Effect of detergents on antibody-antigen interaction

ANALYTICAL

BIOCHEMISTRY

98, 445-451

(1979)

Effect of Detergents

on Antibody-Antigen

GIORGOS

Received

Interaction

J. DIMITRIADIS

February

28. 1979

The effect of the different detergent mixtures on immunodiffusion and immunoprecipitation was studied. The anionic detergent sodium dodecyl sulfate at concentrations above 0.2% (w/v) inhibits the reaction between antigen and antibody by more than 90%. Nonionic detergents at a concentration of 1% (w/v) have little or no detectable effect. In contrast. when we used mixtures of various concentrations of ionic and nonionic detergents the inhibitory effect ofthe ionic detergent decreased.

Some ionic detergents, such as lsodium dodecyl sulfate (SDS)’ in low concentration (0.5% or less), inhibit the antigen-antibody precipitation reaction (I). Conversely, nonionic detergents e.g., Triton X- 100, Nonidet P-40, Tween 80, Brij 58, Berol EMU-043. Tween 20 SD, Lubrol WX (l-5). while effectively solubilizing complexes .such as biological membranes, do not apparently inhibit antigen-antibody reaction. Both SDS and Triton X-100 are used for extraction of proteins from membranes, the former because of its very strong solubilizing properties, the latter because of its very mild action on the extracted proteins. The disadvantages of extraction with SDS. especially when it is used for the extraction of antigenic markers from membranes, is that it inhibits the immunoreaction with the respective antibody. The present work describes the kffect of mixtures of SIDS and nonionic detergents on immunoreaction in solution (immunoprecipitation) as well as in agarose gels (immunodiffusion). The results indicate that the inhibition of the antigenantibody reaction by SDS is greatly rleduced when a nonionic detergent is also present ‘Abbreviations PBS, phosphate

used: SDS, sodium buffered saline.

dodecyl

and in some cases the resolution diffusion is much higher. MATERIALS

by immuno-

AND METHODS

Detergents were obtained from the following sources and were used without further purification: sodium dodecyl sulfate, Triton X-100 (poly (ethyleneglycol) p-isooctylphenylether), Nonidet P-40 (alkyl phenyl ethoxylate), and Brij 58 (polyoxyethylene cetyl ether) were supplied by BDH Chemicals Ltd.; Tween 80 (polyoxyethylene sorbitan mono-oleate) was obtained from Koch-Light Laboratories Ltd.: ChloramineT was from BDH Chemicals Ltd. and Na”“I carrier free (100 mCiim1) from the Radiochemical Centre, Amersham, Bucks, England. Agarose (Indubiose A37) was obtained from L’lndustrie Biologique Francaise S.A. Antigens atltl at~tihodies. Rabbit globin and tobacco mosaic virus coat protein were prepared as described elsewhere (6-7). Antisera were raised in guinea pigs and rabbits, respectively. Both antigens were dissolved in 0.9% NaCl at a concentration of 1 mgiml and mixed with an equal volume of Freund’s complete adjuvant. The immunization procedures described by Avrameas (8) were followed. y-Globulin fractions were

sulfate:

445

0003-2697/79/140445-07$02.00/O Copyght All rIghI

“- 1979 h> Academy Prcr,. Inc of reproductxm m any term ~rvzrved.

446

GIORGOS

J. DlMlTRlADIS

prepared from antisera by 40% ammonium sulfate precipitation and were further purified by using corresponding immunoabsorbents prepared with glutaraldehyde (9). The above antibodies were prepared at The Pasteur Institute, Paris, in the laboratory of Dr. Avrameas. All other antibodies and antigens were generously supplied by Dr. Avrameas and Dr. T. Ternynck. Radioactive antigens were prepared by iodination with chloramine-T (10). Antibody-untigerz reaction. The effect of detergents on antigen-antibody interaction was examined by two methods, immunoprecipitation in solution, and immunodiffusion in agarose gels. Immunoprecipitation was carried out using amounts of antigen and homologous antiserum that were at equivalence in the absence of detergent. Mixtures of antigen, antibody, and appropriate concentrations of detergents were incubated at 37°C for 1 h and subsequently overnight at room temperature. Lower temperatures could not be used because of the high Krafft point (23°C) of SDS (11). Pellets were collected by centrifugation at 3000 rpm at room temperature and washed three times with PBS (phosphate buffered saline). Protein was measured by the method of Lowry et ul. (18) and radioactivity was detected in a Packard 5230 Auto Gamma scintillation spectrometer. Immunodiffusion was carried out in 1% agarose gels as described elsewhere (12). containing the same concentration of the corresponding detergent or mixture of detergents in which the antigens and the antibodies had been dissolved. The antibodies and antigens were solubilized in 0.05 M Verona1 buffer, pH 8.3, containing the appropriate concentration of detergent or mixture of detergents. The immunodiffusion was carried out at room temperature for 24 h and subsequently the gels were washed for 48 h with PBS containing 0.01% sodium azide. The gels were stained with Ponceau S (I g Ponceau S, 450 ml 1 M acetic acid, 450 ml 0.1 M sodium acetate, 100 ml

glycerine) for 30 min and washed with destaining medium (150 ml glycerine, 20 ml acetic acid, 830 ml H,O) for I h. RESULTS

