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  • pg 1
                        R. L. KAHN, FELLOW A. P. H. A.
       Immunologist, Bureau of Laboratories, Michigan Department of Health,
                                Lansing, Michigan
T HIS paper will discuss the author's construction which will hold the test tube
        precipitation test for syphilis as racks employed.
        demonstrated before the Labora-         5. Inactivating bath (560 C.) as well
tory Section of the fifty-second annual as centrifuge and centrifuge tubes may be
meeting of the American Public Health of any make which will be f.ound con-
Association at Boston on October 10, venient in the particular laboratory.
1923. By courtesy of the editors of this
JOURNAL, we are able to include some of II. REAGENTS EMPLOYED IN THE TEST
the results of standardization studies car-     The three ingredients entering into the
ried out since the demonsration and fully test are (1) Antigen, (2) Serum, and
discussed in another paper.' The follow- (3) Physiologic Salt Solution.
ing are included: (1) a simplified and                       1. ANTIGEN t
standardized procedure for the prepara-         Method of Preparation:
tion and titration of antigen, (2) some         The method of antigen preparation has
improvements in the details in carrying recently been standardized with a view of
out the routine test, (3) a special quanti- eliminating several of the variable ele-
tative procedure applicable particularly to ments inherent in the method previously
the study of the serologic effect of anti- used.
syphilitic treatment, (4) a more standard-       The unit amount of
ized technic for the test with spinal fluids. heart used for preparingpowdered isbeef
                                                                          antigen     25
                                              gms. and it is always extracted in a 250
I. GLASSWARE AND APPARATUS REQUIRED c.c. Erlenmeyer flask. The ether extrac-
               FOR THE TEST *                 tion consists of four ether "washings"
   All glassware employed in the test of the powdered muscle at ten minute
must be chemically clean and neutral.         intervals with 100, 75, 75 and 75 c.c.
   1. Test tubes: Standard antigen dilu- ether, respectively. The subsequent alco-
tion tubes are 5.5 cm. in length and 1.5 hol extraction is carried out for three
cm. in diameter.                              days at room temperature (210 C.).
   Standard tubes for performing the test Twenty-five gms. of powdered beef heart
are 7.5 cm. in length and 1 cm. in will yield about 75 c.c. antigen. If the
diameter.                                     preparation of larger amounts is desired,
   2. Test tube racks: Racks should be of as many 25 gm. quantities are employed
such construction as to permit vigorous as needed.
shaking of the tubes.                            Powdered Heart Muscle.-About 400
   3. Pipettes: 10 c.c. pipettes marked in gms. of heart muscle are cut out from at
0.1 c.c. quantities. 1 c.c. pipettes marked least three fresh beef hearts and passed
in 0.01 c.c. quantities. 0.2 c.c. pipettes four times through a meat grinder. The
marked in 0.001 c.c. quantities.              ground material is spread into a thin
   4. Shaking machine: May be of any and drieda porcelain of one or two revolv-
                                              layer on             platter or glass plate
                                                         by means
  4 Glassware and apparatus for the test may be
procured from the Chicago Glass Products Company,     t The Michigan Department of Health furnishes
Chicago, Ill.                                       antigen at a minimum cost.
               THE KAHN PRECIPITATION TEST                FOR   SYPHILIS             499
ing fans. After six or eight hours, when          The moist beef heart is now trans-
the exposed surface is relatively dry, the     ferred for the fourth and last time to tfie
material is turned over and drying con-        flask and 75 c.c. ether added, the mixture
tinued over night. When the layer of           shaken for ten minutes and filtered.
beef heart is in the form of a dry plate,      After gentle pressure with a spatula does
it is broken up into small pieces and dry-     not cause drops of ether to pass through
ing continued until the material is brittle    the funnel-the endpoint employed in
and easily breakable. The material is          each of the four ether filtrations-the
now ground into powder form by means
of a mortar or coffee grinder which is         beef heart is transferred to a sheet of
used for no other purpose.                     white paper and dried either at room tem-
                                               perature or at 370 C. The drying usually
   Powdered beef heart is now obtainable       requires from ten to fifteen minutes.
on the market from the Digestive Fer-
ments Company, Detroit. It is prepared
                                               When no ether odor is detectable, the
essentially as outlined above, except on a     beef heart is ready for extraction with
large scale-as many as seventy-five beef       alcohol. This extraction may be carried
hearts entering into the preparation of a      out in the same flask used for the ether
given lot. This assures a higher degree        extractions, provided the flask is entirely
of uniformity than can be obtained with        freed from ether odor.
three beef hearts. Because of this uni-           Extraction of Powdered Muscle zewth
formity element, the market product is         Alcohol.-The ether extraction being
used in this laboratory almost exclusively.    completed, the dry powdered muscle is
   Extraction of Powdered Muscle with          weighed and placed in a 250 c.c. flask.
