N. R., Kapphahn, J. I., J . Bid. Chem. 224, 1047 (1957). (16) -?:;zjjar, V. d.,“Methods of Enzymology, p. 462, Academic Press, New York, 1957. (17) Praetorius, E., Podsen, H., S c a d . J . Clin. Lab. Invest. 5,273 (1953). (18) Racker, E., J . Bid. Chem. 190, 685 (1951). (15) Lowry, 0. H., Roberts,
(19) Straub, F. B., Biochem. J . 33, 787 (1939). (20) Teller, J. D., Abstracts, Division of Biological Chemistry, ACS, p. 69C, September 1956. (21) Umbreit, W. W., Burris, R. H., Stauffer, J. F., “Manometric Techniques,” Burgess Publishing Co., Minneapolis, Minn., 1957.
(22) Warburg, O., Christian, W., Biochem. Z. 298, 150 (1938). (23) Wartaman, ?* B.,I. Adams, E. Q., Nature 182, 129 (1958). (24) Woodward, G. E., J . Bid. Chem. 109, 1 (1935). RECEIVED for review January 12, 1959. Accepted March 19, 1959.
Some Newer Immunological Techniques JOHN J. MUNOZ Bo cteriology Department, Montana State University, Missoula, Mont. ,Some of the most recent and promising immunological techniques are briefly discussed: gel diffusion, fluorescent antibody, Boyden’s hemagglutination, and passive cutaneous anaphylaxis. The main advantages of these tests are pointed out and key references given.
A
AXTIGEN is a substance that stimulates the formation within an animal of modified globulins capable of combining specifically with the antigen. These modified globulins are knon-n as antibodies. When an antibody reacts with antigen, a visible reaction usually takes place. One can observe either a precipitation of antigens in solution or the agglutination of particulate antigens. I n addition, other well known observable reactions can occur after an antigen has reacted with its corresponding antibody. Among these can be mentioned complement fixation, neutralization of toxins and viruses, protection against infection, anaphylaxis, and opsonization reactions (68). Most proteins, many polysaccharides, lipopolysaccharides, and polysaccharide-lipoprotein complexes have been found to be antigenic (68). Thus, highly specific reagents (antibodies) can be produced to study them. Antibodies to an antigen are prepared by giving an animal a series of injections of the antigenic material. Within a few days after the last injection the animal usually responds with the production of modified globulins (antibodies) that can be demonstrated in the blood stream by their specific combination with the antigen injected. These antibodies are highly stable and can be used, when kept under the proper conditions, for a long time as a reagent to detect the specific antigen. The specificity of antibodies depends to a great extent on the purity of the antigen used for immunization. The sensitivity of the antibody-forming
i s
mechanism is so great, hovever, that it is extremely difficult to prepare antigenic materials pure enough to give rise to the production of antibodies directed to a single antigen. Even 5 times recrystallized egg albumin has been found to give rise to antibodies to various components of egg white (40). Complex antigenic molecules, even when pure, may have more than one type of determinant group or site in the molecule capable of stimulating antibody production, thus giving rise to the formation of more than one specific antibody, each directed to different portions of the same molecule. When
Figure 1. Degradation of pure antigen with production of two serologically distinct substances
the antigen molecule is intact, the multiple antibodies react with the antigen as if they had a single specificity. If, however, the antigen molecule is broken down by enzymatic action or other chemical or physical means, the different antibodies can now detect two or more different antigenic substances in the solution which previously appeared t o be a single entity (33, 34, 40, 53). Figure 1 is a diagrammatic illustration of an Ouchterlony type of gel diffusion test, shelving what may happen to complex antigens when degraded by enzyme treatment, or by certain chemicals and physical agents. I n this diagram a pure antigen A gives only one precipitin line when it reacts with its homologous antiserum. Degraded antigen A gives two bands on reaction with the same antiserum. Antisera can be produced that are highly specific and that react only with the specific antigen and substances very closely related to it. These specific sera are prepared by the u-ell known absorption technique (68). Specific antisera can thus be prepared, even when highly complex mixtures of antigens such as bacteria are injected into an
7
Figure 2.