AND

DISCUSSION

Norlionic Detergrnts Figure 1 and Figs. 3 and 4 (in part) show the effect of different concentrations of SDS and nonionic detergents on immunoprecipitation and immunodiffusion reactions respectively. Triton X-100, as well as the other nonionic detergents, at concentrations from 0.05 to 1% (w/v), have no effect, whereas SDS at a concentration of 0.2%, inhibits the reaction by more than 90%. Figure 1 also shows that SDS at very low concentrations (less than 0.05%) greatly reduces nonspecific binding. The effect of SDS on the elimination of nonspecific binding may be due to the neutralization of strongly charged basic proteins, which appear in the serum and bind nonspecifically to negatively charged immunoglobulins. The effect ofthe different detergents on immunoprecipitation and immunodiffusion reactions is not dependent on the nature of the antibodies or antigens used. Thus there was the same degree of inhibition when polyspecific (Figs. 1A and 3) or monospecific (Figs. 1B and 3) antibodies were used. The different actions of sodium dodecyl sulfate and nonionic detergents must be due to differences in their chemical nature. A common feature of the denaturating detergents (such as sodium dodecyl sulfate) appears to be a combination of a charged head group and a flexible apolar tail. The nature of the charged head group and the length of the detergent alkyl tail are important and influence the critical concentration needed to induce cooperative binding and the resulting conformational change (13- 15). The mild nonionic detergents, Triton X-100 and Nonidet P-40. have rigid and bulky apolar moieties which probably do not penetrate the crevices of the

DETERGENTS

AND

ANTIBODY-ANTIGEN

447

INTERACTION

protein surfaces as efficiently as the flexible alkyl chains. However, the above explanation is not valid for the other nonionic detergents we have used (Brij 58, Tween 80) as they also possess long hydrocarbon chains and it appears that inhibition of

0

-0

0

01

o-2

0.3

cont. of detergent

04

0.5

01

o-2 0.3 0.4 cont. of +r,ton x-100 i%)

0.5

I-O

FIG. 2. Effect of detergent mixtures on the amount of antigen-antibody precipitate. The reaction mixture contained Triton X-100 in the concentrations indicated and SDS in the concentrations shown as follows: A. 0.1% SDS: 0, 0.2% SDS; A 0.5% SDS. The reaction mixture also contained either normal rabbit serum and anti-normal rabbit serum (A) or *z51-labeled globin and anti--globin serum (B). Measurements were carried out as described in the legend for Fig. 1.

i/i

FIG. 1. Effect of increasing concentrations of Triton X-100 and SDS on the immunoprecipitation reaction. (Al Normal rabbit serum was reacted with antinormal rabbit serum in the presence of either Briton X100 (0-O) or SDS (13--13): or with anti-normal mouse serum in the presence of either Trit#on X-100 (O- - -0) or SDS (O- - -cl). (B) ““I-Labeled-globin was reacted with anti-globin serum in the presence of either Triton X-100 (O----e) or SDS (!3--
immunoreaction is predominantly due to the effects of charged repulsion ( 1).

Figures 2 and 4 show the effect of different detergent mixtures on the immunoprecipitation and immunodiffusion, respectively. If in the reaction medium there is a combination either of SDS and Triton X-100 (Figs. 2 and 4) or more generally SDS and nonionic detergent (data not shown) the inhibition by SDS is reduced. Also the resolution in immunodiffusion reaction is much higher at certain concentrations of SDS and Triton

448

GIORGOS

J. DIMITRIADIS

Detergent-%

v Triton

x-100

Nonidet

P-40

FIG. 3. Effect of nonionic detergents and SDS on immunodiffusion reaction. In each detergent of live immunodiffusion plates. the central wells of the top horizontal row contain anti-globin. of the middle row contain anti-normal rabbit serum. and those of the bottom row contain anti-TMV protein. Peripheral wells contain the corresponding antigens in continuous dilutions.

X-100. Thus, in the case of the mouse yI, yz (Fig. 4) at concentrations of 0.2% SDS, 0.2% Triton X-100, and 0.5% SDS, 0.5% Triton X-100, we have the highest resolution, and it seems that the ideal combination of concentrations of SDS and Triton X-100 depend on the concentrations of the antibodies and antigens. Thus, at concentrations of 0.2% SDS, 0.2% Triton X100 the highest resolution for mouse y,, y.. is obtained with low concentration of antigen, while conversely, at concentrations

group those coat

of 0.5% SDS. 0.5% Triton X-100 the highest resolution is obtained with high concentration of antigen. Also the effect of the mixture of detergents is general and is not dependent on the nature of the antibodies or antigens used. It has been demonstrated for a number of detergents that it is the monomeric species that is bound to proteins and not the micellar form, when both ligand and protein are present at relatively low concentrations ( 11.16). It is. therefore. the free monomeric

DETERGENTS

Detergent-%

AND

f\NTIBODY-ANTIGEN

449

INTERACTION

1.0

0.05

‘1 Tween 80

-7

3’

.