Ether.-Twenty-five gm. of powdered             Usually there will be twenty-three gms.
beef heart are placed in a 250 c.c. Erlen-     or less of the powder due to the loss dur-
meyer flask and 100 c.c. ether (anes-          ing the ether extraction. Five c.c. of 95
thesia) added. The flask is shaken from        per cent alcohol are added per gm. of
time to time for an interval of ten min-       powder. The flask is shaken for ten
utes after which the ether is filtered off.    minutes and extraction allowed to con-
The filtration process may be hastened         tinue at room temperature (210 C.) for
somewhat by applying gentle pressure to        three days without shaking. At the end
the beef heart by means of a spatula.          of this period, the mixture is shaken for
Filtration is completed when pressure          five minutes and filtered. The filtrate is
with the spatula does not cause drops of       kept in the dark at room temperature as
ether to pass through the funnel.              stock antigen solution.
   The moist beef heart is now returned           Cholesterinization of Alcoholic Ex-
to the original 250 c.c. extraction flask.     tract.-A given amount of alcoholic ex-
This may be done by first transferring the     tract-likely to be used in a month or
beef heart from the funnel to a sheet of       two-is cholesterinized by adding 6 mg.
white paper and breaking the material          of chemically pure cholesterin per c.c. of
up with a spatula into pieces small enough     extract. The cholesterin is dissolved by
for the mouth of the flask. Seventy-five       rotating the flask in a water bath at 370
c.c. ether are now added and the mixture       C. When all the cholesterin has been dis-
again shaken for a ten-minute interval         solved, the antigen is filtered to remove
and filtered as above.                         impurities and allowed to stand one day.
   After the second filtration of ether, the   It is then ready to be titrated or stand-
beef heart is transferred to the flask for     ardized for the test.
a third time and 75 c.c. ether again added.       It is well to emphasize in connection
The mixture is shaken for a ten-minute         with antigen preparation that the ether
interval and again filtered as described       and alcohol employed should be of high
ahovc.                                         purity and that the latter should be 95
per  cent. At one time we unknowingly                             character of these precipitates, however,
used 80 instead of 95 per cent alcohol                            will vary according to the quantity of salt
with misleading results.                                          solution used. Some of the precipitates
   Tinfoil-covered corks have been found                          will be found to be stable and will not dis-
most satisfactory as stoppers for flasks                          solve when mixed with salt 'solution;
used in antigen preparation and storing.                          other precipitates again will readily dis-
Rubber as well as cork stoppers give off                          solve in salt solution.
soluble elements into the alcohol which                              The solubility in salt solution of each
modify the final product.                                         of the five antigen dilution precipitates
   Method of Antigen Titration:                                   is tested as follows: 0.05, 0.025 and
   The aim of the titration of antigen for                        0.0125 c.c. amounts, respectively, of each
this test is to find the minimum amount of                        antigen dilution are pipetted with a 0.2
physiologic salt solution to use with anti-                       c.c. pipette graduated in 0.001 c.c. into
gen which will result in an antigen-salt                          three tubes (7.5 cm. in length and 1 cm.
solution precipitate that is soluble on fur-                      diameter). These small quantities are
ther addition of salt solution. The titra-                        pipetted in each case to the bottom of the
tion is carried out in the presence of salt                       tubes. 0.15 c.c. quantities of physiologic
solution and not in the presence of serum.                        salt solution are now added to each tube.
solution and not in the presence of serum,                        The rack is shaken vigorously for two
although the latter may be used as a check                        minutes after which 0.5 c.c. salt solution
on the titration, if desired.                                     is added to each tube and observation
   One c.c. amounts of cholesterinized                            made as to whether or not the original
antigen are added to each of five standard                        antigen dilution precipitate has gone back
antigen dilution tubes (5.5 cm. length and                        into solution. The antigen dilution tube
1.5 cm. diameter). To five similar tubes                          containing the smallest amount of salt
are added the following amounts of                                solution in proportion to antigen, having
physiologic salt solution, respectively:                          a precipitate which goes back into solu-
0.8, 0.9, 1.0, 1.1 and 1.2 c.c. Each salt                         tion in salt solution, as shown by this
solution tube is emptied into a given anti                        three tube test, represents the endpoint
gen tube and, without waiting to drain                            of this titration and determines the pro-
the salt solution, the mixture is imme-                           portion in which antigen is to be mixed
diately poured back and forth five or six                         with salt solution in the performance of
times to permit thorough mixing. Each                             the tests.
of the five antigen dilutions will show the                           The accompanying table gives an out-
presence of a definite precipitate. The                           line of a typical antigen titration.
