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The use of tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes
Journal of Immunological Methods, 55 (I 982) 297-307
297
Elsevier Biomedical Press
The Use of Tween 20 as a Blocking Agent in the Immunological Detection of Proteins Transferred to Nitrocellulose Membranes Byron Batteiger *, Wilbert J. Newhall V * and Robert B. Jones *'** • Departments of Medicine, and • * Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46223, U.S.A.
(Received 19 March 1982, accepted 2 June 1982)
The determination of the immunoreactivity of protein antigens in complex mixtures has been greatly facilitated by combining their separation via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with electrophoretic transfer to nitrocellulose membrane (NCM), and probing of bound proteins with specific antisera. Methods using various buffers and blocking agents have been published, but no studies have been published which compare these methods with each other or with others of potential merit. We have performed such a comparative study using protein antigens from Chlamydia trachomatis and Neisseria gonorrhoeae. In addition, we describe a method that blocks unoccupied protein binding sites on NCM with the nonionic detergent Tween 20, rather than proteins. This system proved to be equivalent or superior to other methods evaluated in the detection of immunoreactive proteins, and permitted staining of the NCM for protein after immunological probing. Such staining allowed precise identification of immunoreactive proteins. In addition, individual stained proteins could be excised and assessed for bound antibody in an indirect radioimmunoassay. Key words: chlamydial proteins - - antigenic analysis - - immunoblot - - indirect radioimmunoassay
Introduction S e v e r a l m e t h o d s h a v e b e e n d e v i s e d to d e t e r m i n e the i m m u n o l o g i c a l r e a c t i v i t y of p r o t e i n s a f t e r s e p a r a t i o n b y s o d i u m d o d e c y l s u l f a t e - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s ( S D S - P A G E ) ( B u r r i d g e , 1976; R e n a r t et al., 1979; T o w b i n et al., 1979). P r o t e i n s c a n b e e l e c t r o p h o r e t i c a l l y t r a n s f e r r e d f r o m p o l y a c r y l a m i d e gels to n i t r o c e l l u l o s e m e m b r a n e s ( N C M ) a n d the N C M s t a i n e d to d o c u m e n t t h a t the p o l y p e p t i d e b a n d s of i n t e r e s t h a v e b e e n t r a n s f e r r e d a n d a d s o r b e d ( T o w b i n et al., 1979). P r o t e i n s b o u n d to N C M c a n also be i n c u b a t e d w i t h v a r i o u s a n t i s e r a a n d b o u n d a n t i b o d y localized with a labeled second antibody or staphylococcal protein A followed by a u t o r a d i o g r a p h y o f the N C M . 0022-1759/82/0000-0000/$02.75 ~ 1982 Elsevier Biomedical Press
298 However, it is necessary to block unoccupied protein binding sites on NCM prior to addition of the specific antiserum, and proteins (usually bovine serum albumin or gelatin) are generally used for this purpose (Towbin et al., 1979; Burnette, 1981; Cohen and Falkow, 1981; Vaessen et al., 1981). As a result, the NCM can no longer be stained to identify specific protein bands. Staining for protein after immunological probing would enable one to determine if the electrophoretic pattern is conserved during antibody incubation and washes, and to compare the autoradiogram and the stained NCM from which it was derived for accurate identification of immunoreacrive proteins. Staining would also permit the accurate location of individual immunoreactive protein bands for subsequent quantitation of antibody bound to such bands. In the course of our work with Chlamydia trachomatis and Neisseria gonorrhoeae. we sought to determine an optimal combination of blocking agents and buffers for detection of immunoreactive proteins. A procedure using the detergent Tween 20 as a blocking agent was developed and compared to previously described methods as well as to other techniques of potential merit. This procedure proved to be equivalent or superior to the other methods evaluated in the detection of antibodies bound to NCM-immobilized antigens, and because proteins could be localized by staining after incubation with antiserum, antibody bound to proteins could be quantitated.
Materials Acrylamide, N,N'-methylene-bisacrylamide, SDS, ammonium persulfate, N,N,N',N'-tetramethylethylenediamine (Temed), Coomassie brilliant blue R-250, and amido black 10-B were from Bio-Rad Laboratories, Richmond, CA. Nitrocellulose membrane (NCM), pore size 0.45 ~m, was from Millipore, Bedford, MA. Polyoxyethylene sorbitan monolaurate (Tween 20), sodium N-lauroyl sarcosinate (Sarkosyl), bovine serum albumin (BSA), gelatin (Type IV from calf skin, 60 bloom), purified protein A, N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (Hepes), and tris(hydroxymethyl)aminomethane were from Sigma Chemical Co., St. Louis, MO. Nonidet P-40 T M (NP-40) was from Particle Data Laboratories, Elmhurst, IL. Carrier-free NalZSI was from New England Nuclear Corp., Boston, MA. PercolfTM was from Pharmacia, Uppsala. RenograffinT M was from Squibb, Princeton, NJ.
Methods Bacterial growth and purification Strain L 2 / 4 3 4 / B u (LGV 434) of C. trachomatis was kindly provided by C.-C. Kuo, Seattle, WA. Chlamydia were grown and harvested as described by Kuo et al. (1977). Harvested organisms were separated from host cell debris in 30% Percotl, 25 mM Hepes, 125 mM NaC1, pH 7.4, by centrifugation at 10,000 × g for 30 min at 4°C. The lower band composed of purified elementary bodies (EB) was resuspended in SPG (10 mM sodium phosphate, 250 mM sucrose, 5 mM L-glutamic acid, pH 7.2)
299 and centrifuged at 16,000 x g for 30 min. This wash step was repeated once, and the final EB pellet was suspended in SPG and stored at - 70°C. Outer membranes from 2 variants of strain F62 of N. gonorrhoeae were prepared as previously described (Jones et al., 1980).
Protein content Protein concentrations were estimated by the method of Bradford (1976) with bovine serum albumin as the standard.
SDS-PA GE Protein profiles were obtained on 12.5% polyacrylamide gels (9 cm x 14 cm x 0.15 cm) using the discontinuous buffer system of Laemmli (1970). Purified whole EB or gonococcal outer membranes were prepared for SDS-PAGE by solubilization in a solution of 2.5% ( w / v ) SDS, 1.25% (v/v) 2-mercaptoethanol, and 12.5% (v/v) glycerol in 62.5 mM Tris-HC1, pH 6.8. Solubilization mixtures were incubated at 100°C for 2 min. Samples containing 200/~g of protein in a total volume of 1 ml were underlayed onto a flat 3% polyacrylamide stacking gel. Electrophoresis was at 50 V until the tracking dye reached the separating gel, and then at 100 V until the tracking dye was within 1 cm of the bottom of the separating gel. A 2 cm vertical strip was sliced from each gel for staining with Coomassie brilliant blue (Newhall et al., 1980).
