The 6/2 (AA3) polyclonal antibody identifying a 37 kD keratinocyte protein reacts also with BM-600/nicein, the basement membrane component bound by the monoclonal antibody GB3 Verrando P, Partouche O, Pisani A, Ortonne J P. The 6/2 (AA3) polyclonal antibody identifying a 37 kD keratinocyte protein reacts also with BM-600/nicein, the basement membrane component bound by the monoclonal antibody GB3. Exp Dermatol 1992: 1: 52-58. Abstract: An unexpected finding concerning our previously reported polyclonal antibody raised against an extract from human amnion (pAb 6/2, also termed AA3), and which recognizes an epidermal keratinocyte protein, is presented in this study. Using the immunoblot technique, pAb 6/2 binds to a 37 kD intracellular protein antigen. We have subsequently found that, by radioimmunoprecipitation performed after metabolic labelling with "S-methionine of cultured keratinocytes, pAb 6/2 recognizes the 600 kD epidermal basement membrane component (termed BM-600/nicein) which was reported to be bound by the monoclonal antibody mAb GB3. Specifically, pAb 6/2 reacts with immunoaffinity chromatography-isolated BlVl-600/nicein blotted onto nitrocellulose. The data suggest the existence of two immunological reactivities borne by pAb 6/2, each of them being directed against, respectively, the 37 kD (seen in immunoblots) and the 600 kD protein (seen in immunoprecipitations). The data further suggest possible independent expression of these two proteins in cell culture. In comparison with the staining pattern of normal skin, immunofluorescence was previously noted to be impaired (pAb 6/2) or absent (mAb GB3) in lethal junctional epidermolysis bullosa. Thus, we conclude that mAb GB3, rather than pAb 6/2, is a more appropriate probe for the comprehensive biochemical study of this genodermatosis.
Introduction
Antibodies - polyclonals monoclonals - used as probes for biochemical techniques have proved to be powerful tools for the identification of cell macromolecules and for studying their biological expression. However, antibody-antigen binding requires defined physico-chemical conditions to be optimum, sufficient atnount of antigen to be detected by an assay, easy accessibility to the antigenic site, and, sometimes, particularly for monoclonal antibodies, the conservation of the three-dimensional structure of the protein (conformational This study was supported by I.N.S.E.R.M. grants no. 87.700.6, CJF 90-09, DEBRA Foundation (England)
52
Patrick Verrando, Odile Partouche, Anne Pisani and Jean Paul Ortonne Laboratoire de Recberches Dermatologiques, U.E.R. de Medecine, Nice, France.
Key words: 6/2 (AA3); polyclonal antibody; GB3; monoclonal antibody; BM600/nicein; basement membrane; keratinocyte; epidermolysis bullosa Patrick Verrando, Laboratoire de Recbercbes Dermatologiques, UFR Mfidecine, Avenue de Valombrose, 06107 Nice cedex 2, France Accepted for publication 6 April 1992
binding site). These conditions are not always found in a unique assay system, such as the immunoblotting technique, thus rendering useful, but incomplete, data. Here we report an unexpected finding concerning our polyclonal antibody raised against the human amnion and termed pAb 6/2 (or AA3) (1,2), We have demonstrated, by using the immunoblotting technique, that this antibody recognized a 37 kD protein in human epidermal keratinocytes (2), However, subsequent use of this antibody in radioimmunoprecipitation assays has shown that it also binds the 600 kD epidermal basement membrane protein termed BM-600/nicein (3, 4), recognized by the monoclonal antibody mAb GB3 (4-6), Because the expression of both these antigens was found to be impaired in the lethal
Relationships between 6/2 and GB3 antibodies junctional epidermolysis bullosa (5, 7, 8), providing a valuable basis for the pre- and postnatal diagnosis of the disease, and because these antibodies represent tools for the molecular understanding of the disease, we have focused here on some of the relationships between pAb 6/2 and mAb GB3. Materials and methods Antibodies The preparation and the speeifieity of mAb GB3 has been previously described (5, 9). It recognizes a new basement membrane component of 600 kD (termed BM-600/nieein (3)) which appears to be specifieally involved in lethal junetional epidermolysis bullosa. Some properties of the antigen have been reported by our group (3-6). The polyelonal antibody pAb 6/2, like mAb GB3, was raised against an extract of human amnion and was found to bind a 37 kD protein of human keratinoeytes, thought to be loeated in the epidermal basement membrane (2). Cell culture Human epidennal keratinocytes were obtained from diverse surgeries and were cultured aeeording to the method of Rheinwald & Green (10). Experiments were done at or near confluency (5-12 days after seeding on 78 eni" eulture dishes) without any changes observed in the results. Human dermal fibroblasts were subcultured from outgrowths of trypsinized small pieces of dermis maintained in dishes containing Dulbecco's modified Eagle's medium, plus antiobioties and 10% fetal calf serum (Boehringer Mannheim, Mannheim, FRG). They were used at conlluency. Metabolic labelling and radioimmunopreeipitation The proeedure has been reported previously in detail (5, 6). Briefly, eells growing on 78 em' eulture dishes were labelled for 22-24 h with ''^S-methionine (specific activity > 1100 Ci/mmol, Dupont de Nemours-NEN, Boston, MA, USA) at 70-80 \.iC\l ml. The incubation medium, which was supplemented with 10% fetal calf serum, was made of the following; 35 mM NaHCO,, 1.04 mM NaH.PO4, 1.36 mM CaClj, 1.23 mM MgS04, 5.36 mM KCl, 109.5 mM NaCI, 5.5 mM glucose, 4 mM glutamine. For the time periods used in the metabolic labeling experiments, incubation in this medium had no adverse effect on protein synthesis (data not shown). After centrifugation to remove free cells, dialysis against water for 22-24 h at 4 C and concentration against polyethylene glyeol (Aquaeide II], mol. wt. 20000, Calbiochem, La Jolla, CA,