Prepa-
SUPERNATANT
LfD/MENT
MONOSPECIFIC
ANns€,Runi
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981
animal. Bacterial typing sera are usually prepared by this method (25, 68). In Figure 2 the essential steps in the preparation of a monospecific antiserum are given. An antiserum containing antibodies a, b, e, and d is absorbed with antigens A, B, and C. These antigens react with a, b, and e, respectively. The agglutinated or precipitated antigen-antibody complexes (Aa, Bb, and Cc) can be separated by centrifugation. The supernatant contains only antibody d specific for antigen D. Precipitin and agglutination reactions a s well as the complement fixation test, anaphylaxis, virus neutralization and toxin neutralization tests, opsonization reactions, animal protection tests, and other classical immunological reactions have been used extensively for the assay of antigenic materials. These techniques have been applied mainly as qualitative or semiquantitative tests. The quantitative precipitin reaction, however, was developed aa a truly quantitative test by Heidelberger and coworkers (26). By the use of all the above-mentioned tests, most of the fundamental observations in immunology were made (32, 68). All these techniques are adequately covered in most textbooks of immunology (68). Some newer immunological techniques, such as gel diffusion, fluorescent antibody, Boyden’s hemagglutination, and passive cutaneous anaphylaxis offer advantages because of their greater resolving power, because they are better suited to assay antigens in vivo or because they are more sensitive than the classical immunological techniques. No effort is made here to cover all the published literature on these topics. Instead, the principles involved in each method are discussed and illustrated with a few examples. These techniques are by no means the only ones which hold a great deal of promise. Of necessity some important techniques have been omitted. GEL DIFFUSION
Oudin was the first to realize the full significance of this method (40,51). Many workers had used the principle of reacting antigens and antibodies dissolved in a semisolid medium, but none had interpreted the technique for what it really demonstrated. Oudin showed that when a semisolid antiserum-agar mixture is overlaid with the antigen solution and allowed to incubate for a number of days, a hand of precipitate is formed which moves down the agar layer. Figure 3 is a photograph of four Ondin-type gel diffusion tests, showing in the first tube the reaction given by animpure eggalbuminanti-egg albumin system; in the second tube the reaction given by a pure egg albumin-anti-egg albumin system; in 982
ANALYTICAL CHEMISTRY
Figure 3. test
Oudin type of gel diffusion
n
lony type of gel diffusion test carried out in an agar medium. The central well contains rabbit antibody against Bmdetella pertussis. The outside wells contain, starting a t 12 o’clock and progressing in a clockwise manner, in wells 1 and 2 a fraction obtained from disintegrated B. pertussis cells contaiuing a t least three antigens, and in wells 3,4,5,and 6 a purified iraction from the same bacterium containing only one antigen, which was identified as the B. pertussis agglutinogen. As diffusion of antigen and antibody takes place, they meet in the agar and precipitation occurs. One band is formed for each antigen-antibody system present. Moreover, by this method it is easily
n rigure 4. acnematto representation of formotion of two bonds of precipitate
u the third tube the reaction given by an impure horse serum albumin and its corresponding antibody; and in the fourth tube the reaction given by pure bovine serum albumin and its corresponding antibody (40). For each antigen-antibody system present, one hand is formed. With a pure system, a single hand is obtained; with a complex system, multiple hands develop. Each hand moves independently of the others and has its own characteristic rate and density (41, 61). Figure 4 is a diagrammatical illustration of the formation of two distinct hands of precipitation in the Oudin type of gel diffusion test. Two pure autigen-antibody systems, A and B, when reacting in separate tubes form one band each. When the two systems react in the B), two bands are same tube (A formed which migrate independently of each other. In Figure 4 it is assumed that the antigens, represented as circles, have four combining sites while the antibodies, represented as bars, have only two (40). Ouchterlony (@, 4.9), working independently of Oudin, arrived a t the same conclusions with the aid of a different gel technique from that used by Oudm. Ouchterlony placed the antigen and antiserum in cups cut out in agar contained in a Petri plate. Figure 5 is a photograph of an Ouchter-
+
Figure 5. Ouchterlony type of gel diffusion test in agar medium
established whether two antigens are identical, different, or somewhat related. Identical antigens formed bands that merged completely; different anti, gens formed precipitates that crossed, while hands of related antigens merge8 only partially, forming so-called spurs (2Q, 4.9). Many modifications of the Oudin and Ouchterlony methods have heen devised (26, 25, 45, 4.9, 6.3,hut the fundamental interpretation of the original observations has not been altered.
