Chapter 6 Production of Antisera and Radioimmunoassays for Tubulin

Chapter 6 Prodaction of Antisera and Radioimmunoassays for Tzlbzclin LIVINGSTON V A N DE WATER 111,' SUSAN D. GUTTMAN,2 MARTIN A. GOROVSKY, AND J. B. OLMSTED Department of Biology University of Rochester Rochester. New Yor&

1. Introduction . . . . . . . . . . . . . . . . . . . . 11.Methods . . . . . . . . . . . . . . . . . . . . . A. Production of Tubulin Antisera . . . . . . . . . . . B. Preparation of Protein A Adsorbent (PAA) . . . . . C. Purification and Iodination of Antigens . . . . . . D. Binding Assays . . . . . . . . . . . . . . . . . E. Immunostaining Procedures . . . . . . . . . . . . F. Gel Electrophoresis . . . . . . . . . . . . . . . 111. Results . . . . . . . . . . . . . . . . . . . . . . A. Assay Method . . . . . . . . . . . . . . . . . B. Characterization of Antisera Binding . . . . . . . C. Characterization of Binding Specificity . . . . . . D. Radioimmunoassay . . . . . . . . . . . . . . . IV. Discussion . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

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Introduction

Microtubules are widely distributed organelles that have a variety of functions (for reviews, see Dustin, 1979; Goldman et al., 1976). The amount of mi'Currmr u f j h t i o n : Department of Pathology, Beth Israel Hospital, Boston, Massachusetts *Current uffiliution; Department of Pharmacology, Stanford University School of Medicine, Stanford. California

79 METHODS IN CELL BIOLOGY, VOLUME 24

Copyright 0 1982 by Academic Press. Inc. A11 rights of reproduction in any form reserved ISBN 0-12-5641249

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LIVINGSTON VAN DE WATER Ill ET AL

crotubule protein has been measured in a number of cell types using quantitative gel analyses and drug-binding assays. Recently, antisera to tubulin have been generated for use in both cytochemical and quantitative studies. This chapter discusses the production of antisera raised against electrophoretically purified and SDS-treated Tetrahymena tubulin; these antisera react with tubulin from a number of species. Reports on the reactivity of these antisera with Tetrahymena tubulin (Guttman, 1978; Guttman and Gorovsky, 1979) and mammalian neuronal tubulins (Van De Water, 1979; Van De Water and Olmsted, 1978, 1980) have appeared. This chapter will emphasize the methodology used for raising antisera of this type and the parameters that were found most useful in developing reliable radioimmunoassays.

11. A.

Methods

Production of Tubulin Antisera

Cilia were isolated from Tetrahymena using the ethanol-sucrose procedure of Gibbons (1965). Isolated cilia were pelleted at 16,000 g for 20 min, and axonemes were prepared by dialysis against Tris-EDTA buffer according to procedure 3 of Renaud et ul. (1968). Isolated axonemes were precipitated with 6 vol of acetone and dried under vacuum. Precipitates were dissolved in sample buffer (Laemmli, 1970), boiled for 3 min, and run on SDS-containing polyacrylamide gels (see Section 11,F). Tubulin was localized by scanning gels or gel slices at 280 nm, marking the tubulin band with ink, and excising the strip corresponding to tubulin from the unstained gel. Protein was eluted from the unstained gel slices using a modification of the method of Lazarides (1976). Gel slices were placed in a glass tube to which a dialysis bag was attached; the slices were held in place with absorbent tissue and the tube was filled with Laemmli sample buffer. Electrophoresis was camed out at 240 V for 18-24 hr. Eluted tubulin was dialyzed for 18 hr at room temperature against three I-liter changes of 0.2 M NH,HCO, containing 0.05% SDS. The sample was concentrated in the dialysis bag to a volume of about 1 ml using dry Sephadex G-25 (300 mesh) and was then precipitated overnight with a sixfold volume of acetone. The acetone precipitate was collected by centrifugation at 10,000 rpm for 10 min, and the resulting pellet was dried under vacuum and then redissolved in 0.5-1 .O ml of complete Laemmli sample buffer by boiling for 3 min. The SDS-treated samples were rerun on SDS gels to determine the purity and concentration of the tubulin. Gels run with known amounts of tubulin were stained with Fast Green (Gorovsky et al., 1970) and quantitative densitometry was used to establish a standard curve from which the tubulin concentration of