USA), the ineubation medium was adjusted to the appropriate final salt concentration of an immunopreeipitation buffer (40 mM Tris-HCl pH 7.6, 0.15 M NaCl, 2 mM EDTA, 0.3% Nonidet-P40, 5 mM methionine) eontaining protease inhibitors (1 mM phenylmethylsulfony] fluoride (PMSF), 0.005% pepstatin and 0.04% aprotinin). Unless otherwise stated, 2.5x10'' epm trichloroacetic acid-precipitable material per assay was used as starting amount of total proteins subjected to immunoprecipitation. Experiments carried out on eells themselves were performed by breaking the cells previously washed with a phosphate-buffered saline solution (PBS), using a Konte glass-glass homogeneizer in the presence of the immunoprecipitation buffer with protease inhibitors. The homogenate was then subjected to an 11 000 g eentrifugation, and the supernatant used for immunoprecipitationexperiments(IO X 10''cpm trichloroacetic acidprecipitable material was used per assay as starting pool of proteins). Immunopreeipitations were accomplished overnight at 4 C, with either mAb GB3 (aseitie fluid, final dilution I;50), pAb 6/2 (diluted l;20), NS-1 myeloma culture fluid (diluted l;50), non-immune aseitie fiuid (MOPC 31, Sigma, St Louis, MO, USA, diluted I; 1000; a final immunoglobulin eoneentration found to be elose to that of mAb GB3, when this is used at I;50) or nonimmune rabbit serum (diluted I;20), as indicated in the figures. An incubation was then earried out at room temperature for 50 min, with either Protein A sepharose 6MB-conjugated beads (diluted 1;5 v/ V, Pharmacia, Uppsala, Sweden), when the first antibody was 6/2, or Sepharose 4B beads beads conjugated with goat anti-mouse IgG (diluted 1;5 v/v, Zymed, San Francisco, CA, USA) when the first antibody was GB3. The beads were pelleted by centrifugation and rinsed extensively with rinse buffers, prior to being taken up in an electrophoresis sample buffer (11) containing (redueing conditions) or lacking (non-reducing conditions) 2mercaptoethanol. After boiling, samples were analyzed on SDS-PAGE according to Laemmli (11). Immunoblotting For immunoblotting experiments performed on conditioned medium, the eells were ineubated for 22-24 h in the same incubation medium used for immunoprecipitation experiments, except the eoneentration of fetal calf serum was 1%. Preparation of samples. The eonditioned medium was centrifuged to remove free eell and debris and made 1 mM in PMSF. It was then dialyzed 20-24 h at 4°C and concentrated under vacuum by centrifugation (Speed Vac, Savant instruments Inc., Farmingdale, NY, USA). Usually, one 100 mm-diam-
53
Verrando et al.
eter eulture dish containing 4.5 ml of incubation mediutn was concentrated to 100 pi. Since protein content secreted by the cells could not be evaluated by a chemical assay due to the amount of fetal calf serum, sample deposits onto eleetrophoresis gels were checked to give sitnilar band intensities when colored by Ponceau red after transfer onto nitrocellulose sheet. Unless otherwise stated, when using the cell layers, the cells were recovered in PBS eontaining 1 mM PMSF and broken into a Konte glass-glass homogeneizer at 4 C. The resulting homogenate was used for immunoblotting. Protein content was estimated by the method of Lowry et al. (12). Blotting experiments. SDS-PAGE gels were transferred onto nitocellulose paper (0.45 |im, BA 85 type, Schleicher & Schuell, Dassel, West Gertnany) at 45 V and 4 C overnight using a Iransblot cell (BioRad, Richmond, CA, USA), according to Towbin et al. (13) as already described (2). After visualization of the transferred proteins with Ponceau Red, the itnmunochemistry was performed using a modification of the procedure of Burnette et al. (14). To enhance the sensitivity of the assay, an avidin-biotin system was used to detect the antigen. For this purpose, mAb GB3 was diluted 1:50 in a buffer (blot buffer) made of PBS containing 3% bovine serum albutnin (cat. A7030, Sigma, St Louis, MO, USA) and 0.2% Triton X-100, and then incubated with the nitrocellulose sheet for 3 h at room temperature. After rinsing with the above buffer lacking mAb GB3, an anti-tnouse immunoglobulin serum conjugated to biotin (Vector Laboratories, Burlingatne, CA, USA) diluted 1:500 was incubated with the nitrocellulose sheet, for 45 min at room temperature. After several washes, the sheet was treated for 45 min at room tetnperature with a commercial avidin-biotin-peroxidase assay kit (Vectastain, PK4000, Vector Laboratories) as specified by the firm. After rinsing, the complexes were visualized with diaminobenzidine (0.5 mg/tnl in O.I M Tris-HCl) (pH 7.6) buffer, containing 0.1% HjO,. The same procedure was applied when the nonimmune ascitie fluid (MOPC31, Sigma, diluted 1:1000) was used as control. The polyclonal antibody pAb 6/2 was used diluted 1:20 in the blot buffer and keep in contact with the nitrocellulose sheet for 3 h at room temperature. Immunoaffinity isolation of BM-600/nicein Immunoaffinity isolation of BM-600/nicein was done by coupling GB3 monoclonal antibody to CNBr-Sepharose and by using conditioned medium from cultured keratinocytes. A full manuscript detailing the purification procedure will be
54
submitted. The eluted fraction containing BM600/nicein was dialyzed against distilled water overnight, then concentrated in a Speed Vac apparatus (Savant, Fartningdale, NY, USA). Protein concentration was estitnated by the Lowry method before reduction or not by 2-tnercaptoelhanol and loaded onto SDS-PAGE according to Laemmli. Then immunoblot was performed. Results Clearing radioimmunopreeipitation data When itntnunoprecipitation of the radiolabeled medium of cultured keratinocytes was perfortned with pAb 6/2 and the precipitated products were analyzed by SDS-PAGE under reducing conditions, three polypeptides of 93.5, 125 and 150kD were seen (Fig. I, lane 1). These polypeptides are identical in their size to those precipitated by mAb GB3, when starting frotn the satne initial amount of -"S-labelled proteins (2.5 x 10'' cpm) from an aliquot of the same extract (Fig. I, lane 0). In addition, no 37 kD band could be delected when using a higher percentage of acrylamide for the gel (data not shown). In Fig. 1 it can be seen that.
1
2
^ '•
3
"^ •'
'^
?" '
'^
4
0
ffi
°^ Z - O R
220-
9467-
43
^
J ^'^"^
Figure I. Clearing radioimmunopreeipitation. Traeks I to 4 refer to the number of sueeessive immunopreeipitations pertbrnied on the same aliquot of eonditioned medium extrael, using pAb 6/2. Traek 0 refers lo another aliquot of the same extraet, without immunoprecipitation with pAb 6/2 before. 2.5 X 10'' cpm-proteins were used in both cases. R; rabbit nonimmune serum; N;NS-I myeloma supernatant. Total; Aliquot of the extraet used tor these immunopreeipitations, to visualize total protein content before immunoprecipitation. Or; origin of electrophoretic migration; Hnd; end of eleetrophorelic migration (6'^!) reduced gel). Mr; Molecular weight (kitodaton) of standard proteins.