Electrophoretic transfer The unstained portion of each gel was subjected to electrophoretic transfer of its resolved proteins using an electrophoretic destaining apparatus (E-C Apparatus Corp., St. Petersburg, FL). The procedure for transfer was essentially as described for SDS gels by Towbin et al. (1979) except that the transfer buffer was 50 mM sodium phosphate buffer, pH 7.5 (Bittner et al., 1980), deaerated under reduced pressure for 12 h. A sandwich was constructed consisting sequentially of anode grid, Scotch Brite scouring pad, a sheet of Whatman no. 1 filter paper, NCM, the gel to be transferred, a second sheet of Whatman no. 1, a second Scotch Brite pad, and the cathode grid. The sandwich was constructed while immersed in buffer to exclude bubbles, and was bound tightly together with rubber bands. Transfer was accomplished in a chamber filled with transfer buffer at approximately 27 V (2 A) for 2 h. After transfer, the NCM was sliced vertically into 0.5 cm strips.
Staining of nitrocellulose strips for protein In each experiment, one strip was stained with 0.1% (w/v) amido black 10-B in 45% (v/v) methanol/10% ( v / v ) acetic acid and destained with 90% (v/v) methanol/2% (v/v) acetic acid (Schaffner and Weissmann, 1973). Staining was also frequently performed after immunological probing but before autoradiography to allow superimposition of radiographic film over the NCM strip.
Antigens and antisera EB of LGV 434 and outer membranes of N. gonorrhoeae were solubilized,
300 subjected to SDS-PAGE and transferred to N C M for use as test antigens. Hyperimmune rabbit serum against LGV 434 EB was prepared using EB purified by density gradient centrifugation in Renograffin (Kuo et al., 1977) as the immunogen. EB were diluted to 1 mg of protein per ml in phosphate-buffered saline. The suspension was made 0.02% ( v / v ) with respect to formalin and emulsified with an equal volume of Freund's complete adjuvant (Difco). The rear foot pads of male New Zealand white rabbits were injected with 0.2 ml of this suspension (approximately 100 ~g of chlamydial protein per foot pad). Three weeks later, an identical booster immunization was administered. Sera were harvested by cardiac puncture during the fifth week. Titers were 1 : 2048 against LGV 434 EB in the microimmunofluorescence test of Wang and Grayston (1970). Hyperimmune antigonococcal rabbit sera were prepared as previously described (Jones et al., 1980).
Preparation of protein A Protein A from Staphylococcus aureus was radiolabeled with ~25I by the technique of Fraker and Speck (1978) to a specific activity of 7 ~ C i / ~ g .
Immunologic detection of proteins bound to nitrocellulose After transfer, N C M strips were sequentially subjected to: (1) incubation with a blocking reagent at 37°C for 1 h; (2) incubation with antiserum at 37°C for 2 h; (3) a wash at room temperature for 15 min; (4) incubation with ~25I-protein A at 37°C for 1 h; and (5) 2 final washes at room temperature for 30 min each. Composition of the various blocking, diluent and wash buffers used in each step are given in the legend to Fig. 2. Unstained strips were placed in horizontal 15 ml polystyrene tubes. In the blocking step, strips were incubated with 15 ml of appropriate buffer. Following the blocking step, strips were incubated on a platform rotator at 75 r e v / m i n with antiserum diluted to 3 ml with the appropriate diluent. After removing unbound antibody by incubation in 15 ml of wash buffer, the strips were incubated with 0.25 ~Ci of 12~I-protein A diluted to 3 ml with the appropriate diluent buffer. Excess protein A was removed by rocking the strips in 15 ml of wash buffer. In those experiments where 150 mM NaCI was used as a wash, the wash was changed 5 times over a period of 30 min. Processed strips were air dried and exposed for 7-30 h to Kodak X-Omat A R film with a Dupont Cronex Lightning Plus intensifying screen at - 7 0 ° C (Laskey and Mills, 1977).
Quantitative measurement of bound antibody Purified EB of LGV 434 were subjected to SDS-PAGE and the resolved proteins transferred to NCM. The antigen-bearing N C M sheet was divided into 0.5 cm strips. N C M strips were blocked with PBS-Tween for 1 h at 37°C. Duplicate strips were incubated with serial 2-fold dilutions of rabbit hyperimmune serum in PBS-Tween ( 1 : 2 5 0 - 1 : 128,000). The strips were incubated with excess ~25I-protein A, washed, then subjected to autoradiography, staining with amido black 10-B, and repeat autoradiography. Finally, individual protein bands were sliced from each strip and the 125I cpm bound by that particular band were determined in a g a m m a counter
301
(Searle). Backgrounds were determined using identically sized slices of NCM that were obtained from a portion of each strip where no darkening of the corresponding autoradiographic film was noted.
Results
Blocker, diluent, and wash buffer comparisons Examples of a stained gel and NCM after transfer are shown in Fig. 1. Resolution of polypeptide bands was maintained and all major proteins appear to have transferred to the N C M sheet. A procedure using Tween 20 without protein blocker was compared to 3 published procedures, and to the other combinations of buffers and blockers indicated in Fig. 2. Preimmune serum and PBS without serum were included as controls. Ten procedures were directly compared using identical NCM strips with LGV 434 proteins. The same homologous hyperimmune rabbit serum at a dilution of 1:256 was used for each strip. An autoradiogram of strips processed in various blocking, diluent and wash buffers is shown in Fig. 2. Buffers used for each strip are listed in the legend. No bands of radioactivity were observed in the controls, indicating that protein A did not bind nonspecifically to NCM-immobilized chlamydial proteins, and that the preimmune rabbit serum did not contain IgG or other serum factors that could have bound antigen and protein A to produce false positive results. The autoradiogram shown was overexposed with respect to major immunoreactive bands to permit visualization of minor bands. As a result, the 45,000 and 40,000 dalton major proteins and a minor 38,000 dalton protein appear as a single broad band. These were easily distinguishable as individual bands with shorter exposure times. Strips 3 through 12 were probed with rabbit hyperimmune serum to compare the sensitivity of various procedures. Qualitative observations were made in terms of the number of bands observed and band intensity. For example, strip 5, processed according to Vaessen et al. (1981) with Triton X-100, resulted in fewer, less intense bands when compared to procedures using other detergents (strips 3, 4, 7, 8, 10, 11, and 12). Strips processed with Nonidet P-40 (strip 4) according to Cohen and Falkow (1981), Triton X-100 (strip 5) or Sarkosyl (strip 12) lacked an immunoreactive band that comigrated with the tracking dye. This particular band was identifiable but greatly reduced in strips processed with Tween 20 (3, 7, 8, 10 and 1 l) and conserved in procedures lacking detergents (strips 6 and 9). Otherwise, the PBS-Tween procedure (strip 3) appeared comparable in the identification of major immunoreactive bands to procedures using BSA but no detergent (strip 6). The latter procedure was derived from Towbin et al. (1979) but differed in that protein A was used as the detection reagent and carrier serum therefore was deleted. In addition, autoradiograms of shorter exposure times (not shown) revealed no substantial overall loss of bound radioactivity with any detergent except Triton X-100 (strip 5), confirming the above observations. Strips were also compared in terms of background. Both 3% bovine serum
302
albumin (strip 6) and Tween 20 alone (strips 3, 7 and 10) were effective blockers of unoccupied sites on nitrocellulose. Gelatin (0.25%) alone was not an effective blocker (strip 9) and did not improve background when added to Tween 20 buffers (cf. strips 10 and 11). In procedures using Tween 20 without protein blockers, phosphate and Tris-buffered saline appeared to be comparable (cf. strips 3 and 10). In addition, no loss of protein bands was observed in the PBS-Tween system when
A
B
68-60-45-40--
29--
15-
J
TD
I
I
Fig. 1. A: protein profile of LGV 434 after SDS-PAGE on a 12.5% polyacrylamide gel and staining with Coomassie brilliant blue R-250. The molecular weights in kilodaltons of major polypeptide bands are indicated. The position of the tracking dye is indicated by TD. B: protein profile of LGV 434 after transfer to N C M and staining with amido black 10-B. Major polypeptide bands corresponding to those of the original gel are indicated by dashed lines. The apparent change in position of the bands reflects shrinkage of the N C M and expansion of the gel during staining.
