Electrophoresis 1990, I I , 31-41

Franqois X. Desvaux Bernard David Gabriel Peltre Immuno-Allergy Unit, Pasteur Institute, Paris

Miiltiple successive immunoprinting: A fast blotting technique

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Multiple successive immunoprinting: A fast blotting technique of a single agarose isoelectric focusing gel Multiple successive pressure blottings of a single agaroseisoelectric focusing gel were performed on normal and CNBr-activated nitrocellulose (NC) filters. The results obtained by multiple successive 10s immunoprints were compared to those obtained by a single 10rnin immunoprint. To quantify the transfer efficiency ofthese techniques, a defined amount of radioactive material was separated by isoelectric focusing on agarose gel. After separation and pressure blottings of the gel the N C filters were submitted to autoradiography. The amount of radioactive material bound to the NC filters was determined by scintillation technique. The single 10 rnin pressure blot was more efficient than each of the multiple successive 10s prints. However, the latter procedure allowed equal resolution and resulted in a higher recovery of total radioactivity than the single immunoprint technique. The aim of this paper is to show how to obtain highly reproducible prints of electrophoretic patterns in an agarose gel of heterogeneous samples when accurate multiple immunodetections are to be performed. This technique was tested to characterize the grass pollen specific immunoglobulin classes and subclasses from an allergic patient.

1 Introduction

2 Materials and methods

Ten years ago, Towbin et al. L 1I described a technique for electrophoretic transfer of proteins on a nitrocellulose (NC) sheet from a sodium dodecyl sulfate-polyacrylamide gel (SDSPAGE). Many adaptations of this technique have been made for other types of electrophoresis [21, among them the simple pressure blotting from an agarose gel after isoelectric focusing, first presented in 1980 [3-51. Legocki and Verma [61 described a procedure to obtain three replicas of one polyacrylamide gel on NC filters, and used each replica for screening antigens with different antisera. Each electrophoretic transfer was performed in 1 h. By acidic pH treatment, they eluted antibodies from the filter, allowing antigen recognition by new antibodies, this being done at least 3 times. Manabe et al. 171 used an electroblotting apparatus to produce 5 replicas from microslab two-dimensional PAGE, replacing the NC sheets every 10 min. They showed that many proteins remained in the gel after a 50 rnin transfer, and that small proteins were transferred more rapidly than bigger ones. Both procedures were based on an electrophoretic transfer from PAGE gels. Miribel et al. [S] blotted the same agarose gel with two different filters (NC and nylon), placed on top of each other in order to trap the molecules (mainly hydrophobic or hydrophilic, respectively). In this paper we described a simple, inexpensive and fast pressure blot technique of agarose gels that does not require special equipment, and allows the separation and transfer of large molecules (for instance IgM) as well as smaller ones. In order to quantitate the transfer efficiency in each print, a radioactive sample was focused. The technique was applied to the detection of immunoglobulin classes and sublasses of allergic patient sera directed to the Dactylis glomerata grass pollen components.

2.1 Pollen extracts Fifty mg of Dactylis glomerata pollen were incubated with 450 pL deionized H,O during l h at room temperature on a roller, centrifuged 5 rnin (9000 g). Two hundred pL of supernatant were submitted to electrophoresis. 2.2 Radioactively labelled antibodies '%Rabbit labelled anti-IgE (Rast Kit, Pharmacia Diagnostics, Uppsala, Sweden) were used to quantify the transfer efficiency.

