Chapter 8 Immunochemical Analysis of Nonhistone Proteins F. CHYTIL Departments of Biochemistry and Medicine, Vanderbilt University School of Medicine, Nmhville. Tennessee
I. Introduction This chapter introduces the methodology of immunochemistry of chromosomal nonhistone proteins. Attempts also will be made to review some applications of the immunochemical analysis in chracterization not only of the nonhistone per se, but also in characterization of chromatin. The material discussed here concerns mostly antibody raised against heterogeneous mixtures of these proteins. The immunochemical analysis will certainly become more meaningful when an antibody against a single homogenous nonhistone protein preparation is obtained.
11. Immunogenicity of Nonhistone Proteins In contrast to histones the heterogeneous mixtures of nonhistone proteins are very good immunogens (Z-3). To produce antibody, nonhistone proteins can be injected into the experimental animal complexed with DNA (1,2). Administration of whole chromatin also has been shown to give antibody against nonhistone proteins as well as against histones (3-5). Finally, free (5) as well as sodium dodecyl sulfate (SDS)-treated nonhistone protein preparations (6)are good immunogens. No difficulties have been reported in producing antibody against nonhistone proteins from various eukaryotes. 123
124
F. CHYTIL
111. Choice of Animal for Production of Antihistone Antibody Rabbits as well as chickens are suitable for inducing antibodies. Guinea pigs were not reported to produce antibody against nonhistone proteins, but these animals were used for production of antinuclear antibody without major complications (7). It is therefore no a priori reason to eliminate this animal as a potential antinonhistone antibody producer. Both rabbits and chickens produce and then carry these antibodies without apparent pathological signs. In the past 5 years we have not experienced deaths of immunized animals which could be attributed directly to the immunization by nonhistone proteins.
A.
Rabbits
Rabbits seem to be the choice of most workers because the handling and housing of these animals does not need special skills. Moreover the quantity of blood which can be obtained is usually sufficient for many experiments. The nonhistone protein antibodies have been produced in most instances against heterogeneous mixtures of these proteins. Because the antibody response to such a heterogeneous immunogen may vary from one animal to another, it is not advisable to pool the antisera after immunization of several animals. Also, sera from different bleedings of the same animal should not be pooled since the antibody specificity might undergo variations. But the serum should be tested before immunization for the presence of natural antibody against nuclear material because recently it was found that some nonimmunized rabbit sera contain complement-fixing antibodies directed against nuclear components (8). The most common rabbit strain, New Zealand White, is used. The rabbits usually weigh at least 3 kg; male sex is preferable as it eliminates complications with accidental pregnancies occurring in the animal facilities.
B. Chickens Chickens have been shown to be good producers of nonhistone antibodies when injected with whole chromatin (3,4).This animal might be preferred when precipitation antibody is to be produced. Chicken antibody optimally forms precipitin complexes in the presence of 1.5 M NaC1. This concentration of sodium chloride facilitates the solubility of chromosomal proteins (9).
8.
IMMUNOCHEMICAL ANALYSIS OF NONHISTONEPROTEINS
125
IV. Immunization Procedure A. Preparation of the Immunogen Nonhistone proteins are prepared as immunogens similar to the other proteins. Usually these proteins-regardless of whether injected free, SDStreated, or as the whole chromatin or in the form of complexes with DNAare emulsified with complete Freund adjuvant. An equal volume of Freund adjuvant is stirred with the nonhistone protein preparation in a small beaker using a magnetic stirrer. The resulting white emulsion is administered to the animal. The mixing of the immunogen with the adjuvant also can be accomplished by vigorously shaking the mixture in a syringe.
B. Administration Dose Microgram quantities of the nonhistone protein mixtures (see Table I), are sufficient to elicit antibody response. Some workers have been using milligram quantities. The use of smaller quantities has been adopted in this laboratory for two main reasons. First, the quantity of the nonhistone protein mixture available often represents a limiting factor; second, by using larger quantities of the immunogen there is a higher probability of producing antibody against some undesired impurities.
C. Route of Administration Various laboratories administer the immunogens differently, as is evident from Table I. A subcutaneous, intramuscular, or intradermal injection is usually the starting procedure. Sometimesthe immunization is completed by intravenous injection of the immunogen. In our laboratory we use the following procedure. Fifty to 100 pg of total nonhistone protein mixed with complete Freund adjuvant (1:1 by volume) are injected under the skin of the toe pads of one of the hind legs of the rabbit. Usually 0.25 to 0.5 ml ofthe mixture is injected into the pads, and the rest is administered in small doses intramuscularly over the whole rabbit body. After a week, a second injection is given in the toe pads of the other foot. Then, in weekly intervals (usually two more doses are required), the immunogen is administered intramuscularly in small doses over the whole body. When antibody is detected in the serum taken from these animals an intravenous injection (booster) of the immunogen (usually one-fourth of the original quantity) diluted with saline is given intravenously (major marginal vein of the ear) without adjuvant. Seven days later the animal is bled from the marginal ear vein. Thirty milliliters of the blood can be obtained from a good “bleeder” rabbit in one bleeding.
TABLE I PRODUCTION OF ANTINONHISTONE PROTETEIN W P ) ANITBODY Problem studied Tissue specificity Eu and hetero chromatin
Structure of chromatin Liver development Estrogen-mediated differentiation Malignant growth
Tissue culture Drosophila
Newt oocytes
Immunogen
Animal
Mode of injection"
Dose per injection (mg)
Intervals (days)
Number of injections
NHP-DN A complex NHP-DNA complex Chromatin
Rabbit
SC and IM
0.1
I
3 4
Rabbit
NI
I
3
Chicken Rabbit
ID and SC
Approximately 2 mg
NHP-DNA complex Chromatin NHP-DNA complex NHP-DNA complex NHP-DNA complex Nucleoli
Rabbit
SC and I M
Approximately 8 mg 0.1
Chicken Rabbit
ID and SC SC and IM
0.1
7
34
Rabbit
SC and IM
0.1
I
34
Rabbit
SC and IM
0.1
I
3 4
Rabbit
ID and IM
I
3
Rabbit Chicken Rabbit Rabbit Rabbit
ID and SC ID and SC SC and IP
10,20, 50 total amount
I
5 5
Chromatin NHP and SDS NHP and SDS NHP
NI
sc sc
0.4
0.4
0.4 0.4 9-10 2
NI
3 4
Reference
5
I
3
7
3-6 5
3
3
NI
NI
NI
NI
28
"IV = Intravenously; IP = intrapentoneally; IM = intramuscularly; SC = subcutaneously; ID
Booster dose
=
2
intradermally; NI = not indicated.
8.
IMMUNOCHEMICAL ANALYSIS OF NONHISTONE PROTEINS
127
D. Treatment of the Antisera The blood collected into a centrifuge tube is left to coagulate for about
1 hour at room temperature and then stored in a refrigerator overnight. After centrifugation of the blood at about 2000 g for 10 minutes the serum
is siphoned off and kept frozen at -20°C. To avoid infection, the serum can be sterilized by using Millipore filtration. It is our experience that hemolyzed sera also can be used. Usually the whole serum can be used. When necessary, the immunoglobulin fraction G (IgG) can be purified from the sera by ammonium sulfate precipitation (23) or by 0-(diethylaminoethyl) cellulose (DEAE-cellulose) column chromatography (24).
V. Testing Antigenicity One of the most popular methods for testing for the presence and characteristics of the nonhistone protein antibodies is quantitative microcomplement fixation as described originally by Wasserman and Levine (25). The method used in my laboratory is described below.
A. Quantitative Microcomplement Fixation Principle: The microcomplement fixation method is an adaptation of complement (C') fixation which is based on the capacity of the complement system in fresh guinea pig serum to combine irreversibly with antigen-
TABLE I1 QUANTITATIVE COMPLEMENT-FIXATION ASSAW Stage 1: Fixation of C' by AbAg complex Ab Ag Ab
+ C'
+ C'
+ Ag +
A b + C' A g C' [Ab - A g C'
+
- C']
Stage 2: Lysis of EA by C'
+ EA + EA + EA
Lysis Lysis No lysis
Stage 3: Quantitative measurement of lysis by determining hemoglobin concentration (optical density 413 nm). Results are expressed in percentage of C' fixed. complement (guinea pig); Ab, antibody;Ag = correspondingantigen;E = erythrocytes (sheep); A = antibodies (rabbit) to E (sheep); EA = sensitized erythrocytes (E complexed with A ) .