                                                        TABLE 1.
                                      TYPICAL ANTIGEN TITRATION FOR TEST WITH SERUM

Antigen Dilution Series                      1                  2                  3                  4                 5
Antigen + SaltSolutionc.c.                1 + .8             1 + .9            1 + 1.0             I + 1.1           1 + 1.2
Result of Dilution                              Heavy precipitate in each antigen dilution
Scheme Used in Testing                          Tube No.                  1                  2                3
  Solubility of Precipitate in Each            *Antigen Dilution c.c. .05                  .025             .0125
  Antigen Dilution                              Salt Solution c.c.      .15                .15              .15
                                        Tubes are shaken 2 minutes and 0.5 c.c. salt solution added to each.
                                        All are observed for precipitates.
Solubility of Precipitate as De-        Precipitate      Precipitate        Precipitate         Precipitate       Precipitate
  termined by Three-Tube Test           Not Soluble      Not Soluble        Soluble             Soluble           Soluble
Standard Antigen Dilution                                                Antigen + min-
                                                                            imum amount
                                                                            of salt solution
                                                                         giving precip-
                                                                         itate which dis-
                                                                         solves in salt
      *Each antigen dilution is allowed to stand 30 minutes after mixing antigen and salt solution before solubility test
is made.
               THE KAHN PRECIPITATION TEST                   FOR   SYPHILIS             501
                   2. SERUM                  ing amounts of salt solution by utilizing
   The serums are separated from the the same standard antigen dilution tubes.
clots -by centrifugation in the u sual man-    Procedure: One c.c. antigen is meas-
ner, pipetted off and inactivat ed for 30 ured into an antigen dilution tube. An
minutes at 560 C. The main point re- amount of salt solution usually approxi-
garding serums is that they be free from mating that of the antigen, according to
red cells, fibrin and particles of any kind. the titre of the antigen, is measured into
No difficulty is encountered ir this test a similar tube. The salt solution is
with milky or chylous serum,s or with poured into the antigen and, without wait-
moderately hemolyzed specimens. Only ing to drain the tube, the mixture is im-
such hemolyzed specimens whioch are not mediately poured back and forth five or
fit for a Wassermann test are not fit for six times to insure thorough mixing. For
a precipitation test.                        uniformity, this antigen dilution is per-
                                             mitted to stand ten minutes at room tem-
   Salt solution is prepared by dissolving   perature before pipetting.
8.5 gm. of chemically purle sodium              As a preliminary control of the antigen
chloride per liter of distilled water and    dilution, 0.05 c.c. is measured into a test
filtering. Although sterility of this solu- tube, 1 c.c. salt solution added and the
tion is not essential, the sam e type of tube shaken vigorously for ten or. fifteen
chemical cleanliness usually en inloved in seconds. The mixture should appear
quantitative chemical work is required.      opalescent.
                                                Pipetting of Antigen Diliution.-The
  III. THE ROUTINE TEST WITH SERUM                antigen dilution is always pipetted to the
                                                  bottom of the tubes.       The 0.05    c.c.
  The routine test consists of t                  amounts may be pipetted with a 1 c.c.
containing three different prop horeetubs

serum and antigen dilution in                     pipette in which 0.05 c.c. graduations are
with the following outline:
                                                  indicated with a wax pencil. For the
                                                  0.025 and 0.0125 c.c. amounts of antigen
  Tube No.                  1         2     3     dilution, 0.2 c.c. pipettes are employed
Serum: Antigen Dilution    3:1    6:1     12:1    and the  proper markings may also be in-
Antigen Dilution c.c.      .05    .025    .0125
                                                  dicated with a wax pencil. The antigen
Serum c.c.                 .15    .15     .15
                                                  dilution should be mixed frequently dur-
   Preparation of Standard Anitigen Dilu-         ing the pipetting period to assure a uni-
tion.-It is well when carrying 4out a num-        form mixture. Due to the possibility of
ber of tests to inactivate the s erums and        evaporation of the small amounts of anti-
set up and number the tubes be fore dilut-        gen dilution used in the test, it is neces-
ing antigen with salt solutio:n for the           sary to pipette the serum within several
tests. The antigen dilution is so stand-          minutes after the antigen dilution has
ardized as to necessitate its us within a
half hour after mixing antiger with salt
                                                  been pipetted. If a worker desires to run
                                                  forty precipitation tests, for example, it
solution. In this laboratory, with over           is better to pipette antigen dilution and
200 routine tests per day, enou gh antigen        serums for ten tests at a time, than to
dilution for 60 or 80 tests is, ulsually pre-     pipette the antigen dilution for all the
pared at one time-two e:xperienced                tests first and then follow with the
workers pipetting antigen di]lution and           serums.