2.3 Isoelectric focusing (IEF) IEF of pollen was performed in 1 % agarose, containing 12 % sorbitol, and 2 %of a mixture of carrier ampholytes, forming a pH range of 2-1 1 (Servalyt pH 2-4: 15 pL, pH 4-5: 15 pL, pH 4-9: 60 pL, pH 9- 1 1: 30 pL Serva, Heidelberg, FRG; Pharmalytes, pH 4-6.5: 30 pL, pH 3- 10:60 pL, Ampholines pH 3.5-10: 60 pL,pH 7.9: 30 yL,PharmaciaLKB, Uppsala, Sweden). A flat bed apparatus (FBE 3000, Pharmacia LKB, Uppsala, Sweden) was used with cooling to 10 "C at constant power (4W) for a total of 1500 Vh [ 5 ] . IEF of radioactively labelled antibodies was performedin 1 % agarose gel, containing 12 % sorbitol, and2 %carrier ampholytes,pH 5-8,(Pharmacia) in the flat bed apparatus heated at 28 OC at constant power (4W) for a total of 2000 Vh. 2.4 Print

~

Correspondence:Pr. B. David, Unit&d'Immuno-Allergie, Institut Pasteur. 28 rue du D r Roux, F 75015 Paris, France Abbreviations: BSA, bovine serum albumin; DAB, diaminobenzidine tetrahydrochloride; IEF, isoelectric focusing; Ig, immunoglobulin; NC, nitrocellulose; PAGE, poly acrylamide gel electrophoresis; PBS, phosphate buffered saline; SDS, sodium dodecyl sulfate; SSA, sulfosalicylic acid; TCA, trichloroacetic acid

0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, I990

A single print andlor up to four successive prints were performed with normal or CNBr-activated NC filter (BA 83, Schleicher and Schuell, Dassel, FRG), According to Demeulemester et al. [91. Immediately after the run, the gel was rinsed with deionized water and blotted. Three types of successive prints were performed and compared to a single print: four successive 10sprints; three successive 10s prints followed by a 10 min print; one 10s print followed by a 10 rnin print. 0173-0835/90/0101-0037 $02.50/0

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Electrophoresis 1990.11,37-41

F. X. Desvaux et al.

ing water was determined for 10 rnin in a y-counter (1277 Gammamaster, Pharmacia Diagnostics).

2.4.1 Ten-second print Two mL of deionized water were deposited on the gel prior to blotting with the NC filter. Excess water was drained for 10s by keeping the gel vertically (this water is counted as rinsing water). The NC sheet was covered with a single layer of Schleicher and Schuell GBOO 1 paper and submitted to 2 kg/ dm2pressure for exactly 10s.After blotting, the NC filter was removed from the gel and dried with a fan at room temperature.

3 Results Table 1 shows the distribution of radioactivity recovered after IEF from the electrode strips at the anode and cathode, the Whatman No. 1 paper (sample deposit strip), rinsing water and Schleicher & SchueH GBOOl paper, NC filters and the residual gel after blotting. Comparison of total radioactivity, recovered after transfer (i. e. in N C filters, blotted gel, washing water and absorbing papers ) with the radioactivity contained in the nonblotted gel (i. e. the control gel) is shown in Fig. 1. Based on these experiments, we expressed the transfer efficiency as the ratio between the radioactivity bound to the NC filters and that remaining in the gel after blotting. After blotting for 10 min (type A: 8 experiments) 75 % of the radioactivity were bound to the N C filter and 25 %remained in the gel (Fig. 2a).

2.4.2 Ten-minute print Two mL of deionized water were deposited on the gel prior to blotting, followed by removal of excess water. The NC sheet was covered with a single layer of Schleicher and Schuell GBOOl paper, three layers of absorbing paper, and submitted to 2 kg/dm2 pressure for 5 min. The wet absorbing paper and the Schleicher and Schuell GBOOl paper were replaced, and the print was continued for an additionail 5 min under a 4 kg/ dm2 pressure. After blotting, the NC filter was left on the gel, dried with a fan at room temperature and removed from the gel by incubation in deionized water. The blot was either dried for quantitation, or incubated in phosphate buffered saline (PBS), 0.1 %Tween 20 and 1 %bovine serum albumin (BSA; blocking step: see below) for Ig detection.