303
1
2
3
4
5
6
7
8
9
10 1 1 1 2
68 60 40 29
15 TD Fig. 2. Autoradiogram comparing various blocking, diluent, and wash buffers. Composition of each buffer is given below. The 68, 60, 40, 29 and 15 kilodalton proteins are marked for reference. The immunoreactive band that comigrated with the tracking dye (TD) is indicated by an asterisk for each strip. Strips 1 and 2 are PBS alone and preimmune serum controls, respectively, using PBS-Tween in all steps. Strips 3-12 were probed with hyperimmune rabbit serum. Strip 3: PBS-Tween in all steps; strip 4: TSGAN-1% BSA as blocker and TSGAN as diluent and wash; strip 5: PBS-8% BSA as blocker and diluent, and PBS-Triton as final wash; strip 6: TS-3% BSA as blocker and diluent, and NaCI as wash; strip 7: TS-Tween as blocker and diluent, and NaCI as wash; strip 8: TS-3% BSA as blocker, TS-Tween as diluent and NaCI as wash; strip 9: TSGA as blocker and diluent and NaC1 as wash; strip 10: TS-Tween in all steps; strip I 1: TSGA-Tween in all steps; and strip 12: PBS-Tween as blocker and diluent and PBS-S as final wash. Reagents were prepared as follows: PBS: 4 mM KH2PO 4, 16 mM Na2HPO4, 115 mM NaC1, pH 7.3; PBS-Tween: PBS containing 0.05% (v/v) Tween 20; PBS-8% BSA: PBS containing 8% (w/v) BSA; PBS-Triton: PBS containing 1% (v/v) Triton X-100; TSGAN: 50 mM Tris-HC1, 150 mM NaC1, 0.25% (w/v) gelatin, 0.15% (w/v) sodium azide, 0.1% (v/v) Nonidet P-40, pH 7.5; TSGA-Tween: as with TSGAN, but with 0.05% (v/v) Tween 20 substituted for Nonidet P-40; TSGA: as with TSGAN, but without Nonidet P-40; TS-3% BSA: 10 mM Tris-HC1, 150 mM NaC1, and 3% (w/v) BSA, pH 7.4; TS-Tween: 10 mM Tris-HC1, 150 mM NaC1, and 0.05% (v/v) Tween 20, pH 7.4; PBS-S: PBS containing 0.4% (w/v) Sarkosyl; and NaCh 150 mM NaC1.
NCM
stained for protein after immunologic
immediately
probing was compared
to NCM
stained
after transfer (data not shown).
Identification of immunoreactive proteins Outer membrane p r o t e i n s o f N. gonorrhoeae
were probed
with rabbit
immune
304 s e r u m u s i n g the P B S - T w e e n t r a n s f e r m e t h o d . A f t e r a u t o r a d i o g r a p h y , N C M strips w e r e s t a i n e d w i t h a m i d o b l a c k 10-B a n d a u t o r a d i o g r a p h y was r e p e a t e d . T h e r e w e r e n o q u a l i t a t i v e d i f f e r e n c e s b e t w e e n the a u t o r a d i o g r a m s b e f o r e a n d after staining. H o w e v e r , s h r i n k a g e o c c u r r e d d u r i n g staining, a n d an N C M strip s t a i n e d a f t e r a u t o r a d i o g r a p h y c o u l d n o t b e p r e c i s e l y a l i g n e d w i t h the a u t o r a d i o g r a m , w h e r e a s o n e s t a i n e d b e f o r e a u t o r a d i o g r a p h y c o u l d be. A n N C M strip s t a i n e d b e f o r e a u t o r a d i o g -
A
MAJOR
MAJOR
\
MINOR
TD
B
/
MINOR
i'D
Fig. 3. Outer membrane proteins from two colonial variants of strain F62 of N. gonorrhoeae w e r e resolved on a 12.5% polyacrylamide gel, transferred to NCM, and immunologically probed in the presence of PBS-Tween, as described in the text. A: NCM strip stained with amido black 10-B after immunological probing showing resolved outer membrane proteins. The major outer membrane protein and a minor band are indicated. B: autoradiogram of NCM strip shown in A. The dark band of radioactivity corresponded to a minor protein rather than to the major protein.
305 raphy is shown with the aligned autoradiogram in Fig. 3. A relatively minor outer membrane protein was highly immunoreactive and migrated close to the major outer membrane protein. Had it not been possible to align the stained NCM with the autoradiogram, this immunoreactivity could have been mistakenly attributed to the major protein, which did not bind antibody from this particular serum.
Quantitative measurement of bound antibody The ability to identify individual proteins by staining NCM after immunological probing allowed the development of a radioimmunoassay to indirectly quantitate antibodies against specific polypeptides. A titration of hyperimmune serum against a 40,000 dalton protein of LGV 434 is shown in Fig. 4. Similar titration plots were obtained for other chlamydial polypeptides (not shown). These curves are characteristic of those obtained with conventional radioimmunoassays in other antigen-antibody systems. Thus, slicing individual bands is a reproducible and sensitive method for quantitating antibodies bound to an individual protein.
