SYPHILITIC REAGIN
A PHYSICO-CHEMICAL INVESTIGATION
OSCAR K A N N E R , M.D.
W I T H THE TECHNICAL ASSISTANCE OP F R A N C E S BOYD
Veterans Administration
Hospital,
Oteen, North
Carolina
In a previous paper2 a method was described to explore immunologic constituents of biologic fluids. Evidence was presented tending to show that different
procedures used in the serology of syphilis are dependent upon different aspects
of reagin. The present report deals with the nature of syphilitic reagin and covers
a larger amount of material and a greater variety of serologic procedures. The
results of the initial study were confirmed and, in addition, it was found that
even with the same method, such as the Kahn or Kline test, distinctly different
aspects of reagin are at play, depending upon the degree of reactivity of any
given serum. It was possible to separate the 2 principles.
METHODS
2
In the previous report it was shown that when a strip of purified, dry gelatin
is placed into serum to be tested, the gelatin swells and substances capable of
diffusion, such as water, mineral salts, and other constituents of relatively small
molecular size, in part leave the liquid outside the gelatin strip until a state of
equilibrium is reached. The larger molecules including proteins, lipids, and other
substances, remain in the liquid outside the gelatin because they cannot diffuse
into it. The concentration of diffusible solutes in the outer liquid remains substantially unchanged because the solvent (water) enters the gelatin at about
the same rate as the diffusing elements. Conversely, nondiffusible solutes become
more concentrated in the outside liquid because part of the water enters the
gelatin, as is evident by its swelling.
In a new series of experiments the following procedures were carried out:
Kahn, Kline and VDRL flocculation tests; Kolmer and Eagle complementfixation tests. Both standard and cardiolipin antigens were employed. The following technics were used: Kahn and Kolmer tests were performed according to
directions given in the War Department Technical Manual TM8-227 (p. 305),
with appropriate serum dilutions; Eagle and Kolmer complement-fixation tests,
according to Kilmer and Boerner (Approved Laboratory Techniques, 3rd edition,
p. 855); the VDRL Kline tests according to the VDRL Manual (1949), except
for the fact that cardiolipin antigen, issued and prepared by the Army Medical
School was used for the VDRL test.
EXPERIMENTS
I. In one experiment a set of 130 serums, all of which gave strong or moderately
strong reactions, were investigated. The serums were tested before and after
Received for publication October 29, 1954; accepted, J a n u a r y 14, 1955.
D r . K a n n e r is Chief of the Clinical Laboratory.
494
May 1955
SYPHILITIC
495
REAGIN
TABLE 1
E F F E C T OF G E L A T I N CONCENTRATION ON T I T E R
No.
Concentration
Factor
.1
2
3
4
5
6
7
8
0
LO
11
i\i
3
3
3
2'A
*A
2A
•iA
4
16
17
2K
2^
56
90
120
121
122
123
124
125
126
127
128
12!)
130
Kahr Units
Kl ine
10
40
10
10
30
5
10
80
10
10
10
20
100
30
30
80
20
20
240
40
20
20
+ 1:4
+ 1:6
+ 1:5
+1:4
+1:20
+1:3
+1:2
+1:4
so
40
160
100
v/2
160
6
3
4
2M
4
3
2
•iA
3
2
2
*A
2M
m
Kolmer C. F .
Eagl • C. F .