In experiments with a single 10s print, followed by a 10 rnin print (type B: 7 experiments) an equal amount of radioactive material was recovered in the two successive N C prints (40.6 %; 40.9 %). The radioactive material in the residual gel after blotting was lower than for the type A experiment, namely 18.5 % instead of about 25 % (Fig. 2b). Four complemen-

2.5 Immunoglobulin detection After 90 rnin blocking in PBS, 0.1 % Tween 20, and 1 % BSA at 45 OC, NC was cut in 5 mm wide strips, and each strip was incubated overnight with individual patient sera diluted 1/10 in 1 mL PBS, 0.1 %Tween 20, 1 % BSA at room temperature. After incubation, strips were washed three times (5 min) in saline containing 0.1 % Tween 20. For XgA detection the NC strips were incubated for 1h with horseradish peroxidaselabelled sheep anti-IgA (Institut Pasteur Production, Paris, France), diluted 1/1000 in PBS, 0.1 % Tween 20 and 1 % BSA, washed four times in saline, 0.1 % Tween 20 and detected by diaminobenzidine, tetrahydrochloride (DAB, Sigma, St. Louis,MO,USA): 10mgin20mLO.1 Mphosphate buffer pH 7.4, and 30 pL 30 % H,O,. For the detection of IgG subclasses the NC strips were incubated for lh with monoclonal antibodies from mouse (anti-IgG 1, IgG3, IgG4 from Unipath, Bedford England and anti-IgG2 from BioMakor, Kiryat Weizmann, Rehovot, Israel) diluted 1/1000 in PBS, 0.1 %Tween 20,l % BSA, washed and incubated for another 1h with a second antibody (peroxidase labelled rabbit anti-mouse globulin, Dakopatts, Denmark). Visualization was performed with DAB.

2.6 Radioactivity measurement

4

49.40

50.40 f 6.79

NCfiltor blottd w r t t r +

sum

gel

pqw

A+8+C

(6)

(C)

(A)

I nul

blotted

9Ql

Figure I . Comparison between the amount of radioactivity recovered after transfer (I) and anonblotted gel (11). Meanvalues obtainedfrom 32 transfer experiments after IEF of radiolabelled IgE. (A) NC filter, (B) blotted gel, (C) rinsing water and Schleicher & Schuell GB 001 paper.

The amount of radioactive material bound to NC filters, Schleicher and Schuell papers, gels, and contained in the rinsTable 1. Distribution of radioactive material after transferal -

Anode strip

Cathode strip

Whatman No. 1 paper

Rinsing water and Schleicher & Schuell paper

N C filters

blotted gels

20.13f5.34

0.15+0.11

24.6523.83

5.0620.98

38.602517

11.58k3.54

a) Results expressed in % as mean values f standard deviation

I1 -

1

95

Sum of

Electrophoresis 1990,11, 31-41

Multiple successive immunoprinting: A fast blotting technique

39

75.03*2.68%

16.% *2.1€1%

2c

rn

D

lnNC18 U N C f M

a

14.43 5.98%

0 6860*5 97X

40 93f 3.74%

f

3.38%

24 I r t N C laS 2ndNC 10s 3 r d N C lcls 4 t h N C 10s blotted gel

ed

:396% I0 79*292%

I~~NCIOE 2ndNCIomln bloltsdgel

1092 *2.13%

IslNCIo5 2ndNClaS 3rdNCIQ

Figure 2 . Distribution of radioactive material (%) bound to NC filters and in the residual gels after blotting. (a) Single 10 minblot;(b)twosuccessiveblots:1 x 10 s; 1 x 10 min; (c) as in (b) but with 4 layers of absorbing paper instead of one; (d) four successive blots: 4 x 10 s; (e) four successive blots: 3 x 10s; 1 x 10 min. Results expressed as mean values ? standard deviation.

Figure 3. Comparison between a single 10 min immunoprint (A) and four successive 10s immunoprints (B1, B2, B3, B4), applied to immunoglobulin detection from one patient serum (a) IgG3; (b) IgGl ; (c) IgA; (d) IgG2; (e) IgG4. These immunoglobulins are directed against pollen components of Dactylis glomerata, separated by IEF and blotted. Arrows indicate bands which disappear faster than others from the 10sB 1 print to the B4 print. Almost all the bands detected asIgG3 on thefirst 10simmunoprint are weakly visualized on the three consecutive prints.