I
I
I
I
l
E
E
•
•
•
I
I
I
I
4 d
A
g o x
E a o 2-
u3
o4
r~
•
RECIPROCAL DILUTION x
10 .3
Fig. 4. Quantitation of antibodies contained in homologous rabbit hypefimmune serum against a 40,000 dalton protein of L G V 434. Antiserum dilution is given on the abscissa and radioactivity in 125] cpm on the ordinate. Bars indicate ] standard deviation of the mean of duplicate determinations. Background determinations for each dilution are shown as dosed circles.
306 Discussion
The technique of SDS-PAGE followed by transfer to NCM and immunological probing is a powerful tool for antigenic analysis (Anderton and Thorpe, 1980). However, the quality of permanent record reported in published studies (Towbin et al., 1979; Burnette, 1981; Cohen and Falkow, 1981; Vaessen et al., 1981) has been variable, .and there have been no published studies directly comparing different techniques in terms of detection of antibody bound to NCM-immobilized proteins. We have performed such a comparison and have developed a procedure using PBS containing the nonionic detergent Tween 20 as a blocking agent. A non-detergent method similar to that described by Towbin et al. (1979) allowed identification of an immunoreactive band that comigrated with the tracking dye which was lost or greatly reduced in the presence of detergents. However, staining of N C M strips after processing in PBS-Tween showed no reduction in intensity of amido black 10-B staining material at the dye front, suggesting that this additional low molecular weight band is probably not a polypeptide. Thus, the PBS-Tween method appears suitable for detection of protein antigens adsorbed to NCM. All of the methods evaluated, except the one in which gelatin was used as a blocker, provided acceptable backgrounds with radioiodinated protein A as the detection reagent. However, for certain applications a labeled immunoglobulin may be a more suitable detection reagent (Towbin et al., 1979; Glass et al., 1981; Karcher et al., 1981). In preliminary experiments using goat anti-human IgM as a detection reagent, the backgrounds obtained with PBS-Tween were substantially lower than those obtained when large amounts of BSA or carrier serum were used as blocking reagents (data not shown). One of the major advantages of the PBS-Tween system is that it provides effective blocking of unoccupied protein binding sites on NCM and thereby obviates the need for proteins as blockers and carriers. Thus, after immunological probing, proteins on the NCM can be stained and precisely aligned with immunoreactive bands on the resultant autoradiogram. The utility of such alignment is illustrated by the experiment depicted in Fig. 3. Therefore, the ability to superimpose an autoradiogram on the stained NCM from which it was derived allows convenient and unambiguous identification of immunoreactive proteins, eliminating the uncertainties inherent in comparing the autoradiogram with stained gel or NCM side strips. In addition, staining after immunological probing circumvents any inhibition of antibody binding that may occur if NCM is stained before reaction with antiserum (Anderton and Thorpe, 1980). Staining of NCM can also aid in the interpretation of a negative result on the autoradiogram. Conditions whicl~ could result in the absence of immunoreactivity of an individual protein band are: (1) failure of the antigen to migrate out of the SDS gel; (2) failure of the antigen to adsorb to NCM; and (3) loss of antigen from NCM during any of the blocking, incubation, or wash steps. Comparison of gel strips stained for protein before and after transfer, and NCM strips stained before and after immunological probing can distinguish among these possibilities. Under circumstances where the antigen is a protein and can be demonstrated on the NCM
307
after processing, other conditions that may be responsible for observed non-reactivity are: (1) denaturation of antigenic determinants by SDS; (2) dissociation of antibody or protein A during processing; or (3) lack of specific antibody in the test serum. S t a i n i n g also allows simple, accurate identification of i n d i v i d u a l p r o t e i n b a n d s for s u b s e q u e n t slicing a n d indirect q u a n t i t a t i o n of b o u n d antibody. The feasibility of this a p p r o a c h was illustrated by the q u a n t i t a t i o n of a n t i b o d y to a chlamydial p r o t e i n (Fig. 4) in serial dilutions of h y p e r i m m u n e r a b b i t serum. Therefore, i m m u n o l o g i c a l p r o b i n g of a n t i g e n - b e a r i n g N C M strips in the presence of P B S - T w e e n can be used to p e r f o r m what is in effect a r a d i o i m m u n o a s s a y for a n y p r o t e i n resolvable b y S D S - P A G E . This technique has proven useful in the evaluation of the h u m a n serological response to specific polypeptides d u r i n g infection with C. trachomatis (W.J. Newhall V, m a n u s c r i p t in p r e p a r a t i o n ) a n d should have general applicability.
Acknowledgements This investigation was supported in part by Grant AI-17502 from the National Institutes of Health. We thank C.E. Wilde III and K. Fife for their critical review of the manuscript.
References Anderton, B.H. and R.C. Thorpe, 1980, Immunol. Today 1, 122. Bittner, M., P. Kupferer and C.F. Morris, 1980, Anal. Biochem. 102, 459. Bradford, M.M., 1976, Anal. Biochem. 72, 248. Burnette, W.E., 1981, Anal. Biochem. 112, 195. Burridge, K., 1976, Proc. Natl. Acad. Sci. U.S.A. 73, 4457. Cohen, M.L. and S. Falkow, 1981, Science 211,842. Fraker, P.J. and J.C. Speck, Jr., 1978, Biochem. Biophys. Res. Commun. 80, 849. Glass II, W.F., R.C. Briggs and L.S. Hnilica, 1981, Science 211, 70. Jones, R.B., J.C. Newland, D.A. Olsen and T.M. Buchanan, 1980, J. Gen. Microbiol. 121,365. Karcher, D., A. Lowenthal, H. Thormar and M. Noppe, 1981, J. Immunol. Methods 43, 175. Kuo, C.-C., S.-P. Wang and J.T. Grayston, 1977, in: Nongonococcal Urethritis and Related Infections, eds. D. Hobson and K.K. Holmes (Amer. Soc. Microbiol., Washington, DC) p. 328. Laemmli, U.K., 1970, Nature (London) 227, 680. Laskey, R.A. and A.D. Mills, 1977, FEBS Lett. 82, 314. Newhall, W.J.V., C.E. Wilde III, W.D. Sawyer and R.A. Haak, 1980, Infect. Immun. 27, 475. Renart, J., J. Reiser and G.R. Stark, 1979, Proc. Natl. Acad. Sci. U.S.A. 76, 3116. Schaffner, W. and C. Weissmann, 1973, Anal. Biochem. 56, 502. Towbin, H., T. Staehelin and J. Gordon, 1979, Proc. Natl. Acad. Sci. U.S.A. 76, 4350. Vaessen, R.T.M.J., J. Kreike and G.S.P. Groot, 1981, FEBS Lett. 124, 193. Wang, S.-P. and J.T. Grayston, 1970, Am. J. Ophthalmol. 70, 367.