serum for about 80 tests in less than               Pipetting of Serum.-The 0.15         c.c.
twenty minutes.                                   amounts of each serum are added to the
   One c.c. antigen diluted wilth one c.c.        antigen dilution by means of a 1 c.c.
salt solution gives sufficient anttigen dilu-     pipette graduated in 0.01 c.c. In pipetting
tion for about 18 tests. Two o r three c.c.'      these amounts of serum it is not neces-
antigen may be diluted with correspond-           sary to lower the pipette to the bottom of
502           THE AMERICAN JOURNAL                OF   PUBLIC HEALTH
 the tube. As soon as the serums have          slanted to spread the fluid into a thin
 been added for ten tests, or less, the rack   layer and examined for a precipitate.
 is shaken sufficiently to insure thorough        In this laboratory, precipitation tests.
mixing of the serum with antigen dilution.     are read at night with the same ease as.
    Shaking of Tests.-After the serums         during the day. Light is furnished by a
 have been mixed with the antigen dilu-        300-watt daylight bulb in an overhead
tion, the tests are shaken for a two min-      indirect fixture.
ute interval. A shaking machine is of the
utmost importance for this purpose, par-         NOTE: If it is desired to make a check read--
ticularly in the examination of compara-       ing of the results at some later period, it is
                                               well to keep the tests at icebox temperature.
tively large numbers of specimens at a         Practically all positive reactions will be
given time.                                    slightly stronger at the second reading. Occa-
                                               sionally, however, even at icebox temperature,.
    Effect of Incubation.-Although the         a serum which gave a   -,   +, ++ at the first
final results may be read immediately          reading may be found to be negative during
after the shaking period, different            the second reading. We have observed these
                                               reversible precipitation reactions especially in
workers have observed that a 15 minute         early primary and in highly treated cases. The
incubation period in the water bath at         reading made immediately after the addition
370 C. produces sufficient clumping of         of 0.5 c.c. salt solution to each tube is the
                                               standard reading and is always the one
the precipitates to make the reading of        reported.
the results easier, particularly in the case
of weak reactions. In studying the effect          The Control System.-1. Antigen Coni-
of 15 minute incubation on this test, no       trol.   When pipetting antigen dilution
tendency for false positive reactions has      for a series of tests, the last set-up of
been observed, and on the basis of easier      three regular antigen amounts receives
reading we consider the employment of          0.15 c.c. salt solution instead of serum
this incubation period of some advantage.      and is read with the regular tests. All
   Addition of Salt Solution and Reading       three tubes should show freedom from a
of Results.-After the shaking of the           precipitate.
tests as well as after the 15 minute incu-        2. Serum Controls. In the case of each
bation period, if employed, the serum-         positive reaction, the serum used in the
antigen mixtures appear uniformly              test is examined for particles that might
cloudy. In order to render the negative        give the appearance of a specific precipi-
reactions clear and thus simplify the          tate.
reading of results, 0.5 c.c. salt solution        3. Positive and Negative Controls.
is added to each tube. The tests should        One or more such controls are included
be read immediately after the addition of      with each series of tests.
salt solution as an occasional weak pre-          Interpretation of Results.-A definite
cipitate may go back into solution on          precipitate suspended in a clear medium
standing.                                      is considered a complete reaction and is
   Readings are best made in front of a        read four plus. Proportionally weaker
window with a darkened background.             reactions are read three, two and one
The negative serums appear opalescent          plus, respectively. The final result in
and readily distinguishable without lift-      each test is the average finding of the
ing the tubes from the racks. The              three tubes. Thus, if the precipitation
strongly positive serums show heavy pre-       reaction is four plus in each of the three
cipitates which are also easily read           tubes, the final result is four plus. If
directly.                                      the reaction is -, +++, ++++, the
   Only the tubes showing weak reactions       final result is two plus. A number of
need to be removed from the rack and           typical reactions with this method and
examined individually. Each tube is            the final result in each case, are illus-
lifted from the rack above the eye level,      trated in Table 2.