The Oudin technique has been called “simple diffusion” because the antibody and antigen are adjacent to each other and the reaction starts a t the interface of the two reagents. The Ouchterlony technique has been called the double diffusion technique because both reagents migrate through a layer of gel before they come in contact with each other. When in simple diffusion the migration of the band is plotted against the square root of time, a straight line is obtained (41, 51). The slope of this line is mainly dependent on the antigen and antibody concentrations, the diffusion coefficient of the antigen, the temperature, and agar concentration. As the migration of the band depends on antigen and antibody concentrations, the technique can be employed as a quantitative tool for measuring antigens and antibodies (51, 58). Many complications, however, limit the usefulness of this technique as a truly quantitative tool. The concentration of protein (50, 56, 58), the concentration of sodium chloride and other salts (58), the position of the tubes (57), the constancy of the temperature (41, 51), probably the presence or absence of nonprecipitating antibodies, and other factors (42) affect the migration of the band and thus interfere with quantitative determinations of antigens or antibodies. Simple diffusion as well as double diffusion has been utilized for the estimation of diffusion coefficients of antigens with amazingly good results (2, 4 7 ) . The gel diffusion techniques have proved most useful in establishing the complexity of antigens and in following the purification of certain substances. TT’ith these techniques, the antigenic complexity of sera has been studied (61) as well as that of innumerable antigenic mixtures (1, 17, SO, 69). It has also been possible to follow the development of certain antigens during metamorphosis of insects (64), to demonstrate the nonreactivity of certain toxins after the treatment with formalin (59),and to study various tissue antigens ( 3 ) . These are but a few of the many uses which have been made of these techniques during the last few years. Immuno-electrophoresis (23, 64, 55, 67) is another method that offers a great deal of promise. Herein, antigens are separated electrophoretically in a semisolid medium and then immune serum is allowed to diffuse through one side of the strip. Each antigenic component reacts with its corresponding antiserum, forming a visible arc of precipitation. Immune electrophoresis thus combines the electrophoretic resolution with the serological resolution of the gel precipitin reaction, producing a highly effective method of resolving different antigenic mixtures. Grabar
under study has been called “direct staining” technique. Figure 6 illustrates the basic steps involved in the “direct” method of staining antigen with fluorescent antibody. Another method, known as the “indirect” or “layering” technique (IO), is based on the fact that antibodies are antigenic 7-globulin for which antibodies can be prepared. This anti-r-globulin is combined with a fluorescent dye and used to stain the ?-globulin (antibody) attached to the antigen under investigation. Figure 7 illustrates the basic steps involved in the “indirect” method of staining antigen (including y-globulin or antibody) with fluorescent FLUORESCENT ANTIBODY anti-?-globulin. Because antigen molecules have several reactive sites capable Coons and coworkers (10, 11) deof combining with specific antibody, and scribed a very useful technique for the the staining techniques are carried out detection of antigenic materials in in antibody excess, the use of the indirect tissue cells. This technique consists of method intensifies the staining by combining an antibody nith a fluoresallowing more fluorescent anti-rcent dye such as fluorescein isocyanate globulin molecules to react per original (11,S6), tetramethylrhodamine (24, G I ) , or 1-dimethylaminonaphthalene-5-sul- antigen molecule. For this reason, this method is more sensitive. The possifanyl chloride ( 6 ) . The fluorescent antibility of nonspecific reactions is, howbody thus produced is employed as a