128
F. CHYTIL
antibody complexes. If the antigen is associated with the sheep erythrocyte cell surface, such a combination may result in lysis of the erythrocyte. The lysis of hemolysin-sensitized sheep erythrocyte determined quantitatively by measuring hemoglobin spectrometrically is an excellent indicator system for C' activity (the principle of this method is outlined in Table 11). If C' is allowed to incubate with the antigen-antibody system under study, then its combination can be estimated by the residual hemolytic activity it possesses when a known quantity of antibody-coated (hemolysin-sensitized) erythrocyte is added at a later time. The method is not only very sensitive for detecting the presence of antibody, but also by using the same antibody against two or more antigens, one can determine the extent of similarity or conformational differences of the protein molecules by comparing the amount of each protein which elicits maximum fixation (26). 1. MATERIALS
a. Diluent. Isotris stock buffer: To 81.6 gm of NaCl add 12.1 gm ofTris (hydroxymethyl)aminoethane base and dissolve in about 800ml ofdeionized H,O. Add 6.6 ml of concentrated HCl, mix, and add 33 ml of 0.15 M MgSO,. 7 H,O (3.7 gm/100ml)and 15 ml ofO.l MCaCI,(1.1 gm/lWml). Dilute to 1 liter after adjusting the pH to 7.4. b. Isotris diluent- workingsolutions. To 100ml of the above stock buffer add 1 gm of bovine serum albumin and mix with a magnetic stirrer until dissolved. Make up the volume to lo00 ml with water. c. 0.1% Na,CO,. d. Sheep cells. These can be purchased commercially (50% whole blood, 50% Alsevers solution). They are kept refrigerated and can be stored up to 1 month. e. Anti-sheep hemolysin. This is purchased commercially, frozen at -2O"C, and can be stored over 4 years. The commercial hemolysin is in 50% glycerol solution. Two milliliters of this preparation are diluted to 50 ml with isotris diluent and stored frozen in 0.5 ml aliquots. The 1/1OOO dilution is prepared fresh daily: 0.2 ml of 1/50 stock dilution plus 3.8 ml of isotris diluent. 1: Guinea pig complement. Complement (C')should be kept cold all the time (not exceeding 4 "C). The commercial product (lyophilized guinea pig serum) is reconstituted with 5 ml of the supplied diluent. The serum is kept frozen in 0.25-ml aliquots. Once an aliquot is unfrozen for use, any complement remaining is discarded. Complement will retain its original titer approximately 14 months if stored at -2OOC (-40°C is preferable). When commercial frozen preparation of complement is used, the sample is first thawed and kept frozen in aliquots of 0.25 ml or more,
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IMMUNOCHEMICAL ANALYSIS OF NONHISTONE PROTEINS
129
2. PROCEDURES a. Complement titer determination. With any new batch of complement it is necessary to determine the titer as follows: (1) dilute the frozen complement aliquots MOO (0.1 ml is diluted to 10 ml with isotris diluent); (2) prepare different dilutions of C’according to Table 111. Remember that all dilutions must be made in an ice bath. Use disposable glass culture tubes (13 x 100 mm). Pipette different dilutions of C‘ according to Table IV. Again, all operations have to be performed in an ice bath. After mixing the above components, store the tubes in a cold room on ice overnight. Next day add 0.2 ml of sensitized sheep cells to each tube (see Section V,A,
2,b).
After the addition of sensitized cells the tubes are placed in a constanttemperature water bath and incubated 40-60minutes at 37°C.Thelast tube is the sheep cell blank (lysis in the absence of C). The tubes are then transferred to an ice bath for 10 minutes to stop hemolysis and centrifuged 10 minutes at 2000 rpm in a clinical centrifuge. The optical density (OD) of the supernatants is measured at 413 nm against isotris. The highest dilution of complement that gives 90% lysis is used for the fixation procedure (see Fig. I). We find that the titer of the complement fluctuates between 1/125 and 1/225,depending on the batch and the supplier. b. Standardization of sheep cells (prepare daily). 1. Three milliliters of cell suspension are centrifuged at 1500 rpm for 5-10 minutes in a clinical centrifuge (refrigerated).
TABLE 111 DILUTION OF COMPLEMENT C’ C‘ dilution C’(11100)(ml)
Isotris (ml)
1/125 1 0.25
11100 1
-
11150 1
0.50
11175 1 0.75
11200 1 1
11225 1 1.25
1/250 1 1.50
11275 11300 1 1 1.75 2
TABLE IV COMPLEMENT TITER 11100 1/125
C’dilution ml Isotris (ml)
0.2 1
11150 11175 11200 11225 11250 11275 11300
0.2
0.2
1
I
0.2 1
0.2 1
0.2 1
0.2
0.2
I
1
0.2 1
Cell blank 0.0 1.2
130
F. CHYTIL 0.6-
0.5m T
0.40,30.20.1 -
&&&7$3.&Tki& DILUTION OF COMPLEMENT
FIG. I . Determination of the complement titer. A,,,
=
optical density at 413 nm.
2. The supernatant is aspirated off, and the cells are resuspended in
4-5 ml of isotris diluent. Repeat twice more or until the supernatant is
clear. 3. Next 0.5 ml of washed, packed cells is suspended in 9.5 ml of isotris diluent . 4. Then 1 ml of this 5% cell suspension is added to 14 ml of 0.1% Na,CO, ; 5 minutes are allowed for complete hemolysis. 5. At 541 nm the desired optical density is 0.680. The range of acceptability is 0.6654.695. 6. The concentration of sheep cells is adjusted to the proper value by isotris diluent. The formula for adjusting is OD
0.680
x y, = y,.
Y , = initial volume of the preparation after removing 1 ml for estimating optical density; V , = adjusted final volume. c. Sensitization of sheep cells (preparefresh daily). 1. In a 50-mlErlenmeyer flask, to 1 ml of standardized sheep cells add with a swirling motion 1 ml of hemolysin (1/1OOO) [0.2 ml of the stock solution (1/50) of hemolysin is diluted with 3.8 ml of isotris diluent]. 2. Incubate for 15 minutes at 37°C to allow maximum sensitization. Then 18 ml of isotris diluent is added. 3. The sensitized cells may be kept 4 day if they are refrigerated. d. C' fixation procedure. The nonhistone protein preparation of known protein content to be tested for antigenicity should be kept frozen in small aliquots. Before use in complement fixation the antibody serum is incubated at
8.
131
IMMUNOCHEMICAL ANALYSIS OF NONHISTONE PROTEINS
60°C for 20 minutes to inactivate the endogenous
C'. The dilution of antibody is to be determined by complete complement fixation run on each dilution (1/200, 1/400, 1/800, 1/1600, etc.). The antibody is stored in the freezer at not less than 1/200 dilution. 1. To a series of disposable glass culture tubes (13 x 100 mm) add in the following order: 0.6 ml of diluent; 0.2 ml of diluted serum; 0.2 ml of antigen solution serially diluted as shown in Table V; 0.2 ml of diluted C' (see C' titer determination). Table V shows an example of this determination. 2. Appropriate dilutions of antigen and C', antibody and C', diluent and C',and diluent in a total volume of 1.2 ml are included in every experiment (Table V, tubes 7-17). 3. After incubation at 2"-4"C for 16-18 hours, 0.2 ml of sensitized cells is added and hemolysis allowed to proceed, with occasional swirling
TABLE V COMPLEMENT FIXATION Tube no.
Isotris (ml) 0.6 0.6 0.6 0.6 0.6 0.6 0.6
1 2 2 3 4 5 6 7 8 9
0.8 0.8 0.8 0.8 0.8 0.8
I0 11 12 13 14a 15 16 17
0.8 I .o 0.8 1.2 1.2 Na,CO, ~~~
Antisera (ml) 0.2 0.2 0.2
Antigen (ml) and content of nonhistone protein 0.2 (10 pg) 0.2 ( 5 pg) 0.2 ( 5 pg) 0.2 (2.5 pg) 0.2 (1.25 pg) 0.2 (0.62 pg) 0.2 (0.31 pg) 0.2 (10 PP) 0.2 (5 pg) 0.2 (2.5 pg) 0.2 (1.25 pg) 0.2 (0.62 pg) 0.2 (0.31 pg)
0.2 0.2
0.2 0.2
-
0.2 -
-
~~
aOD obtained in tube 14 is used as 100% of lysis.
C' (ml)
Cells (ml)
0.2
0.2 0.2
0.2 0.2 0.2 0.2
0.2 0.2 0.2 0.2
0.2 0.2 0.2 0.2
0.2 0.2 0.4
-
0.2 0.2 0.2 0.2 0.2 0.2 0.2
0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2
132
F. CHYTIL
in a water bath at 3So-37OC until controls are estimated to be 80-90% hemolysed. This requires about 40-60 minutes. 4. After immersion in an ice bath to stop hemolysis reaction (10 minutes), the tubes are centrifuged 10 minutes at 2000 rpm in a clinical centrifuge. 5. The optical density of the supernatant is determined at 413 nm. e. Calculation. OD sample x 100 = percent lysis OD 100% lysis The OD obtained in tube 14 is used as 100% of lysis. Percent C’ fixed 100 - percent lysis.