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the immunogen was determined. In a typical preparation, acetone-precipitated axonemes from I liter of Tetruhymena (2-3 x 10” cells/ml) were dissolved in 1 . 1 ml of SDS sample buffer, and 0 . 8 ml was run on a 4-mm-thick 7.5% acrylamide slab gel for elution. The final yield of purified eluted tubulin was between 500 and 800 p g . Preparations that showed lower-molecular-weight contaminants that might be indicative of breakdown products were not used for immunization. For injection, 350 p g of tubulin in 0 . 7 ml of SDS sample buffer was mixed with 0.7 ml of complete Freund’s adjuvant. This suspension was emulsified by 20-30 vigorous passes through a glass syringe, although a typical emulsion was not formed because of the presence of SDS. Rabbits (New Zealand White) were injected with a total of 1.4 ml of the emulsion (350 pglrabbit) placed subcutaneously in a total of two sites on either side of the backbone (0.7 ml/site). Rabbits were boosted one week later with the same amount of protein in complete Freund’s adjuvant prepared as described earlier. Animals were first bled two weeks after the second injection and were subsequently bled every 5-7 days. Preimmune serum was obtained from animals one week prior to the first injection. The data presented in this paper were obtained using antisera from a total of four rabbits injected according to this procedure. An alternate injection schedule was used in earlier experiments (Guttman, 1978; Guttman and Gorovsky, 1979; Van De Water, 1979; Van De Water and Olmsted, 1980). Animals were injected subcutaneously on days 1 and 8 as above, followed by intravenous injections in the upper marginal ear vein with 150 p g tubulin in 0 . 2 ml SDS sample buffer on days 22, 29, 36, 38, and 40. They were also boosted biweekly with intravenous injections. The animals were first bled 45 days after the initial injection and 5-7 days after each booster injection. These antisera had lower titers than the antisera produced by the preceding procedure.

B.

Preparation of Protein A Adsorbent (PAA)

Quantitation of immune complexes was carried out using protein-A-bearing strains of Staphylococcus aureus as an immunoadsorbent. The Cowan I strain of S. uureus was grown, harvested, fixed, and heat-treated after 16-17 hr of exponential growth as described by Kessler (1976). Stock solutions of the protein A adsorbent (PAA) were stored at 4°C in phosphate-buffered saline (PBS: 0.14 M NaCl, 3 mM KCI, 1.5 mM KH,P04, 6 . 7 mM Na,HPO, . 7 H,O, pH 7.2) containing 0.2% sodium azide. As described by Kessler (1976), PAA was washed and incubated for 15 min at room temperature in NET buffer (0.15 M NaCl, 5 mM EDTA, 0.05 M Tris, 0.02% sodium azide, pH 7.4) containing 0.5% Nonidet P-40, followed by washing in NET containing 0.05% NP-40. Samples were resuspended to 10% v/v in NET-0.05% NP-40, 5 mg/ml BSA for use in the

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LIVINGSTON V A N DE WATER 111 ET AL.

Oh P A A (v/v) FIG. I , Standard curve for determining PAA concentration. Serial dilutions of PAA were prepared in NET-BSA containing 0.05% NP-40 and the absorbance at 550 nm was determined. Dilutions were then centrifuged in hematocrit tubes to obtain a viv measure of PAA content.

assay. The concentration of this solution was adjusted by determining the OD,,, of diluted samples (Fig. 1).

C. Purification and Iodination of Antigens Microtubule protein was purified from hog brain by repetitive cycles of assembly-disassembly (Borisy et al., 1975). Tubulin was purified from microtubule-associated proteins by DEAE-Sephadex (Murphy et al., 1977) or phosphocellulose (Sloboda et al., 1976) chromatography. Purity was assessed by quantitative densitometry of gels stained with Fast Green (Gorovsky et al., 1970) or Coomassie Blue (Fairbanks et al., 197 1). Competitor tubulin and iodinated tubulin were prepared from samples determined by quantitative gel analyses to be greater than 95% pure. Iodinations were carried out using the method of Hunter and Greenwood (1962). Typically, 10 p g of protein in 10 p1 or less was mixed with 25 p1 of 0.5 M sodium phosphate buffer, pH 7.2. To the sample was added 250 pCi of NaI29 and 10 p1 of 5 mg/ml chloramine T in 0.05 M sodium phosphate buffer. After 60 sec the reaction was stopped by the addition of 25 p1 of 2.5 mg/ml sodium metabisulfite in 0.05 M sodium phosphate buffer, pH 7.2. Free iodine was separated by chromatography of the sample on a 0.7 cm I.D. x 13 cm (3-25 (150 mesh) Sephadex column equilibrated in 0.05 M sodium phosphate buffer and prerun with 1 ml of 2%