ever, increased. The anti-7-globulin specific histochemical stain. The procedure of preparing fluorescein isoused for staining must not cross-react cyanate-antibody complex (10, 11) with the 7-globulin of the animal in which the experiments are performed, consists briefly of producing an antiserum with high antibody content, conas nonspecific staining then could not be jugating it with fluorescein isocyanate, avoided. The main advantages of the indirect dialyzing and purifying the conjugated method are increase of sensitivity, antibody, and then absorbing the preparation with normal ground tissue use of same fluorescent antibody to study many antigens, elimination of to rid the preparation of most of the nonspecific staining material. Sections need for high titer antiserum, thus allowing use of naturally occurring of the tissues under study are made antisera or convalescent sera, and and then treated with the fluorescent detection of antibody as well as antigen. antibody. After proper washing and Recently (18) this technique was drying, the sections are examined under employed to demonstrate in the serum the fluorescence microscope using a of patients with disseminated lupus good source of ultraviolet light (10). Tissues containing the specific antigen erythematosus the presence of a globulin component that reacts with the nuclei will react and bind the fluorescent antiof tissue cells. The tissue sections were body, thus showing a bright green first treated with the serum from lupus fluorescence under ultraviolet light. erythematosus patients and a specific Many precautions are required to obfactor reacted with the nuclei of cells. tain reliable results with this technique. Fluorescent antihuman globulin serum Coons (10) pointed out that nonspecific was then applied. Where any human staining was one of the most persistent difficulties encountered with this pro?-globulin had been deposited, anticedure. By precipitation and dialysis globulin was also deposited, with the of the conjugates, most of the nonresultant staining of the specific sites specific staining material could be rewith the fluorescent material. moved. The inclusion of proper controls is Shaking the dialyzed conjugates with most important in these techniques. a washed and dried suspension of ground Proof must be definite that staining is liver removed most of the nonspecific due to the specific antigen-antibody staining, but did not eliminate all nonsystem under study and not to some other nonspecifically stained material. specific staining material. Absorption wTith red bone marrow further removed This proof may be provided by specific materials which nonspecifically stained inhibition with unconjugated homolpolymorphonuclear leucocytes and eoogous antiserum; by specific removal sinophiles (12, 60). Absorption of the of antibody from an aliquot of the conantisera with the normal tissue under jugate by precipitation with the antigen; investigation also should eliminate nonby demonstrating that the conjugate specific staining. does not stain normal tissues; and by The staining of tissues with fluoresshowing that conjugates prepared from cent antibody directed to the antigen normal serum or heterologous antiand coworkers (21, 22, 66) have made extensive use of this technique. They have devised methods of staining and preserving the agar preparations in a dry state for permanent record (65, 66), which should prove extremely valuable. The immuno-electrophoresis has an added advantage over the two previous agar techniques, in that it can be utilized to separate reasonably pure antigenic components as in paper chromatography or starch electrophoresis. It can also separate antigenically similar substances possessing different electrophoretic mobility.
VOL. 31, NO. 6, JUNE 1959
* 983
serum purified in the same way do not stain the tissue being studied (10). By the fluorescent antibody technique, the presence of various antigens in tissues and specific cells of animals has been demonstrated (13, 27, 28, 37, 38). The presence of viruses in infected cells and the distribution of viruses within a given cell have been shown (14, 44).