=
3. COMMENTS The quantitative complement fixation assay has been the most widely used method for determination of antigenicity of nonhistone proteins. It is very reproducible, sensitive, and allows the detection of conformational changes of antigens (26). Some problems may be encountered, however. As shown in Table 11, one of the prerequisites for quantitative evaluation of the interaction of the antigens with antibody is that the antisera, at the dilution used, should not bind complement in the absence of the antigen. However, some antisera exert this type of binding. This becomes a complicating factor mainly when low dilutions of antisera have to be employed. To minimize this problem it is advisable to screen the sera before immunization of the animals by determining the amount of complement fixed by various dilutions of the serum starting at a 1/200 dilution followed by 1/400, 1/800, etc. Usually those animals whose sera shows binding of complement (anticomplementarity) at 1/400 dilution are not used for immunization. Occasionally, this anticomplementarity develops during the immunization of the animal. There are several ways to remedy this complication. Purification of the IgG fraction might help. Sometimes centrifugation of the serum diluted with 9 volumes of 0.14 M sodium chloride at 105,000 g at 4OCfor 1 hour removes the anticomplementarity. Finally, the amount of C’used in the assay can be raised by an increment which eliminates the binding by antiserum (27). A more severe problem is the anticomplementarity of the antigens, which might be concentration dependent. Whole chromatin preparations usually do not show anticomplementarity. But nonhistone proteins as well as nonhistone protein-DNA complexes may show limited binding C’.Then the amount of C‘ fixed in the fixation assay in the absence of antiserum (see Table V) can be subtracted from that observed in the presence of antigen-
8.
IMMUNOCHEMICAL ANALYSIS OF NONHISTONE PROTEINS
133
antibody complexes. We have noted that repeated thawing and freezing of chromatin preparations sometimes induces anticomplementarity. Storing antigens in small aliquots is therefore advisable. Chromatin preparations treated with acid to remove histones usually show high anti-complementarity. Removing histones with a high concentration of urea and salt seems to have little effect. A typical complement fixation curve when antinonhistone proteinDNA antisera are reacted with the preparation used for immunization and with the corresponding whole chromatin preparation is shown in Fig. 2. The fact that the nonhistone protein antisera also react with the whole chromatin lets one use such an antibody in the characterization of chromatin preparations. An example of this use is plotted in Fig. 3. The amount of antigen can be expressed in the amount of DNA or nonhistone protein in the assay.
B. Other Methods of Testing Antigenicity Recently a double diffusion technique was employed (13,28). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis also has been used (6).
C. Applications By the use of the quantitative complement fixation method tissue specificity of antigenic properties of nonhistone protein-DNA complexes from chick oviduct nuclei has been described (1). Wakabayashi and Hnilica (2) have shown that an antiserum against nonhistone protein-DNA complexes isolated from Novikoff hepatoma will distinguish between this tumor and similar complexes isolated from rat liver and calf thymus. Furthermore, Zardi et al. ( 4 ) found that whole chromatin, when used as an immunogen, can elicit complement-flxing antibodies which recognize differences in chromatin and nonhistone proteins not only between WI-38 human diploid fibroblast and 3T6 mouse fibroblasts grown in culture, but also between chromatin of WI-38 cells and their SV-40 transformed counterparts, the 2-RA cells (3). Changes in antigenicity of nonhistone protein-DNA complexes during perinatal development of rat liver were noted (18)as well as during estrogeninduced growth and differentiation of chick oviduct (14,15). Regeneration of rat liver apparently induces changes in antigenic properties of these complexes (19).Bush et al. (29) have described production of antibody showing specificity of nucleolar proteins. Considerable success has been had in analyzing antigenic properties of
134
F. CHYTIL 1
a
O0
r
W
x
LL t-
.1
.5 1 pg ANTIGEN ( D N A )
5
10
FIG. 2. Complement fixation by varying quantities of chick oviduct chromatin (0) and nonhistone protein-DNA complex ( 0 )in the presence of antiserum against chick oviduct nonhistone protein-DNA complex. From Chytil and Spelsberg (I).
nonhistone proteins during malignant growth. Wakabayshi and Hnilica (2) have found that anti-Novikoff-hepatoma nonhistone protein-DNA antiserum also reacted with chromatins prepared from Walker carcinoma or AS-30D hepatomas. Furthermore, it was reported (20) that chromatins from fast-growing and poorly differentiated Morris hepatoma 7777 and 3924-A
p g DNA
FIG. 3. Complement fixation of chromatin preparations from Novikoff hepatoma ( 0or ) and normal rat liver ( 0 or 0 ) in the presence of antiserum against Novikoff hepatoma nonhistone protein-DNA complex. The antiserum was absorbed with normal rat liver chromatin (+or 0 ). Assays ( O o r 0 )were performed in the presence of antiserum against nonhistone protein-DNA complex from Novikoff hepatoma absorbed with Novkioff hepatoma chromatin. From Chiu et al. (IZ),with permission.
8.
IMMUNOCHEMICAL ANALYSIS OF NONHISTONE PROTEINS
135
are more immunoreactive than better differentiated and slow-growing hepatomas 7800 and 7787 when anti-Novikoff-nonhistone protein-DNA antibody was employed. Normal liver chromatin differs antigenically from tumor chromatins; for instance, from transmissible sarcoma and Ehrlich ascitic tumor (20).Immunochemically specific sites inherent to nonhistone proteins can be induced by administration of a carcinogen-N-N-diamethylp-(m-tolylazo) aniline (20). The character of the antigenic sites belonging to nonhistone protein is still obscure. McClure and Mayer (31)have found a nonuniform distribution of the antigenic sites within chromatin isolated from oviduct of estrogentreated chicks. A tissue-specificimmunochemically reactive fraction of nonhistone proteins which has very high affinity to the homologous DNA (rat) showing two major and one minor band (12,OOO-15,OOO MW) has been described (11). Low molecular fractions of nonhistone proteins with a tissuespecific antigenic characteristics binding very tightly to the homologous DNA has been recently found in rat liver and Novikoff hepatoma (30). Experiments using reconstituted chromatins have shown that tissue-specific antigenic sites belonging to nonhistone proteins can be transferred from one DNA to another. Using antisera specific for the nonhistone-DNA complexes from a differentiated oviduct, it was found that the conformation of nonhistone proteins after transfer from chick oviduct to DNA of other organs is similar to that found in native chromatin (14).Wakabayashi and Hnilica (2) have used complement fixation to show that transfer of antigenic nonhistone proteins from Novikoff hepatoma chromatin requires presence of homologous (rat) DNA. Reconstitution of nonhistone proteins to other anions such as sea urchin DNA, yeast RNA, dextran sulfate, polyethylene sulfonate, or polyglutamic acid did not produce complexes reactive immunospecifically with anti-Novikoff-hepatoma nonhistone protein-DNA antibody; this suggests that the antinonhistone antibody can see only the conformation of nonhistone proteins influenced by the presence of the homologous DNA (20).These results were in contrast to Zardi et al. ( 3 4 ) who found that free nonhistone protein can react with antichromatin antibody. No presence of DNA was therefore necessary to the antigen-antibody reaction. Recent results obtained by Zardi ( 9 ) suggest that immunization with chromatin might induce a spectrum of nonhistone protein antibodies, some of which have higher affinity to the nonhistone protein-DNA complex than to free nonhistones. It is quite possible that individual differences among animals of the same species might produce various types of antibody. The complement fixation method has been also used in assessing conformational changes which occur in chromatin after removing histone with acids (16).
136
F. CHYTIL
VI. Immunohistochemical Localization of Nonhistone Proteins A. Horse Radish Peroxidase Method Principle: Tissue slices are incubated with a nonhistone protein antiserum produced, for instance, in rabbit. Then an immunoglobulin fraction (IgG) isolated from sheep (goat) serum immunized with rabbit serum IgG fraction conjugated with horse radish peroxidase (HRP) is added. The attachment of antinonhistone antibody to the cell components is localized by light microscopy after adding to the slide diaminobenzidine and hydrogen peroxide. Horse radish peroxidase attached to the second antibody catalyzes oxidation of diaminobenzidine by hydrogen peroxide. The reaction product exhibits a gold-brown color. 1. PREPARATION OF PEROXIDASELABELED ANTIRABBIT IgG
The methods described here were introduced by Nakane and Pierce (32) and reviewed recently by Nakane (33). The method of labeling antirabbit IgG by HRP was worked out by Nakane and Kawaoi (34). Principle: The antirabbit IgG fraction is conjugated with HRP in two steps. First the carbohydrate portion of HRPisoxidized to aldehyde. Sodium periodate is employed to react with HRPpreviously treated with fluorodinitrobenzene to block amino groups. The resulting HRP-aldehyde is then coupled with IgG. a. Materials 1. Horse radish peroxidase (Sigma Type VI). 2. Freshly prepared sodium bicarbonate solution (0.3 M). 3. Fluorodinitrobenzene (1 % in absolute alcohol). 4. Sodium periodate (0.06 M in water). 5 . Ethylene glycol solution (0.14 M-dilute 0.9 ml of ethylene glycol, spe gr 1.113, to 100 ml with water). 6. 0.01 M sodium bicarbonate (PH 9.5). 7. 0.01 M phosphate-buffered saline (PBS): To 42 gm of NaCl add 6.89 gm of NaH,PO; H,O; dissolve in water, adjust pH to 7.1-7.2, and dilute to 5 liters. b. Procedure Preparation of horse radish peroxidase-aldehyde: 1. Ten milligrams of HRP are dissolved in a 30-ml centrifuge tube in2 ml of freshly prepared sodium bicarbonate solution (0.3 M).A brownish solution is formed. 2. Then 0.2 ml of the fluorodinitrobenzene solution is added. If a precipitate is formed, it should be removed by centrifugation at 10,000 g for 5 minutes.