Relationships between 6/2 and GB3 antibodies despite starting with the same number of total cpm, the amount of precipitated antigen by mAb GB2 is higher than that with pAb 6/2 and showed a faint 130 kD accompanying band as noted in a previous work (6). When, after the first immunoprecipitation with pAb 6/2, a second one was done on the same aliquot of the conditioned medium, the three proteins could again be detected, but in a lower amount (Fig. I, lane 2). At the third immunoprecipitation on the same extract, the labeled protein bands were only barely seen, indicating they had been totally removed fVom the extract (Fig. I, lane 3). If then, on this same aliquot, an additional immunopreeipitation was performed with mAbGB3, this monoclonal antibody no longer immunopreeipitated any antigen (Fig. 1, lane 4) as it did on this extract before the suecessive immunopreeipitations with pAb 6/2 (Fig. 1, lane 0). This suggests strongly that the protein constituted by the three polypeptides and recognized by pAb 6/2 is the same as those recognized by mAb GB3. pAb 6/2 reacts with immunoaffinity-purified BM-600/nicein bv immunoblot Fig. 2a shows clearly that pAb 6/2 reacts with the reduced 125, 130 kD (a subunit-species found in concentrated preparations of BM-600/nicein (6))
NIL 6/2
Mr
and 150 kD subunit of BM-600/nicein, Its specificity is not directed against the smaller 93.5 kD polypeptide subunit. It also reacts with the unreduced whole BM-600/nicein protein (Fig. 2b). Differential expression of the 37 kD protein and BM-600/nicein As previously reported (2) in immunoblot analysis, pAb 6/2 recognizes a 37 kD protein from total protein extracts of cultured keratinocytes (Fig. 3g). However, no band of peroxidase reactivity could be found when conditioned medium from the same dish of keratinocytes was used (Fig, 3e). It is unlikely that this can be attributed to too little protein, since the lanes were so intensely colored by Ponceau red before the immunochemical reaetion that they even gave background staining which can be seen in Fig. 3 (a ehemieal assay for protein quantitation in the medium was impossible due to the fetal calf serum added to the culture medium). Fig. 3h shows that pAb 6/2 recognizes the 37 kD protein in the protein extraet from dermal fibroblasts in culture, indicating production by these eells. However, as in the case of the keratinocytes, this protein is not secreted in the incubation medium from the same eulture dish (Fig. 30In a same starting volume of conditioned keratinocyte medium as in the preeeeding case, BM600/nicein could be detected by immunoblotting with mAb GB3, providing the separation of total proteins was done using nonredueed SDS-PAGE (Fig. 4c), Note two additional lower molecular weight bands that reaet faintly with the antibody;
NiL 6/2 Mr
-170
Mr 94.
e
f
67_ 4330-.
Figure 2. Immunobiotling reactivity of pAti 6/2 wilh alTinity chi'omalography-piirined BM-600/niccin. About 0.6 ^ig of isolated and reduced BM-600/iiiccin was blotted onto nitrocellulose sheet atid teacted witli pAb 6/2 diluted 1:20. An avidiiibiotin atnplincation system was used. NIL: uon-ininiune rabbit serum. 6/2: pAb 6/2 serum, a) reduced SDS-PAGE (6"/o acrylamide gel). The antibody reacts mainly with the 150 kD and 125 k D subunits, but also with a 130 kD BM-600/nieein-related subunit, usually found in concentrated pteparations of the anligen (6). b) unreduced SDS-PAGE (4% acrylamide gel).
Figure 3. Immunobiol analysis using pAb 6/2 o( both cell extract and conditioned medium from cultured libroblasts and keratinoeytes. The same amount of protein and the same volume of medium sample were used from botli cell types, a-d: noninimuiie rabbit serum (control), e-li: pAb 6/2 serum, a and e: medium from keratinocytes; b atid f: medium from fibroblasts; c and g: cell honiogenale from keratinocytes; d and h: eell homogenate from fibrobUists. Arrowhead indieates the 37 kD protein. Mr: tnolecular weight (kilodalton) of standard proteins. Reduced SDS-PAGE, \0% aerylamide gel.
55
Verrando et al.
they possibly represent partial degradation products of the antigen, or partial reduced form of it (15). In contrast, again no antigen could be detected in the same starting volume of conditioned dermal flbroblast medium (Fig. 4d). This result is confirmed by radioimmunoprecipitation experiments on both epidermal keratinocytes and dermal fibroblasts in culture (Fig. 5): BM-600/nicein could not be found in the conditioned incubation medium from cultured human dermal fibroblasts. This was true whether we started with the same amount of total "S-labelled proteins found in the keratinocyte-conditioned medium or used 3-fold more of these proteins (data not shown). These data show that, in a given cell type, the 37 kD protein could be present even if BM-600/ nicein is absent. Discussion
Mr
J669
J33O
In this paper we show some relationships existing between two antisera made by our group: the 6/2 polyclonal antibody and the GB3 monoclonal antibody, both being raised against human amnion proteins. When using the immunoblotting technique, pAb 6/2 reacts with a 37 kD protein of cultured human keratinocytes (2). Unexpectedly, we found that this antibody, when used for immunoprecipitation on the same cells, also reacts with the 600 kD protein recognized by mAb GB3
b e d
J20
Figttre 5. Radioimmunoprecipitalioii from conditioned medium from bolli cultured fibrobhists (F) keratinocyles (K) using mAb GB3. N: non-immune ascitic lluid (control). G: mAb GB3. Non-reduced SDS-PAGE (3'^ acrylamide gel). The same amount of starting labelled proteins was used in both cases (see Material and Methods). Mr: reduced thyroglobulin used as standard molecular weights.
Mr -660
-330
Figure 4. Immunoblot analysis of conditioned medium from both cultured libroblasts and keratinocytes. using mAb GB3. The same volumes of medium sample as in Fig. 3 were used from both the two cell types. a,b: non-immune ascitic fluid (MOPC 31. Sigma), c.d: mAb GB3 monoclonal antibody, a and c: medium from keratinocytes; b and d: medium from fibroblasts. Arrowhead shows BM-6()0/nicein. Non-tcduced SDS-PAGE. 3% acrylamide gel. Mr: molecular weight (kilodalton) of thyroglobulin (and its subunits) used as a standard
protein.
