fulness of such a n approach has been demonstrated in immunology (1, 2), where determinations of the diffusion coefficient have yielded reliable values for the molecular weight. There is still much progress to be made in understanding the vitamin assays, m-here zones of exhibition are formed. Further developments will no doubt extend the applicability of diffusion methods to other types of trace analysis in analytical chemistry. LITERATURE CITED
(1) Bechtle, R. &I.,Scherr, C. H., Antibiotics & Chemotherapy 7, 243 (1957). (2) Becker, E. L., Munoz, J., Lapresle, C., LeBeau, L. J., J . Immunol. 67, 501 (1951). (3) Becker, E. L., Nef, J. C., Zbzd., 78, 5 (1957). (4) Brimley, R. C., ,Vatwe 163, 215 (1949). (5) Brock, T. D., Snttbiotics R. Chemotherapy 8 , 599 (1958). (6) Cooper, K. E., Xature 176,510 (1955).
Table
I.
Effect of Variations in Cup Diameter on Size of Zone
Inside Cup Diam., Lfm. 2 6 3 8 5 0 6 4 8 2
a
Zone Diam." 11m.
Distance Diffused,b Mm.
10 1 11 8 13 3 15 4 17 4
3 8 4 0 4 2 4.5 4 6
.%\rerage of four replicates, each read
to 0.1 mni
b Equivalent to diametei ).
(zone diameter
- cup
( i )Cooper, K. E., Gillespic,
W. -4., J. Gen. M i c r o b i d . 7, 1 (1952). ( 8 ) Cooper, I(.E., Linton, A. H., Ibid., 7, 8 (1952). (9) Cooper, II., J . Biol. Chem. 167,
429 (1947).
(11) Kaplan, N. O., “Methods of Enzymology,” p. 107, Academic Press, New
York, 1957. (12) Kaplan, N. O., Ciotti, hI. >I., Ibid., p. 253. (13) Keilin. D.. Hartree. E. F.. Biochem. 1. ‘ 39, 293 (1945). (14) Kramer, D. N., Gamson, R. M., AKAL.C n m . 29, 21A (1957).
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). RECEIVEDfor 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 a n 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
VOL. 31, NO. 6, JUNE 1959
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