297
Elsevier Biomedical Press
The Use of Tween 20 as a Blocking Agent in the Immunological Detection of Proteins Transferred to Nitrocellulose Membranes Byron Batteiger *, Wilbert J. Newhall V * and Robert B. Jones *'** • Departments of Medicine, and • * Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46223, U.S.A.
(Received 19 March 1982, accepted 2 June 1982)
The determination of the immunoreactivity of protein antigens in complex mixtures has been greatly facilitated by combining their separation via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with electrophoretic transfer to nitrocellulose membrane (NCM), and probing of bound proteins with specific antisera. Methods using various buffers and blocking agents have been published, but no studies have been published which compare these methods with each other or with others of potential merit. We have performed such a comparative study using protein antigens from Chlamydia trachomatis and Neisseria gonorrhoeae. In addition, we describe a method that blocks unoccupied protein binding sites on NCM with the nonionic detergent Tween 20, rather than proteins. This system proved to be equivalent or superior to other methods evaluated in the detection of immunoreactive proteins, and permitted staining of the NCM for protein after immunological probing. Such staining allowed precise identification of immunoreactive proteins. In addition, individual stained proteins could be excised and assessed for bound antibody in an indirect radioimmunoassay. Key words: chlamydial proteins - - antigenic analysis - - immunoblot - - indirect radioimmunoassay
Introduction S e v e r a l m e t h o d s h a v e b e e n d e v i s e d to d e t e r m i n e the i m m u n o l o g i c a l r e a c t i v i t y of p r o t e i n s a f t e r s e p a r a t i o n b y s o d i u m d o d e c y l s u l f a t e - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s ( S D S - P A G E ) ( B u r r i d g e , 1976; R e n a r t et al., 1979; T o w b i n et al., 1979). P r o t e i n s c a n b e e l e c t r o p h o r e t i c a l l y t r a n s f e r r e d f r o m p o l y a c r y l a m i d e gels to n i t r o c e l l u l o s e m e m b r a n e s ( N C M ) a n d the N C M s t a i n e d to d o c u m e n t t h a t the p o l y p e p t i d e b a n d s of i n t e r e s t h a v e b e e n t r a n s f e r r e d a n d a d s o r b e d ( T o w b i n et al., 1979). P r o t e i n s b o u n d to N C M c a n also be i n c u b a t e d w i t h v a r i o u s a n t i s e r a a n d b o u n d a n t i b o d y localized with a labeled second antibody or staphylococcal protein A followed by a u t o r a d i o g r a p h y o f the N C M . 0022-1759/82/0000-0000/$02.75 ~ 1982 Elsevier Biomedical Press
298 However, it is necessary to block unoccupied protein binding sites on NCM prior to addition of the specific antiserum, and proteins (usually bovine serum albumin or gelatin) are generally used for this purpose (Towbin et al., 1979; Burnette, 1981; Cohen and Falkow, 1981; Vaessen et al., 1981). As a result, the NCM can no longer be stained to identify specific protein bands. Staining for protein after immunological probing would enable one to determine if the electrophoretic pattern is conserved during antibody incubation and washes, and to compare the autoradiogram and the stained NCM from which it was derived for accurate identification of immunoreacrive proteins. Staining would also permit the accurate location of individual immunoreactive protein bands for subsequent quantitation of antibody bound to such bands. In the course of our work with Chlamydia trachomatis and Neisseria gonorrhoeae. we sought to determine an optimal combination of blocking agents and buffers for detection of immunoreactive proteins. A procedure using the detergent Tween 20 as a blocking agent was developed and compared to previously described methods as well as to other techniques of potential merit. This procedure proved to be equivalent or superior to the other methods evaluated in the detection of antibodies bound to NCM-immobilized antigens, and because proteins could be localized by staining after incubation with antiserum, antibody bound to proteins could be quantitated.
Materials Acrylamide, N,N'-methylene-bisacrylamide, SDS, ammonium persulfate, N,N,N',N'-tetramethylethylenediamine (Temed), Coomassie brilliant blue R-250, and amido black 10-B were from Bio-Rad Laboratories, Richmond, CA. Nitrocellulose membrane (NCM), pore size 0.45 ~m, was from Millipore, Bedford, MA. Polyoxyethylene sorbitan monolaurate (Tween 20), sodium N-lauroyl sarcosinate (Sarkosyl), bovine serum albumin (BSA), gelatin (Type IV from calf skin, 60 bloom), purified protein A, N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (Hepes), and tris(hydroxymethyl)aminomethane were from Sigma Chemical Co., St. Louis, MO. Nonidet P-40 T M (NP-40) was from Particle Data Laboratories, Elmhurst, IL. Carrier-free NalZSI was from New England Nuclear Corp., Boston, MA. PercolfTM was from Pharmacia, Uppsala. RenograffinT M was from Squibb, Princeton, NJ.
Methods Bacterial growth and purification Strain L 2 / 4 3 4 / B u (LGV 434) of C. trachomatis was kindly provided by C.-C. Kuo, Seattle, WA. Chlamydia were grown and harvested as described by Kuo et al. (1977). Harvested organisms were separated from host cell debris in 30% Percotl, 25 mM Hepes, 125 mM NaC1, pH 7.4, by centrifugation at 10,000 × g for 30 min at 4°C. The lower band composed of purified elementary bodies (EB) was resuspended in SPG (10 mM sodium phosphate, 250 mM sucrose, 5 mM L-glutamic acid, pH 7.2)
299 and centrifuged at 16,000 x g for 30 min. This wash step was repeated once, and the final EB pellet was suspended in SPG and stored at - 70°C. Outer membranes from 2 variants of strain F62 of N. gonorrhoeae were prepared as previously described (Jones et al., 1980).
Protein content Protein concentrations were estimated by the method of Bradford (1976) with bovine serum albumin as the standard.
SDS-PA GE Protein profiles were obtained on 12.5% polyacrylamide gels (9 cm x 14 cm x 0.15 cm) using the discontinuous buffer system of Laemmli (1970). Purified whole EB or gonococcal outer membranes were prepared for SDS-PAGE by solubilization in a solution of 2.5% ( w / v ) SDS, 1.25% (v/v) 2-mercaptoethanol, and 12.5% (v/v) glycerol in 62.5 mM Tris-HC1, pH 6.8. Solubilization mixtures were incubated at 100°C for 2 min. Samples containing 200/~g of protein in a total volume of 1 ml were underlayed onto a flat 3% polyacrylamide stacking gel. Electrophoresis was at 50 V until the tracking dye reached the separating gel, and then at 100 V until the tracking dye was within 1 cm of the bottom of the separating gel. A 2 cm vertical strip was sliced from each gel for staining with Coomassie brilliant blue (Newhall et al., 1980).
