SEPTEMBER, 1948]
JOHN
B.
POLDING
149
The theory and practice of the standard agglutination test is as follows:
Antigen : Fundamentally, a Brucella antigen consists of a suspension of Brucella organisms in
saline. It may therefore vary, (1) as to the character of the organisms used, (2) as to the density
of the bacterial suspension, and (3) as to the concentration of NaCl in the vehicle. The character
of the organisms employed is important in one outstanding respect; the strain must be smooth, but
contrary to the belief of many in this country, it is not particularly important which variant is used,
and some antigens are made from a combination of one, or more, strains of each of the variants abortus
and melitensis, whilst others are prepared from a mixture of several strains of the same variant. In
the present work, tests for variation in antigenic sensitivity have been made on numerous Indian
and European strains (p. 146), and it has been found that, within the limits of experimental error,
they all react to the same end-titre against a standard serum. Consequently, it does not appear to
be very important from the point of view of origin, or type, which strain is selected, whilst the use
of a multiplicity of strains complicates the work and leaves more loopholes for error. Actually, a
single English Br. abortus strain is used for preparing standard abortus antigen and a single Maltese
melitensis strain for making a melitensis antigen. The latter antigen has hitherto been offered merely
to satisfy an apparent demand from persons wishing to make comparative tests, but it serves no useful
purpose for antigens of either variant will react to approximately the same titre with their hetero-
logous or homologous antisera.
The density of an antigen is a matter of considerable importance, for variation of bacterial
concentration will materially alter the end-point of a titration. In practice, antigens are encountered
that vary from the transparency of water to nearly the turbidity of milk and while many complain
of inconsistent results, there seems to be no satisfactory explanation why any individual adopts
any particular density. The writer believes that a bacterial suspension should, in the first place,
be sufficiently visible to make the detection of agglutination possible without the use of agglutinos-
copes and for this, the final opacity, after dilution with serum, should not be much less than one half
of the density of Brown's tube I. Secondly, within this limit, the density should be such as to be
in nearly optimum proportion with the major antibody, when a serum is diluted to near its end-
point. Some optimum proportion tests have been made to discover the antigenic density that gives
an optimum reaction with serum, so diluted. In optimum proportion tests conducted near a serum's
end-titre, the first appearance of agglutination is so trivial that the readings of different observers
may disagree and the results of such tests must, therefore, be accepted cautiously. In each of the
present tests, the selected dilution of serum was kept constant and the density of antigen varied.
The systems were placed in a water-bath at 60°C. and observations were made at 5 minute intervals
from the commencement of visible agglutination. The results of some of these tests are shown in
Table (XXXI). The master titres of individual sera were obtained by testing them against antigen
diluted to between tubes 1 and 2 of the Brown's scale.
TABLE XXXI
Optimum proportion tests of Brucella antigens
Time
Density of antigen*
5
>4
<4
3
2
<2
1
<1
½
<½
Serum (1) Stableforth's reconstructed serum, diluted 1/320 (titre +1/1280).
11.15 A.M. . .
1.25 A.M. . .
11.32 A.M. . .
—
—
—
—
—
—
—
—
—
—
—
—
—
—
+
±
+
+
—
+
++
—
—
±
—
—
±
—
—
—
Optimum primary dilution of antigen= between 2 & 1 on Brown's scale.
E 2