8.
IMMUNOCHEMICAL ANALYSIS OF NONHISTONE PROTEINS
137
3. The clear solution is shaken for 1 hour at room temperature and 2 ml of sodium periodate solution are then added. 4. The solution is again shaken for 1 hour at room temperature. 5. Then 2 ml of the ethylene glycol solution are added and shaking is continued for another 1 hour. 6. The solution is dialyzed against four changes of 100-200 volumes of the sodium bicarbonate buffer (0.01 M), pH 9.5. Conjugation of IgG with HRP-aldehyde: 1. To 5 mg of the IgG fraction 3 ml of the above HRP-aldehyde are added. 2. The mixture is then shaken for 2-3 hours and dialyzed against four changes of 100-200 volumes of 0.01 M phosphate-buffered saline (PBS). 3. The conjugate is then stored in aliquots at -2OOC. 2.
LOCALIZATION PROCEDURE
a. Materials 1. 0.01 M phosphate-buffered saline (PBS), pH 7.2. 2. 0.05 M Tris-HC1 buffer (PH 7.6). 3. Diaminobenzidine staining solution [90 mg of diaminobenzidine hydrochloride are dissolved in 300 ml of 0.05 M Tris buffer (PH 7.6) and0.05 ml of 30% hydrogen peroxide is added]. The solution is used immediately. 4. Other reagents: Ames O.C.T. Compound (Ames Company, Elkhart, Indiana), acetone, denatured ethyl alcohol, ethyl alcohol (50, 70, 95, and loo%), xylene, permount, Dry Ice, aluminium foil. b. Procedure Tissue preparation: 1. A piece of tissue is placed in a small cylinder prepared from the aluminum foil. 2. The tissue is covered by slowly pouring the O.C.T. compound into the cylinder. 3. The sample is then frozen by immersing the cylinder in a mixture of denatured alcohol and Dry Ice. 4. Sections 6 pm thick are prepared with acryostat,placedonmicroscopic slides, and left for 30 minutes at room temperature to dry. Staining procedure: The following steps are performed on ice. 1. The slides with the tissue sections are immersed in ice-cold acetone for 10 minutes and removed. 2. Then they are washed 3 times in fresh PBS buffer, each time for 3 minutes. 3. The liquid around the tissue sections is wiped off with a piece of cotton.
138
F. CHYTIL
4. The slides are .then transferred to a wet chamber which is held moist by putting on the bottom a thick filter paper soaked with PBS. 5. Then a drop of sheep serum is applied on each section and left for 20 minutes. This treatment lowers the nonspecific staining. 6. The slides are again washed 3 times in PBS (as under 2). 7. A drop of the nonhistone antibody is then applied (usually diluted 1:lO or 1:20 with PBS) for 15-20 minutes. 8. The slides are again washed 3 times, as under 2. 9. Now a drop of HRP-conjugated IgG is placed on each slide for 1530 minutes. 10. Washing is repeated as under 2. The next steps are performed at room temperature. 11. The slides are placed in freshly prepared diaminobenzidine solution for 10 seconds-10 minutes. 12. The slides are washed 3 times in fresh PBS for 5 minutes and in deionized water for 5 minutes once. 13. Then the slides are placed for 5 minutes each time sequentially into 50,70,95% and absolute alcohol. 14. The slides are immersed in xylene for clearing and mounted with permount.
3. COMMENTS The analysis of the attachment of antinonhistone antibody to the cell components by the above method requires careful experimental assessment of the incubation times employed. This depends on the antibody, sometimes on the batch of diaminobenzine, or on the actual concentration of hydrogen peroxide. The absence of naturally occurring antimembrane antibodies in some nonimmunized animals (8) always should be determined prior to the immunization of the animal. The optimum dilution of nonhistone protein antibody is a prerequisite for successful staining. As can be seen from Fig. 4, this method can show good interaction of the antisera with the nucleus with no signs of appreciable cytoplasmic staining. This method is also suitable for electron microscopy of the interaction of the antinonhistone protein antibody with nuclear components. The staining of the nucleus will be refined by using antibody against a single nonhistone protein species. The preparation of the HRP-IgG conjugate does not require special skills. Though Nakane and Kawaoi (34) have recommended the use of conjugate separated by gel filtration from the free HRP, we have found that this separation is not necessary to obtain good staining.
8.
IMMUNOCHEMICAL ANALYSIS OF NONHISTONE PROTEINS
I39
FIG.4. Histochemical localization of anti-rat liver nonhistone protein-DNA antisera by the horse radish peroxidase technique. (A) Liver slice incubated with serum before immunization (B) Liver slice incubated with antiserum against liver nonhistone protein-DNA complex. From Chytil (5).
B. Other Methods of Localization Coleman and Pratt (35)have compared the use of the method of Steinberger et al. (36) which employs antiperoxidase-peroxidase complex to localize nonhistone protein antibody. A fluorescein-labeled antibody technique has been used by Okita and Zardi (37), Scott and Sommerville (22), Silver and Elgin (21), and Elgin et al. (38). This method is also described by Silver et al., this volume, Chapter 10.
C . Applications Unlike the quantitative complement fixation technique, the localization methods have not yet gained popularity. The horse radish peroxidase method has been used to assess possible cytoplasmic contaminations of nonhistone protein preparations. Okita and Zardi (37) have used the fluoresceinlabeled antibody method to localize antibodies against chromatin from 3T6 mouse fibroblasts and WI-38 human diploid fibroblasts. Species specificity of these antibodies was demonstrated. The nucleus was diffusely stained, and a diffuse fluorescence in the cytoplasm was observed which the authors interpreted as a suggestion of the presence of a cytoplasmic pool of chromosomal proteins. Because localization of antibody raised against nonhistone protein-DNA complex failed to show any cytoplasmic localization ( 5 ) , the
140
F. CHYTIL
above results also could be interpreted to mean that the chromatin preparations, unlike the nonhistone protein-DNA complexes, did contain a cytoplasmic contamination. The indirect immunofluorescence method has been used very elegantly for in situ localization of Drosophila nonhistone proteins (21.38). Localization of nuclear proteins on chromosomes of newt oocytes has been demonstrated (22). The fluorescence technique facilitated the localization of rabbit antibodies in nucleoli of normal rat liver and Novikoff hepatoma (29).
ACKNOWLEDGMENTS This work was supported by U.S. Public Health Service grants HD-05384, HD-09105, and HL-15341. I wish to thank L. Chytil for collaboration and discussions.
REFERENCES 1. Chytil, F., and Spelsberg, T. C., Nature (London) New Biol. 233, 215 (1971). 2. Wakabayashi, K., and Hnilica, L. S., Nature (London) New Biol. 242, 153 (1973). 3. Zardi, L., Lin, J. C., and Baserga, R., Nature (London) New Biol. 245.21 1 (1973). 4. Zardi, L., Lin, J. E., Peterson, R. O., and Baserga, R., Cold Spring Conf: CellProlifea-
tion, p. 729, 1974. 5. Chytil, F. in “Methods in Enzymology,” Vol. 40: Hormone Action, Part E, Nuclear Structure and Function (B. W. O’Malley and J. G. Hardman, eds.), p. 191. Academic Press, New York, 1975. 6. Stumpf, W. E., Elgin, S. C. R., and Hood, L., J. Immunol. 113, 1752 (1974). 7. Miescher, P., Cooper, N. S., and Benacerraf, B., J. Immunol. S , 2 7 (1960). 8. Wilson, E. M., and Chytil, F., Biochim. Biophys. Acta426, 88 (1976). 9. Zardi, L., Eur. J. Biochem. 55, 231 (1975). 10. Wakabayashi, K., Wang, S., Hord, G., and Hnilica, L. S., FEBS Lett. 32,46 (1973). 11. Wakabayashi, K., Wang, S., and Hnilica, L. S., Biochemistry 13, 1027 (1974). 12. Chiu, J. F., Hunt, M., and Hnilica, L. S., Gamer Ref. 35, 913 (1975). 13. Ashmarin, I. P., Gavriljuk, I. P.,Konarev, V. G., Resnik, S. E., Sidorova, V. V., and Fedorova, N. A., Cytologia 16, 1488 (1974). 14. Spelsberg, T. C., Steggles, A. W., Chytil, F., and O’Malley, B. W., J. Biol. G e m . 247, 1368 (1972). 15. Spelsberg, T. C., Mitchell, W. M., Chytil, F., Wilson, E. M., and OMalley, B. W., Biochem. Biophys. Acta 312,765 (1973). 16. Spelsberg, T. C., Mitchell, W. M., and Chytil, F., Mol. Cell. Biochem. 1, 243 (1973). 17. Nicolini, C., Physiol. Chem. Phys. 7. 571 (1975). 18. Chytil, F., Glasser, S. R., and Spelsberg, T. C., Dev.Biol. 37,295 (1973). 19. Chiu, J. F., Wakabayashi, K., Craddock, C., Morris, J. P., and Hnilica, L. S., in “Cell Cycle Controls” (G. M. Padilla, I. L. Cameron, and A. Zimmerman, 4s.). Academic Press, New York, 1974. 20. Chiu, J. F., Craddock, C., Morris, H. P., and Hnilica, L. S., FEBS Lert. 42, 94 (1974). 21. Silver, L. M., and Elgin, S. C. R., Proc. Natl. Acad. Sci. U.S.A.73, 423 (1976). 22. Scott, S. E. M., and Sommerville, J., Nature (London) 250, 680 (1974). 23. Fudenberg, H. H., in “Methods in Immunology and Immunochemistry” (C. A. Williams, and M. W. Chase, eds.), Vol. 1, p. 307. Academic Press, New York, 1967. 24. Fahey, J. L., in “Methods in Immunology and Immunochemistry” (C. A. Williams, and M. W. Chase, eds.), Vol. 1, p. 321. Academic Press, New York, 1967.
