UNIVERSAL SEROLOGIC REACTION WITH LIPID ANTIGEN
I. I N NORMAL PERSONS*
REUBEN L. KAHN, Sc.D.
From the Serological Laboratory, University Hospital, University of Michigan, Ann Arbor,
Michigan
The serologic reaction to be considered in this series of articles is unique in
several respects. It is a precipitation reaction which lias been found in all human
beings and animals tested thus far; yet, it is differentiable both in different human
beings and in animals. Each normal person gives a reaction of an individual
serologic pattern and indications are that this pattern remains unchanged
in health and assumes new characteristics in disease. This reaction has been
studied thus far in syphilis, leprosy, malaria and tuberculosis. The results in
normal human beings, in animals, in these four diseases and in others, as well
as in special animal experiments, will be presented in this series of six articles
and in later publications. These six articles will be brief and preliminary in
nature because it is aimed to determine first the general behavior of the reaction
in health and disease before undertaking fundamental studies of the reaction
and of its detailed applicability to any given disease. In this article, the report
will be limited to universal serologic findings in normal human beings.
The reaction under consideration is referred to as "universal serologic reaction" 16, IS because experimental indications "are that it is a widespread biologic
manifestation. The antigen employed in the reaction, which consists of an alcoholic extract of heart muscle, is the same as that used in tests for syphilis;
the technical steps, however, are entirely different. The reaction is based on
a precipitation technic which combines different concentrations of sodium chloride, serum dilutions and incubation periods at cold temperature as variables.
These three variables form the basis of the different serologic patterns noted
under conditions of health and disease.
HISTORICAL,
The foundation of the universal serologic technic began to be laid in this
laboratory in .1922.° We then reported on the employment in serologic precipitation studies in syphilis (1) of a salt solution range extending from 0 to 2 per cent
NaCl and (2) of incubation at cold temperature, 6 two basic steps in the universal
technic. Those studies, however, were limited to syphilis. Among the several
factors affecting precipitation which we investigated at that time was the effect
of dilution on the formation of precipitates in mixtures of syphilitic serum and
tissue extract antigen and we observed that NaCl solutions ranging from 0 to 2
per cent, when added in sufficient quantity to these mixtures, will interfere with
* This study has been conducted with the aid of a grant from the Office of Naval Research,
Medical Sciences Branch, Navy Department, Washington, D. C.
Received for publication, October 28, 194S.
347
348
KAHN
precipitation. In 1925 we observed7 that the sensitivity of standard Kahn reactions with syphilitic serums is considerably reduced if the diluent of physiologic
salt solution added to the serum-antigen mixture is replaced by water. This
observation suggested that an increase in the sensitivity might be attained by
the employment of salt concentrations above the physiologic level. This surmise proved true, and in 1928 we reported10 that increasing the concentration of
the salt solution above the physiologic level increases the sensitivity of the precipitation reactions with syphilitic serums until about 3 per cent NaCl is reached,
when non-syphilitic serums begin to give weak reactions, while decreasing the
salt concentration below 0.7 per cent decreases the sensitivity of the reactions
with syphilitic serums. Yet, the increase in the NaCl concentration in the
standard Kahn test in practice was limited to 0.9 per cent 9 in order to prevent
oversensitive reactions.
Dunlop and Sugden2 reported precipitation reactions with nonsyphilitic serum
and Sachs-Georgi antigen by employing NaCl concentrations below 0.2 per cent,
while with concentrations extending from 0.5 to 1.0 per cent, they obtained
reactions only with syphilitic serums. Mackie and Anderson20 obtained precipitation with syphilitic serums and various tissue extract antigens when employing NaCl concentrations below 2.5 per cent but not with nonsyphilitic
serums. These workers were thus unable to corroborate the nonspecific precipitation findings reported by Dunlop and Sugden with low salt concentration.