Recently the technique has been applied to the identification of specific bacterial cells from direct mixed smears (19, 39). Whether this latter application Kill have practical value remains to be determined. BOYDEN’S HEMAGGLUTINATION
Although the classical immunological methods as well as those just described are extremely sensitive, certain antibodies are not easily detected by these methods. Accordingly, various hemagglutinating techniques have been developed. Two excellent reviews on this subject hare been recently made (4, 43). Red blood cells can be directly coated with products from many bacteria without inducing agglutination of the red blood cells. When these coated cells are suspended in antiserum to the bacterial substance, the coated red cells specifically agglutinate. This reaction has proved to be of considerable value in serological studies of many bacteria, rickettsiae, and protozoa (43). Red blood cells can be coated directly with only relatively few substances, usually polysaccharide in nature (32). Proteins, however, usually cannot be adsorbed on red blood cells unless the surface of the red cell is modified. Boyden’s method of modifying the surface of red blood cells has perhaps proved most successful ( 5 ) . In Boyden’s technique red blood cells are treated with a very dilute solution of tannic acid, which changes the surface in such a way as to allow the proteins to be adsorbed onto the tanned cell. Tanned cells which have adsorbed the proteins are susceptible to agglutination
U+ RYE ANT/. GAMMA GLOB U L I ,4
+
by specific antiprotein serum. Figure 8 illustrates the steps involved in Boyden’s technique for coating red blood cells with protein antigens. This method is one of the most sensitive in vitro serological techniques. With the use of ovalbumin-antiovalbumin system, 0.005 y of antibody nitrogen can be detected (20). Recently (62) this method has been employed successfully in the study of the production of antibody in vitro. A method of preparing stable coated red blood cells which can be stored for many months in the frozen state or in a lyophilized form has been reported (35). This method should prove extremely valuable because it avoids the need to prepare freshly coated cells for each experiment. Other methods of adsorbing proteins on red cells may also prove valuable (7-9). PASSIVE CUTANEOUS ANAPHYLAXIS
Passive cutaneous anaphylaxis (PCA reaction) is a highly sensitive in vivo test for detection of antibodies (52). As little as 0.003 y of antibody nitrogen (20) can be detected by this procedure. The technique is simple and consists of injecting the properly diluted antibody intradermally into the experimental animal. Three hours later the animal receives an intravenous injection of the antigen mixed with a solution of dye (Evans Blue can be used). Fifteen minutes thereafter the animals are killed, the skins are removed, and the inner surface a t the site of antibody injection is examined for staining with the dye. The presence of a local antigen-antibody reaction increases the
D+ DYE ANTIBODY
capillary permeability at the site, allowing the dye t o concentrate in that location. With this test one can investigate antigen-antibody reactions in vivo. As a result this technique can be used not only to detect small amounts of antibody but also to study the effect of various substances on the inflammatory reactions induced in the animal body by the combination of an antigen with its corresponding antibody. This technique has been recently used to demonstrate the possible role of complement on this form of local anaphylaxis in the rat (46). CONCLUSIONS
The techniques mentioned are representative of some newer methods employed in immunological n-ork. No effort was made to discuss any of them in detail. It is hoped, however, that the presentation will help by bringing to the attention of persons not well versed in immunology these highly useful methods of detecting antigens and antibodies. LITERATURE CITED
( 1 ) Becker, E. L., Munoz, J., Proc. SOC. Exptl. Bid. Med. 72,287 (1949). (2) Becker, E. L., Munoz, J., Lapresle, C., LeBeau, L. J., J . Immunol. 67, 501 ( 1951 ). (3) Bjoiklund, B., Proc. SOC.Exptl. Biol. Med. 79,324 (1952). (4) Boyden, S. V., International Sym-
posium on Mechanisms of Hypersensitivity, Henry Ford Hospital, 1958, in press. (5) Boyden, S. V., J . Exptl. Med. 93, 107 (1951).
3 FL UOREJCE4 T ANTi8ODY
Figure 6.
FIL/ORESCENr ANTlIBOPY
Direct staining
+a+I:I:a AN r/ m!
F L UOUFSLENT LOMPLEX
n
r
’.
FLUORESCENT ANTI. GAMMA
Gl06Uf/N
RBC Ah’TFMDi
AN~IGEN
T A N N f O RBG
ANTIBODY- ANTIGFN COMPLEX
G L 06LJLfN
ANTIBODY.ANTf6EN COMPLEX
fUORESCEHT
ANTI-GAMMA GLOBULIN
Figure 7.