8.
IMMUNOCHEMICAL ANALYSIS OF NONHISTONE PROTEINS
141
25. Wasserman, E., and Levine, L., J. Immunol. 87,290 (1961). 26. Levine, L., in “Handbook of Experimental Immunology” (D. M. Klir, ed.), p. 701. Davis, Philadelphia, Pennsylvania, 1967. 27. Cikes, M., J. Immunol. Met& 8, 89 (1975). 28. Roberts, D. B., and Andrews, P. W., Nucleic Acids Res. 2, 1291 (1975).
29. Bush, R. K., Daskal, I., Spohn, W. H., Kellermayer, M., and Bush, H., Cancer Res. 34, 2362 (1974).
30. 31. 32. 33.
Chiu, J. F., Wang, S.,Fujitani, H., Hnilica, L. S., Biochemistry 14, 4552 (1975). McClure, M. E., and Mayer, J. L., J. Cell. Eiol. 63, 215 (1974). Nakane, P. K . , and Pierce, G.B. Jr., J. Cell. Eiol. 33, 307 (1%7). Nakane, P. K., in “Methods in Enzymology,” Vol. 37: Hormone Action, Part B, Peptide Hormones (B. W. O’Malley and J. G. Hardman, eds.), p. 133. Academic Press, New
York, 1975. 34. Nakane, P. K., and Kawaoi, A., J. Hixrochem. Cytochem. 22, 1084 (1974). 35. Coleman. R. A., and Pratt, L. H., J. Hisrochem. Cytochem. 22, 1039 (1974). 36. Steinberger, L. A,, Hardy, P. H., Jr., Cucullis, J. J., and Meyer, H. G., J. Hisrochem. Cytochem. 28, 315 (1970). 37. Okita, K., and Zardi, L., Exp. Cell Res. 86, 59 (1974). 38. Elgin, S . C . R., Silver, L. M., and Wu, C. E. C., in “The Molecular and Biological Mammalian Genetic Apparatus” (P. 0. P. Ts’o, ed.), Vol. 1, p. 127. North-Holland, Amsterdam, 1977.
I. Introduction This chapter introduces the methodology of immunochemistry of chromosomal nonhistone proteins. Attempts also will be made to review some applications of the immunochemical analysis in chracterization not only of the nonhistone per se, but also in characterization of chromatin. The material discussed here concerns mostly antibody raised against heterogeneous mixtures of these proteins. The immunochemical analysis will certainly become more meaningful when an antibody against a single homogenous nonhistone protein preparation is obtained.
11. Immunogenicity of Nonhistone Proteins In contrast to histones the heterogeneous mixtures of nonhistone proteins are very good immunogens (Z-3). To produce antibody, nonhistone proteins can be injected into the experimental animal complexed with DNA (1,2). Administration of whole chromatin also has been shown to give antibody against nonhistone proteins as well as against histones (3-5). Finally, free (5) as well as sodium dodecyl sulfate (SDS)-treated nonhistone protein preparations (6)are good immunogens. No difficulties have been reported in producing antibody against nonhistone proteins from various eukaryotes. 123
124
F. CHYTIL
111. Choice of Animal for Production of Antihistone Antibody Rabbits as well as chickens are suitable for inducing antibodies. Guinea pigs were not reported to produce antibody against nonhistone proteins, but these animals were used for production of antinuclear antibody without major complications (7). It is therefore no a priori reason to eliminate this animal as a potential antinonhistone antibody producer. Both rabbits and chickens produce and then carry these antibodies without apparent pathological signs. In the past 5 years we have not experienced deaths of immunized animals which could be attributed directly to the immunization by nonhistone proteins.
A.
Rabbits
Rabbits seem to be the choice of most workers because the handling and housing of these animals does not need special skills. Moreover the quantity of blood which can be obtained is usually sufficient for many experiments. The nonhistone protein antibodies have been produced in most instances against heterogeneous mixtures of these proteins. Because the antibody response to such a heterogeneous immunogen may vary from one animal to another, it is not advisable to pool the antisera after immunization of several animals. Also, sera from different bleedings of the same animal should not be pooled since the antibody specificity might undergo variations. But the serum should be tested before immunization for the presence of natural antibody against nuclear material because recently it was found that some nonimmunized rabbit sera contain complement-fixing antibodies directed against nuclear components (8). The most common rabbit strain, New Zealand White, is used. The rabbits usually weigh at least 3 kg; male sex is preferable as it eliminates complications with accidental pregnancies occurring in the animal facilities.
B. Chickens Chickens have been shown to be good producers of nonhistone antibodies when injected with whole chromatin (3,4).This animal might be preferred when precipitation antibody is to be produced. Chicken antibody optimally forms precipitin complexes in the presence of 1.5 M NaC1. This concentration of sodium chloride facilitates the solubility of chromosomal proteins (9).
8.
IMMUNOCHEMICAL ANALYSIS OF NONHISTONEPROTEINS
125
IV. Immunization Procedure A. Preparation of the Immunogen Nonhistone proteins are prepared as immunogens similar to the other proteins. Usually these proteins-regardless of whether injected free, SDStreated, or as the whole chromatin or in the form of complexes with DNAare emulsified with complete Freund adjuvant. An equal volume of Freund adjuvant is stirred with the nonhistone protein preparation in a small beaker using a magnetic stirrer. The resulting white emulsion is administered to the animal. The mixing of the immunogen with the adjuvant also can be accomplished by vigorously shaking the mixture in a syringe.
B. Administration Dose Microgram quantities of the nonhistone protein mixtures (see Table I), are sufficient to elicit antibody response. Some workers have been using milligram quantities. The use of smaller quantities has been adopted in this laboratory for two main reasons. First, the quantity of the nonhistone protein mixture available often represents a limiting factor; second, by using larger quantities of the immunogen there is a higher probability of producing antibody against some undesired impurities.
C. Route of Administration Various laboratories administer the immunogens differently, as is evident from Table I. A subcutaneous, intramuscular, or intradermal injection is usually the starting procedure. Sometimesthe immunization is completed by intravenous injection of the immunogen. In our laboratory we use the following procedure. Fifty to 100 pg of total nonhistone protein mixed with complete Freund adjuvant (1:1 by volume) are injected under the skin of the toe pads of one of the hind legs of the rabbit. Usually 0.25 to 0.5 ml ofthe mixture is injected into the pads, and the rest is administered in small doses intramuscularly over the whole rabbit body. After a week, a second injection is given in the toe pads of the other foot. Then, in weekly intervals (usually two more doses are required), the immunogen is administered intramuscularly in small doses over the whole body. When antibody is detected in the serum taken from these animals an intravenous injection (booster) of the immunogen (usually one-fourth of the original quantity) diluted with saline is given intravenously (major marginal vein of the ear) without adjuvant. Seven days later the animal is bled from the marginal ear vein. Thirty milliliters of the blood can be obtained from a good “bleeder” rabbit in one bleeding.
TABLE I PRODUCTION OF ANTINONHISTONE PROTETEIN W P ) ANITBODY Problem studied Tissue specificity Eu and hetero chromatin
Structure of chromatin Liver development Estrogen-mediated differentiation Malignant growth
Tissue culture Drosophila
Newt oocytes
Immunogen
Animal
Mode of injection"
Dose per injection (mg)
Intervals (days)
Number of injections
NHP-DN A complex NHP-DNA complex Chromatin
Rabbit
SC and IM
0.1
I
3 4
Rabbit
NI
I
3
Chicken Rabbit
ID and SC
Approximately 2 mg
NHP-DNA complex Chromatin NHP-DNA complex NHP-DNA complex NHP-DNA complex Nucleoli
Rabbit
SC and I M
Approximately 8 mg 0.1
Chicken Rabbit
ID and SC SC and IM
0.1
7
34
Rabbit
SC and IM
0.1
I
34
Rabbit
SC and IM
0.1
I
3 4
Rabbit
ID and IM
I
3
Rabbit Chicken Rabbit Rabbit Rabbit
ID and SC ID and SC SC and IP
10,20, 50 total amount
I
5 5
Chromatin NHP and SDS NHP and SDS NHP
NI
sc sc
0.4
0.4
0.4 0.4 9-10 2
NI
3 4
Reference
5
I
3
7
3-6 5
3
3
NI
NI
NI
NI
28
"IV = Intravenously; IP = intrapentoneally; IM = intramuscularly; SC = subcutaneously; ID
Booster dose
=
2
intradermally; NI = not indicated.