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BSA. Fractions (0.5 ml) were collected into tubes containing 50 p1 of 2% BSA in 0.05 M phosphate buffer, and those containing bound radioactivity were aliquoted, frozen in liquid nitrogen, and stored at -80°C. The specific activity of the labeled tubulin was routinely 10,000 c p d n g , corresponding to approximately 0.5- 1 .O iodine molecules/l 10,000 dalton tubulin dimer.

D.

Binding Assays 1.

SERUMDILUTIONASSAYS

The extent of binding of antisera to labeled tubulin and the relative binding capacity of various antisera were tested by carrying out serum dilution experiments. Dilutions of sera were obtained by serial passage of a fixed volume of serum (usually 10, 25, or 50 p l ) into reaction test tubes (12 x 75 mm) that contained 25 p1 of SBA-BSA (0.15 M NaCI, 0.05 M sodium borate, 0.02% sodium azide, pH 7.4 containing 5 mg/ml BSA). Iodinated tubulin diluted in SBA-BSA to contain 10,000 c p d 2 5 p1 was added, and incubation carried out for 2 hr at 0°C. A volume of 10% PAA sufficient to absorb the highest concentration of serum was then added (usually 50 or 100 pl), and incubation at 0°C continued for 10 min. The samples were mixed with 2 ml SBA-BSA and centrifuged at 3000 rpm for 10 min. The supernatant was decanted, and the pellet resuspended in 2 ml SBA-BSA. Following centrifugation, the pellet was resuspended in 2 ml NET-BSA. The resuspended pellets were poured onto 0.2-pm cellulose acetate filters that had been precoated with 1 ml NET-BSA. The tubes were rinsed twice with 1 ml of NET-BSA, and suction was then applied. Filters were subsequently washed twice with 1 ml of NET-BSA under suction, dried, and counted in toluene-based fluor in a liquid scintillation counter. Controls included incubations of labeled tubulin with serial dilutions of preimmune serum and with buffer alone (background). The number of input countshube was determined by TCA precipitation of an aliquot of the diluted tubulin. The percent bound was calculated as the fraction of the TCA precipitable counts bound at a given serum concentration, after subtraction of background counts from both samples. 2.

COMPETITION ASSAYS

For competition assays, serial dilutions of a known amount of tubulin were prepared in the reaction tubes as described earlier. Typically, unlabeled tubulin at concentrations from 1 ng to 1 p g was used to establish a standard curve. To 25 p1 of the diluted protein was added 25 p1 of iodinated tubulin (10,000 cpm, 1 ng), followed by 50 pl of antiserum at a dilution that bound 50% of the input counts. Incubation of the reaction mixture, addition of 10%PAA (50 p l ) , and processing

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LlVINGSTON VAN DE WATER 111 ET AL.

of the filters were carried out as previously described. Controls included samples in which unlabeled tubulin was deleted (uncompeted samples) and in which preimmune serum or buffer and labeled tubulin were incubated (background samples). To determine tubulin content in cellular extracts, serial dilutions of the experimental samples were made and incubated with labeled tubulin and antiserum as described earlier. Values for the experimental samples were computed from the standard curves using the logit transformation of Rodbard ef al. (1969).

E. Immunostaining Procedures 1. GELS The distribution of antigenic species fractionated on SDS gels was determined using the immunostaining procedure of Burridge ( 1 978). Typically, gels were fixed overnight in 10% acetic acid, 50% methanol, agitated for 4 hr in several changes of the same solution, and then equilibrated in washing buffer (WB: 0.15 M NaCl, 10 mM Tris, 0.1% azide, pH 7.4) until a pH of 7.4 was attained. Strips of gel were overlayered with antiserum (l/lO dilution in WB containing 10 mg/ml BSA) and left at room temperature for 24 hr. The gel strips were then washed for four days with several changes of WB before overlayering with iodinated protein A (Amersham; 30 mCi/mg; final of 2-5 x lo6 c p d m l diluted in WB containing 10 mg/ml BSA). Washing continued for four additional days before gels were stained with Coomassie Blue (Fairbanks et al., 1971) and autoradiographed with X-Omat XR-5 film.