Electrophoretic transfer The unstained portion of each gel was subjected to electrophoretic transfer of its resolved proteins using an electrophoretic destaining apparatus (E-C Apparatus Corp., St. Petersburg, FL). The procedure for transfer was essentially as described for SDS gels by Towbin et al. (1979) except that the transfer buffer was 50 mM sodium phosphate buffer, pH 7.5 (Bittner et al., 1980), deaerated under reduced pressure for 12 h. A sandwich was constructed consisting sequentially of anode grid, Scotch Brite scouring pad, a sheet of Whatman no. 1 filter paper, NCM, the gel to be transferred, a second sheet of Whatman no. 1, a second Scotch Brite pad, and the cathode grid. The sandwich was constructed while immersed in buffer to exclude bubbles, and was bound tightly together with rubber bands. Transfer was accomplished in a chamber filled with transfer buffer at approximately 27 V (2 A) for 2 h. After transfer, the NCM was sliced vertically into 0.5 cm strips.
Staining of nitrocellulose strips for protein In each experiment, one strip was stained with 0.1% (w/v) amido black 10-B in 45% (v/v) methanol/10% ( v / v ) acetic acid and destained with 90% (v/v) methanol/2% (v/v) acetic acid (Schaffner and Weissmann, 1973). Staining was also frequently performed after immunological probing but before autoradiography to allow superimposition of radiographic film over the NCM strip.
Antigens and antisera EB of LGV 434 and outer membranes of N. gonorrhoeae were solubilized,
300 subjected to SDS-PAGE and transferred to N C M for use as test antigens. Hyperimmune rabbit serum against LGV 434 EB was prepared using EB purified by density gradient centrifugation in Renograffin (Kuo et al., 1977) as the immunogen. EB were diluted to 1 mg of protein per ml in phosphate-buffered saline. The suspension was made 0.02% ( v / v ) with respect to formalin and emulsified with an equal volume of Freund's complete adjuvant (Difco). The rear foot pads of male New Zealand white rabbits were injected with 0.2 ml of this suspension (approximately 100 ~g of chlamydial protein per foot pad). Three weeks later, an identical booster immunization was administered. Sera were harvested by cardiac puncture during the fifth week. Titers were 1 : 2048 against LGV 434 EB in the microimmunofluorescence test of Wang and Grayston (1970). Hyperimmune antigonococcal rabbit sera were prepared as previously described (Jones et al., 1980).
Preparation of protein A Protein A from Staphylococcus aureus was radiolabeled with ~25I by the technique of Fraker and Speck (1978) to a specific activity of 7 ~ C i / ~ g .
Immunologic detection of proteins bound to nitrocellulose After transfer, N C M strips were sequentially subjected to: (1) incubation with a blocking reagent at 37°C for 1 h; (2) incubation with antiserum at 37°C for 2 h; (3) a wash at room temperature for 15 min; (4) incubation with ~25I-protein A at 37°C for 1 h; and (5) 2 final washes at room temperature for 30 min each. Composition of the various blocking, diluent and wash buffers used in each step are given in the legend to Fig. 2. Unstained strips were placed in horizontal 15 ml polystyrene tubes. In the blocking step, strips were incubated with 15 ml of appropriate buffer. Following the blocking step, strips were incubated on a platform rotator at 75 r e v / m i n with antiserum diluted to 3 ml with the appropriate diluent. After removing unbound antibody by incubation in 15 ml of wash buffer, the strips were incubated with 0.25 ~Ci of 12~I-protein A diluted to 3 ml with the appropriate diluent buffer. Excess protein A was removed by rocking the strips in 15 ml of wash buffer. In those experiments where 150 mM NaCI was used as a wash, the wash was changed 5 times over a period of 30 min. Processed strips were air dried and exposed for 7-30 h to Kodak X-Omat A R film with a Dupont Cronex Lightning Plus intensifying screen at - 7 0 ° C (Laskey and Mills, 1977).
Quantitative measurement of bound antibody Purified EB of LGV 434 were subjected to SDS-PAGE and the resolved proteins transferred to NCM. The antigen-bearing N C M sheet was divided into 0.5 cm strips. N C M strips were blocked with PBS-Tween for 1 h at 37°C. Duplicate strips were incubated with serial 2-fold dilutions of rabbit hyperimmune serum in PBS-Tween ( 1 : 2 5 0 - 1 : 128,000). The strips were incubated with excess ~25I-protein A, washed, then subjected to autoradiography, staining with amido black 10-B, and repeat autoradiography. Finally, individual protein bands were sliced from each strip and the 125I cpm bound by that particular band were determined in a g a m m a counter
301
(Searle). Backgrounds were determined using identically sized slices of NCM that were obtained from a portion of each strip where no darkening of the corresponding autoradiographic film was noted.
Results
Blocker, diluent, and wash buffer comparisons Examples of a stained gel and NCM after transfer are shown in Fig. 1. Resolution of polypeptide bands was maintained and all major proteins appear to have transferred to the N C M sheet. A procedure using Tween 20 without protein blocker was compared to 3 published procedures, and to the other combinations of buffers and blockers indicated in Fig. 2. Preimmune serum and PBS without serum were included as controls. Ten procedures were directly compared using identical NCM strips with LGV 434 proteins. The same homologous hyperimmune rabbit serum at a dilution of 1:256 was used for each strip. An autoradiogram of strips processed in various blocking, diluent and wash buffers is shown in Fig. 2. Buffers used for each strip are listed in the legend. No bands of radioactivity were observed in the controls, indicating that protein A did not bind nonspecifically to NCM-immobilized chlamydial proteins, and that the preimmune rabbit serum did not contain IgG or other serum factors that could have bound antigen and protein A to produce false positive results. The autoradiogram shown was overexposed with respect to major immunoreactive bands to permit visualization of minor bands. As a result, the 45,000 and 40,000 dalton major proteins and a minor 38,000 dalton protein appear as a single broad band. These were easily distinguishable as individual bands with shorter exposure times. Strips 3 through 12 were probed with rabbit hyperimmune serum to compare the sensitivity of various procedures. Qualitative observations were made in terms of the number of bands observed and band intensity. For example, strip 5, processed according to Vaessen et al. (1981) with Triton X-100, resulted in fewer, less intense bands when compared to procedures using other detergents (strips 3, 4, 7, 8, 10, 11, and 12). Strips processed with Nonidet P-40 (strip 4) according to Cohen and Falkow (1981), Triton X-100 (strip 5) or Sarkosyl (strip 12) lacked an immunoreactive band that comigrated with the tracking dye. This particular band was identifiable but greatly reduced in strips processed with Tween 20 (3, 7, 8, 10 and 1 l) and conserved in procedures lacking detergents (strips 6 and 9). Otherwise, the PBS-Tween procedure (strip 3) appeared comparable in the identification of major immunoreactive bands to procedures using BSA but no detergent (strip 6). The latter procedure was derived from Towbin et al. (1979) but differed in that protein A was used as the detection reagent and carrier serum therefore was deleted. In addition, autoradiograms of shorter exposure times (not shown) revealed no substantial overall loss of bound radioactivity with any detergent except Triton X-100 (strip 5), confirming the above observations. Strips were also compared in terms of background. Both 3% bovine serum
302
albumin (strip 6) and Tween 20 alone (strips 3, 7 and 10) were effective blockers of unoccupied sites on nitrocellulose. Gelatin (0.25%) alone was not an effective blocker (strip 9) and did not improve background when added to Tween 20 buffers (cf. strips 10 and 11). In procedures using Tween 20 without protein blockers, phosphate and Tris-buffered saline appeared to be comparable (cf. strips 3 and 10). In addition, no loss of protein bands was observed in the PBS-Tween system when
A
B
68-60-45-40--
29--
15-
J
TD
I
I
Fig. 1. A: protein profile of LGV 434 after SDS-PAGE on a 12.5% polyacrylamide gel and staining with Coomassie brilliant blue R-250. The molecular weights in kilodaltons of major polypeptide bands are indicated. The position of the tracking dye is indicated by TD. B: protein profile of LGV 434 after transfer to N C M and staining with amido black 10-B. Major polypeptide bands corresponding to those of the original gel are indicated by dashed lines. The apparent change in position of the bands reflects shrinkage of the N C M and expansion of the gel during staining.