                 THE KAHN PRECIPITATION TEST                         FOR   SYPHILIS               503
                                                  TABLE 2.
                              INTRPRTATION OF RzsuLrs o0 RourNzr Tzsr WITR SERumL

          Tube N6.                     1                     2               3
                                                                                    Average of Reaction in
                                                                                     the Three Tubes
Serum: Antigen Dilution                3:1                6:1                12:1        Final Result
Antigen Dilution c.c.                 .05                .025               .0125
Serum Undiluted c.c.                  .15                .15                .15

             Reaction No.
                      I   *         ++++
                      3              ++               ++++               ++++
                      4               +               ++++               ++++              +++
   Some               5                               ++++               ++++

                                      -+                                 + +
                                                                         ++++               ++
                      8                                 +                ++++
 Reactions            9                                                  ++++
                     10               -                  -                                    +
                          11          -                 ~~~~~~~+          ++
                     12               -
                                      _                                   ++
                     13               -                  -                ++
                     14               -                  :                  +

   Correlation of Results with Clinical                  also gives no false reactions. Yaws, yel-
Findinigs.-When the average result with                  low fever and pellagra, which give occa-
this test is four plus, it will usually be               sional false Wassermann reactions, are
found that we are dealing with untreated                 still to be studied with this method.
or little treated syphilis. When the re-                    While strong reactions give every in-
sult is less than four plus, the clinical                dication of being associated with syphi-
condition is usually early primary or in-                litic infection, weak reactions-particu-
tensively treated syphilis. As a general                 larly when the average finding is one
rule, the average result corresponds with                plus-appear to have the same diagnostic
a cholesterinized antigen Wassermann ex-                 significance as weak Wassermann reac-
cept that the precipitation test is possibly             tions. Needless to say, as is true with
more specific-due to the elimination of                  any laboratory method, the finding with
the hemolytic system.                                    this test should be considered but one
   In reporting the results with this test               factor in the diagnostic syndrome in a
to physicians, it is well to give the actual             given case.
precipitation findings in each of the three
tubes as well as the final results. This                     IV. SPECIAL QUANTITATIVE TEST WITH
gives the physician a more correct pic-                                     SERUM
ture of the serologic condition of the                      The routine test being quantitative in
patient than the average result alone.                   only a limited degree, a special quanti-
   Clinical studies carried out in conjunc-              tative procedure has been devised for the
tion with the Department of Derma-                       purpose of determining the relative
tology and Syphilology of the University                 potency of serums in terms of a unit
of Michigan Medical School indicate that                 system.
this precipitation test is highly specific for              The Unit Reaction.-In the quantita-
syphilis. Tuberculosis, scarlet fever and                tive procedure, a constant amount of
other febrile diseases which occasionallv                standard antigen dilution is employed
give falsely positive reactions with the                 with varying dilutions of serum. In the
Wassermann test appear to give no such                   derivation of the unit reaction, 0.0125 c.c.
reactions with this method. Pregnancy                    antigen dilution and 0.15 c.c. undiluted
504           THE AMERICAN JOURNAL               OF     PUBLIC HEALTH
serum are     the unit quantities employed.   each tube containing 0.0125 c.c. standard
 If complete precipitation is obtained when   antigen dilution with 0.15 c.c. quantities
0.0125 c.c. antigen dilution is used with     of serum dilutions ranging frQm 1:1 to
 0.15 c.c. serum, only in undiluted form,     1:60.
 the reaction represents four reacting          The serum dilutions may be prepared
 units. If precipitation is complete with     as follows:
 certain dilutions of serum, then the
 potency of the serum in terms of react-      1 :1 = undiluted       serum
 ing units (S) is equal to four times the     1:5   =   0.2 undiluted serum + 0.8 c.c.
                                                              c.c.                              salt
 maximum dilution of the serum giving         1:10 =0.6 c.c. 1:5 dilution + 0.6 c.c.            salt
 complete precipitation (D). The po-                   solution
 tency of any serum may be expressed by       1:20= 0.2 c.c. 1:10 dilution + 0.2 c.c.           salt
the formula                                   1:30 =0.2 c.c. 1:10 dilution +0.4 c.c.            salt
                  S      4D                   1:40 =0.1 c.c. 1:10 dilution + 0.3 c.c.           salt
 Thus, if 1:10 represents the highest dilu-            solution
                                              1:50=0.1 c.c. 1:10 dilution + 0.4 c.c.            salt
 tion of serum capable of giving complete              solution
precipitation, the serum contains 4 x 10      1:60=0.1 c.c. 1:10 dilution + 0.5 c.c.            salt
or 40 reacting units. If 1:60 represents
the highest dilution in which a serum            NOTE: If it is desired to make more exact deter.
gives a complete reaction, the serum con-     minations of the number of reacting units in certain
                                              cases, serum dilutions in between those already indi-
tains 4 x 60 or 240 units. Similarly the      cated may be included. Serums of high potency,
                                              which give complete precipitation in a 1:60 dilution
potency of any serum may be determined.       may be diluted still further until a negative reaction
                                              is obtained.