In 1942 it was reported12 that, under proper technical conditions, syphilitic
and nonsyphilitic (false positive) serums employed with Kahn antigen suspension are affected differently by different NaCl concentrations; that an increase in
salt concentration from 0 to 2.5 per cent increases the intensity of syphilitic
reactions and decreases the intensity of nonsyphilitic reactions. This observation formed the basis of a practical technic as an aid in the differentiation between
syphilitic and nonsyphilitic reactions. In the same year, Green and Shaughnessy4 reported that nonsyphilitic human serums, giving negative reactions with
the standard Kahn test, showed strong precipitation with Kahn antigen in a
salt range of 0 to 0.3 per cent and weak precipitation in a salt range of 6 to 14
per cent NaCl after the tests had stood overnight; the reactions in salt ranges
above 0.3 per cent and below 6 per cent they found to be negative. Syphilitic
serums in their hands gave maximal precipitation between 2 and 8 per cent NaCl
and practically no precipitation between 20 and 25 per cent NaCl. These
authors also stated that, by the use of different salt concentrations, they obtained
characteristic aggregation and dispersion patterns of the Kahn precipitate with
serums from different animal species. Their experimental data are yet to be
published.
In 1943 it was reported16 from this laboratory that Kahn reactions given by
rabbit, pig and cow serums became progressively stronger the lower the salt
concentration employed. The NaCl concentration range was from 0 to 10 per
cent. This behavior of animal serums was in contradistinction to that of syphilitic serums which had been found to give stronger Kahn reactions with increasing
salt concentration. In 1947 it was also reported17 that, by employing reduced
SEROLOGIC REACTIONS IN NORMAL PERSONS
349
NaCl concentration such as 0.3 per cent in the Kahn technic, the serums from
patients with malaria exhibit a far greater tendency toward precipitation than
normal serums.
With regard to studies of the effect of incubation at cold temperature on precipitation with lipid antigen, we reported 6 in 1922 that mixtures of syphilitic
serum and antigen suspension show least precipitation when incubated at ice
box temperature, more marked precipitation when incubated at room temperature and most marked precipitation when incubated at 37 C. The period of
incubation employed was four hours. A similar effect was noted in connection
with Sachs-Georgi reactions by Sachs.28
The favorable effect of a short incubation period at warm temperature on
syphilitic reactions was utilized for some time in the routine Kalui procedure. 3
Muckenfuss and Ebel24 also reported on the effect of brief incubation periods
on the sensitivity of Kahn reactions. At a temperature of 37 C. the sensitivity
was increased, but not at ice box temperature. Prolonged incubation at ice box
temperature tended to give false positives. Nagle and Lazarov reported25 on a
comparative study of overnight incubation at three temperatures of completed
Kahn tests. They found that at 37 C. the precipitates tended to disperse, at
room temperature they were unaffected, while ice box temperature rendered the
tests oversensitive.
In 1940, we showed11 that syphilitic and nonsyphilitic serologic reactions require different temperatures for optimal results; that warm temperature (37 C.)
favors specific precipitation, while cold temperature, such as 1 C , favors nonspecific precipitation both with human and animal serums. This observation
formed the basis of a verification technic as an aid in the detection of false
positives. We then showed15 that, by the use of an excessively sensitive tissue
extract antigen and by the performance of the tests at 1 C , close to 100 per cent
precipitation reactions in normal human beings and in different animals (hog,
chicken, horse, cow and rabbit) could be obtained. Quantitative differential
temperature studies in different animals were reported by Marcus, 23 and Carter 1
suggested the use of quantitative determinations in the author's verification
technic.
In 1946 we reported13 that it was possible to elicit nearly 100 per cent positive
serologic reactions in normal human beings with antigens that gave specific
reactions in tests for syphilis, such as Kolmer, Kahn and cardiolipin antigens,
by certain technical steps which included low salt concentration combined with
prolonged incubation at cold temperature. It was then decided to combine
different NaCl concentrations ranging from 0 to 2.1 per cent, different serial
dilutions of serum and different incubation periods at cold temperature in one
serologic technic with a view of determining the type of reactions which would
be given by human beings and animals. These efforts led to the standardized
universal serologic technic described below.