2.

CELLS

Cells plated on coverslips were fixed for 15-30 min in 3.7% formalin in PBS, washed with several changes of PBS, and then extracted with -20°C acetone for 30-60 sec. Coverslips were incubated sequentially with immune serum (1/30 dilution in PBS) and fluoroisothiocyanate-labeled goat antirabbit IgG antibody (Miles; 1/30 in PBS) for 30 min at 37°C with intervening washes in PBS. Coverslips were mounted in water on pieces of coverslip and sealed to slides with nail polish. Cells were photographed using a Zeiss narrow-band FITC filter and a 40 or 63 x planapochromat objective, and using Tri-X film developed with Diafine at an ASA of 1600.

F. Gel Electrophoresis Electrophoresis was carried out according to the method of Laemmli (1970), using a 7.5% acrylamide running gel and a 3% stacking gel. Analytical slab gels

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were 1.3 mm thick, and preparative slab gels were 4 mm thick. Electrophoresis was normally carried out at 30 mA for 2-6 hr, and gels were stained either with Coomassie Blue (Fairbanks er al., 1971) or with Fast Green (Gorovsky et al., 1970).

111. Results A.

Assay Method

Because of our interest in developing antisera to use for measuring tubulin content in cell extracts, assay methods were sought that would give accurate quantitation of antibody-antigen binding. Double immunodiffusion and precipitin curve analyses were used for characterization of the initial antiserum and its reaction with Tetruhymena tubulin (Guttman, 1978; Guttman and Gorovsky, 1979). However, these methods required large amounts of serum, and precipitation of immune complexes did not occur as readily with mammalian cell extracts. Antibody-antigen binding was therefore measured by an indirect precipitation method in which protein-A-bearing strains of S . uureus were used as the precipitating agent. This precipitation method was as efficient in binding immune complexes as conventional second antibody techniques, but resulted in lower backgrounds and was more economical. The following discusses the general conditions for the optimization of this assay. 1.

PROTEIN-A-ADSORBENT (PAA) CONCENTRATION

For a given concentration of serum to be used in serum dilution curves or the radioimmunoassay, it was necessary to establish the optimum amounts of PAA needed to quantitatively absorb the immune complexes. Figure 2 shows a typical PAA saturation curve in which fixed amounts of labeled tubulin and antibody were incubated with a fixed volume of PAA diluted to various concentrations. For each preparation of PAA, this type of curve was established. Typically, a volume of 10% PAA was used that would give binding in the middle of the plateau region of the curve.

2.

PREPARATION OF LABELED TUBULIN

Iodination by the chloramine T method was carried out using Na"9 that had been commercially prepared within two weeks. If older iodine was used, less efficient incorporation of label and lower binding to tubulin was obtained. Iodination using the Bolton-Hunter reagent (Bolton and Hunter, 1973) was not satis-

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LIVINGSTON V A N DE WATER 111 ET A L .

i r

*-cP-o-o-o

0

5

I

0-11-0-

10

1%-

% PAA FIG.2 . PAA saturation curve. Twenty-five p1 of immune serum (1/100 dilution) (0)or SBAwas incubated with 25 pI (2500 cpm) of iodinated tubulin for 2 hr at 0°C; 50 pI of PAA, BSA (0) serially diluted between 0.1 and 50% viv, was added to each sample, and the samples were then processed as described in Section 1I.D.

factory, presumably because of reaction with sites with which the antibody complexed. Iodinated proteins were stable for up to three months at -8O"C, with less than 10-15% loss of binding to antibody.

3.

ASSAYPROCEDURES

Procedures were optimized such that greater than 90% binding of antibody to antigen occurred under conditions of antibody excess, and background levels were routinely less than 10% of the input counts. Inclusion of BSA in all incubation buffers was essential to minimize nonspecific absorbtion. Background levels were dependent on the number of washes and the age of the PAA preparation used. A minimum of one 2-ml wash by centrifugation and two rinses of the filter were essential to obtain quantitative recovery and low backgrounds. Precoating the filters with 1 ml of NET-BSA also reduced nonspecific trapping. Backgrounds increased as the PAA preparation increased in age, although binding capacity did not appear to change. Routinely, PAA was prepared immediately before use by incubation with the solutions indicated in Section 11,B; if less than 24 hr elapsed before the next assay, PAA was only rewashed with NET containing 0.05% NP 40 and BSA.