    In the quantitative procedure only com-
plete precipitation reactions are consid-
ered in determining the concentration of         The antigen dilution is prepared as for
reacting units in a given serum. The          the routine test and pipetted in 0.0125 c.c.
interpretation of intermediary reactions      quantities to the bottom of the tubes in
must be left to the judgment of individual    the usual manner. This is followed by
workers. A reaction giving the appear-        0.15 c.c. amounts of the various serum
ance of a three plus might be included        dilutions, in order, beginning with the
among complete reactions, while weaker        undiluted serum. The rack is shaken for
reactions are considered negative. The        two minutes, 0.5 c.c. salt solution added
results of the quantitative procedure, fur-   to each tube, and the results read and
thermore, are always expressed in units       recorded. If a fifteen minute incubation
and not in plus signs. By thus adhering       period at 370 C. is employed, it should
to the use of plus signs in connection        be carried out as suggested in connection
with the routine test with serum and of       with the regular test, namely, after the
units in connection with the special          shaking period and prior to the addition
quantitative test, confusion of the re-       of salt solution.
sults with the two procedures will be           Table 3 gives some typical reactions
                                              with the quantitative procedure.
   Outline of Quantitative Test.-The
quantitative procedure is applicable only        Correlation of Quantitative Procedure
to strongly positive serums, since only       with Routine Serum Test.-Since the
such serums are capable of giving pre-        last proportion of serum and antigen dilu-
cipitation reactions after dilution. A        tion of the routine test is the basis for
routine test, therefore, is always made       determining the reacting unit, the results
before employing the quantitative test        of the routine procedure can be readily
with a given serum.                           interpreted in terms of reacting units.
   Eight tubes are employed in this test,     The three types of reactions in the routine
                      THE KAHN PRECIPITATION TEST                     FOR     SYPHILIS                505
                                                 TABLE 3.

Serum: Salt 1:1          1:5     1:10       1:20    1:30      A:40    1:50     1:60    Four times Serum
  Solution                                                                             the maxi- potency
Serum Dilu-                                                                            mum dilu- in terms
  tionc.c.    .15       .15       .15       .15     .15       .15     .15      .15     tion of so-   of
Antigen Dilu-                                                                          rum giving reacting
  tion c.c.   .0125     .0125     .0125     .0125   .0125     .0125   .0125    .0125   Precipita-   units
                                                                                        tion (4D)    (S)

 Serum No.
     1        ++++*                                                                          -1 -44
     2        ++++       ++++-     -    -    _    _    -                                  4x5    20
      3       ++++       ++++ ++++-     -    _    _   -                                   4x10   40
      4       ++++       ++++ ++++++++-      -    -   -                                   4x20   80
      5       ++++       ++++ ++++ ++++ ++++-     _    -                                  4 x30 120
      6       ++++       ++++ ++++ ++++ ++++ ++++      _                                  4x40  160
      7       ++++       ++++ ++++ ++++ ++++ ++++ ++++-                                   4x 50 200

    *+ + + + = Complete precipitation reaction.

procedure of especial interest in this con-                antigen titration for the test with serum
nection are:                                               in that the solubility of the precipitates in
             Standard Antigen Dilution c.c.                the various antigen dilutions is tested
              .05         .025       .0125                 with an ammonium sulphate solution ap-
   Reaction        Undiluted Serum c.c.                    proximating 10 per cent, instead of
     No.      .15          .15         .15                 physiologic salt solution. This is neces-
      1                           ++++
     2     -           ++++ ++++                           sary because the concentrated spinal fluid
      3    ++++ ++++ ++++                                  to be tested for specific reacting sub-
  The first reaction, in which the third                   stances contains approximately 10 per
tube only gives a four plus, represents                    cent ammonium sulphate.
four reacting units. The second, in                           Five 1 c.c. quantities of standard anti-
which a four plus results from the use                     gen are measured into five standard anti-
of double the amount of antigen dilution,                  gen dilution tubes. Into five similar tubes
indicates that the serum in a dilution of                  are measured 1.1, 1.2, 1.3, 1.4 and 1.5 c.c.