303
1
2
3
4
5
6
7
8
9
10 1 1 1 2
68 60 40 29
15 TD Fig. 2. Autoradiogram comparing various blocking, diluent, and wash buffers. Composition of each buffer is given below. The 68, 60, 40, 29 and 15 kilodalton proteins are marked for reference. The immunoreactive band that comigrated with the tracking dye (TD) is indicated by an asterisk for each strip. Strips 1 and 2 are PBS alone and preimmune serum controls, respectively, using PBS-Tween in all steps. Strips 3-12 were probed with hyperimmune rabbit serum. Strip 3: PBS-Tween in all steps; strip 4: TSGAN-1% BSA as blocker and TSGAN as diluent and wash; strip 5: PBS-8% BSA as blocker and diluent, and PBS-Triton as final wash; strip 6: TS-3% BSA as blocker and diluent, and NaCI as wash; strip 7: TS-Tween as blocker and diluent, and NaCI as wash; strip 8: TS-3% BSA as blocker, TS-Tween as diluent and NaCI as wash; strip 9: TSGA as blocker and diluent and NaC1 as wash; strip 10: TS-Tween in all steps; strip I 1: TSGA-Tween in all steps; and strip 12: PBS-Tween as blocker and diluent and PBS-S as final wash. Reagents were prepared as follows: PBS: 4 mM KH2PO 4, 16 mM Na2HPO4, 115 mM NaC1, pH 7.3; PBS-Tween: PBS containing 0.05% (v/v) Tween 20; PBS-8% BSA: PBS containing 8% (w/v) BSA; PBS-Triton: PBS containing 1% (v/v) Triton X-100; TSGAN: 50 mM Tris-HC1, 150 mM NaC1, 0.25% (w/v) gelatin, 0.15% (w/v) sodium azide, 0.1% (v/v) Nonidet P-40, pH 7.5; TSGA-Tween: as with TSGAN, but with 0.05% (v/v) Tween 20 substituted for Nonidet P-40; TSGA: as with TSGAN, but without Nonidet P-40; TS-3% BSA: 10 mM Tris-HC1, 150 mM NaC1, and 3% (w/v) BSA, pH 7.4; TS-Tween: 10 mM Tris-HC1, 150 mM NaC1, and 0.05% (v/v) Tween 20, pH 7.4; PBS-S: PBS containing 0.4% (w/v) Sarkosyl; and NaCh 150 mM NaC1.
NCM
stained for protein after immunologic
immediately
probing was compared
to NCM
stained
after transfer (data not shown).
Identification of immunoreactive proteins Outer membrane p r o t e i n s o f N. gonorrhoeae
were probed
with rabbit
immune
304 s e r u m u s i n g the P B S - T w e e n t r a n s f e r m e t h o d . A f t e r a u t o r a d i o g r a p h y , N C M strips w e r e s t a i n e d w i t h a m i d o b l a c k 10-B a n d a u t o r a d i o g r a p h y was r e p e a t e d . T h e r e w e r e n o q u a l i t a t i v e d i f f e r e n c e s b e t w e e n the a u t o r a d i o g r a m s b e f o r e a n d after staining. H o w e v e r , s h r i n k a g e o c c u r r e d d u r i n g staining, a n d an N C M strip s t a i n e d a f t e r a u t o r a d i o g r a p h y c o u l d n o t b e p r e c i s e l y a l i g n e d w i t h the a u t o r a d i o g r a m , w h e r e a s o n e s t a i n e d b e f o r e a u t o r a d i o g r a p h y c o u l d be. A n N C M strip s t a i n e d b e f o r e a u t o r a d i o g -
A
MAJOR
MAJOR
\
MINOR
TD
B
/
MINOR
i'D
Fig. 3. Outer membrane proteins from two colonial variants of strain F62 of N. gonorrhoeae w e r e resolved on a 12.5% polyacrylamide gel, transferred to NCM, and immunologically probed in the presence of PBS-Tween, as described in the text. A: NCM strip stained with amido black 10-B after immunological probing showing resolved outer membrane proteins. The major outer membrane protein and a minor band are indicated. B: autoradiogram of NCM strip shown in A. The dark band of radioactivity corresponded to a minor protein rather than to the major protein.
305 raphy is shown with the aligned autoradiogram in Fig. 3. A relatively minor outer membrane protein was highly immunoreactive and migrated close to the major outer membrane protein. Had it not been possible to align the stained NCM with the autoradiogram, this immunoreactivity could have been mistakenly attributed to the major protein, which did not bind antibody from this particular serum.
Quantitative measurement of bound antibody The ability to identify individual proteins by staining NCM after immunological probing allowed the development of a radioimmunoassay to indirectly quantitate antibodies against specific polypeptides. A titration of hyperimmune serum against a 40,000 dalton protein of LGV 434 is shown in Fig. 4. Similar titration plots were obtained for other chlamydial polypeptides (not shown). These curves are characteristic of those obtained with conventional radioimmunoassays in other antigen-antibody systems. Thus, slicing individual bands is a reproducible and sensitive method for quantitating antibodies bound to an individual protein.