+1:6
+ 1:20
+1:8
+1:10
+ 1:60
+ 1:8
+1:3
nc
+ 1:10
+ 1.40
+1:5
+1:20
+ 1:80
+l:S
+1:10
+1:60
+1:10
+1:2
+1:2
nc*
nc
+ 1:3
nc
nc
+ 1:9
+1:8
nc
+ 1:S
nc
nc
+ 1:10
+ 1:2
+1:3
+1:10
+ 1:8
+ 1:8
+1:60
+1:10
+1:2
+ 1:5
nc
nc
nc
+ 1:20
+ 1:10
+ 1:10
nc
nc
nc
nc
+ 1:5
+ 1:5
+1:20
+1:5
+1:10
+1:5
nc
+ 1:20
+1:20
+1:40
+ 1:S0
+ 1:S0
640
+ 1:20
+1:80
+1:10
+1:80
100
640
+1:5
+ 1:20
+ 1:40
+1:200
+ 1:40
+ 1:100
20
640
30
10
20
20
60
80
40
160
10
60
2580
SO
40
60
50
200
160
80
320
20
+
+
+
+
(4)
1:5
(4)
(4)
+ 1:5
+ 1:20
+1:5
+ 1:5
+1:10
+1:100
+1:10
+ (4)
nc
nc
nc
nc
+1:5
+1:40
+1:5
±
nc
nc
nc
+ (2)
+
+
+
+
+
(4)
1:10
1:2
1:3
1:5
+1:5
+ 1:30
+ 1:5
+ 1:10
+1:10
+1:10
+1:40
+1:5
+ (4)
+1:100
+1:10
+1:20
nc
nc
nc
nc
nc
+1:10
+1:4
+ 1:10
+ (4)
+1:20
nc
nc
nc
nc
nc
—
—
—
—
+1:20
nc
* nc indicates no change.
gelatin concentration; the concentrations varied between 2-fold and G-fold with a
mean of 3.4 ± 0.8 standard deviation. Standard antigens were used in this series.
Quantitative Kahn tests were performed on all of the serums; the Kline test
with 115 serums, the Kolmer complement-fixation test with 128, and the Eagle
complement-fixation test with 125. An analysis of the results, represented in
Table 1, reveals the following: With the 2 complement-fixation tests the results
were not significantly influenced by the concentration except for serums No.
17, 56, and 90 with the Kolmer test, and No. 16 and 90 with the Eagle test, where
the titer increased considerably following concentration. The 2 fiocculation tests
led to results of a different nature; here the titer increased nearly in proportion
with the concentration obtained by the gelatin treatment. The correlation be-
496
KANNER
Vol. 25
tween the number indicating how much larger the volume of the serum was
before the gelatin was introduced, and the corresponding relative increase in
titer are apparent with both the Kahn and Kline methods. There seems to be a
little more precision with the Kahn test, indicating that the quantitative Kahn
test was better for reproducibility in our hands than the Kline. A statistical
analysis of the figures pertaining to the Kahn procedure yields pertinent information. Under the hypothesis that the titer increases in the same proportion as
the concentration of the serum by the gelatin treatment, the mean factor by
which the original titer should be multiplied to obtain the new one is calculated as
3.4 ± 0.8 standard deviation (S.D.). Actually, the mean factor found by comparing the original and subsequent titer was 3.2 ± 0.9 S.D. The difference of the means
between the calculated and observed titers following concentration is 0.2 ± 0.05
S.D. The coefficient of correlation between the 2 sets of values is 0.961: When
fiducial limits at the 1 per cent level are applied, it is found that the coefficient
of correlation lies between 0.939 and 0.975. Statistical tables* show that with a
sample as large as ours (130) a correlation coefficient of only 0.228 would already
be significant at the 1 per cent level.
Comment
The observed data support the idea that the titer of the Kahn and Kline fiocculation tests varies nearly in proportion with the serum concentration, while
the Kolmer and Eagle complement-fixation tests tend to retain their original
titer under the same conditions. The findings suggest a behavior depending upon
whether the performed test is a flocculation test or involves complement fixation;
however, we have previously found2 that with respect to gelatin concentration,
another flocculation test, the Vernes test, sides with the complement-fixation
group because its reproducible results remain unaffected by gelatin concentration
(Table 2). In a few cases, we have performed the VDRL flocculation (tube) test
before and after gelatin concentration; the results show that this test behaves
like the standard Kahn and Kline; a proportional increase in titer occurs with
gelatin concentration. For instance, with a four-fold concentration the titers
changed from 2 to 8 dils, and from 8 to 32 dils.
It is suspected that at least 2 reagin principles are at play. One is diffusible
into swelling gelatin and therefore is of a relatively small molecular size; the
Kolmer and Eagle complement-fixation tests and the Vernes flocculation test
depend upon it. Apparently they are not affected by the other principle which
is incapable of penetrating the gelatin by diffusion and therefore is assumed to
be of rather large molecular size; its presence is revealed in the Kahn, Kline and
VDRL flocculation tests. However, the picture becomes more complex when one
considers weaker reactors. Table 3 depicts the behavior of 20 cases with 4 Kahn
units or less. As in the first experiment, the Kahn, Kline, Kolmer and Eagle
tests were performed before and after gelatin concentration. The Kahn test remained unchanged in 19 of the 20 serums; one serum changed from doubtful to
* For instance, Snedecor, Statistical Methods, 4th edition, The Iowa State College Press,
Table 7.3, p. 149.