56
(BM-600/nicein). This has been ascertained by the "preclearing" immunoprecipitation strategy employing first pAb 6/2 and, second, mAb GB3, on the same biological aliquot putatively containing the BM-600/nicein protein (cultured keratinocyte conditioned medium). This strategy allowed us to conclude that pAb 6/2 displays two immunological reactivities: one against the 37 kD protein (seen on immunoblots) and the other against the 600 kD protein (seen by immunoprecipitation) which is recognized by mAb GB3. To clarify further the nature of the reactivity of pAb 6/2 and BM-6()0/ nicein, we isolated, by immunochromatography, the protein from conditioned media of cultured keratinocytes and blotted it onto nitrocellulose. We confirmed unequivocally that pAb 6/2 is able to recognize the larger subunits of BM-600/nicein (150 kD and 125 kD), although no reactivity was detected with the smaller 93.5 kD subunit. On the other hand, the absence of reactivity of pAb 6/2
Relationships between 6/2 and GB3 antibodies in immunoblots with BM-600/nicein in homogenates of cells or medium could in fact be explained by the extremely low amount of antigen in the extract and, additionally, by different affinity/tiler between pAb 6/2 and mAb GB3. Indeed, we reported earlier that the pool of antigen in the cell is very low (6), BM-600/nicein being secreted immediately into the medium. Furthermore, the radioimmunopreeipitation technique concentrates the antigen in contrast to the immunoblot technique, which would tend to require a large amount of homogenate not suitable for the eleetrophoresis step prior to the blotting. In the medium, the main problem is to deal with the large amounts of albumin from serum used for cell culture that impair the concentration needed for loading extracts SDSPAGE. In that regard, the affinity chromatography represents an ideal tool and allowed us to show the actual binding of pAb 6/2 to the isolated BM600/nicein from keratinocyte conditioned media. In order lo understand better the dual-specificity of the 6/2 polyclonal antibody and the possible relationship between the 37 kD protein and BM600/nicein, we have studied here the expression of these two proteins in cell culture. For example, if there were co-expression, one hypothesis would be that one of these two proteins might represent either a degradation product or a precursor form of the other. This would explain why pAb 6/2 reacted with both of them. If the antigenie site is common, the 37 kD protein is a part of the larger species. The experimental data given in this paper suggest this is unlikely, tn contrast to BM-600/ nicein, the 37 kD protein could not be found in the medium of cultured keratinocytes or fibroblasts. Thus, it seems unlikely that the 37 kD protein is a degraded form of BM-600/nicein, unless the 37 kD is rapidly hydrolyzed by proteases secreted by the cells. Additionally, the 37 kD protein is expressed by the human dermal fibroblasts, thus again making it unlikely that the 37 kD protein is a degraded form of the larger. Nevertheless, this does not prove that the 37 kD protein is not a precursor of BM-600/nicein and additional experiments will be required to investigate this possibility. Finally, taking all of the (admittedly somewhat indirect) results together, we are tempted to suggest that there is probably no relationship between the two proteins. The two immunologic specificities detected by pAb 6/2 may be explained by the fact that, obviously, severai proteins were present in the collected chromatographic peak from solubilized amnion which was used for the immunization procedure described previously (1, 2). Although the immunogenic extract was similar (1, 9), the dual specificity lias been avoided when mAb GB3 was made, because of the clonal screening performed for
monoclonal antibody production. Thus we conclude that mAb GB3 represents a more useful and appropriate probe than pAb 6/2 for the biochemical and bioclinical studies concerning the new basement membrane component BM-600/nicein (3-6). Its potential for unlimited production and its monospecificity can be applied for diagnosis, including prenatal diagnosis of lethal junctional epidermolysis bullosa where mAb GB3 displays a clear-cut absence of signal rather than a diminished one as is the case for pAb 6/2 (8). A eknowledgments We wish 10 express our graliuide to Pr Eugene Bauer (Stanford University Department ol" Dennatology) for helpful dis-
References 1. Hsi B L. Yeh C J. Faulk P W. Characterization of antibodies to antigens of the human amnion. Placenta 1985: 5: 513-522. 2. Verrando P. Ortonne .1 P. Pautrat G. Hsi B L. Yeh C J: Identification of a 37 kilodalton protein at the epidermal basement membrane by an antiserum to human amniotT. J Invest Dermatol 1986:'87: 190 1%. 3. Verrando P. Pisani A, Partouche O. Ortonne .1 P BM(lOO/nicein, a putative new basement membrane component involved in lethal junctional epidermolysis bullosa. In: Priestly G C, Tidnian M } , Weiss J B. Eady R A .), eds. Epidermolysis bullosa: a comprehensive review of classification, management and laboratory studies. Crowthorne. Berkshire. U.K.: DEBRA. 1990; pp. 137-140. 4. Verrando P. Partouche O, Pisani A. Ortonne J P. New molecular and cellular tools for studying BM-600/nicein. the "GB3" basement membrane antigen involved in lethal junctional epidermolysis bullosa. .1 Invest Dermatol (abstr.) 1989: 92: 538. 5. Verrando P, Hsi B L, Yeh C .1. Pisani A, Serieys N. Ortonne J P. Monoelonal antibody GB3. a new probe lor the study of human basement membianes and heniidesmosomes. Exp Cell Res 1987: 170: 116-128. 6. Verrando P, Pisani A, Ortonne .1 P. The new basement membrane antigen recognized by the monoclonal antibody GB3 is a large size glycoprotein: modulation of its expression by retinoic acid. Biochitn Biophys Acta 1988: 942: 45-56. 7. Heagerty A H M, Kennedy A R. Eady R A .1, et al. GB3 nionoclonal antibody for diagnosis of junctional epidennolysis bullosa. Lancet 1986: 8485: 860." 8. Kennedy A R. Heagerty A H M. Ortonne .1 P. Hsi B L, Yeh C .1 G, Eady R A .1. Abnormal binding of an antiamnion antibody to epidermal basement metnbrane provides a novel diagnostic probe for junctional epidermolysis bullosa. Br J Dermatol 1985: 113: 651-659. 9. Hsi B L. Yeh C .1 G. Monoclonal antibodies to human amnion. J Repro Immunol 1986: 9: 1 1-21. 10. Rheinwald .1 G. Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 1975: 6: 331-344. 11. Laemmli U. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 1970: 277: 680-685. 12. Lowry O H, Rosebrough N .1. Farr A L, Randall R .1.
