration of vWf to be 10 pg/mL [18], this dilution would correspond to a quantity of 400 pg vWf in 20 pL
Figure 3. HIV Western blots of a patient who seroconverted after transfusion of a retrospectively contaminated blood unit. (a) Luminography detected seroconversion considerably earlier (day 28 post transfusion) than either the chrornogenic assay or the ELISA screening test (day 57 post transfusion). (b) Comparison of sensitivity between luminography, chromogenic assay, and ELISA screening test. Luminography detected antibodies against HIVat by the chromogenic assay or by ELISA. Exposure time forluminoconcentrations two orders of magnitude lower, at a serum dilution of lo-*versus graphy was 3 min. 3*= same blot as -1g of serum dilution = 3, but exposure time was reduced to 3 s to optimize luminography. Results of the ELISA screening test are stated as '+' or '-'.
E I ~ c t r o p h o r e s i1991, ~
12, 367-372
of sample to be applied to the gel. This quantity is then separated by electrophoresis into a set of up to 20 multimers, each of them resolved as a triplet by our electrophoretic system. By slot blot analysis of different plasma concentrations of vWf, we could estimate the sensitivity of luminography to be down to 10 pg of protein (results not shown). Furthermore, the different types and subtypes of vWd can be reliably discriminated by luminography. There was virtually no difference in the two methods with respect to the banding patterns observed in patients with vWd types IIA, IIB, IIC, IID, IIE, and IIF (Fig. 2b). The multimeric patterns can also be evaluated by densitometry, as shown in Fig. 2c.
3.3 HIV Western blot Figure 3 shows the results of testing for HIV antibodies in human sera. By luminography the detection of antibodies to HIV in one patient was made possible 28 days after the assumed infection by a retrospectively contaminated blood transfusion. Neither the chromogenic Western blot nor the enzyme-linked immunosorbent assay (ELISA) screening test were reactive prior to day 57 after transfusion. Sensitivity with respect to the detection of antibodies was shown to be 100 times higher in luminography than in the conventional chromogenic assay (Fig. 3b).
3.4 Heterophilic antibodies Immunoassays of the “sandwich type, performed directly on serum, can yield aberrant results due to heterophilic antibodies in the specimen, which may bind to reagent mouse antibodies, causing false positive results. These interferences are potentially serious pitfalls. Acceptance of spuriously elevated results leads to a great deal of unnecessary followup testing or, even worse, of superfluous therapy. Pre-
Figure 4. Detection of heterophilic antibodies reactive to the immunostimulant Wobenzym, with crossreactivity to mouse immunoglobulins. Comparison between luminography and chromogenic visualization subsequent to SDS-PAGE. (A) molecular weight markers; (B)-(E) Wobenzym, (B) unspecific silver stain of Wobenzym, (C) chromogenic and (D) luminographic detection after incubation with patient’s serum, (E) negative luminographic control incubated only with second antibody without prior incubation with patient’s serum.
Luminography
37 1
viously, we reported on a patient who underwent adjuvant chemotherapy because of a spuriously elevated tumor marker-alphafetoprotein (AFP) [19].The factor which was responsible for this false increase could be purified and was identified as an IgG molecule 1201. In addition to the patient’s recommended cancer therapy, he used 18 different preparations of alternative anti-cancer drugs. To obtain clues pertaining to the etiology of the heterophilic antibody, we analyzed these preparations. All of them were immune-stimulating substances of partly plant, partly animal origin. Of all 18 preparations tested, only “Wobenzym”, a protein extract from bovine pancreatic tissue (Mucos Pharma, Geretsried, Germany), was reactive with the IgG fraction of the patient’s serum. As shown in Fig. 4, detection of heterophilic antibodies was questionable, using the chromogenic assay, although the sandwich assay was clearly positive. However, using luminography, a strong signal can be obtained, which is even more intense than the unspecific silver stain, indicating clearly the existence of IgG antibodies to Wobenzym.
3.5 Human IgG antibodies against gliadin Elevated titers of IgG and IgA antibodies against gliadin are of diagnostic value in coeliac disease of childhood [21]. Figure 5 shows the detection of antigliadin IgG antibodies in a child with coeliac disease by means of a gliadin Western blot subsequent to SDS-PAGE. The diagnosis was confirmed by small bowel biopsy and an ELISA, specific for antigliadin antibodies (results not shown). Incubation of the Western blot with sera of healthy control persons failed to visualize any protein bands.