984
F L U O R ~ J C F N T cohm EX
Indirect staining
ANALYTICAL CHEMISTRY
TANNED R 6 C
COATED
RBC
Figure 8. Boyden technique for adsorbing proteins onto red blood cells
(6) Clayton, R. hI,, Xature 174, 1059 (1954). (7) R. R. A,. Fiset. M. L.. ~, Coombs. ‘ Brit. J . Exptl. Pathol. 35,472 (1954). (8) Coombs, R. R. A., Howard, A. N., Mynors, L. S., Ibid., 34,525 (1953). (9) Coombs, R. R. A., Howard, A. N., Wild, F., Ibid., 33, 390 (1952). (10) Coons, A. H., “General Cytochemical Methods,” Vol. 1, p. 399, ed. by J. F. Danielli, hcademic Press, New York, 1958. (11) Coons, -4.H., Kaplan, H., J . Exptl. iMed. 91, 1 (1950). (12) Coons, 8.H., Leduc, E. H., Connolly J. LI., Ibzd., 102, 49 (1958). (13) Coone, -4.H., Leduc, E. H., Kaplan, hI. H., Ibid., 93, 173 (1951). (14) Coons, -4.H., Snyder, J. C., Cheever, F. S., Murray, E. S., Ibid., 91,31 (1950). (15) Edwards, P. R., Bruner, D. >Tr., Univ. of Kentucky Agr. Expt. Station, Circ. 54 (1942). (16) Elek, S. D., Brit. J . Erptl. Pathol. 30, 484 119491. (17) Eiek, S . D., Levy, E., Ibid., 31, 358
(27) Kaplan, LISH., J . Immunol. 80, 254 (1958). (28) Kaplan, M. H., Coons, A. H., Deane, H. W., J . Exptl. Med. 91, 15 (1950). (29) Korngold, L., J. Immunol. 77, 119 (1956). (30) Korngold, L., Lipari, R., Cancer 9, 183 (1956). (31) Landsteiner, K., “Specificity of Serological Reactions,” rev. ed., Harvard Univ. Press, Cambridge, Mass., 1945. (32) Landy, AI., Am. J . Public Health 44, 1059 (1954). (33) Lapresle, C., Bull. SOC. chim. biol. 37, 969 (1955); Ann, inst. Pasteur 89, 654 f 1955). (34) Lapresie, C., Durieux, J., Ibid., 94,38 (1957). (35) McKenna, J. M., Proc. SOC.Exptl. Biol. X e d . 95, 519 (1957). (36) Marshall, J. D., Eveland, W. C., Smith, C. IT.,Ibid., 98, 898 (1958). (37) Marshall, J. M.,Exptl. Cell Research 6,240 (1954). (38) Marshall, J. AI., J . Exptl. X e d . 94,
(18) Fdou, G. J., Finch, S. C., Detre, K. D., J . Immunol. 80,324 (1958). 119) Gordon. M. A.. J . Invest. Dermatitis 31, 123 (1958). ‘ (20) Grabar, P., Atti V I congress0 intern. rnicrobiol., Roma 2, 169 (1953). (21) Grabar, P., Courcon, J., Ilberg, L. T., Loutit, J. F., Merrill, J. P., Compt. rend. 245,950 (1957). (22) Grabar, P., Sowinski, N. W., Generaux, B. D., Nature 178,430 (1956). (23).Grabar, P., Williams, C. A., Biochem. Bzovhvs. Acta 17,67 (1955). (24) Hiramoto, R.; Engel, K., Pressman, D., Proc. SOC.Exptl. Biol. Med. 97, 611 (1958). (25) Jennings, R. K., Malone, F., J . Immunol. 72. 411 (1954). (26) Kabat, E. A:, Mayer, 11. M., ’ ‘Experimental Immunochemistry,” Charles C Thomas, Springfield, Ill., 1948.
(39) Mood M. D., Ellis, E. C., Updyke, E. I,., J. 8acteriol. 75, 553 (1958). (40) Munoz, J., “Serological Approaches to Studies of Protein Structure and Metabolism,” p. 55, Rutgers University Press, New Brunswick, h’. J., 1954. (41) Munoz, J., Becker, E. L., J . Immunol. . 65,47 (1950): (42) Neff, J. C., Becker, E. L., Ibid, 78, 5 (1957). (43) Neter, E., Bacteriol. Rev. 20, 166 (1956). (44) Noyes, W. F., Mellors, R. C., J . Exvtl. Med. 106. 555 (1957). (45) Oakley, C. ‘L., Fulthorpe, A. J., J . Path. Bacteriol. 65, 49 (1953). (46) Osler, -4.S., Hawrisiak, &I. M., Ovary, Z., Siqueira, M., Bier, 0. G.; J . E r d . M e d . 106.811 f 1957). (47) O&hterlony, O:, Acta Paihol. Microbid. Scand 25, 187 (1948). (48) Ouchterlony, O., Arkiv Kemi,
(\ 1- -w,n) - -
i ~ n ~ i \
01
Yl
(1JC)l).