57
Verrando et al. Protein measurement with the Folin phenol reagent. J Biol Chem 1951: 193: 265-275. 13. Towbin H, Staehelin T, Gordon J. Electrophorelic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979: 76: 4350-^354. 14. Burnette N. Eleclrophoretic transfer of proteins from SDS
58
polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 1981: 112: 195-203. 15. Verrando P, Partouche O, Pisani A, Ortonne J P. Antibodies to basement membrane protein nicein (BM-6()0/nicein) give new clues in lethal junctional epidermolysis bullosa. 1992 (submilted).
Introduction
Antibodies - polyclonals monoclonals - used as probes for biochemical techniques have proved to be powerful tools for the identification of cell macromolecules and for studying their biological expression. However, antibody-antigen binding requires defined physico-chemical conditions to be optimum, sufficient atnount of antigen to be detected by an assay, easy accessibility to the antigenic site, and, sometimes, particularly for monoclonal antibodies, the conservation of the three-dimensional structure of the protein (conformational This study was supported by I.N.S.E.R.M. grants no. 87.700.6, CJF 90-09, DEBRA Foundation (England)
52
Patrick Verrando, Odile Partouche, Anne Pisani and Jean Paul Ortonne Laboratoire de Recberches Dermatologiques, U.E.R. de Medecine, Nice, France.
Key words: 6/2 (AA3); polyclonal antibody; GB3; monoclonal antibody; BM600/nicein; basement membrane; keratinocyte; epidermolysis bullosa Patrick Verrando, Laboratoire de Recbercbes Dermatologiques, UFR Mfidecine, Avenue de Valombrose, 06107 Nice cedex 2, France Accepted for publication 6 April 1992
binding site). These conditions are not always found in a unique assay system, such as the immunoblotting technique, thus rendering useful, but incomplete, data. Here we report an unexpected finding concerning our polyclonal antibody raised against the human amnion and termed pAb 6/2 (or AA3) (1,2), We have demonstrated, by using the immunoblotting technique, that this antibody recognized a 37 kD protein in human epidermal keratinocytes (2), However, subsequent use of this antibody in radioimmunoprecipitation assays has shown that it also binds the 600 kD epidermal basement membrane protein termed BM-600/nicein (3, 4), recognized by the monoclonal antibody mAb GB3 (4-6), Because the expression of both these antigens was found to be impaired in the lethal
Relationships between 6/2 and GB3 antibodies junctional epidermolysis bullosa (5, 7, 8), providing a valuable basis for the pre- and postnatal diagnosis of the disease, and because these antibodies represent tools for the molecular understanding of the disease, we have focused here on some of the relationships between pAb 6/2 and mAb GB3. Materials and methods Antibodies The preparation and the speeifieity of mAb GB3 has been previously described (5, 9). It recognizes a new basement membrane component of 600 kD (termed BM-600/nieein (3)) which appears to be specifieally involved in lethal junetional epidermolysis bullosa. Some properties of the antigen have been reported by our group (3-6). The polyelonal antibody pAb 6/2, like mAb GB3, was raised against an extract of human amnion and was found to bind a 37 kD protein of human keratinoeytes, thought to be loeated in the epidermal basement membrane (2). Cell culture Human epidennal keratinocytes were obtained from diverse surgeries and were cultured aeeording to the method of Rheinwald & Green (10). Experiments were done at or near confluency (5-12 days after seeding on 78 eni" eulture dishes) without any changes observed in the results. Human dermal fibroblasts were subcultured from outgrowths of trypsinized small pieces of dermis maintained in dishes containing Dulbecco's modified Eagle's medium, plus antiobioties and 10% fetal calf serum (Boehringer Mannheim, Mannheim, FRG). They were used at conlluency. Metabolic labelling and radioimmunopreeipitation The proeedure has been reported previously in detail (5, 6). Briefly, eells growing on 78 em' eulture dishes were labelled for 22-24 h with ''^S-methionine (specific activity > 1100 Ci/mmol, Dupont de Nemours-NEN, Boston, MA, USA) at 70-80 \.iC\l ml. The incubation medium, which was supplemented with 10% fetal calf serum, was made of the following; 35 mM NaHCO,, 1.04 mM NaH.PO4, 1.36 mM CaClj, 1.23 mM MgS04, 5.36 mM KCl, 109.5 mM NaCI, 5.5 mM glucose, 4 mM glutamine. For the time periods used in the metabolic labeling experiments, incubation in this medium had no adverse effect on protein synthesis (data not shown). After centrifugation to remove free cells, dialysis against water for 22-24 h at 4 C and concentration against polyethylene glyeol (Aquaeide II], mol. wt. 20000, Calbiochem, La Jolla, CA,
USA), the ineubation medium was adjusted to the appropriate final salt concentration of an immunopreeipitation buffer (40 mM Tris-HCl pH 7.6, 0.15 M NaCl, 2 mM EDTA, 0.3% Nonidet-P40, 5 mM methionine) eontaining protease inhibitors (1 mM phenylmethylsulfony] fluoride (PMSF), 0.005% pepstatin and 0.04% aprotinin). Unless otherwise stated, 2.5x10'' epm trichloroacetic acid-precipitable material per assay was used as starting amount of total proteins subjected to immunoprecipitation. Experiments carried out on eells themselves were performed by breaking the cells previously washed with a phosphate-buffered saline solution (PBS), using a Konte glass-glass homogeneizer in the presence of the immunoprecipitation buffer with protease inhibitors. The homogenate was then subjected to an 11 000 g eentrifugation, and the supernatant used for immunoprecipitationexperiments(IO X 10''cpm trichloroacetic acidprecipitable material was used per assay as starting pool of proteins). Immunopreeipitations were accomplished overnight at 4 C, with either mAb GB3 (aseitie fluid, final dilution I;50), pAb 6/2 (diluted l;20), NS-1 myeloma culture fluid (diluted l;50), non-immune aseitie fiuid (MOPC 31, Sigma, St Louis, MO, USA, diluted I; 1000; a final immunoglobulin eoneentration found to be elose to that of mAb GB3, when this is used at I;50) or nonimmune rabbit serum (diluted I;20), as indicated in the figures. An incubation was then earried out at room temperature for 50 min, with either Protein A sepharose 6MB-conjugated beads (diluted 1;5 v/ V, Pharmacia, Uppsala, Sweden), when the first antibody was 6/2, or Sepharose 4B beads beads conjugated with goat anti-mouse IgG (diluted 1;5 v/v, Zymed, San Francisco, CA, USA) when the first antibody was GB3. The beads were pelleted by centrifugation and rinsed extensively with rinse buffers, prior to being taken up in an electrophoresis sample buffer (11) containing (redueing conditions) or lacking (non-reducing conditions) 2mercaptoethanol. After boiling, samples were analyzed on SDS-PAGE according to Laemmli (11). Immunoblotting For immunoblotting experiments performed on conditioned medium, the eells were ineubated for 22-24 h in the same incubation medium used for immunoprecipitation experiments, except the eoneentration of fetal calf serum was 1%. Preparation of samples. The eonditioned medium was centrifuged to remove free eell and debris and made 1 mM in PMSF. It was then dialyzed 20-24 h at 4°C and concentrated under vacuum by centrifugation (Speed Vac, Savant instruments Inc., Farmingdale, NY, USA). Usually, one 100 mm-diam-
53
Verrando et al.