I
I
I
I
l
E
E
•
•
•
I
I
I
I
4 d
A
g o x
E a o 2-
u3
o4
r~
•
RECIPROCAL DILUTION x
10 .3
Fig. 4. Quantitation of antibodies contained in homologous rabbit hypefimmune serum against a 40,000 dalton protein of L G V 434. Antiserum dilution is given on the abscissa and radioactivity in 125] cpm on the ordinate. Bars indicate ] standard deviation of the mean of duplicate determinations. Background determinations for each dilution are shown as dosed circles.
306 Discussion
The technique of SDS-PAGE followed by transfer to NCM and immunological probing is a powerful tool for antigenic analysis (Anderton and Thorpe, 1980). However, the quality of permanent record reported in published studies (Towbin et al., 1979; Burnette, 1981; Cohen and Falkow, 1981; Vaessen et al., 1981) has been variable, .and there have been no published studies directly comparing different techniques in terms of detection of antibody bound to NCM-immobilized proteins. We have performed such a comparison and have developed a procedure using PBS containing the nonionic detergent Tween 20 as a blocking agent. A non-detergent method similar to that described by Towbin et al. (1979) allowed identification of an immunoreactive band that comigrated with the tracking dye which was lost or greatly reduced in the presence of detergents. However, staining of N C M strips after processing in PBS-Tween showed no reduction in intensity of amido black 10-B staining material at the dye front, suggesting that this additional low molecular weight band is probably not a polypeptide. Thus, the PBS-Tween method appears suitable for detection of protein antigens adsorbed to NCM. All of the methods evaluated, except the one in which gelatin was used as a blocker, provided acceptable backgrounds with radioiodinated protein A as the detection reagent. However, for certain applications a labeled immunoglobulin may be a more suitable detection reagent (Towbin et al., 1979; Glass et al., 1981; Karcher et al., 1981). In preliminary experiments using goat anti-human IgM as a detection reagent, the backgrounds obtained with PBS-Tween were substantially lower than those obtained when large amounts of BSA or carrier serum were used as blocking reagents (data not shown). One of the major advantages of the PBS-Tween system is that it provides effective blocking of unoccupied protein binding sites on NCM and thereby obviates the need for proteins as blockers and carriers. Thus, after immunological probing, proteins on the NCM can be stained and precisely aligned with immunoreactive bands on the resultant autoradiogram. The utility of such alignment is illustrated by the experiment depicted in Fig. 3. Therefore, the ability to superimpose an autoradiogram on the stained NCM from which it was derived allows convenient and unambiguous identification of immunoreactive proteins, eliminating the uncertainties inherent in comparing the autoradiogram with stained gel or NCM side strips. In addition, staining after immunological probing circumvents any inhibition of antibody binding that may occur if NCM is stained before reaction with antiserum (Anderton and Thorpe, 1980). Staining of NCM can also aid in the interpretation of a negative result on the autoradiogram. Conditions whicl~ could result in the absence of immunoreactivity of an individual protein band are: (1) failure of the antigen to migrate out of the SDS gel; (2) failure of the antigen to adsorb to NCM; and (3) loss of antigen from NCM during any of the blocking, incubation, or wash steps. Comparison of gel strips stained for protein before and after transfer, and NCM strips stained before and after immunological probing can distinguish among these possibilities. Under circumstances where the antigen is a protein and can be demonstrated on the NCM
307
after processing, other conditions that may be responsible for observed non-reactivity are: (1) denaturation of antigenic determinants by SDS; (2) dissociation of antibody or protein A during processing; or (3) lack of specific antibody in the test serum. S t a i n i n g also allows simple, accurate identification of i n d i v i d u a l p r o t e i n b a n d s for s u b s e q u e n t slicing a n d indirect q u a n t i t a t i o n of b o u n d antibody. The feasibility of this a p p r o a c h was illustrated by the q u a n t i t a t i o n of a n t i b o d y to a chlamydial p r o t e i n (Fig. 4) in serial dilutions of h y p e r i m m u n e r a b b i t serum. Therefore, i m m u n o l o g i c a l p r o b i n g of a n t i g e n - b e a r i n g N C M strips in the presence of P B S - T w e e n can be used to p e r f o r m what is in effect a r a d i o i m m u n o a s s a y for a n y p r o t e i n resolvable b y S D S - P A G E . This technique has proven useful in the evaluation of the h u m a n serological response to specific polypeptides d u r i n g infection with C. trachomatis (W.J. Newhall V, m a n u s c r i p t in p r e p a r a t i o n ) a n d should have general applicability.
Acknowledgements This investigation was supported in part by Grant AI-17502 from the National Institutes of Health. We thank C.E. Wilde III and K. Fife for their critical review of the manuscript.
References Anderton, B.H. and R.C. Thorpe, 1980, Immunol. Today 1, 122. Bittner, M., P. Kupferer and C.F. Morris, 1980, Anal. Biochem. 102, 459. Bradford, M.M., 1976, Anal. Biochem. 72, 248. Burnette, W.E., 1981, Anal. Biochem. 112, 195. Burridge, K., 1976, Proc. Natl. Acad. Sci. U.S.A. 73, 4457. Cohen, M.L. and S. Falkow, 1981, Science 211,842. Fraker, P.J. and J.C. Speck, Jr., 1978, Biochem. Biophys. Res. Commun. 80, 849. Glass II, W.F., R.C. Briggs and L.S. Hnilica, 1981, Science 211, 70. Jones, R.B., J.C. Newland, D.A. Olsen and T.M. Buchanan, 1980, J. Gen. Microbiol. 121,365. Karcher, D., A. Lowenthal, H. Thormar and M. Noppe, 1981, J. Immunol. Methods 43, 175. Kuo, C.-C., S.-P. Wang and J.T. Grayston, 1977, in: Nongonococcal Urethritis and Related Infections, eds. D. Hobson and K.K. Holmes (Amer. Soc. Microbiol., Washington, DC) p. 328. Laemmli, U.K., 1970, Nature (London) 227, 680. Laskey, R.A. and A.D. Mills, 1977, FEBS Lett. 82, 314. Newhall, W.J.V., C.E. Wilde III, W.D. Sawyer and R.A. Haak, 1980, Infect. Immun. 27, 475. Renart, J., J. Reiser and G.R. Stark, 1979, Proc. Natl. Acad. Sci. U.S.A. 76, 3116. Schaffner, W. and C. Weissmann, 1973, Anal. Biochem. 56, 502. Towbin, H., T. Staehelin and J. Gordon, 1979, Proc. Natl. Acad. Sci. U.S.A. 76, 4350. Vaessen, R.T.M.J., J. Kreike and G.S.P. Groot, 1981, FEBS Lett. 124, 193. Wang, S.-P. and J.T. Grayston, 1970, Am. J. Ophthalmol. 70, 367.