UNIVERSAL SEROLOGIC TECHNIC
The universal technic consists of nine quantitative procedures, referred to as
"set-ups", of different NaCl concentrations. Blood specimens are obtained in
350
KAHN
the same manner as for serologic tests for syphilis. The serum is separated from
the clot by centrifugation and is heated for 30 minutes at 56 C. before use. Nine
NaCl solutions of the following concentrations are prepared: 0, 0.15, 0.3, 0.6,
0.9, 1.2, 1.5, 1.8 and 2.1 per cent. Serial serum dilutions are then prepared with
each of these nine salt solutions in the following ratios: 1:1, 1:2.5, 1:5, 1:10,
1:20, 1:40, 1:80, 1:160, 1:320 and 1:640. In some instances, the ratios of
serum to salt solution employed extended to 1:200.
Tests are set up as for nine quantitative Kahns, but in which each quantitative
employs serum dilutions with a solution of a given NaCl concentration. The
first quantitative is carried out with serial dilutions of serum with water; the
second, with 0.15 per cent NaCl solution and so on; and the ninth quantitative
is carried out with serial dilutions of serum with 2.1 per cent NaCl solution.
Kahn antigen suspension is prepared with 0.9 per cent NaCl solution in accordance with the Kahn standard technic. The ratio of serum dilution to antigen
employed is 6:1. The antigen suspension is pipetted to the bottom of the tubes
in 0.025 ml. amounts, followed by 0.15 ml. amounts of the dilutions of each
serially diluted serum. The mixtures of serum dilutions and antigen suspension
are then agitated for three minutes in the Kahn shaker after which 0.5 ml. of
diluent is added to each tube, the contents mixed and the results read.
The diluent in each quantitative set-up consists of salt solution of the same
concentration as was used in the preparation of the serum dilutions. The
method of reading is similar to that employed in the Kahn test. Three readings
are made. The first is made after mixing the reagents following the addition of
the diluent to each tube. The second reading is made after four hours' incubation
in the ice box (about 5 C.) and a third, after 24 hours in the ice box. Four plus,
three plus or two plus readings are recorded as positive reactions, while one plus
and borderline readings are recorded as doubtful reactions. In charting the
results graphically, only the positive reactions were recorded.
When reading the results after incubation, it is well to remove one rack at a
time from the ice box. This step will help retain the cool state of the serumantigen mixtures until the actual reading time, as some precipitates will redisperse when the mixtures are unduly exposed to the warmth of room temperature. As each rack is taken from the ice box and brought to the reading table,
a film of moisture will cover the tubes. To remove this film each tube is dipped
in cold 95 per cent alcohol immediately before reading the precipitation results.
It should be added that, in the quantitative set-up of the universal technic in
which the serums are serially diluted with water, we may be dealing with a situation in which the separation of globulin in the high dilutions might be a factor in
precipitation with lipids. The interesting finding in that particular set-up is the
absence of precipitation noted in the controls.
The universal serologic technic embraces a serodiagnostic technic for syphilis.
Thus, the quantitative set-up with 0.9 per cent NaCl solution in which the results are read without incubation, is similar to the quantitative Kahn test.
Hence, universal reactions can be readily correlated with serodiagnostic reactions
for syphilis. If this quantitative set-up with 0.9 per cent salt solution shows precipitation with a given serum, that serum may be considered as giving also a
SEROLOGIC REACTIONS IN NORMAL PERSONS
351
positive reaction with a test for syphilis. No suggestion of positivity for syphilis
is noted in the table and charts presented in this article.
The universal serologic technic for the present is intended for research purposes. Indications are that it has potential value in serologic studies in health
and disease. I t is recognized that in its present form, the universal technic is
quite elaborate, involving nine quantitative set-ups. To reduce the technical
steps, five quantitative set-ups are being employed in this laboratory for investigative purposes in certain situations, as, for example, in tuberculosis. The
salt concentrations used in these five set-ups are: 0, 0.3, 0.9, 1.5, and 2.1 per cent.
Only actual trial can best determine the optimal number of quantitative setups to employ in the investigation of the universal serologic response in a given
disease. It is well, when beginning the investigation of the serologic response in
a disease, first to employ the nine quantitative set-ups in order to note the more
complete serologic pattern. A reduction in the number of quantitative set-ups
can then be brought about without reducing the value of the results obtained.