8.
IMMUNOCHEMICAL ANALYSIS OF NONHISTONE PROTEINS
127
D. Treatment of the Antisera The blood collected into a centrifuge tube is left to coagulate for about
1 hour at room temperature and then stored in a refrigerator overnight. After centrifugation of the blood at about 2000 g for 10 minutes the serum
is siphoned off and kept frozen at -20°C. To avoid infection, the serum can be sterilized by using Millipore filtration. It is our experience that hemolyzed sera also can be used. Usually the whole serum can be used. When necessary, the immunoglobulin fraction G (IgG) can be purified from the sera by ammonium sulfate precipitation (23) or by 0-(diethylaminoethyl) cellulose (DEAE-cellulose) column chromatography (24).
V. Testing Antigenicity One of the most popular methods for testing for the presence and characteristics of the nonhistone protein antibodies is quantitative microcomplement fixation as described originally by Wasserman and Levine (25). The method used in my laboratory is described below.
A. Quantitative Microcomplement Fixation Principle: The microcomplement fixation method is an adaptation of complement (C') fixation which is based on the capacity of the complement system in fresh guinea pig serum to combine irreversibly with antigen-
TABLE I1 QUANTITATIVE COMPLEMENT-FIXATION ASSAW Stage 1: Fixation of C' by AbAg complex Ab Ag Ab
+ C'
+ C'
+ Ag +
A b + C' A g C' [Ab - A g C'
+
- C']
Stage 2: Lysis of EA by C'
+ EA + EA + EA
Lysis Lysis No lysis
Stage 3: Quantitative measurement of lysis by determining hemoglobin concentration (optical density 413 nm). Results are expressed in percentage of C' fixed. complement (guinea pig); Ab, antibody;Ag = correspondingantigen;E = erythrocytes (sheep); A = antibodies (rabbit) to E (sheep); EA = sensitized erythrocytes (E complexed with A ) .
128
F. CHYTIL
antibody complexes. If the antigen is associated with the sheep erythrocyte cell surface, such a combination may result in lysis of the erythrocyte. The lysis of hemolysin-sensitized sheep erythrocyte determined quantitatively by measuring hemoglobin spectrometrically is an excellent indicator system for C' activity (the principle of this method is outlined in Table 11). If C' is allowed to incubate with the antigen-antibody system under study, then its combination can be estimated by the residual hemolytic activity it possesses when a known quantity of antibody-coated (hemolysin-sensitized) erythrocyte is added at a later time. The method is not only very sensitive for detecting the presence of antibody, but also by using the same antibody against two or more antigens, one can determine the extent of similarity or conformational differences of the protein molecules by comparing the amount of each protein which elicits maximum fixation (26). 1. MATERIALS
a. Diluent. Isotris stock buffer: To 81.6 gm of NaCl add 12.1 gm ofTris (hydroxymethyl)aminoethane base and dissolve in about 800ml ofdeionized H,O. Add 6.6 ml of concentrated HCl, mix, and add 33 ml of 0.15 M MgSO,. 7 H,O (3.7 gm/100ml)and 15 ml ofO.l MCaCI,(1.1 gm/lWml). Dilute to 1 liter after adjusting the pH to 7.4. b. Isotris diluent- workingsolutions. To 100ml of the above stock buffer add 1 gm of bovine serum albumin and mix with a magnetic stirrer until dissolved. Make up the volume to lo00 ml with water. c. 0.1% Na,CO,. d. Sheep cells. These can be purchased commercially (50% whole blood, 50% Alsevers solution). They are kept refrigerated and can be stored up to 1 month. e. Anti-sheep hemolysin. This is purchased commercially, frozen at -2O"C, and can be stored over 4 years. The commercial hemolysin is in 50% glycerol solution. Two milliliters of this preparation are diluted to 50 ml with isotris diluent and stored frozen in 0.5 ml aliquots. The 1/1OOO dilution is prepared fresh daily: 0.2 ml of 1/50 stock dilution plus 3.8 ml of isotris diluent. 1: Guinea pig complement. Complement (C')should be kept cold all the time (not exceeding 4 "C). The commercial product (lyophilized guinea pig serum) is reconstituted with 5 ml of the supplied diluent. The serum is kept frozen in 0.25-ml aliquots. Once an aliquot is unfrozen for use, any complement remaining is discarded. Complement will retain its original titer approximately 14 months if stored at -2OOC (-40°C is preferable). When commercial frozen preparation of complement is used, the sample is first thawed and kept frozen in aliquots of 0.25 ml or more,
8.
IMMUNOCHEMICAL ANALYSIS OF NONHISTONE PROTEINS
129
2. PROCEDURES a. Complement titer determination. With any new batch of complement it is necessary to determine the titer as follows: (1) dilute the frozen complement aliquots MOO (0.1 ml is diluted to 10 ml with isotris diluent); (2) prepare different dilutions of C’according to Table 111. Remember that all dilutions must be made in an ice bath. Use disposable glass culture tubes (13 x 100 mm). Pipette different dilutions of C‘ according to Table IV. Again, all operations have to be performed in an ice bath. After mixing the above components, store the tubes in a cold room on ice overnight. Next day add 0.2 ml of sensitized sheep cells to each tube (see Section V,A,
2,b).
After the addition of sensitized cells the tubes are placed in a constanttemperature water bath and incubated 40-60minutes at 37°C.Thelast tube is the sheep cell blank (lysis in the absence of C). The tubes are then transferred to an ice bath for 10 minutes to stop hemolysis and centrifuged 10 minutes at 2000 rpm in a clinical centrifuge. The optical density (OD) of the supernatants is measured at 413 nm against isotris. The highest dilution of complement that gives 90% lysis is used for the fixation procedure (see Fig. I). We find that the titer of the complement fluctuates between 1/125 and 1/225,depending on the batch and the supplier. b. Standardization of sheep cells (prepare daily). 1. Three milliliters of cell suspension are centrifuged at 1500 rpm for 5-10 minutes in a clinical centrifuge (refrigerated).
TABLE 111 DILUTION OF COMPLEMENT C’ C‘ dilution C’(11100)(ml)
Isotris (ml)
1/125 1 0.25
11100 1
-
11150 1
0.50
11175 1 0.75
11200 1 1
11225 1 1.25
1/250 1 1.50
11275 11300 1 1 1.75 2
TABLE IV COMPLEMENT TITER 11100 1/125
C’dilution ml Isotris (ml)
0.2 1
11150 11175 11200 11225 11250 11275 11300
0.2
0.2
1
I
0.2 1
0.2 1
0.2 1
0.2
0.2
I
1
0.2 1
Cell blank 0.0 1.2
130
F. CHYTIL 0.6-
0.5m T
0.40,30.20.1 -
&&&7$3.&Tki& DILUTION OF COMPLEMENT
FIG. I . Determination of the complement titer. A,,,
=
optical density at 413 nm.
2. The supernatant is aspirated off, and the cells are resuspended in
4-5 ml of isotris diluent. Repeat twice more or until the supernatant is
clear. 3. Next 0.5 ml of washed, packed cells is suspended in 9.5 ml of isotris diluent . 4. Then 1 ml of this 5% cell suspension is added to 14 ml of 0.1% Na,CO, ; 5 minutes are allowed for complete hemolysis. 5. At 541 nm the desired optical density is 0.680. The range of acceptability is 0.6654.695. 6. The concentration of sheep cells is adjusted to the proper value by isotris diluent. The formula for adjusting is OD
0.680
x y, = y,.
Y , = initial volume of the preparation after removing 1 ml for estimating optical density; V , = adjusted final volume. c. Sensitization of sheep cells (preparefresh daily). 1. In a 50-mlErlenmeyer flask, to 1 ml of standardized sheep cells add with a swirling motion 1 ml of hemolysin (1/1OOO) [0.2 ml of the stock solution (1/50) of hemolysin is diluted with 3.8 ml of isotris diluent]. 2. Incubate for 15 minutes at 37°C to allow maximum sensitization. Then 18 ml of isotris diluent is added. 3. The sensitized cells may be kept 4 day if they are refrigerated. d. C' fixation procedure. The nonhistone protein preparation of known protein content to be tested for antigenicity should be kept frozen in small aliquots. Before use in complement fixation the antibody serum is incubated at
8.
131
IMMUNOCHEMICAL ANALYSIS OF NONHISTONE PROTEINS
60°C for 20 minutes to inactivate the endogenous
C'. The dilution of antibody is to be determined by complete complement fixation run on each dilution (1/200, 1/400, 1/800, 1/1600, etc.). The antibody is stored in the freezer at not less than 1/200 dilution. 1. To a series of disposable glass culture tubes (13 x 100 mm) add in the following order: 0.6 ml of diluent; 0.2 ml of diluted serum; 0.2 ml of antigen solution serially diluted as shown in Table V; 0.2 ml of diluted C' (see C' titer determination). Table V shows an example of this determination. 2. Appropriate dilutions of antigen and C', antibody and C', diluent and C',and diluent in a total volume of 1.2 ml are included in every experiment (Table V, tubes 7-17). 3. After incubation at 2"-4"C for 16-18 hours, 0.2 ml of sensitized cells is added and hemolysis allowed to proceed, with occasional swirling
TABLE V COMPLEMENT FIXATION Tube no.