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The times necessary for maximum binding were also investigated. With these sera, maximum binding of 90% of the input iodinated antigen to saturating amounts of antibody required a minimum of 2 hr at 0°C. In contrast, the interaction of PAA with immune complexes occurred rapidly and with high affinity; no change was observed in the amount of immune complex precipitated for incubation times of 10 min to 2 hr at 0°C. The reproducibility from assay to assay was very high. However, in order to minimize variation within each assay, glass capillary pipets were used for all dilutions and additions of reagents; use of automatic pipeting devices significantly decreased the accuracy of the assay.

B.

Characterization of Antisera Binding

Bleeds of the four immunized rabbits were initially assayed using serum dilution curves. A previously tested antiserum was assayed simultaneously to allow normalization of the data from the various bleeds. Figure 3 shows a serum dilution curve typical of the animals immunized by the methods outlined, in which greater than 90% of the labeled tubulin is bound under conditions of antibody excess. Sera obtained from three of the animals two weeks after the

IOC

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n

z

3 6C

0

m

8

40

20

FIG. 3.

Serum dilution curve. Serial dilutions (25 p l ) of immune serum were incubated with 25

p l of purified iodinated hog brain tubulin (10,000 cpm, 1 ng) as described in Section 11,D.l.

Background counts for preimmune controls were 220 cpm (2.2%of input counts) and those for buffer controls were 150 cpm ( I .5%).

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final injection all showed maximum binding to tubulin, and the fourth serum bound approximately 70% of the labeled tubulin. After an additional week, and for the succeeding two months, all four sera bound greater than 90% of the labeled antigen, and all showed 50% binding at serum dilutions of 1/500 to 1/1000. At the time of sacrifice (four months post injection), tubulin binding capacities of the sera had started to decrease, but 50% binding still ranged between dilutions of 1/200 and 1/1000, depending on the animal; all animals still bound greater than 90% of the labeled antigen. Because the binding to tubulin remained high over a long period, no additional booster injections were given. In fact, antibody titers decreased rapidly when the earlier protocol utilizing frequent booster injections was employed.

C.

Characterization of Binding Specificity

The specificity of the antisera for binding to tubulin was characterized in a number of ways. Although direct precipitation of immune complexes with similar antisera had been reasonably efficient for Tetrahymena tubulin (Guttman, 1978; Guttman and Gorovsky, 1979), mammalian tubulins did not form precipitating complexes. It was therefore felt that Ouchterlony analyses and rocket immunoelectrophoresis could not be routinely used as indicators of antibody specificity and titer. Instead, the binding of the antisera to labeled tubulin under conditions of antibody excess was routinely monitored. All of the sera maximally bound purified labeled tubulin at levels of 90% or greater. This was a qualitative indication not only that the sera contained tubulin antibodies, but also the sera did not react with a subset of tubulin, as had previously been found for another tubulin antiserum (Van De Water and Olmsted, 1978). As described below, the specificity of antiserum binding to tubulin was also demonstrated by experiments in which tubulin from Tetrahymena, hog, mouse, and neuroblastoma cells competed the binding of iodinated hog brain tubulin to background levels. The use of antibodies for immunofluorescent staining has demonstrated the network of microtubules that exists in various cell types. As shown in Fig. 4, the antisera raised against Tetrahymena tubulin stained the microtubules of neuroblastoma cells. The background levels of staining with these sera were very low, indicating little nonspecific absorption, and staining was abolished if preimmune serum or immune serum absorbed with tubulin was used in the primary incubation. These data also demonstrated that the antisera reacted well with aldehydefixed microtubules. To determine the protein species to which the antisera bound, cell extracts to be assayed (usually brain or neuroblastoma cells) were fractionated on SDS gels, and “stained” with the antibody and iodinated protein A (Burridge, 1978). As shown in Fig. 5 , only the tubulin doublet in a complex extract of mouse neuroblastoma cells reacted with the antiserum; no bands were observed with preim-

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FIG.4. lmmunofluorescent staining of microtubules in neuroblastoma cells. Mouse neuroblastoma cells (Nb2a-AB-I) were cultured on coverslips for 24 hr before being processed for immunofluorescent staining as described in Section II.E.2. Magnification: X 1120. Bar = 20 p m .