1:2 would give complete precipitatiotn                     physiologic salt solution, respectively.
with 0.0125 c.c. antigen dilution, thus                    Each of the saline amounts is poured into
representing approximately eight react-                    an antigen tube and the mixture, in each
ing units. On the same basis, the third                    case, immediately poured back and forth
reaction indicates that the serum would                    several times. These five antigen-saline
give complete precipitation in a dilution                  dilutions contain precipitates and, after
of 1 :4 and therefore represents at least                  twenty minutes' standing, the solubility
sixteen reacting units.                                    of these precipitates is tested with an
                                                           ammonium sulphate solution prepared by
     V. THE TEST WITH SPINAL FLUIDS                        mixing 1 c.c. of saturated solution with
   The test with spinal fluids requires pre-               9 c.c. normal saline. These tests are car-
liminary concentration of the reacting                     ried out in duplication employing 0.01 c.c.
substances in these fluids with a saturated                amounts of the five antigen dilutions with
solution of ammonium sulphate.* This                       0.15 c.c. quantities of the approximately
in turn necessitates a special antigen                      10 per cent ammonium sulphate solution.
titration. The test consists of one pro-                    The proportion of ammonium sulphate
portion of concentrated fluid and antigen                  solution to antigen dilution is 15 :1. The
dilution and is carried out in duplication.                antigen dilutions are pipetted to the bot-
   Titration of Antigen for Spinal Fluid                   tom of the tubes with a 0.2 c.c. pipette
Test.-This titration differs from the                      graduated in 0.001 c.c. The ammonium
                                                           sulphate solution is then added, the tubes
    Merck's Reagent Ammonium Sulphate has been             shaken for two minutes and 0.2 c.c. saline
found to give good results.
 506                THE AMERICAN JOURNAL                          OF    PUBLIC HEALTH
added to each tube to render easier the                       the inner wall of the tube. The precipi-
reading of the results. The dilution of                       tate immediately dissolves and the result-
antigen and saline which contains the                         ing solution is tested with antigen dilution
smallest amount of saline in proportion to                    in a manner to be described.
antigen, giving a precipitate which is                           Preparation of Antigen Dilution.-
soluble in ammonium sulphate solution                         Antigen is diluted with salt solution as
represents the titre of the antigen for                       described under the test with serum, using
spinal fluids. A typical titration is given                   the proportion indicated by the special
in Table 4.                                                   titration method. The antigen dilution is
                                                       TABLE 4.

Antigen Dilution Series                  1                2                 3                 4                5
Antigen + Salt Solution c.c.          1 + 1.1          1 + 1.2           1 + 1.3           1 + 1.4          1 + 1.5
Result of Dilution                           Heavy precipitate in each antigen dilution
                                             Tube No.                                     1                 2
Scheme Used in Testing                      *Antigen Dilution c.c.                       .01               .01
  Solubility of Precipitate in                10% Sol. Ammonium Sulyhate c.c.            .15               .15
  Each Antigen Dilution               Tubes are shaken 2 minutes and 0.2 c.c. salt solution added to each.
                                      All are observed for precipitates.
Solubility of Precipitate          Precipitate        Preci itate        Precipitate       Precipitate        Precipitate
                                    Not Soluble       Not Soluble        Not Soluble       Soluble            Soluble
                                                                                        Antigen + min-
                                                                                           imum amount
                                                                                           of normal sa-
                                                                                          line giving pre-
Standard Antigen Dilution                                                                  cipitate which
                                                                                           dissolves    in
                                                                                            10% ammon-
                                                                                           ium sulphate
     *Each antigen dilution is allowed to stand 20 minutes after mixing antigen and salt solution before solubility test
is made.

   Concentration of Fluid zewith Am-                          allowed to stand for ten minutes before
monium Sulphate.-Three c.c. of spinal                         use  in the test.
fluid, previously rendered free from cel-                       Pipetting of Antigen Dilution.-For
lular material by centrifugation, are                         each fluid to be tested, 0.01 c.c. of antigen
pipetted into a centrifuge tube and 2 c.c.                    dilution is pipetted to the bottom of each
of saturated solution ammonium sulphate                       of two tubes, using a 0.2 c.c. pipette.
added (Herrold). This is mixed, per-                             Pipetting of Concentrated Fluid.-
mitted to stand for one hour at room tem-                     Concentrated spinal fluid in 0.15 c.c.