Isotris (ml) 0.6 0.6 0.6 0.6 0.6 0.6 0.6
1 2 2 3 4 5 6 7 8 9
0.8 0.8 0.8 0.8 0.8 0.8
I0 11 12 13 14a 15 16 17
0.8 I .o 0.8 1.2 1.2 Na,CO, ~~~
Antisera (ml) 0.2 0.2 0.2
Antigen (ml) and content of nonhistone protein 0.2 (10 pg) 0.2 ( 5 pg) 0.2 ( 5 pg) 0.2 (2.5 pg) 0.2 (1.25 pg) 0.2 (0.62 pg) 0.2 (0.31 pg) 0.2 (10 PP) 0.2 (5 pg) 0.2 (2.5 pg) 0.2 (1.25 pg) 0.2 (0.62 pg) 0.2 (0.31 pg)
0.2 0.2
0.2 0.2
-
0.2 -
-
~~
aOD obtained in tube 14 is used as 100% of lysis.
C' (ml)
Cells (ml)
0.2
0.2 0.2
0.2 0.2 0.2 0.2
0.2 0.2 0.2 0.2
0.2 0.2 0.2 0.2
0.2 0.2 0.4
-
0.2 0.2 0.2 0.2 0.2 0.2 0.2
0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2
132
F. CHYTIL
in a water bath at 3So-37OC until controls are estimated to be 80-90% hemolysed. This requires about 40-60 minutes. 4. After immersion in an ice bath to stop hemolysis reaction (10 minutes), the tubes are centrifuged 10 minutes at 2000 rpm in a clinical centrifuge. 5. The optical density of the supernatant is determined at 413 nm. e. Calculation. OD sample x 100 = percent lysis OD 100% lysis The OD obtained in tube 14 is used as 100% of lysis. Percent C’ fixed 100 - percent lysis.
=
3. COMMENTS The quantitative complement fixation assay has been the most widely used method for determination of antigenicity of nonhistone proteins. It is very reproducible, sensitive, and allows the detection of conformational changes of antigens (26). Some problems may be encountered, however. As shown in Table 11, one of the prerequisites for quantitative evaluation of the interaction of the antigens with antibody is that the antisera, at the dilution used, should not bind complement in the absence of the antigen. However, some antisera exert this type of binding. This becomes a complicating factor mainly when low dilutions of antisera have to be employed. To minimize this problem it is advisable to screen the sera before immunization of the animals by determining the amount of complement fixed by various dilutions of the serum starting at a 1/200 dilution followed by 1/400, 1/800, etc. Usually those animals whose sera shows binding of complement (anticomplementarity) at 1/400 dilution are not used for immunization. Occasionally, this anticomplementarity develops during the immunization of the animal. There are several ways to remedy this complication. Purification of the IgG fraction might help. Sometimes centrifugation of the serum diluted with 9 volumes of 0.14 M sodium chloride at 105,000 g at 4OCfor 1 hour removes the anticomplementarity. Finally, the amount of C’used in the assay can be raised by an increment which eliminates the binding by antiserum (27). A more severe problem is the anticomplementarity of the antigens, which might be concentration dependent. Whole chromatin preparations usually do not show anticomplementarity. But nonhistone proteins as well as nonhistone protein-DNA complexes may show limited binding C’.Then the amount of C‘ fixed in the fixation assay in the absence of antiserum (see Table V) can be subtracted from that observed in the presence of antigen-
8.
IMMUNOCHEMICAL ANALYSIS OF NONHISTONE PROTEINS
133
antibody complexes. We have noted that repeated thawing and freezing of chromatin preparations sometimes induces anticomplementarity. Storing antigens in small aliquots is therefore advisable. Chromatin preparations treated with acid to remove histones usually show high anti-complementarity. Removing histones with a high concentration of urea and salt seems to have little effect. A typical complement fixation curve when antinonhistone proteinDNA antisera are reacted with the preparation used for immunization and with the corresponding whole chromatin preparation is shown in Fig. 2. The fact that the nonhistone protein antisera also react with the whole chromatin lets one use such an antibody in the characterization of chromatin preparations. An example of this use is plotted in Fig. 3. The amount of antigen can be expressed in the amount of DNA or nonhistone protein in the assay.
B. Other Methods of Testing Antigenicity Recently a double diffusion technique was employed (13,28). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis also has been used (6).
C. Applications By the use of the quantitative complement fixation method tissue specificity of antigenic properties of nonhistone protein-DNA complexes from chick oviduct nuclei has been described (1). Wakabayashi and Hnilica (2) have shown that an antiserum against nonhistone protein-DNA complexes isolated from Novikoff hepatoma will distinguish between this tumor and similar complexes isolated from rat liver and calf thymus. Furthermore, Zardi et al. ( 4 ) found that whole chromatin, when used as an immunogen, can elicit complement-flxing antibodies which recognize differences in chromatin and nonhistone proteins not only between WI-38 human diploid fibroblast and 3T6 mouse fibroblasts grown in culture, but also between chromatin of WI-38 cells and their SV-40 transformed counterparts, the 2-RA cells (3). Changes in antigenicity of nonhistone protein-DNA complexes during perinatal development of rat liver were noted (18)as well as during estrogeninduced growth and differentiation of chick oviduct (14,15). Regeneration of rat liver apparently induces changes in antigenic properties of these complexes (19).Bush et al. (29) have described production of antibody showing specificity of nucleolar proteins. Considerable success has been had in analyzing antigenic properties of
134
F. CHYTIL 1
a
O0
r
W
x
LL t-
.1
.5 1 pg ANTIGEN ( D N A )
5
10
FIG. 2. Complement fixation by varying quantities of chick oviduct chromatin (0) and nonhistone protein-DNA complex ( 0 )in the presence of antiserum against chick oviduct nonhistone protein-DNA complex. From Chytil and Spelsberg (I).
nonhistone proteins during malignant growth. Wakabayshi and Hnilica (2) have found that anti-Novikoff-hepatoma nonhistone protein-DNA antiserum also reacted with chromatins prepared from Walker carcinoma or AS-30D hepatomas. Furthermore, it was reported (20) that chromatins from fast-growing and poorly differentiated Morris hepatoma 7777 and 3924-A
p g DNA
FIG. 3. Complement fixation of chromatin preparations from Novikoff hepatoma ( 0or ) and normal rat liver ( 0 or 0 ) in the presence of antiserum against Novikoff hepatoma nonhistone protein-DNA complex. The antiserum was absorbed with normal rat liver chromatin (+or 0 ). Assays ( O o r 0 )were performed in the presence of antiserum against nonhistone protein-DNA complex from Novikoff hepatoma absorbed with Novkioff hepatoma chromatin. From Chiu et al. (IZ),with permission.
8.
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are more immunoreactive than better differentiated and slow-growing hepatomas 7800 and 7787 when anti-Novikoff-nonhistone protein-DNA antibody was employed. Normal liver chromatin differs antigenically from tumor chromatins; for instance, from transmissible sarcoma and Ehrlich ascitic tumor (20).Immunochemically specific sites inherent to nonhistone proteins can be induced by administration of a carcinogen-N-N-diamethylp-(m-tolylazo) aniline (20). The character of the antigenic sites belonging to nonhistone protein is still obscure. McClure and Mayer (31)have found a nonuniform distribution of the antigenic sites within chromatin isolated from oviduct of estrogentreated chicks. A tissue-specificimmunochemically reactive fraction of nonhistone proteins which has very high affinity to the homologous DNA (rat) showing two major and one minor band (12,OOO-15,OOO MW) has been described (11). Low molecular fractions of nonhistone proteins with a tissuespecific antigenic characteristics binding very tightly to the homologous DNA has been recently found in rat liver and Novikoff hepatoma (30). Experiments using reconstituted chromatins have shown that tissue-specific antigenic sites belonging to nonhistone proteins can be transferred from one DNA to another. Using antisera specific for the nonhistone-DNA complexes from a differentiated oviduct, it was found that the conformation of nonhistone proteins after transfer from chick oviduct to DNA of other organs is similar to that found in native chromatin (14).Wakabayashi and Hnilica (2) have used complement fixation to show that transfer of antigenic nonhistone proteins from Novikoff hepatoma chromatin requires presence of homologous (rat) DNA. Reconstitution of nonhistone proteins to other anions such as sea urchin DNA, yeast RNA, dextran sulfate, polyethylene sulfonate, or polyglutamic acid did not produce complexes reactive immunospecifically with anti-Novikoff-hepatoma nonhistone protein-DNA antibody; this suggests that the antinonhistone antibody can see only the conformation of nonhistone proteins influenced by the presence of the homologous DNA (20).These results were in contrast to Zardi et al. ( 3 4 ) who found that free nonhistone protein can react with antichromatin antibody. No presence of DNA was therefore necessary to the antigen-antibody reaction. Recent results obtained by Zardi ( 9 ) suggest that immunization with chromatin might induce a spectrum of nonhistone protein antibodies, some of which have higher affinity to the nonhistone protein-DNA complex than to free nonhistones. It is quite possible that individual differences among animals of the same species might produce various types of antibody. The complement fixation method has been also used in assessing conformational changes which occur in chromatin after removing histone with acids (16).