mune serum. However, it has been proposed that some antigens may lose reactivity after fractionation on SDS gels (Burridge, 1978). Therefore, in separate experiments, extracts from neuroblastoma cells labeled in vivo were incubated with saturating amounts of antiserum, treated with PAA, and the number of counts bound determined. It was found that the bound fraction was equivalent to the percentage of counts in the alpha and beta tubulin bands isolated from gels. In addition, these bound counts could be displaced by incubation with purified tubulin. These experiments taken together demonstrate that the antisera are spe-

FIG.5 . Gel electrophoresis of neuroblastoma extract. A postmitochondrial supernatan1 from neuroblastorna cells was electrophoresed on an SDS-polyacrylamide gel, and incubated with immune serum and protein A as described in Section I I , E , I , (A) Gel stained with Coomassie Blue. (B) Autoradiogram of same gel. Lines denote tubulin bands.

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LIVINGSTON VAN DE WATER 111 ET A L .

cific for tubulin in mammalian extracts and that the antisera react with both alpha and beta tubulins. Cross reactivity of the antisera with mammalian tubulin was also demonstrated by the adsorption of the immune IgG fraction to an affinity column prepared with purified tubulin isolated from hog brain. This affinity purified material bound efficiently to hog and neuroblastoma tubulins, and also to Terruhymena tubulin (F. Calzone, unpublished observations).

D . Radioimmunoassay In order to develop a reliable radioimmunoassay (RIA) for quantitation of tubulin, a number of parameters were explored. In all the experiments described, purified hog brain tubulin was used as the iodinated antigen (tracer). For the RIA, dilutions of serum were chosen at which 50% binding of input tracer occurred; this value was determined from a serum dilution curve, such as that shown in Fig. 3 , and usually corresponded to a 1/500 to 1/1000 dilution of serum. It has been reported that the sensitivity of a RIA may be increased by prolonged incubations with lower serum concentrations (Haber and Poulson, 1974). However, the majority of experiments for which the RIA was developed involved incubation of cell extracts, and although proteolysis did not appear to be a problem (Van De Water and Olmsted, 1980), attempts were made to keep incubation times to a minimum. Figure 6 shows a typical standard curve in which purified hog brain tubulin was used as unlabeled competitor. The variable Y (or % bound) was calculated for samples with standard and unknown amounts of tubulin by the equation

Y

=

[ ( B - N ) / ( B , , - N ) ] x 100

where the amount of radioactivity bound to the antibody in the presence ( B ) or absence ( B , ) of competitor is normalized for background counts ( N ) . Figure 6 also demonstrates the same data recalculated using the logit function of Rodbard et al. (1969), which converts the sigmoidal binding curve to a linear function. The logit function was calculated as logit Y = ln(Y/100 - Y ) and results in a straight line with the equation: logit Y = a

+ b(1og x)

where x is amount of competitor tubulin and u and b are derived empirically. The line derived from the logit function was fitted using a linear least squares computer program and was used for calculating the amounts of tubulin in the experimental samples. Typically, experimental samples containing between 0.5 and 50 pg/ml of tubulin could be assayed reliably. In order to demonstrate that tubulin from various sources would compete

PRODUCTION OF ANTISERA AND RADIOIMMUNOASSAYS FOR TUBULIN I

I

91

I

0

I

0

-I

>

-a I-

-2 0 -I

-3

PROTEIN (np) FIG. 6 . Standard competition curve. Purified hog brain tubulin (25 pi) was diluted (lil.5) over a range of 1 ng to 1 p g ( x axis). Iodinated tubulin (25 p l , 10,OOO cpm, 1 ng) was added to each dilution, followed by 50 p1 of antiserum (1/1OOO dilution). Samples were processed as described in Section II,D,2. Left y axis and closed circles: % bound. Right y axis and open circles: logit Y .

effectively in the RIA, studies were carried out to examine the characteristics of the antisera for binding to tubulin from various species and in different polymeric states. As previously shown for the original antiserum raised with Tetrahymena tubulin (Van De Water and Olmsted, 1980), the sera described in this report showed broad species reactivity. Tubulin derived from Tetrahymena, hog and mouse brain, and neuroblastoma cells competed iodinated hog brain tracer to background and had similar competition curves. Fixed microtubules and monomeric tubulin were also essentially equivalent in competition of labeled tubulin. These properties make these antisera generally useful for quantitating tubulin in a variety of cell types. Although soluble materials can be readily assayed in most buffers, it is often problematical to assay cell extracts because of the presence of particulate or insoluble material that does not dilute reproducibly enough for quantitation in the RIA. Under conditions where buffers other than those usually employed in the RIA were used, it was necessary to determine whether they affected the RIA by preparing standard curves in the same solutions in which the cell extracts were prepared. A number of buffers have now been tested, the majority in the pH range of 7.0-7.4. The presence of up to 10%glycerol, 0.1% spermidine, or high magnesium (25 mM) had no effect on the slope of the competition curve; 2%