eter eulture dish containing 4.5 ml of incubation mediutn was concentrated to 100 pi. Since protein content secreted by the cells could not be evaluated by a chemical assay due to the amount of fetal calf serum, sample deposits onto eleetrophoresis gels were checked to give sitnilar band intensities when colored by Ponceau red after transfer onto nitrocellulose sheet. Unless otherwise stated, when using the cell layers, the cells were recovered in PBS eontaining 1 mM PMSF and broken into a Konte glass-glass homogeneizer at 4 C. The resulting homogenate was used for immunoblotting. Protein content was estimated by the method of Lowry et al. (12). Blotting experiments. SDS-PAGE gels were transferred onto nitocellulose paper (0.45 |im, BA 85 type, Schleicher & Schuell, Dassel, West Gertnany) at 45 V and 4 C overnight using a Iransblot cell (BioRad, Richmond, CA, USA), according to Towbin et al. (13) as already described (2). After visualization of the transferred proteins with Ponceau Red, the itnmunochemistry was performed using a modification of the procedure of Burnette et al. (14). To enhance the sensitivity of the assay, an avidin-biotin system was used to detect the antigen. For this purpose, mAb GB3 was diluted 1:50 in a buffer (blot buffer) made of PBS containing 3% bovine serum albutnin (cat. A7030, Sigma, St Louis, MO, USA) and 0.2% Triton X-100, and then incubated with the nitrocellulose sheet for 3 h at room temperature. After rinsing with the above buffer lacking mAb GB3, an anti-tnouse immunoglobulin serum conjugated to biotin (Vector Laboratories, Burlingatne, CA, USA) diluted 1:500 was incubated with the nitrocellulose sheet, for 45 min at room temperature. After several washes, the sheet was treated for 45 min at room tetnperature with a commercial avidin-biotin-peroxidase assay kit (Vectastain, PK4000, Vector Laboratories) as specified by the firm. After rinsing, the complexes were visualized with diaminobenzidine (0.5 mg/tnl in O.I M Tris-HCl) (pH 7.6) buffer, containing 0.1% HjO,. The same procedure was applied when the nonimmune ascitie fluid (MOPC31, Sigma, diluted 1:1000) was used as control. The polyclonal antibody pAb 6/2 was used diluted 1:20 in the blot buffer and keep in contact with the nitrocellulose sheet for 3 h at room temperature. Immunoaffinity isolation of BM-600/nicein Immunoaffinity isolation of BM-600/nicein was done by coupling GB3 monoclonal antibody to CNBr-Sepharose and by using conditioned medium from cultured keratinocytes. A full manuscript detailing the purification procedure will be
54
submitted. The eluted fraction containing BM600/nicein was dialyzed against distilled water overnight, then concentrated in a Speed Vac apparatus (Savant, Fartningdale, NY, USA). Protein concentration was estitnated by the Lowry method before reduction or not by 2-tnercaptoelhanol and loaded onto SDS-PAGE according to Laemmli. Then immunoblot was performed. Results Clearing radioimmunopreeipitation data When itntnunoprecipitation of the radiolabeled medium of cultured keratinocytes was perfortned with pAb 6/2 and the precipitated products were analyzed by SDS-PAGE under reducing conditions, three polypeptides of 93.5, 125 and 150kD were seen (Fig. I, lane 1). These polypeptides are identical in their size to those precipitated by mAb GB3, when starting frotn the satne initial amount of -"S-labelled proteins (2.5 x 10'' cpm) from an aliquot of the same extract (Fig. I, lane 0). In addition, no 37 kD band could be delected when using a higher percentage of acrylamide for the gel (data not shown). In Fig. 1 it can be seen that.
1
2
^ '•
3
"^ •'
'^
?" '
'^
4
0
ffi
°^ Z - O R
220-
9467-
43
^
J ^'^"^
Figure I. Clearing radioimmunopreeipitation. Traeks I to 4 refer to the number of sueeessive immunopreeipitations pertbrnied on the same aliquot of eonditioned medium extrael, using pAb 6/2. Traek 0 refers lo another aliquot of the same extraet, without immunoprecipitation with pAb 6/2 before. 2.5 X 10'' cpm-proteins were used in both cases. R; rabbit nonimmune serum; N;NS-I myeloma supernatant. Total; Aliquot of the extraet used tor these immunopreeipitations, to visualize total protein content before immunoprecipitation. Or; origin of electrophoretic migration; Hnd; end of eleetrophorelic migration (6'^!) reduced gel). Mr; Molecular weight (kitodaton) of standard proteins.
