An immunoblot assay for the simultaneous quantification of several antigens

177,203-206

ANALYTICALBIOCHEMISTRY

(1989)

An lmmunoblot Assay for the Simultaneous Quantification of Several Antigens’ Karl

&hilling

Abteilung

Received

and M. Ciicilie

Anatomie

August

und Zellbiologie

Aletsee-Ufrecht der Universitiit

Ulm, D- 7900 Urn, Federal Republic

1,1988

A radioimmunologic assay method that allows for the simultaneous quantification of several antigens in one sample is described. Polypeptide antigens are resolved electrophoretically and electroblotted to nitrocellulose. The nitrocellulose is then reacted with a mixture of several antisera simultaneously, and antibody-binding proteins are visualized by incubation with 12’1-protein A and by autoradiography. Antigens are identified according to their molecular weights and quantified by counting the bound radioactivity. The sensitivity of the assay is in the low nanogram range and can be adjusted individually for each antigen by appropriately diluting the first antiserum. The procedure is presently applied to the detection of three neural antigens, neural cell adhesion molecule, neuron-specific enolase, and synaptophysin, in adult brain tissue and to the assessment of expression of the latter two during development of brain cells in primary culture. The method is fast, comparatively cheap, and associated with a low radiation exposure. It should prove especially useful when only scarce amounts of sample are available. a 1989 Academic Press,

of Germany

Inc.

The combination of SDS-PAGE’ and immunoblotting is now widely used in many fields of biomedical research (for reviews, see Refs. (l-3)). This technique has been successfully adopted to quantify a variety of antigens with a sensitivity comparable to classic liquidphase radioimmunoassay (e.g., Refs. (4-9)). Howe and Hershey (4) pointed out two distinct advantages of immunoblotting over liquid-phase RIA; i.e., immuno1 This work was supported by the Deutsche Forschungsgemeinschaft (Grant Schi 271/l-2) and by special funds from the State of Baden-Wiirttemberg (Forschungsschwerpunkt No. 24). ’ Abbreviations used: NC, nitrocellulose; N-CAM, neural cell adhesion molecule; NSE, neuron-specific enolase; PAGE, polyacrylamide gel electrophoresis; ~38, synaptophysin; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline; TX-100, Triton X-100. 0003-2697/89 $3.00 Copyright 0 1989 by Academic Press, All rights of reproduction in any form

blotting can distinguish different molecular forms of an antigen, and it can also tolerate considerable levels of antibody impurity if the antigens recognized are separable electrophoretically. We felt that the separation of antigens provided by SDS-PAGE prior to immunodetection may also be useful for establishing an assay suited to quantify several antigens simultaneously. The procedure, described here, may prove especially valuable when only limited amounts of sample are available. MATERIALS

AND

METHODS

Sample preparation. Primary dissociated cultures of fetal rat diencephalon were established as described previously (10). After cultivation for 2-12 days, cells were lysed in TX-100 buffer (10 mM NaH2P04, 110 mM NaCl, 5 mM EDTA, 0.5% (v/v) Triton X-100; pH 6.5). Insoluble material was removed by centrifugation (12,OOOg,, 15 min, 4”C), and soluble proteins were cleared of detergent (ll), resuspended in electrophoresis sample buffer (62.5 mM Tris-HCl; 2 mM EDTA; 2% (w/v) SDS; 5% (v/ v) 2-mercaptoethanol; 0.005% (w/v) bromphenol blue; pH 6.8), and heated to 100°C for 5 min. Adult rat cerebral cortex, which served as a standard for quantification (see below), was homogenized in 10 vol of TX-100 buffer by 10 strokes in a Teflon-to-glass homogenizer. The soluble proteins were prepared for electrophoresis as described above. Electrophoresis and blottingprocedures. SDS-PAGE was done according to the method of Laemmli (12), under reducing conditions, in l-mm-thick X 130-mm-wide X lOO-mm-long slab gels of 10% acrylamide/0.267% bisacrylamide. The stacking gel was 10 mm long and contained 3.1% acrylamide/0.083% bisacrylamide. Usually, samples were loaded in 5-mm-wide sample wells. In some experiments, the sample was layered across the entire top surface of the stacking gel in a preparative manner, taking care that the surface was level and that the sample formed a zone of uniform thickness across the gel. Proteins were transfered to 0.45~pm pore size nitro203

Inc. reserved.