In certain situations in animals, it may seem desirable to use special quantitative set-ups. For example, in experimental serologic studies in mice and guinea
pigs, quantitative set-ups of 0, 0.15,1.8 and 2.1 per cent NaCl solution might best
be employed, since, as will be seen in the following article, the central zone of the
universal reaction has consistently given negative precipitation results in these
animals. However, these animals have as yet not been studied under pathologic conditions.
In recording the results of the universal serologic technic, it is suggested that
the serologic readings be illustrated by graphs. Tabular data, it seems to us,
do not bring out the characteristics of the serologic patterns as clearly as graphs.
UNIVERSAL SEROLOGIC REACTIONS IN DIFFERENT HUMAN BEINGS
The universal serologic technic was developed in this laboratory in the spring
of 1946, and since then it has been continuously in use in human beings in health
and in certain diseases and in animals under normal and experimental conditions.
More than 400 universal reactions have been obtained in normal human beings
but for reasons of economy of space only six reactions will be illustrated in this
preliminary report.
Table 1 presents a universal reaction given by a normal individual. The nine
quantitative precipitation set-ups employed, are arbitrarily divided into three
zones. The three precipitation set-ups of the lowest salt concentrations (0,
0.15 and 0.3 per cent NaCl) are grouped under Zone I. The three precipitation
set-ups close to physiologic salt concentrations (0.6, 0.9 and 1.2 per cent NaCl)
are grouped under Zone II, while the three set-ups of the highest salt concentrations (1.5, 1.8 and 2.1 per cent NaCl), under Zone III.
As is shown in the table, no precipitation was obtained in the quantitative setups when the readings were made without incubation, with the exception of the
three highest serum dilutions of the quantitative set-up in which water was used
as diluent. After four hours' incubation, precipitation was obtained mainly in
the quantitative set-ups of 0 and 0.15 per cent salt concentrations on the one
352
IvAHN
hand, and of 1.8 and 2.1 per cent salt concentrations on the other. After 24
hours' incubation, precipitation reached the quantitative set-ups of 0.3 and 1.5
TABLE 1
UNIVERSAL SEROLOGIC REACTIVITY WITH LIPID ANTIGEN
Quantitative precipitation results with increasing concentrations of NaCl solutions
ranging from 0 to 2.1 per cent, employing normal (Kahn-negative) human serum.
CONCENTRATIONS OF N a C l (PER CENT) USED IN PREPARATION OF SERIAL DILUTIONS OF SERUM
Zone I
0.15
Zone I I I
Zone II
0.3
0.6
Readings made without incubation of serum-antigen suspension mixture
—
-
-
-
-
-
-
-
1
1 -H
.1
:2.5
:5
10
•20
:40
80
•160
:320
640
±
4
4
4
-
-
-
-
-
-
-
—
±
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
R e a d i n g s after 4 h o u r s ' incubati on in ice box
1
1
1
1
1
1
1
1
1
1
1
2.5
5
10
20
40
80
160
320
640
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
2
—
2
±
±
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
_
—
—
—
—
—
—
—
—
—
_
—
—
—
—
—
—
—
—
2
±
—
—
—
—
—
—
—
4
4
2
2
2
2
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
' 4
4
4
4
4
4
4
4
4
4
4
Readings after 24 h o u r s ' i n c u b a t i o n in ice box
1:1
1:2.5
1:5
1:10
1:20
1:40
1:80
1:160
1:320
1:640
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
±
±
4
4
4
—
—
—
—
—
—
_
—
—
—
—
—
—
—
_
—
—
—
—
—
—
_
—
—
—
—
—
2
2
2
±
—
per cent salt concentration. These precipitation reactions are graphically illustrated in Figure 1.
SEROLOGIC REACTIONS IN NORMAL PERSONS
353
Figure 2 exhibits a serologic pattern similar to that of Figure 1 and is here
presented to show that, while serologic patterns of different normal individuals
generally differ from one another, now and then two serologic patterns are observed which are closely similar. A common characteristic of Figures 1 and 2
is that the degree of precipitation after four and 24 hours' incubation at cold
temperature is approximately the same.