Relationships between 6/2 and GB3 antibodies despite starting with the same number of total cpm, the amount of precipitated antigen by mAb GB2 is higher than that with pAb 6/2 and showed a faint 130 kD accompanying band as noted in a previous work (6). When, after the first immunoprecipitation with pAb 6/2, a second one was done on the same aliquot of the conditioned medium, the three proteins could again be detected, but in a lower amount (Fig. I, lane 2). At the third immunoprecipitation on the same extract, the labeled protein bands were only barely seen, indicating they had been totally removed fVom the extract (Fig. I, lane 3). If then, on this same aliquot, an additional immunopreeipitation was performed with mAbGB3, this monoclonal antibody no longer immunopreeipitated any antigen (Fig. 1, lane 4) as it did on this extract before the suecessive immunopreeipitations with pAb 6/2 (Fig. 1, lane 0). This suggests strongly that the protein constituted by the three polypeptides and recognized by pAb 6/2 is the same as those recognized by mAb GB3. pAb 6/2 reacts with immunoaffinity-purified BM-600/nicein bv immunoblot Fig. 2a shows clearly that pAb 6/2 reacts with the reduced 125, 130 kD (a subunit-species found in concentrated preparations of BM-600/nicein (6))
NIL 6/2
Mr
and 150 kD subunit of BM-600/nicein, Its specificity is not directed against the smaller 93.5 kD polypeptide subunit. It also reacts with the unreduced whole BM-600/nicein protein (Fig. 2b). Differential expression of the 37 kD protein and BM-600/nicein As previously reported (2) in immunoblot analysis, pAb 6/2 recognizes a 37 kD protein from total protein extracts of cultured keratinocytes (Fig. 3g). However, no band of peroxidase reactivity could be found when conditioned medium from the same dish of keratinocytes was used (Fig, 3e). It is unlikely that this can be attributed to too little protein, since the lanes were so intensely colored by Ponceau red before the immunochemical reaetion that they even gave background staining which can be seen in Fig. 3 (a ehemieal assay for protein quantitation in the medium was impossible due to the fetal calf serum added to the culture medium). Fig. 3h shows that pAb 6/2 recognizes the 37 kD protein in the protein extraet from dermal fibroblasts in culture, indicating production by these eells. However, as in the case of the keratinocytes, this protein is not secreted in the incubation medium from the same eulture dish (Fig. 30In a same starting volume of conditioned keratinocyte medium as in the preeeeding case, BM600/nicein could be detected by immunoblotting with mAb GB3, providing the separation of total proteins was done using nonredueed SDS-PAGE (Fig. 4c), Note two additional lower molecular weight bands that reaet faintly with the antibody;
NiL 6/2 Mr
-170
Mr 94.
e
f
67_ 4330-.
Figure 2. Immunobiotling reactivity of pAti 6/2 wilh alTinity chi'omalography-piirined BM-600/niccin. About 0.6 ^ig of isolated and reduced BM-600/iiiccin was blotted onto nitrocellulose sheet atid teacted witli pAb 6/2 diluted 1:20. An avidiiibiotin atnplincation system was used. NIL: uon-ininiune rabbit serum. 6/2: pAb 6/2 serum, a) reduced SDS-PAGE (6"/o acrylamide gel). The antibody reacts mainly with the 150 kD and 125 k D subunits, but also with a 130 kD BM-600/nieein-related subunit, usually found in concentrated pteparations of the anligen (6). b) unreduced SDS-PAGE (4% acrylamide gel).
Figure 3. Immunobiol analysis using pAb 6/2 o( both cell extract and conditioned medium from cultured libroblasts and keratinoeytes. The same amount of protein and the same volume of medium sample were used from botli cell types, a-d: noninimuiie rabbit serum (control), e-li: pAb 6/2 serum, a and e: medium from keratinocytes; b atid f: medium from fibroblasts; c and g: cell honiogenale from keratinocytes; d and h: eell homogenate from fibrobUists. Arrowhead indieates the 37 kD protein. Mr: tnolecular weight (kilodalton) of standard proteins. Reduced SDS-PAGE, \0% aerylamide gel.
55
Verrando et al.
they possibly represent partial degradation products of the antigen, or partial reduced form of it (15). In contrast, again no antigen could be detected in the same starting volume of conditioned dermal flbroblast medium (Fig. 4d). This result is confirmed by radioimmunoprecipitation experiments on both epidermal keratinocytes and dermal fibroblasts in culture (Fig. 5): BM-600/nicein could not be found in the conditioned incubation medium from cultured human dermal fibroblasts. This was true whether we started with the same amount of total "S-labelled proteins found in the keratinocyte-conditioned medium or used 3-fold more of these proteins (data not shown). These data show that, in a given cell type, the 37 kD protein could be present even if BM-600/ nicein is absent. Discussion
Mr
J669
J33O
In this paper we show some relationships existing between two antisera made by our group: the 6/2 polyclonal antibody and the GB3 monoclonal antibody, both being raised against human amnion proteins. When using the immunoblotting technique, pAb 6/2 reacts with a 37 kD protein of cultured human keratinocytes (2). Unexpectedly, we found that this antibody, when used for immunoprecipitation on the same cells, also reacts with the 600 kD protein recognized by mAb GB3
b e d
J20
Figttre 5. Radioimmunoprecipitalioii from conditioned medium from bolli cultured fibrobhists (F) keratinocyles (K) using mAb GB3. N: non-immune ascitic lluid (control). G: mAb GB3. Non-reduced SDS-PAGE (3'^ acrylamide gel). The same amount of starting labelled proteins was used in both cases (see Material and Methods). Mr: reduced thyroglobulin used as standard molecular weights.
Mr -660
-330
Figure 4. Immunoblot analysis of conditioned medium from both cultured libroblasts and keratinocytes. using mAb GB3. The same volumes of medium sample as in Fig. 3 were used from both the two cell types. a,b: non-immune ascitic fluid (MOPC 31. Sigma), c.d: mAb GB3 monoclonal antibody, a and c: medium from keratinocytes; b and d: medium from fibroblasts. Arrowhead shows BM-6()0/nicein. Non-tcduced SDS-PAGE. 3% acrylamide gel. Mr: molecular weight (kilodalton) of thyroglobulin (and its subunits) used as a standard
protein.