May
1955
SYPHILITIC REAGIN
497
TABLE 2
VERNES TEST
Original
Concentrate
1
2
119
52
120
3
4
10
42
No.
51
10
40
17
14
5
6
7
29
25
13
32
28
28
15
8
9
10
31
91
90
TABLE 3
No.
1
2
Concentr.
Factor
2
4
3K
3
4
4
5
6
7
VA
V/2
•A'A
8
4
9
3H
10
11
12
13
14
15
16
17
18
19
20
3
4
3
4
4
4
•iA
4
4
4
4
Kahn
±
±
±
±
±
±
±
±
±
±
4
4
4
4
4
4
4
4
4
4
Kl ne
nc
±
Kolmer C. F.
nc
10
nc
nc
nc
nc
nc
nc
nc
nc
nc
nc
nc
nc
nc
nc
nc
nc
nc
nc
±
±
±
±
±
nc
1:1
nc
nc
nc
__
+1:2
nc
+1:3
+1:7
±
±
+ (4)
±
±
±
±
ac
+ (4)
±
+1:5
+1:3
+1:12
+1:3
+1:10
+1:5
+1:10
+1:3
+1:4
Eagle C F.
nc
+ (4)
nc
nc
nc
nc
nc
ac
nc
nc
nc
nc
nc
nc
nc
nc
nc
nc
nc
+ (2)
+ (2)
±
±
nc
nc
nc
nc
+ (2)
+ (2)
nc
+1:20
nc
+1:3
nc
+1:12
+ 1:5
nc
nc
nc
10 Kahn units. No definite change occurred with the other tests. This experiment
shows that in general the postulated nondiffusible reagin of large size is not
manifest with the weak reactors and that the weak reactions may depend upon
the other type which is smaller and diffusible. Incidentally, the findings remove
the hope that gelatin concentration could be used to increase the sensitivity of
the tests with weak reactors.
II. Another series of experiments was then performed with the intent of confirming or discrediting the idea of the complex nature of what has been called
reagin. A separation was attempted. The experimental design was based upon
the following consideration: If it is true that the Kolmer and Eagle complementfixation tests depend upon a form of reagin that diffuses into the gelatin, some of
498
KANNEH
Vol.
25
TABLE 4
K.U.
No. .1
K.U.
N o . 18
No. 19
K.U.
Orig.
Cone.
4X
40
160
Orig.
Cone.
2J4X
SO
200
Orig.
Cone.
3X
160
500
Orig.
Cone.
2X
20
40
100
80
80
nu.
Redil.
Redil.
4X
and
and
and
Reconc. Reconc.
Reconc.
160
.160
160
Dil.
Redil. Redil.
2'AX
and
and
and
Reconc. Reconc.
Reconc.
160
160
200
Redil.
Dil.
Redil.
3X
3X
and
and
and
Reconc. Reconc. Reconc.
500
500
500
SO
Redil.
and
Reconc.
160
Redil.
and
Reconc.
200
Redil.
and
Reconc.
480
Redil.
Dil.
Redil. Redil.
and
2X
2X
and
Reand
and
Reconc. conc.
Reconc. Reconc.
40
40
40
40
Dilution
io
Orig.
Cone. 2'/zX
Dil. 2\iX Reconc.
Redil. & Reconc.
Redil. & Reconc.
Redil. & Reconc.
Dilution
Orig.
Cone. 3X
Dil. 3X & Reconc.
Redil. 3X & Reconc.
Redil. & Reconc.
Redil. & Reconc.
Dilution
Orig.
Cone. 2X
Dil. 2X & Reconc.
Redil. 2X & Reconc.
Redil. & Reconc.
Redil. & Reconc.
©
-
o o
o
oo oo
++
++
+
Dilution
Orig.