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cellulose (Schleicher & Schiill BA 85; Dassel, West Germany) as described by Towbin et al. (13). The transfer buffer contained 25 mM Tris, 192 mM glycine, and 20% (v/v) methanol, and blotting took 3 h at 8.5 V/cm. Antigen detection and quantification. The blotted NC membrane was washed for 20 min with four changes of TBS-Tween (50 mM Tris-HCI, 150 mM NaCl, 0.05% (v/v) Tween 20; pH 7.6) containing 2.5% (w/v) nonfat dry milk. After a final 5-min wash in TBS-Tween/0.5% nonfat dry milk, the NC was incubated overnight at 4°C with primary antiserum diluted in TBS-Tween. In order to assessthe impact of antibody dilution on the sensitivity of quantification, we incubated strips of NC containing equal amounts of antigen with serial 1:l dilutions of primary antibodies. The dilutions used ranged from 1: 1250 to 1:160,000 for anti-NSE, and 1:500 to 1:64,000 for anti-p38. When the NC was to be incubated with several antisera simultaneously, these were mixed in TBSTween, taking care that the final dilution of each antiserum was appropriate. After reaction with first antiserum (antisera), the NC was washed for 1 h with three changes of TBS-Tween, followed by incubation with 1251-labelled protein A (1 &i/ml) in TBS-Tween for 1 h. Again, the NC was washed with three changes of TBS-Tween for 3 h. All incubations/washing steps were done at room temperature, unless otherwise specified. After a final rinse in TBS-Tween, the NC was dried between filter paper and exposed to Kodak XAR X-ray film for 6-72 h. For quantification, the localized antigens were cut from the NC, and the bound radioactivity was counted. Extracts of adult rat cortex were used as standards. The amounts of NSE or ~38, respectively, associated with 1 /*g of soluble rat cortex protein were defined as 1 unit. Based on data from the literature, one may estimate that this is equivalent to approximately lo-15 ng of NSE and 3.2 ng p38 (14,15). Standards and samples were always blotted simultaneously in order to eliminate possible errors in quantification resulting from incomplete transfer. The antisera used in this study were directed against neuron-specific enolase, synaptophysin, and the neural cell adhesion molecule. They were all raised in rabbits, and their specificities have been described (16-19). Protein A was radioiodinated (20) to a specific activity of 15.7 &i/pg protein and used for up to 3 months after preparation. RESULTS

AND

DISCUSSION

The ~38 and NSE antisera each stained a single protein band on blots prepared from electrophoretically separated proteins of either adult rat brain cortex (Fig. 1, lanes A and B) or cell cultures (not shown). The immunoreactive bands displayed the molecular weights reported for the respective proteins (i.e., 38,000 for ~38 and 49,000 for NSE). The antibody to N-CAM reacted

*I*r _ NSE

*cd

- ~38

A

B

C

D

FIG. 1.

Immunological identification of soluble proteins of adult rat cortex. Blots were incubated with antibodies to ~38 (A; 1:15,000), NSE (B; l:lO,OOO), or N-CAM (C; 1:lOOO). Strip D was incubated with all three antibodies simultaneously.

with three proteins of apparent M,‘s 120,000, 140,000, and 180,000-200,000 (Fig. 1, lane C), and, in addition, with a sharp band migrating at 65 kDa. This band probably represents the amino-terminal N-CAM fragment that is released from N-CAM spontaneously in solution (21). The background staining caused by the three antibodies was moderate. Moreover, the background staining caused by any of these antibodies did not fall within those regions of the blot where the antigens recognized by the other two antibodies were located. Simultaneous staining of electrophoretically separated extracts with all three antibodies resulted in six well separated bands which corresponded to ~38, NSE, the 65-kDa N-CAM fragment, N-CAM 120, N-CAM 140, and N-CAM 180 (Fig. 1, lane D). We (9,lO) and others (6,8) have shown a linear, or, at higher antigen concentrations, a logarithmic relationship between the amount of a given antigen on Western blots and the radioactivity bound following incubation with appropriate antibodies. However, as the amounts of antigens present in a sample may vary independently of each other, it was important to determine whether the sensitivity of the assay can be adjusted for each antigen individually. This can in fact be achieved by varying the antibody dilution. As shown in Fig. 2 for NSE and ~38, the amount of radioactivity bound to a given amount of antigen varies inversely with the antibody dilution. These results agree well with previous data showing a similar relationship between sensitivity and antibody dilution in a dot-blot assay for p38 (22). An apparently sigmoidal relationship between log antibody dilution

QUANTIFICATION

OF

SEVERAL

‘, ANTIBODY

DILUTION

(log,,)