56
(BM-600/nicein). This has been ascertained by the "preclearing" immunoprecipitation strategy employing first pAb 6/2 and, second, mAb GB3, on the same biological aliquot putatively containing the BM-600/nicein protein (cultured keratinocyte conditioned medium). This strategy allowed us to conclude that pAb 6/2 displays two immunological reactivities: one against the 37 kD protein (seen on immunoblots) and the other against the 600 kD protein (seen by immunoprecipitation) which is recognized by mAb GB3. To clarify further the nature of the reactivity of pAb 6/2 and BM-6()0/ nicein, we isolated, by immunochromatography, the protein from conditioned media of cultured keratinocytes and blotted it onto nitrocellulose. We confirmed unequivocally that pAb 6/2 is able to recognize the larger subunits of BM-600/nicein (150 kD and 125 kD), although no reactivity was detected with the smaller 93.5 kD subunit. On the other hand, the absence of reactivity of pAb 6/2
Relationships between 6/2 and GB3 antibodies in immunoblots with BM-600/nicein in homogenates of cells or medium could in fact be explained by the extremely low amount of antigen in the extract and, additionally, by different affinity/tiler between pAb 6/2 and mAb GB3. Indeed, we reported earlier that the pool of antigen in the cell is very low (6), BM-600/nicein being secreted immediately into the medium. Furthermore, the radioimmunopreeipitation technique concentrates the antigen in contrast to the immunoblot technique, which would tend to require a large amount of homogenate not suitable for the eleetrophoresis step prior to the blotting. In the medium, the main problem is to deal with the large amounts of albumin from serum used for cell culture that impair the concentration needed for loading extracts SDSPAGE. In that regard, the affinity chromatography represents an ideal tool and allowed us to show the actual binding of pAb 6/2 to the isolated BM600/nicein from keratinocyte conditioned media. In order lo understand better the dual-specificity of the 6/2 polyclonal antibody and the possible relationship between the 37 kD protein and BM600/nicein, we have studied here the expression of these two proteins in cell culture. For example, if there were co-expression, one hypothesis would be that one of these two proteins might represent either a degradation product or a precursor form of the other. This would explain why pAb 6/2 reacted with both of them. If the antigenie site is common, the 37 kD protein is a part of the larger species. The experimental data given in this paper suggest this is unlikely, tn contrast to BM-600/ nicein, the 37 kD protein could not be found in the medium of cultured keratinocytes or fibroblasts. Thus, it seems unlikely that the 37 kD protein is a degraded form of BM-600/nicein, unless the 37 kD is rapidly hydrolyzed by proteases secreted by the cells. Additionally, the 37 kD protein is expressed by the human dermal fibroblasts, thus again making it unlikely that the 37 kD protein is a degraded form of the larger. Nevertheless, this does not prove that the 37 kD protein is not a precursor of BM-600/nicein and additional experiments will be required to investigate this possibility. Finally, taking all of the (admittedly somewhat indirect) results together, we are tempted to suggest that there is probably no relationship between the two proteins. The two immunologic specificities detected by pAb 6/2 may be explained by the fact that, obviously, severai proteins were present in the collected chromatographic peak from solubilized amnion which was used for the immunization procedure described previously (1, 2). Although the immunogenic extract was similar (1, 9), the dual specificity lias been avoided when mAb GB3 was made, because of the clonal screening performed for
monoclonal antibody production. Thus we conclude that mAb GB3 represents a more useful and appropriate probe than pAb 6/2 for the biochemical and bioclinical studies concerning the new basement membrane component BM-600/nicein (3-6). Its potential for unlimited production and its monospecificity can be applied for diagnosis, including prenatal diagnosis of lethal junctional epidermolysis bullosa where mAb GB3 displays a clear-cut absence of signal rather than a diminished one as is the case for pAb 6/2 (8). A eknowledgments We wish 10 express our graliuide to Pr Eugene Bauer (Stanford University Department ol" Dennatology) for helpful dis-
References 1. Hsi B L. Yeh C J. Faulk P W. Characterization of antibodies to antigens of the human amnion. Placenta 1985: 5: 513-522. 2. Verrando P. Ortonne .1 P. Pautrat G. Hsi B L. Yeh C J: Identification of a 37 kilodalton protein at the epidermal basement membrane by an antiserum to human amniotT. J Invest Dermatol 1986:'87: 190 1%. 3. Verrando P. Pisani A, Partouche O. Ortonne .1 P BM(lOO/nicein, a putative new basement membrane component involved in lethal junctional epidermolysis bullosa. In: Priestly G C, Tidnian M } , Weiss J B. Eady R A .), eds. Epidermolysis bullosa: a comprehensive review of classification, management and laboratory studies. Crowthorne. Berkshire. U.K.: DEBRA. 1990; pp. 137-140. 4. Verrando P. Partouche O, Pisani A. Ortonne J P. New molecular and cellular tools for studying BM-600/nicein. the "GB3" basement membrane antigen involved in lethal junctional epidermolysis bullosa. .1 Invest Dermatol (abstr.) 1989: 92: 538. 5. Verrando P, Hsi B L, Yeh C .1. Pisani A, Serieys N. Ortonne J P. Monoelonal antibody GB3. a new probe lor the study of human basement membianes and heniidesmosomes. Exp Cell Res 1987: 170: 116-128. 6. Verrando P, Pisani A, Ortonne .1 P. The new basement membrane antigen recognized by the monoclonal antibody GB3 is a large size glycoprotein: modulation of its expression by retinoic acid. Biochitn Biophys Acta 1988: 942: 45-56. 7. Heagerty A H M, Kennedy A R. Eady R A .1, et al. GB3 nionoclonal antibody for diagnosis of junctional epidennolysis bullosa. Lancet 1986: 8485: 860." 8. Kennedy A R. Heagerty A H M. Ortonne .1 P. Hsi B L, Yeh C .1 G, Eady R A .1. Abnormal binding of an antiamnion antibody to epidermal basement metnbrane provides a novel diagnostic probe for junctional epidermolysis bullosa. Br J Dermatol 1985: 113: 651-659. 9. Hsi B L. Yeh C .1 G. Monoclonal antibodies to human amnion. J Repro Immunol 1986: 9: 1 1-21. 10. Rheinwald .1 G. Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 1975: 6: 331-344. 11. Laemmli U. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 1970: 277: 680-685. 12. Lowry O H, Rosebrough N .1. Farr A L, Randall R .1.
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Verrando et al. Protein measurement with the Folin phenol reagent. J Biol Chem 1951: 193: 265-275. 13. Towbin H, Staehelin T, Gordon J. Electrophorelic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979: 76: 4350-^354. 14. Burnette N. Eleclrophoretic transfer of proteins from SDS
58
polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 1981: 112: 195-203. 15. Verrando P, Partouche O, Pisani A, Ortonne J P. Antibodies to basement membrane protein nicein (BM-6()0/nicein) give new clues in lethal junctional epidermolysis bullosa. 1992 (submilted).