perature and centrifuged at high speed                        quantities is added to each of the tubes
for about fifteen minutes-a period                            containing the antigen dilution.
usually sufficient to completely throw                           Shaking of Tests.-After the concen-
down the globulins. The supernatent fluid                     trated fluids have been mixed with the
is poured off as completely as possible, the                  antigen dilution, the tests are shaken for
last drop adhering to the lip of the tube                     a two minute period.
being taken up with filter paper. The                            Incubation.-The tests may be read
globulin precipitate is found to be                           immediately after the shaking period. As
suspended in a small drop of ammonium                         in the case of serums, however, a fifteen
sulphate solution. Three-tenths c.c. of                       minute incubation period in the water
salt solution (one-tenth of the original                      bath may be employed, if desired.
amount of spinal fluid used) is now added                        Addition of Salt Solution and Reading
to the tube. This is added by lowering                        of Results.-Immediately before reading
the pipette into the tube and running the                     the tests, 0.2 c.c. salt solution is added to
saline down in the center so as to avoid                      each tube in order to render the negatives
washing down ammonium sulphate from                           clear and reading easier.
               THE KAHN PRECIPITATION TEST                  FOR   SYPHILIS                 507
   The results are read as described under     pose   independently of any other labora-
the test with serum.                           tory  procedure provided (1) the workers
   The Control System.-The controls are        performing it have had both training and
of particular importance in the spinal         experience in bacteriology and immunity
fluid test in view of the fact that certaini   and that (2) each test is performed in
concentrations of ammonium sulphate            duplication.
are capable of giving precipitates with           2. Special Quantitative Procedure.-
standard antigen dilution. The control         This procedure is highly quantitative in
of particular importance is carried out as     character, consisting of eight tubes, each
follows: Three c.c. normal saline are          containing a constant amount of antigen
added to a centrifuge tube and 2 c.c. of a     dilution with different dilutions of serum.
saturated solution of ammonium sulphate        The results with this procedure are al-
thoroughly mixed with it. The tube is          ways expressed in reacting units and are
now emptied, in the same manner as the         in this way distinguished from the results
spinal fluid tube is emptied after centrifu-   with the routine test which are expressed
gation, and 0.3 c.c. salt solution added.      on a plus sign basis.       This procedure
We thus have here a concentration of am-       enables one to determine the relative num-
monium sulphate approaching that in the        ber of reacting substances in syphilitic
concentrated globulin solution. A regu-        serum and is of especial value when it is
lar test performed with 0.15 c.c. of this      desired to study the quantitative effect of
control ammonium sulphate solution and         specific treatment on the number of these
0.01 c.c. standard antigen dilution should     reacting substances.
show no precipitation.
   An antigen dilution control, 0.01 c.c.         3. Test with Spinal Fluids.-In this
mixed with 0.15 c.c. salt solution, is also    procedure, the globulins of the fluid are
included with the regular tests. Further-      precipitated with ammonium sulphate and
more, every concentrated fluid to be           the precipitate subsequently dissolved in a
tested is examined to establish total free-    minimum amount of salt solution-
dom from foreign particles. Finally,           thereby concentrating the reacting sub-
positive and negative spinal fluid controls    stances of the fluid. This concentrated
should accompany each series of tests.         fluid is tested with antigen dilution in the
   Interpretation of Results.-Reactions        usual manner. The test is essentially
are read on the basis of four, three, two      qualitative, its quantitative character be-
and one plus, respectively, depending          ing limited to the relative intensity of the
upon the distinctness of the precipitates.
                                               precipitate formed in one proportion of
Since the two tubes used for testing each      concentrated fluid and antigen dilution.
fluid are duplicates, the reactions in the     The results with this method appear to
two should be identical.                       conform with the findings of a conserva-
                                               tive Wassermann test.
               VI. SUMMARY
   1. Routine Test with Serum.-This
test is quantitative to a limited degree-         The writer desires to express his thanks
consisting of three tubes, each containing     to Dr. W. W. Duemling of this laboratory
different proportions of serum and anti-       for assistance in the demonstration, and
gen dilution. The final result is the aver-
                                               particularly to Dr. Wm. A. Hinton of the
age of the precipitation findings in the
                                               Harvard Medical School for furnishing
                                               serums and glassware for the demonstra-
three tubes, and is expressed in plus
signs. The test is of value both in the        tion.
diagnosis of syphilis and as a check on           1. Kahn, R. L. The Serology of Syphilis by   Pre-
treatment. It may be used for this pur-        cipitation. (In press.)

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