Figure 3 exhibits a serologic pattern different from the first two figures, especially in the fact that precipitation is most pronounced after 24 hours' incubation.
No precipitation is noted without incubation. After four hours' incubation,
precipitation is limited to Zone I. After 24 hours' incubation, precipitation is
noted in all three zones. No increase in precipitation is noted in Zone I. In
Zone II, precipitation reaches the serum dilution ratio of 1:2.5, while in Zone III,
precipitation reaches the serum dilution ratio of 1:640.
A still different serologic pattern is illustrated in Figure 4. In Zone I, a similar degree of precipitation is noted both before and after incubation. In each
of the three readings, precipitation reaches the serum dilution ratio of 1:640.
No precipitation is noted in Zone II on immediate reading and after four hours'
incubation. After 24 hours' incubation, precipitation reaches the serum dilution ratio of 1:2.5. In Zone III, considerable precipitation is noted immediately
and on incubation, but only in relatively low dilutions. Precipitation reaches a
dilution ratio of 1:10 without incubation, 1:20 after four hours' incubation and
1:40 after 24 hours.
Figure 5 is characterized by practically no precipitation without incubation,
by limited precipitation in Zones I and III after four hours' incubation and by
considerable precipitation in all three zones after 24 hours' incubation. A still
different serologic pattern is noted in Figure 6. Some precipitation without incubation is noted in Zone I. A relatively marked increase in precipitation is
noted after four hours' incubation and only a slight increase after 24 hours' incubation. Of interest is the fact that precipitation after 24 hours' incubation
illustrated in this chart is approximately the same as that illustrated in Figure 5.
Yet, the precipitations on immediate reading and after four hours' incubation
are altogether different in the two charts.
I t must not be assumed that the universal reaction herewith considered is
elicited only by standard Kahn antigen. Sensitized Kahn antigen, which is
about 10 per cent more sensitive than standard antigen with syphilitic serums,
gives broadly similar universal reactions. Somewhat more marked precipitation is obtained with this antigen than with standard antigen. An antigen
prepared from powdered liver, instead of from powdered beef heart, was found to
give reduced precipitation. Various lipid antigens are under investigation as
well as various technics. The main reason the universal technic was standardized with Kahn antigen is that methods are available for maintaining the antigen
at a standard level of sensitivity. This antigen has been employed in the universal technic for nearly three years with consistent results. Different lots of
Kahn antigen have been found to give the same serologic patterns. Furthermore, each individual, under conditions of health, examined from time to time,
354
KAHN
NORMAL(KAHN-NEGATIVE)HUMAN SERUM
AFTER 31 HOURS INCUBATION
IN ICEBOX
AFTER 4 HOURS INCUBATION
IN ICEBOX
WITHOUT INCUBATION
640r
|
320
o
c
o
o
o
o
'
O
n
c
(LOGARITHMIC SCALE)
SERUM DILUTIONS WITH NaCl SC
_1
.6
.9
1.2 1.5 1.8 2.1
0
.3
.6
.9
1.2
1.5
0
1.8 2.1
.3
.6
.9
1.2
1.5
1.8 2.1
CONCENTRATION OF NaCl (?•)
X////A
] ZONE OF NEGATIVE
ZONE OF P O S I T I V E . REACTIONS
REACTIONS
FIG. 1. Zones of precipitation, with NaCl concentrations ranging from 0 to 2.1 per
cent, without incubation and after 4 and 24 hours' incubation in ice box.
,..; ,,
WITHOUT
<0
z
o
i-J
O
is
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3
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NORMAL (KAHN-NEGATIVE)HUMAN
INCUBATION
AFTER 4 HOURS INCUBATION
IN ICEBOX
200M-,
200
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100
80
60
100
80
60
40
40
20
20
10
10
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5
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2.5
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AFTER 2 4 HOURS INCUBATION
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1
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I .
SERUM
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.9
1.2
11
1.5
1.8
2.1
6
.9
1.2 1.5 1.8 2.1
CONCENTRATION OF NaCl [1.)