Figure 5. Luminographic detection of human antigliadin antibodies by means of immunoblotting subsequent to SDS-PAGE of gliadin. MW, molecular weight markers, P, visualization after incubation with serum from a patient with coeliac disease, C, incubation with serum from a healthy control. Exposure time for luminography, P = 3 s, C = 3 min.
372
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3.6 Factor XIIla polymorphim Factor XIIIA, a transglutaminase, catalyzes the last step in the blood coagulation cascade, the crosslinking of the fibrin clot at y-glutamylle-lysine sites to a stable product. Polymorphism at the FXIIIAlocus is responsible for the existence oftwo common alleles (FXIIIA-I and FXIIIA-2) with reported allele frequencies of 0.8 and 0.2, respectively [22]. This polymorphism makes FXIIIA a useful marker in studies of human population genetics, linkage analysis, and forensic medicine. A typical presentation of FXIIIA electromorphs is shown in Fig. 6. FXIIIA, a dimeric enzyme, displays a 3-banded pattern in the heterozygous state and one band in the homozygous state. Among 218 individuals, only the two common alleles (FXIIIA-1, FXIIIA-2) were detected. Allele frequencies were 0.773 for FXIIIA-1 and 0.227 for FXIIIA-2.The distribution ofphenotypes was consistent with random mating expectations according to the Hardy-Weinberg principle [23] k:=O.128;p=O.74). Studies on 3 families confirmed autosomal codominant inheritance of these alleles.
exposed “hard copies” as permanent records can be obtained from one gel to optimize the result. (v) After elution of the antibodies, even multiple probing for the same or different antigens is possible. (vi) Using non-fat milk as blocking agent for the nitrocellulose membranes, avery low background results, even after prolonged exposure. (vii) Reagents are extremely cheap, which makes the method economical. These advantages make luminography a superior tool for the evaluation of protein electrophoresis. It combines ideally the high sensitivity of autoradiography with the relatively nonhazardous nature of chromogenic assays. It is tempting to also apply this method to the analysis of DNA; however, several experiments with different nonradioactive labeling techniques (e.g. digoxigenin labeling of DNA probes in combination with peroxidase-labeled antidigoxigenin antibodies) failed to show higher sensitivity of DNA luminography when compared with chromogenic assays employing peroxidase or alkaline phosphatase labels.
We thank Mrs. H. El Abd Miiller and Mrs. R . Rosenberg f o r their excellent technical assistance and Mrs. Reimers f o r the photographs. We greatly appreciate suggestions made by J. Ingerslev on electrotransfer methods j o r vW$ This work was supported in part by the Deutsche Forschungsgemeinschaft (grant No. Bu 608/1-1 and grant No. Schn 325/1-1). Received November 7, 1990
5 References
figure 6. Luminographic identification offactorXIII.4 polymorphism following agarose IEF and capillary transfer to nitrocellulose. 1-1, homozygous phenotype ofthe common allele 1; 2-2, homozygous phenotype of allele 2; 1-2, heterozygous phenotype. The three-banded pattern displayed by heterozygotes are consistent with the dimeric structure of FXIIIA.
4 Concluding remarks The potential value of luminography as a highly sensitive visualization method based on peroxidase-mediated light emission has been proven by its successful application to a variety of electrophoretic systems. Peroxidase itself was the subject of electrophoresis in our isozyme study. However, the widest applicability of this enzyme is seen in analyzing antigens and antibodies. Thanks to the availability of specific antibodies to many relevant proteins, adequate visualization assays after electrophoresis of these antigens can easily be designed. Furthermore, membrane-immobilized particular antigens can be used to detect specific antibodies in a sample. The method has many advantages: (i) Luminography is as sensitive as ‘*’I-autoradiographyin the analysis of multimers of vWf without the potential hazards and disadvantages of radioactive isotopes. (ii) The sensitivity is two orders of magnitude higher than the conventional chromogenic peroxidase assay in the detection of specific antibodies to HIV, which is of great clinical relevance. (iii) The method is reliable and reproducible in analyzing particular electrophoretic patterns of proteins at low concentrations, e.g. in vWd and in forensic medicine, as shown by the study of FXIIIA polymorphism. (iv) The method is fast. Expocure times vary between ceconds and minutes, compared with days in the case of autoradiography. Many differently
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