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Triton X- 100 depressed the slope slightly, but concentrations of 1 % or less had little effect. In addition, as previously described for immunoprecipitation (Lingappa et a/., 1978), samples can be treated with SDS sample buffer lacking mercaptoethanol, and then diluted into Triton X- 100 containing buffer such that the final ratio (%) of SDS to Triton X-100 is 1:lO. This procedure works reliably in the RIA and allows solubilization of most cellular fractions such that dilutions can be accurately made.

IV. Discussion Tubulin is apparently a highly conserved protein (Luduena and Woodward, 1975), and production of antisera, even with moderate titers, has proved problematical. A number of different immunogen preparations have been used; these include (1) native purified tubulins (Bhattacharyya and Wolff, 1975; Crawford et a/., 1976; Frankel, 1976; Gozes e t a / . , 1977; Hiller and Weber, 1978; Ikeda and Steiner, 1976; Joniau et a/., 1977; Meier and Jorgensen, 1977; Van De Water and Olmsted, 1978); (2) glutaraldehyde cross-linked tubulin (Fuller et al., 1975; Gordon et a/., 1977; Karsenti et al., 1978; Morgan et al., 1977, 1978); (3) SDS-denatured species, either injected directly (Zenner and Pfeuffer, 1976) or purified from gels (Aubin et al., 1976; Cande et a / . , 1977; Connolly et a/., 1977; Eckert and Snyder, 1978; Guttman, 1978; Koehn and Olsen, 1978; Piperno and Luck, 1977; Wiche and Cole, 1976); and (4) other tubulincontaining preparations [e.g., Arabacia axonemes (Fulton et a / . , 1971); Naegleria axonemes (Kowit and Fulton, 1974); and vinblastine-tubulin crystals from cultured cells (Dales, 1972) and sea urchin eggs (Fujiwara and Pollard, 197811. The majority of these preparations have been used for immunofluorescent localization of tubulin; a few have been used for radioimmunoassays (Bhattacharyya and Wolff, 1975; Gozes et a/., 1977; Hiller and Weber, 1978; Joniau et a/., 1977; Koehn and Olson, 1978; Kowit and Fulton, 1974; Morgan et al., 1977; Van De Water, 1979; Van De Water and Olmsted, 1978, 1980). The majority of antisera have been tested for specificity by double immunodiffusion tests, immunoelectrophoresis, or staining of microtubule networks by indirect immunofluorescence. These assays have given variable results. Some immune sera have given high backgrounds in immunofluorescence, and further purification by affinity chromatography has been necessary to obtain satisfactory images. In addition, the results that depend on precipitation of immune complexes may be difficult to analyze because of the apparent weak precipitation properties of most tubulin antisera; this property may make assessment of the presence of minor contaminants in either the immunogen or test antigens difficult.