Cone. 2V2X
Dil. 2}iX & Reconc.
Redil. & Reconc.
Redil. & Reconc.
Redil. & Reconc.
©
++
++
++
+
Dilution
Orig.
Cone. 4X
Dil. 4X & Reconc.
Redil. & R e c o n c .
Redil. & Reconc.
Redil. & Reconc.
o
o
-
©
©
oo o©
+++
+++
+++
+
o
©
©
©
++
++
++
++
+
-
©
o
©
©
-*
+++ 1 1 1
K.U.
100
Redil.
Dil.
Redil.
Redil.
and
2iiX
and
and
and
ReReconc. Reconc. Reconc. conc.
1:100
|
No. 2
40
Cone.
2H X
1 1 ++++
K.U.
Orig.
++++++
No. 1
|
REAGIN'
1:200
S E P A R A T I O N OF " K O I . M E R R E A G I N " FROM " K A H N
©
+
+
+-
©
oo
May 1955
499
SYPHILITIC REAGIN
T A B L E 4—Continued
No. 20
K.U.
Orig.
Redil. Redil. Redil.
Dil.
and
Cone.
3X
3X
and
Reand
and
3X
Reconc. Reconc. Reconc. conc.
320
1280
1280
1280
1000
640
Dilution
Orig.
Cone. 3X
Dil. 3X & Reconc.
Redil. 3X & Reconc.
Redil. & Reconc.
Redil. & Reconc.
o
++++
++++
++++
+++
+++
++
oo oo oo
•*•
-
it must be present in the swelled gelatin and be removed with it, leaving the
concentration of this reagin unchanged in the reduced volume of serum. If now
the remaining serum is rediluted with saline, and reconcentrated with gelatin,
and if this cycle is repeated a number of times, the remaining serum would lose
most of the diffusible reagin because each fresh gelatin strip removes some of it.
Hence, a positive serum would eventually become negative with respect to the
Kolmer and Eagle tests. Conversely, if the Kahn and Kline tests depend upon
nondiffusible reagin, the titers should remain unchanged. Such experiments were
performed with 20 reactors. The results are tabulated in Table 4. Each serum
was first concentrated with gelatin and then rediluted with physiologic saline
and reconcentrated 4 consecutive times; Kahn aiid Kolmer tests were performed
on the original specimen, also following the first concentration, and following
each reconcentration preceded by redilution; each serum was tested in this
manner 6 times altogether. Again we found the same contrast between the Kahn
and .the Kolmer tests. The results show that the Kolmer titer diminishes progressively following the cycles of redilutions and reconcentrations; in 12 of the
serums the Kolmer test became entirely negative. The results with the Kahn
test resemble the ones of the first experiment which were given in Table 1. The
titer increases proportionally with the concentration of each serum; it had a
slight tendency only to drop following the cycles of dilution and concentration,
A statistical analysis yields the following information, similar to the findings in
the first experiment: The concentrations accomplished with the gelatin had a
mean value of 3.0 ± 0.8 S.D. Following the first concentration the mean remained at 3.0 ± 0.8 S.D. At the end of all cycles this mean value was still 2.3
± 0.9 S.D. The coefficient of correlation between the set of original and final
titers was found to be 0.888, and, with fiduciary limits at the 1 per cent level
between 0.651 and 0.966. In this series of 20 cases a coefficient of only 0.575 would
have been significant at the 1 per cent level.* The coefficients of correlation may
contain some bias owing to experimental error, in this series as well as before,
but they are consistently high enough to be regarded as safe.
DISCUSSION
The experimental evidence indicates that the syphilitic reagin apparently has
2 components. One is of relatively large molecular size, necessary and sufficient
* Cf. footnote on p. 496.
500
KANNBR
Vol. 25
for the appearance of moderately strong or strong reactions with the Kahn,
Kline and VDRL flocculation tests, but not sufficient to cause positivity with
the Kolmer and Eagle complement-fixation tests or the Vernes flocculation test.