-I I

Brii

58

.TJ

/ SDS

1

450

GIORGOS

TritonX-100 I

J. DIMITRIADIS

(%I O

0.05

t

SDS(%) 0

0.05

0.1

0.2

0.5

FIG. 4. Effect of Triton X-100 and SDS mixtures on immunodiffusion reaction. Each immunodiffusion plate contains a mixture of detergent as indicated. In each plate the top central well contains anti-normal rabbit serum and the bottom central well contains anti-normal mouse serum. The top peripheral wells contain normal rabbit serum and the bottom peripheral wells contain yI. yr mouse globulins in continuous dilutions.

concentration of the detergent that determines the amount bound to the protein. Little further binding is observed upon increasing the free detergent concentration beyond the critical micelle concentration. This also means that the binding of detergents to proteins has to compete with the self-association of detergent molecules to micelles. Thus, there are two possible explanations for the elimination by nonionic detergents of the SDS-induced inhibition of the antigen-antibody reaction. The first is

the direct interaction between nonionic and ionic detergent, which results in the formation of mixed micelles and therefore in the decrease of free SDS monomers. This means that there are less molecules of SDS for binding to the proteins, and hence reduced conformational changes. Such formation of mixed micelles has been observed between some amphiphiles with the same or different head groups (17). The second is a competitive binding of the anionic and nonionic detergents for the hydrophobic sites of the

DETERGENTS

AND

ANTIBODY-ANTIGEN

proteins. Of course it is possible that both mechanisms take place at the same time, although the second one seems more favorable as the mixture of detergents solubilizes more effectively than the detergents themselves (unpublished results). Further work is required to determine which mechanism is correct. The data show that the use of detergent mixtures for solubilization of proteins or protein complexes (membranes) may be very useful because they combine strong solubilizing properties with high resolution immunochemical analysis. Also, use of low concentrations of SDS (less than 0.05%) allows one to circumvent the problems of nonspecific binding probably caused by the presence of basic proteins in the serum. Detergent mixtures were used very successfully for the solubilization of the membranes of Schistosorm munsoni and for the following immunoprecipitation with the corresponding antibodies (L. Brink, personal communication) as well as to improve the reaction between the frog vitellogenin and albumin and corresponding antibodies (D. F. Smith, personal communicatilon).

drawings G. J. D. is the recipient term fellowship.

of an EMBO

long-

REFERENCES

4.

6. 7. 8. 9. IO. 11. 12. 13. 14

IS.

ACKNOWLEDGMENTS 16. I would like to thank Dr. Stratis Avrameas and Dr. Therese Ternynck for donating many materials used in this work, Dr. G. Allen for helpful discussions, Ms. M. Layton for the excellent technical a:,sistance, Mrs. E. Heather for typing. Mr. M. Tatham for excellent photographic work. and Mrs. R. Harris for

451

INTERACTION

17. 18.

Crumpton. M. J., and Parkhouse. R. M. E. I 1972) FEB.5 Lett. 22, 210-212. Dehhnger. P. J., and Schimke. R. T. ( 1971 ).I. Bicd. Chrm. 246. X74-2583. Lerner. R. A.. McConahey. P. J. and Dixon. F. J. ( 1971) Sckrrr 173, 60-62. Schwartz. B. D.. and Nathanson, J. (1971) .I. Ir,~muno/. 107. 1363- 1367. Bjerrum. 0. J.. and Lundahl. P. ( 1973) Sccrrrtl. .I. Inrmuno/. 2(Suppl. I), 139- 143. Dimitriadis. G. J.. and Georgatsos. J. G. (19741 FEES Lctt. 46, 96-100. Dimitriadis. G. J.. and Georgatsos, J. G. (19751 Nucleic, Acids Rcs. 2. 1719- 1726. Avrameas. S. (19691 Ilnmunochrmi.\t~~ 6, 43-52. Ternynck. T., and Avrameas. S. (1972) FEBS Lett. 23, 24-28. Bray, B., and Brownlee. S. M. (1973) Anrrl. Eiochenr. 55, 213-221. Helenius, A., and Simons, K. (1975) Bkrhrm. Biophys. A(.tu 415, 29-79. Dimitriadis, G. J. (1974) Biochrm. Biophx.s. N. Lv/t. 7. 4. Tanford. C.. (1973) The Hydrophobic Effect, Wiley. New York. Steinhardt, J.. and Reynolds. J. A.. (1969) Multiple Equilibria in Proteins, pp. 10-80. Academic Press, New York. Nakaya, K.. Yamada, K., Onozawa, M., and Nakamura. Y. ( 1971) Biwhem. Biuphy.s. Acto 251, 7- 13. Makino, S.. Reynolds, J. A., and Tanford. C. ( 1973) .I. Bird. Chem. 248, 4926-4932. Becher. P.. (1967) in Non-Ionic Surfactants (Schick, M. J.. ed.). Dekker, New York. Lowry. 0. H., Rosebrough. N. J., Farr. A. L., and Randall. R. J. ( 1951) .I. Bid. C’hem. 193. 265-275.