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For those sera that precipitate antigens efficiently, the properties of immune complexes can be tested directly. These sera have been useful in demonstrating different species reactivities (Fulton et al., 1971 ; Gordon et af., 1977; Joniau et af., 1977) and in quantitating tubulin synthesis (Guttman, 1978; Guttman and Gorovsky, 1979; Kowit and Fulton, 1974; Piperno and Luck, 1977). However, the titers of the tubulin antisera produced usually have been low, necessitating the use of large amounts of serum even in those cases where precipitation reactions could be used. In addition, animals have not generally appeared to respond well to booster injections, further limiting the amount of serum that might be produced. It therefore seemed desirable to develop techniques for the production of higher titer antisera. The basic technique of purifying the immunogen off of SDS gels was originally used to produce actin antibodies (Lazarides, 1976) and, as noted previously, has also been successful for tubulin. In the case described here, the excellent response of all four animals to immunogen prepared in this manner suggests that the technique may be generally useful for antibody production. The strong immune reaction may also be due to the use of tubulin isolated from an organism distantly related to the injected animal; this is consistent with other reports that demonstrated that tubulin derived from lower eukaryotes (e.g., sea urchin, Fujiwara and Pollard, 1978; Naegferia, Kowit and Fulton, 1974; Chfumydomonas, Piperno and Luck, 1977) appear to produce more highly reactive sera. In addition, isolation of gel-purified Tetrahyrnena tubulin reduces the probability of eliciting antibodies to contaminating protein species that would then cross-react with proteins present in mammalian cell extracts. In those cases where antiserum binding has been measured quantitatively (by binding to labeled antigen), the efficiency of binding has varied widely, with 50% binding of labeled antigen (usually 0.5-1 .O ng) occurring over serum dilutions from 1/25 (Zenner and F'feuffer, 1976) to 1/30,000 (Kowit and Fulton, 1974). The majority of antisera appear to have 50% binding to nanogram amounts of tubulin in the dilution range of 1/50-1/100. The antisera described here show 50% binding at dilutions of 11500 to 1/1000. However, normalization of these data is difficult, since not all reports demonstrate serum dilution curves with a typical sigmoidal shape, or binding that reached a maximum of 100% under conditions of antibody excess. Tubulin antisera have been produced that show generalized binding to a variety of tubulins or that are more reactive with particular species. Under the conditions used for immunofluorescence, most tubulin antisera appear to react with a number of cell types and species. With some antisera, more limited reactivities have been shown by Ouchterlony tests (Fulton et ul., 1971), precipitin tests (Gordon el al., 1977), and radioimmunoassays (Hiller and Weber, 1978; Morgan et a l . , 1978). For example, antiserum raised to lamb brain tubulin has been shown to discriminate between various mammalian brain tubulins (Morgan

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et al., 1978), and a chick brain tubulin antiserum reacted differently with tubulins from various tissues and cultured cells (Hiller and Weber, 1978). In contrast, the antisera described here, as well as others (Dales, 1972; Joniau et al., 1977), react equally well with tubulin from diverse species. It is not possible to generalize about which preparation of immunogen results in either narrow or broad antigenic reactivity. However, in the cases reported, the use of SDSdenatured, electrophoretically purified tubulins seems to result in antisera that recognize determinants common to several tubulins. The types of variable properties with which tubulin antiserum reacts with various antigens indicates the care with which these reactions must be characterized in order to develop a reliable RIA. The sensitivity of the RIA has allowed discrimination between antigenic determinants that are distinct from species to species (Morgan et al., 1978) and from one cell type to the next (Hiller and Weber, 1978). However, these examples make clear the necessity for characterizing the binding properties of the antisera carefully in order to assay various tissue types. For example, if the reactivity of a cellular tubulin is markedly different from the tracer or competitor used for obtaining the standard curve, uninterpretable measurements could be obtained. This is especially true in those cases where heterologous tubulin does not compete the tracer to background levels, indicating that the tubulins may have different antigenic determinants. The resulting measurements may reflect only a subset of the tubulin in the extract. In addition, it is particularly difficult to normalize these data if the competition curves with homologous competitor and heterologous experimental material have different slopes over either part or all of the curves. It is also important to establish the reactivity of the antisera with different oligomeric forms of tubulin. Previous work (Meier and Jorgensen, 1977; Van De Water and Olmsted, 1978) suggested that some tubulin antisera react preferentially with aggregated or polymerized forms of tubulin. Under solution conditions where tubulin might be largely in subunit form, RIA utilizing these sera would underestimate actual tubulin content. The antisera described here react with both SDS-denatured tubulin and glutaraldehyde-fixed microtubules equally efficiently. It therefore appears that these antibodies react with determinants exposed on the subunit regardless of whether it is in monomeric or oligomeric form. In summary, in order to determine tubulin content accurately in heterogeneous solutions by RIA, a number of parameters must be investigated. These include ( 1) demonstrating the efficient reaction of the labeled tracer with the antibody, (2) measuring the competability of the tracer with homologous antigen for a standard curve, and (3) assessing the cross-species and oligomeric reactivity of the antibody. With these factors taken into consideration, the use of tubulin antibodies should greatly increase the sensitivity with which tubulin distribution and metabolism in a number of cell types can be measured.

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ACKNOWLEDGMENTS Special thanks go to F. Calzone and C. Kenny, who participated in obtaining the antisera described in this chapter. This work has been supported by grants from the NIH and ACS to J.B.O. and M.A.G.

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