The other is of relatively small molecular size and necessary for the appearance
of positive reactions with the Kolmer, Eagle and Vernes tests; however, the
question remains whether it is also sufficient. In an attempt to answer this question, gelatin strips swelled in positive serums were introduced into negative
serums. As it had been demonstrated that the reagin of small molecular size
enters the gelatin, it may be assumed that those strips would then contain it,
and that it possibly could be eluted into the negative serum which thereby may
become positive. However, numerous attempts in this direction failed to change
negative serums to positive ones. This behavior suggests that either the reagin
of small molecular size is not sufficient to cause positive reactions or that elution does not occur. Similar results were noticed when the swelled gelatin strips
were reinserted into positive serums, made negative with respect to Kolmer and
Eagle tests. No reactivation was observed. Therefore, it appears probable that
elution was not obtained. The reagin in question may have greater affinity for
gelatin than for serum. The problem could perhaps be settled by means of ultrafiltrates.
A change in terminology is now proposed. We should like to call "large molecule reagin" simply reagin and to designate "small molecule reagin" as coreagin.
The behavior of the weak reactors requires an explanation. So far, we have
silently assumed that the activity of reagin in a serum is proportional to its concentration. There seems to be little doubt that such proportionality exists over
considerable ranges, as is evident by the experiments with several flocculation
tests. But we have no experimental justification to assume that such proportionality holds over all ranges, in particular the low ones. For instance, assumed
hydrogen ion concentration was used to define the pH. This simplifying assumption cannot always be justified in practice. Instead, we speak now of hydrogen
ion activity and define the pH in terms of certain electrode potentials. Similar
considerations hold for other constituents of solutions. In the light of a concept
of similar activity, the results obtained with the weak reactors could be accounted
for if one assumes the activity to be relatively small with low concentrations of
reagin. Figure 1 illustrates this idea. Let the abscissa represent the hypothetical
reagin concentration and the ordinate the hypothetical activity. Assume that a
segment of the ordinate, between a' and b', represents the region equivalent to
4 Kahn units, for instance. The corresponding points on the abscissa are a and b.
If the functional relationship between activity and concentration resembles the
one depicted by the curve, a relatively large increment in concentration of reagin
will correspond to a relatively small increment of activity. Hence it would be
plausible that as much as 4-fold concentration, as was applied (cf. Table 4) for
the weak reactors, would not affect the quantitative result of the test. The experimental evidence would support such a theory. Also, it should be assumed
that to the right of the point b the curve is linear and has a sharper slope than
to the left.
May
1955
SYPHILITIC REAGIN
a
501
i>-^REAGIN CONCENTRATION
FIG.
1
Our findings would help the understanding of discrepancies, observed by serologists, in the results obtained with different methods. Baylis and associates1
believe that the Vernes test depends, in part, upon other features in the patient's
blood than does the Wassermann reading. Quite recently Thomas 3 suggested
that reagin in the blood serum consists of more than one presumed antibody.
The gelatin experiments not only support such ideas but reveal actual mechanisms to account for differences between various tests.
SUMMARY
Experimental work suggests a complexity of the reagin principle. In particular,
evidence for the existence of a coreagin is presented. Its presence was found necessary to explain the findings with the Kolmer and Eagle complement-fixation
tests and the Vernes flocculation test; its sufficiency was not ascertained. Its
absence did not influence the results of the Kahn, Kline and VDRL flocculation
tests. It is possible to separate the postulated coreagin from the reagin. In this
way, serums, originally positive with Kolmer and Kahn methods, lost all of their
reactivity with respect to the Kolmer method, while at the same time the Kahn
titers remained substantially intact.
The evidence tends to show that with weaker reactors the relationship between
concentration of reagin and activity of reagin is not the same as with stronger
reactors, and that the coefficient of activity of reagin is smaller with low concentrations of reagin.
REFERENCES
1. B A Y L I S , A. B . , SHKPLAR, A. E., AND M A C N B A L , W. J . : T h e Vernes flocculation test as an
accessory serological guide in t h e combat against syphilis. Am. J. Syph., 10: 29S-337,
1926.
2. K A N N E R , 0 . : Method for immunological and chemical investigations of body fluids by
means of purified gelatin. Science, 104: 253-254, 1946.
3. THOMAS, E. W.: Blood and spinal fluid tests for reagin after t r e a t m e n t of neurosyphilis.
J. A. M. A., 153: 718-722, 1953.