40

F. X. Desvaux et al.

tary experiments were made to determine the role of filter papers. The importance of their absorbing capacity was evaluated by layering three absorbing papers onto the Schleicher & Schuell GBOOl paper. In this case, 68.6 % were bound to the first 10s immunoprint, only 14.4 % t o the second 10 rnin print and 17 %remained in the blotted gel (Fig. 2c). In experiments with four successive 10s prints (type C: 6 experiments) the percentage of radioactive material bound to the successive N C were, respectively, 3 1.0 %, 14.9 %, 10.8 % and 7.0 %. The radioactivity remaining in the gel after blotting was higher in these experiments than in the two preceding types, namely 36.2 % (Fig. 2c). With three successive 10s prints, followed by a 10 rnin print (type D: 7 experiments), the percentage of radioactive material bound to the successive NC were, respectively, 3 1.7 %, 16.9 %., 10.9 % and 23.5 % (Fig. 2e). The radioactivity left on the gel after blotting (1 7.0 %)waslowerthanthevaluesobservedin the threeothers types of experiments. Radioactivity measurements showed that only small amounts of sample were transferred if more than four prints were made (up to ten), so that these blots were useless (data not shown). Figure 3 shows four successive immunoprints from the same gel compared to one single 10 min pressure blot. Grass pollen extract was focused, blotted and recognized by one patient serum. In a, b, c, d and e are shown the respective specificities of IgG3, IgG1, IgA, IgG2 and IgG4. The first 10s print was the most intense and band detection decreased throughout the successive blots. Some bands indicated Iby an (arrow) did not fade as fast as the others. It was also noticeable that IgG3 detection was relatively strong on the first 10s immunoprint but rather weak on the others. For the other Ig classes and subclasses, especially IgG4 which is not (quantitativelyimportant, auseful signal was stilldetectableom the lastNC filter.

Electrophoresis 1990.11, 31-41

When the transfer efficiency onto normal and CNBr-activated NC sheets was compared, no significant differences between these two types offilters were found (datanot shown). The advantage of the CNBr-activated NC filter is to more firmly bind the blotted constituents, probably partially by covalent bonds throughout the subsequent incubation and washing steps, requiring up to 24h in a typical immunoprint procedure [ 51. In the experiments described in this article, no additional incubation or washing steps were performed after transfer of the radioactive material. This may explain why the blotting capacity of CNBr-activated NC and ordinary N C filters are here equivalent. Our results were obtained with a radioactive immunoglobulin sample under the described experimental conditions, but obviously their quantitative aspects depend upon the nature of the sample used. We have applied these techniques to the detection of grass pollen specific Ig classes and subclasses in allergic patient sera with only qualitative results. The fact that not all the pollen constituents could be recognized with the same intensity from one blot to another may be due to several factors: (i) differential ability of pollen components to bind the CNBr-activated N C filter and to resist the washing steps (whereas the experiments with radioactive material does not need these washing steps); (ii) different sensitivity of the Ig detectability by each of the specific monoclonal or polyclonal antibodies used. (iii) For IgG3 detection, the signal decreased rapidly from the first 10s blot to the next (Fig. 3, B la-B4a). In this case, a weaker detection could be due to the rather low affinity of anti-IgG3 monoclonal antibodies whose binding is more altered by decreasing amounts of antigen present in the successive prints. Only major components such as Dac g l 101 were always recognized by patient antibodies.