Xineral. Geol. 26B, No. 7, 43 (1949). (49) Ouchterlony, O., Progr Allergy 5, 1 f1958). (50) Oudin, J., Discussions Faraday SOC. 18,351 (1954). (51) Oudin, J., “Methods in Medical Research,” Vol. 5, p. 335, Year Book Publishers, Chicago, 1952. (52) Ovary, Z., Intern. Arch. Allergy and Applied Immunol. 3,293 (1952). (53) Pope, C. G., Stevens, M. F., Brit. J . Exptl. Pathol. 39, 150 (1958). (54) Poulik, AI. D., Can. J . Mea. Sci. 30,417 (1952). (55) Poulik. >I. D., Suture 177, 982
1
(1954). (59) Schuchardt, L. F., Munoz, J., Verwey, T I T . F., Ibzd., 80, 237 (1958). (60) Sheldon, IT. H , Proc. SOC. Erptl. Biol. Med 84, 165 (1953). (61) Silverstein, A. M., J . Histochem. 5, 94 (1957). (62) Stevens, K. RI., McKenna, J. M., J . Erptl. Med. 107,537 (1958). (63) Surgalla, RI. J., Bergdoll, M. S . Dack, G. M., J . Immunol. 69, 357 (1952). (64) Telfer, W. H., Killiams, c. M., J . Gen. Physiol. 36,389 (1953). (65) Uriel, J , Grabar, P., Ann. inst. Pasteur 90,427 (1956) (66) Uriel. J., Scheidegger, J. J., bull. SOC. chim. biol. 37, 165 (1955). (67) Williams, C. A., Grabar, P., J Immwnol. 74, 158 (1955). (68) Wilson, G. S., Miles, A. “Topley and Wilson’s Principles of Bacteriology and Immunity,” 4th ed., Williams and Wilkins, Baltimore, Md., 1955. (69) Wodehouse, R. P., Ann. Allergy 14, 121 (1956). RECEIVED for review January 12, 1959. hccepted April 6, 1959.
END OF SYMPOSIUM
Nonaqueous Titration of Organic Acids, Anhydrides, Acyl Halides, Strong Inorganic Acids, and Reactive Alkyl Halides in Various Mixtures ABRAHAM PATCHORNIK and SARAH EHRLICH ROGOZlNSKl Department of Biophysics, The Weizmann Institute o f Science, Rehovoth, Israel
( A method is presented for the determination of milligram quantities of the individual components of complex mixtures of organic and inorganic acids, acyl halides, anhydrides, and alkyl halides. The accuracy of individual determinations compares favorably with that obtained b y simple titrations in pure solutions. All titrations are carried out in nonaqueous media, using inexpensive readily available apparatus. Three standard solutions of bases are used with a single
indicator. The method has proved valuable for routine as well as research purposes.
T
increasing interest in acid-base titrations in nonaqueous media is evidenced by the large number of papers published in recent years. An excellent review of this literature has appeared recently ( 7 ) . Procedures developed during work on the synthesis of substituted N-carboxyHE
histidine anhydrides (6) permit the selective determination of hydrochloric acid, acid chlorides, acid anhydrides, organic acids, and reactive alkyl halides such as benzyl chloride (CYchlorotoluene) in mixtures. They have proved useful in determining the purity of acid chlorides and acid anhydrides in general. When pure alkyl halides were analyzed, results were comparable to those obtained by the classical Carius method. All titrations are carried out in anVOL. 31, NO. 6, JUNE 1959
@
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