FIG. 2. Relationship between antibody dilution and radioactivity bound by a fixed amount of antigen. Soluble proteins of adult rat cortex were separated on a preparative 10% gel and transferred to NC. The NC was cut into strips of equal width, and each strip was incubated with a different dilution of antibody to NSE (0) or ~38 (D). After incubation with “‘I-protein A, the immunoreactive bands were localized by autoradiography, cut from the NC, and counted. Each point is the mean of two measurements which differed by less than 15%.

and the amount of antibody bound in solid-phase radioimmunoassay has also been described for numerous other antigens (e.g., (5,23,24)). Although the theoretical foundations underlying this relationship are not clearly understood (24), such antibody titrations are helpful in selecting the most appropriate sensitivity for each individual antigen. Figure 3 illustrates an application of our assay to the simultaneous quantification of NSE and ~38 in primary diencephalic cultures of different cultivation times. In this system, sufficient sensitivity was obtained by diluting anti-NSE l:lO,OOO, and anti-p38 1:15,000. This relation was chosen to ensure that the amounts of radioactivity associated with NSE or p38 were within the same order of magnitude. This results in comparable exposure times and counting errors and is thus the most convenient approach. Counting of the radioactivity bound by the individual bands shown in Fig. 3 revealed that NSE increased from initial values of 0.47-O-62 unit in freshly prepared cells to a maximum value of 52.1-57.5 units after 12 days of cultivation. During the same period, the ~38 content rose from 0.50-0.60 unit to 32.3-38.1 units. A detailed analysis of the expression of NSE and p38 under varying experimental conditions will be presented elsewhere (18). The ability to measure several antigens simultaneously in a single sample by our procedure has several obvious advantages when compared to the measurement of each antigen in an individual sample. First, the number of samples needed is considerably reduced, saving both time and material. Both the amount of radioactivity and the time needed to handle the radioactive solu-

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tions are at least cut in half, resulting in less potentially hazardous waste and reduced radiation exposure. Second, reduced sample handling should lead to increased assay accuracy, as potentially error-prone steps are eliminated. Finally, measuring several antigens in one electrophoretically separated sample eliminates all variability resulting from sample handling prior to electrophoresis (e.g., interdish variability in cell cultures, dilutionlpipetting errors) and creates an internal standard which allows the values obtained to be normalized. An illustration of this last advantage can also be seen in Fig. 3. The values for both NSE and ~38 determined from the two cultures of Day 4 varied by more than 50%. Nevertheless, the p38/NSE ratios calculated for these two cultures were 0.57 and 0.67, the variation of which is well within the limits of biological variability in our culture system (18). Previously two methods suited to demonstrate several antigens in one electrophoretically separated sample have been described. These methods include either the preparation of several blots (replicas) from one gel (2527) or the sequential probing of one NC with several antibodies (28). The latter method involves removing of the first step reagents by exposure of the NC to buffers of low pH or to buffers containing detergents, prior to application of the second step reagents. With regard to antigen quantification, these methods suffer from two main disadvantages. In the first method, it is generally difficult to ensure equal transfer of proteins to all NC sheets. Consequently, quantitative comparison of antigens measured on different sheets of NC may prove difficult. In the second method, it cannot be excluded that erasing of first step reagents does not result in a loss of antigen from the NC. These drawbacks are eliminated by the procedure described presently. In conclusion, we have presented a modified method of Western blotting, suited to simultaneously quantify several antigens in one sample. The method has a sensi-

0

DIV

I2

I

41

8

112 -.

NSEp38

-

FIG. 3. Developmental changes in the amounts of NSE and ~38 in primary diencephalic cell cultures. After different times of cultivation (days in t&o, DIV), soluble proteins were extracted from the cultures, separated electrophoretically, and transferred to NC. The NC was then incubated with anti-NSE (l:lO,OOO) and anti-p38 (1:15,000) simultaneously, and bound antibody was visualized by reaction with ‘?-protein A and autoradiography. Immunoreactive bands are well separated from each other and can be readily cut and counted.

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tivity comparable to that of classic liquid-phase radio immunoassay. It is fast, comparatively cheap, and associated with a relatively low radiation exposure. The new method is especially suited in situations where sample size is limited as, e.g., in cell culture preparations. ACKNOWLEDGMENTS We thank Dr. R. Jahn (Munich, FRG), Dr. P. J. Marangos (San Diego, CA), and Dr. G. Rougon (Marseille, France) for the generous gift of antibodies. S. Keck provided skilled technical assistance, J. Fassberg helped to word the manuscript, and B. Mader typed it. REFERENCES 1. Towbin, 34a. 2. Gershoni, 3. Gershoni, 15.

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