Quantitative data about transfer efficiency are scarce. Vaessen et al. [ 111 studied quantitative aspects of protein blotting, 4 Discussion using immunodetection with specific antisera and lZ5I-labelled protein A. They showed that the amount of radioactivity Radioactivity measurements showed that an important part bound to each antigen increased with the amount of protein of the radioactive material was not submitted to electrophore- present in the gel. They reported that the transfer efficiency of sis since 24.6 % of radioactivity is found, in the Whatman No, a given protein from an SDS-PAGE to an NC sheet is depen1 paper strip used for application samplt: and 20.1 % is recov- dent on its molecular size. Bittner et al. [ 121 determined the ered in the anode strip (Table 1). The paper-bound radio- quantitation of eluting force for optimum results in transfer activity found at the anode is certainly clue to free anioniclZ5I and gave data on the kinetics of electrophoretic transfer of and the radioactivity bound to the Whatman No. 1 paper DNA. They also showed that smaller fragments were transferstrip could be due to denatured labelled antibodies. Some red with greater efficiency than larger ones. Legocki and discrepancies between results of the same types of experi- Verma [61 in their multiple immuno-replica technique noticed ments can be explained by the extreme sensitivity of the that some proteins were transferred more efficiently than transfer. After soaking the NC in deionized water the total others, but without correlation between the velocity of elecduration for all steps before and after the 10s print, namely trophoretic transfer and molecular size of the proteins. layering the NC filter, the GBOO 1 Schleicher & Schuell paper, Manabe et at. [71 also noticed that the amount of protein and their removal after blotting, is about 1 min. Especially for transferred to each replica was not uniform, and showed that the first blot, only one extra second of pressure (that is 10 % their multiple replica technique was suited for qualitative, not more of the total pressure time) can affect the results. Another quantitative, purposes. Unlike Legocki and Verma, they critical issue is to apply uniform pressure on the successive showed that the rate of protein transfer was higher for low blots, particularly if small or narrow agarose gels are pressed. molecular weight proteins. Our results showed that these Shorter pressure time (less than 10s) were poorly repro- techniques (successive 10s or single 10 min print) are comducible. The absorbing capacity of blotting papers is also plementary. The single 10 min print procedure is preferable crucial. Our experiments showed that in successive prints, it is when high sensitivity is required, whereas the multiple sucimportant to apply only one sheet of Schleicher & Schuell cessive prints, especially the two successive blots, are an expaper on the NC filter. Three additional sheets of absorbing cellent way to compare different immune detections of the paper inducedmore water flow throughtheNC in the 10sprint same components. than only one sheet, resulting in a more efficient transfer (compare Figs. 2b and c). Received May 22, 1989

Electrophoresis 1990. I ! . 41-45

Rapid detection of proteins by enzyme-linked immunofiltration assay

5 References Towbin, H., Staehelin, T. andGordon,J.,Proc. Natl. Acad. Sci. USA 1976,9,4350-4354. Anderson, N.L., Nance, S. L., Pearson, T. W. and Anderson, N. G., Electrophoresis 1982,3,135-142. Peltre, G.,Weyer, A,, Le Mao, J., Lapeyre, J. and David. B.. Abstracts 4~hInternationalCongressofImmunology,Paris, 1980,No.13.1.21. Peltre, G.,Lapeyre,J. andDavid,B.,in:Molina,C.(Ed.).Proceedings of the European Academy of Allergology and Clinical Immunology Meeting, Lavoisier, 1982,Vol. 1, pp. 92-97. Peltre, G., Lapeyre, J . and David, B., Immunol. Lett. 1982, 5 , 127-131. Legocki, R. P. and Verma, D. P. S., Anal. Biochem. 1981, 1 1 1 , 385-392.