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VI. Immunohistochemical Localization of Nonhistone Proteins A. Horse Radish Peroxidase Method Principle: Tissue slices are incubated with a nonhistone protein antiserum produced, for instance, in rabbit. Then an immunoglobulin fraction (IgG) isolated from sheep (goat) serum immunized with rabbit serum IgG fraction conjugated with horse radish peroxidase (HRP) is added. The attachment of antinonhistone antibody to the cell components is localized by light microscopy after adding to the slide diaminobenzidine and hydrogen peroxide. Horse radish peroxidase attached to the second antibody catalyzes oxidation of diaminobenzidine by hydrogen peroxide. The reaction product exhibits a gold-brown color. 1. PREPARATION OF PEROXIDASELABELED ANTIRABBIT IgG
The methods described here were introduced by Nakane and Pierce (32) and reviewed recently by Nakane (33). The method of labeling antirabbit IgG by HRP was worked out by Nakane and Kawaoi (34). Principle: The antirabbit IgG fraction is conjugated with HRP in two steps. First the carbohydrate portion of HRPisoxidized to aldehyde. Sodium periodate is employed to react with HRPpreviously treated with fluorodinitrobenzene to block amino groups. The resulting HRP-aldehyde is then coupled with IgG. a. Materials 1. Horse radish peroxidase (Sigma Type VI). 2. Freshly prepared sodium bicarbonate solution (0.3 M). 3. Fluorodinitrobenzene (1 % in absolute alcohol). 4. Sodium periodate (0.06 M in water). 5 . Ethylene glycol solution (0.14 M-dilute 0.9 ml of ethylene glycol, spe gr 1.113, to 100 ml with water). 6. 0.01 M sodium bicarbonate (PH 9.5). 7. 0.01 M phosphate-buffered saline (PBS): To 42 gm of NaCl add 6.89 gm of NaH,PO; H,O; dissolve in water, adjust pH to 7.1-7.2, and dilute to 5 liters. b. Procedure Preparation of horse radish peroxidase-aldehyde: 1. Ten milligrams of HRP are dissolved in a 30-ml centrifuge tube in2 ml of freshly prepared sodium bicarbonate solution (0.3 M).A brownish solution is formed. 2. Then 0.2 ml of the fluorodinitrobenzene solution is added. If a precipitate is formed, it should be removed by centrifugation at 10,000 g for 5 minutes.
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3. The clear solution is shaken for 1 hour at room temperature and 2 ml of sodium periodate solution are then added. 4. The solution is again shaken for 1 hour at room temperature. 5. Then 2 ml of the ethylene glycol solution are added and shaking is continued for another 1 hour. 6. The solution is dialyzed against four changes of 100-200 volumes of the sodium bicarbonate buffer (0.01 M), pH 9.5. Conjugation of IgG with HRP-aldehyde: 1. To 5 mg of the IgG fraction 3 ml of the above HRP-aldehyde are added. 2. The mixture is then shaken for 2-3 hours and dialyzed against four changes of 100-200 volumes of 0.01 M phosphate-buffered saline (PBS). 3. The conjugate is then stored in aliquots at -2OOC. 2.
LOCALIZATION PROCEDURE
a. Materials 1. 0.01 M phosphate-buffered saline (PBS), pH 7.2. 2. 0.05 M Tris-HC1 buffer (PH 7.6). 3. Diaminobenzidine staining solution [90 mg of diaminobenzidine hydrochloride are dissolved in 300 ml of 0.05 M Tris buffer (PH 7.6) and0.05 ml of 30% hydrogen peroxide is added]. The solution is used immediately. 4. Other reagents: Ames O.C.T. Compound (Ames Company, Elkhart, Indiana), acetone, denatured ethyl alcohol, ethyl alcohol (50, 70, 95, and loo%), xylene, permount, Dry Ice, aluminium foil. b. Procedure Tissue preparation: 1. A piece of tissue is placed in a small cylinder prepared from the aluminum foil. 2. The tissue is covered by slowly pouring the O.C.T. compound into the cylinder. 3. The sample is then frozen by immersing the cylinder in a mixture of denatured alcohol and Dry Ice. 4. Sections 6 pm thick are prepared with acryostat,placedonmicroscopic slides, and left for 30 minutes at room temperature to dry. Staining procedure: The following steps are performed on ice. 1. The slides with the tissue sections are immersed in ice-cold acetone for 10 minutes and removed. 2. Then they are washed 3 times in fresh PBS buffer, each time for 3 minutes. 3. The liquid around the tissue sections is wiped off with a piece of cotton.
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4. The slides are .then transferred to a wet chamber which is held moist by putting on the bottom a thick filter paper soaked with PBS. 5. Then a drop of sheep serum is applied on each section and left for 20 minutes. This treatment lowers the nonspecific staining. 6. The slides are again washed 3 times in PBS (as under 2). 7. A drop of the nonhistone antibody is then applied (usually diluted 1:lO or 1:20 with PBS) for 15-20 minutes. 8. The slides are again washed 3 times, as under 2. 9. Now a drop of HRP-conjugated IgG is placed on each slide for 1530 minutes. 10. Washing is repeated as under 2. The next steps are performed at room temperature. 11. The slides are placed in freshly prepared diaminobenzidine solution for 10 seconds-10 minutes. 12. The slides are washed 3 times in fresh PBS for 5 minutes and in deionized water for 5 minutes once. 13. Then the slides are placed for 5 minutes each time sequentially into 50,70,95% and absolute alcohol. 14. The slides are immersed in xylene for clearing and mounted with permount.
3. COMMENTS The analysis of the attachment of antinonhistone antibody to the cell components by the above method requires careful experimental assessment of the incubation times employed. This depends on the antibody, sometimes on the batch of diaminobenzine, or on the actual concentration of hydrogen peroxide. The absence of naturally occurring antimembrane antibodies in some nonimmunized animals (8) always should be determined prior to the immunization of the animal. The optimum dilution of nonhistone protein antibody is a prerequisite for successful staining. As can be seen from Fig. 4, this method can show good interaction of the antisera with the nucleus with no signs of appreciable cytoplasmic staining. This method is also suitable for electron microscopy of the interaction of the antinonhistone protein antibody with nuclear components. The staining of the nucleus will be refined by using antibody against a single nonhistone protein species. The preparation of the HRP-IgG conjugate does not require special skills. Though Nakane and Kawaoi (34) have recommended the use of conjugate separated by gel filtration from the free HRP, we have found that this separation is not necessary to obtain good staining.
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FIG.4. Histochemical localization of anti-rat liver nonhistone protein-DNA antisera by the horse radish peroxidase technique. (A) Liver slice incubated with serum before immunization (B) Liver slice incubated with antiserum against liver nonhistone protein-DNA complex. From Chytil (5).
B. Other Methods of Localization Coleman and Pratt (35)have compared the use of the method of Steinberger et al. (36) which employs antiperoxidase-peroxidase complex to localize nonhistone protein antibody. A fluorescein-labeled antibody technique has been used by Okita and Zardi (37), Scott and Sommerville (22), Silver and Elgin (21), and Elgin et al. (38). This method is also described by Silver et al., this volume, Chapter 10.
C . Applications Unlike the quantitative complement fixation technique, the localization methods have not yet gained popularity. The horse radish peroxidase method has been used to assess possible cytoplasmic contaminations of nonhistone protein preparations. Okita and Zardi (37) have used the fluoresceinlabeled antibody method to localize antibodies against chromatin from 3T6 mouse fibroblasts and WI-38 human diploid fibroblasts. Species specificity of these antibodies was demonstrated. The nucleus was diffusely stained, and a diffuse fluorescence in the cytoplasm was observed which the authors interpreted as a suggestion of the presence of a cytoplasmic pool of chromosomal proteins. Because localization of antibody raised against nonhistone protein-DNA complex failed to show any cytoplasmic localization ( 5 ) , the
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above results also could be interpreted to mean that the chromatin preparations, unlike the nonhistone protein-DNA complexes, did contain a cytoplasmic contamination. The indirect immunofluorescence method has been used very elegantly for in situ localization of Drosophila nonhistone proteins (21.38). Localization of nuclear proteins on chromosomes of newt oocytes has been demonstrated (22). The fluorescence technique facilitated the localization of rabbit antibodies in nucleoli of normal rat liver and Novikoff hepatoma (29).
ACKNOWLEDGMENTS This work was supported by U.S. Public Health Service grants HD-05384, HD-09105, and HL-15341. I wish to thank L. Chytil for collaboration and discussions.
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