Jean-Michel Pinon Dominique Puygauthier-Toubas Herd Lepan Cathy Marx Annie Bonhomme Joselyne Boulant RCgine Geers HervC Dupont Laboratoire de ParasitologieMycologie C.H.R.U., Reims

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[71 Manabe, T. Takahashi, Y. and Okuyama, T., Anal. Biochem. 1984, 143,39-45. Lsl Miribel,L., Peltre,G., Amaud, P. and David, B.,in: Dunn.M. J.(Ed.), Proceedings of the Fifth Meeting of the International Electrophoresis Society, VCHVerlagsgesellschaft, Weinheim 1986,pp. 703-706. 191 Demeulemester, C., Peltre, G., Laurent, M. and David. B.. Electrophoresis 1987,8,71-73. lol Mecheri, S., Peltre, G. and David, B., lnt. Arch. Allergy Appl. Immunol. 1985, 78,283-289. I' Vaessen, R.T. M. J., Kreike, J. and Groot, G. S . P., FEBS Leu. 198I,

124, 193-196. [12] Bittner, M., Kupferer, P. and Morris, C. F.,Anal. Biochem. 1980,102, 459-471.

Rapid detection of proteins by enzyme-linked immunofiltrationassay after transfer onto nitrocellulose membranes Enzyme-linked immunofiltration assay (ELIFA) for labeling transferred proteins is an interesting and powerful technique for the rapid specific detection (1 5 min) of proteins immobilized on nitrocellulose or nylon membranes (0.20 and 0.45 pn).ELIFA does not require fastidious handling of the membranes. Saturation, specific labeling and washing procedures are achieved by filtration, controlled by a monitoring unit which regulates the flow rate and ensures excellent specificity, repetition and reproducibility. The recycling by closed circuit or by repetetive inversion of the flow direction offers the advantage of reducing the volumes of expensive reagents while simultaneously increasing the sensitivity of the technique. The detection limit is at least as low as 1-5 ng using directly or indirectly enzymatically labelled probes. ELIFA may be extended to the identification of glycoproteins using specific ligands such as lectins or to the immunocapture of an antigen using specific antibodies immobilized on an activated membrane. ELIFA complements fast separation, by e.g., isoelectric focusing, polyacrylamide gel electrophoresis, or sodium dodecyl sulfatepolyacrylamide gel electrophoresis and accelerated electrotransfer to membranes with rapid detection reducing the total time for separation transfer and detection to less than 2 h.

1 Introduction Described by Renart [ 11 and Towbin [21 in 1979, the transfer of proteins onto immobilizing membranes has opened up a considerable field of biological applications. At present, in most cases blotting is the intermediate step of a three-step methodology including electrophoretic separation and finally protein detection using labelled probes, ligands or specific Correspondence: Dr. J . M. Pinon, Laboratoire de Parasitologie-Mycologie, CHRU HBpital Maison Blanche, 45 rueCognacq Jay, F-5 1092Reims, France Abbreviations: 2-DE, two-dimensional electrophoresis; ELIFA, enzymelinked immunofiltration assay; IEF, isoelectric focusing; Mab, monoclonal antibody; NC, nitrocellulose: PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; Tg, Tomplasma gondii; TTBS, Tris-Tween buffered saline 0VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990

antibodies [3,41. This analytical approach wouldhave aneven greater impact in research or in biodiagnostics if the three steps, preferably standardized, were simple and fast, and the consumption of expensive reagents could be reduced. The recently described techniques of horizontal electrophoresis in miniaturized gels 151 and of accelerated electrotransfer 161 onto nitrocellulose (NC) membranes can easily be carried out in less than 1 h. By contrast, the last visualization step, including saturation or inactivation of the membranes, followed by specific antigen detection, has not been developed in parallel and still requires at least several hours [3,7]. Therefore, it seemed challenging to adapt the potential of rapid immunodetection, offered by the enzyme-linked immunofiltration assay (ELIFA), to immobilizing membranes. Initially, this technique was developed for the characterization of antibodies (IgG, IgM, IgA or IgE) implicated in electro-immunoprecipitating reactions carried out on cellulose acetate membranes 0 1 73-0835/90/0 101-004 I %02.50/0

Multiple successive immunoprinting: a fast blotting technique of a single agarose isoelectric focusing gel.

Multiple successive pressure blottings of a single agarose isoelectric focusing gel were performed on normal and CNBr-activated nitrocellulose (NC) fi...
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