X/////X
Z O N E OF P O S I T I V E
REACTIONS
J
ZONE OF N E G A T I V E
REACTIONS
FIG. 2. Zones of precipitation, with NaCl concentrations ranging from 0 to 2.1 per
cent, without incubation and after 4 and 24 hours' incubation in ice box.
SEROLOGIC REACTIONS IN NORMAL PERSONS
355
NORMAL(KAHN-NEGATIVE)HUMAN SERUM
AFTER 4 HOURS INCUBATION
IN ICE80X
WITHOUT INCUBATION
«1
Z
o
640
640
320
320
640
160
160 \
80
80
40
40
20
20
o<
10
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33
5
5
2.5
2.5
o
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in
AFTER 24 HOURS INCUBATION
IN ICEBOX
'',
I
.6
.9
V///A
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1.8 2.1
<
0
3 .6 .9 12 1.5 18 2.1
C O N C E N T R A T I O N OF NaCI (•/.)
ZONE OF POSITIVE REACTIONS
ZONE OF NEGATIVE REACTIONS
FIG. 3. Zones of precipitation, with NaCI concentrations ranging from 0 to 2.1 per
cent, without incubation and after 4 and 24 hours' incubation in ice box.
NORMAL (KAHN-NEGATIVE) HUMAN SERUM
WITHOUT
AFTER 4 HOURS INCUBATION
IN ICEBOX
INCUBATION
AFTER 2 4 HOURS INCUBATION
IN ICEBOX
640*
640*
320
160
80
.3
.6
X////A
.9
1.2 1.5
1.8 2.1
0 .3 .6 .9 1.2 1.5 1.8 2.1
C O N C E N T R A T I O N OF NaCI (f.)
ZONE OF POSITIVE REACTIONS
2 ZONE OF NEGATIVE REACTIONS
FJG. 4. Zones of precipitation, with NaCI concentrations ranging from 0 to 2.1 per
cent, without incubation and after 4 and 24 hours' incubation in ice box.
356
KAHN
NORMAL (KAHN-NEGATIVE) HUMAN SERUM
AFTER 24 HOURS INCUBATION
IN ICEBOX
AFTER 4 HOURS INCUBATION
IN ICEBOX
WITHOUT INCUBATION
Z
o
60
o
ui
80
40
*i
20
£o<S
10
3.3.
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1.8 2.1
0
.3
.6
.9
1.2 1.5
1.8 2.1
.3
.6
.9
I.2
I.5
I.8 2.
CONCENTRATION OF NaCl (1-)
X////A
] ZONE OF NEGATIVE REACTIONS
ZONE OF POSITIVE REACTIONS
FIG. 5. Zones of precipitation, with NaCl concentrations ranging from 0 to 2.1 per
cent, without incubation and after 4 and 24 hours' incubation in ice box.
N O R M A L ( K A H N - N E G A T I V E ) HUMAN SERUM
WITHOUT
tD
_l
O
«0—
H
fa
320
160
80
/
40
20
(01-
o<
10
•-£
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5
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640*
<0
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o
AFTER 24 HOURS INCUBATION
IN ICEBOX
AFTER 4 HOURS INCUBATION
IN ICEBOX
INCUBATION
2.5
ft
.6
.9
1.2 1.5
IB 2.1
3
.6
9
1.2 1.5
1.8 2.1
I.2
I.5
18 2.I
CONCENTRATION OF NaCl (1-)
AL I
X////A
ZONE OF POSITIVE REACTIONS
ZONE OF NEGATIVE REACTIONS
FIG. 6. Zones of precipitation, with NaCl concentrations ranging from 0 to 2.1 per
cent, without incubation and after 4 and 24 hours' incubation in ice box.
SEROLOGIC REACTIONS IN NORMAL PERSONS
357
months apart, with different lots of Kahn antigen, has been found to give the
same serologic pattern.
NATURE OF UNIVERSAL SEROLOGIC REACTION
A consideration of the nature of the universal serologic reaction with lipid
antigen takes us into the realm of theory. At first, we believed that the reaction
represented a colloid-chemical interaction between serum and lipids, perhaps unrelated to antigen-antibody reactions. Indeed, having devoted many years to
the study of serologic reactions in syphilis with lipid antigen, it seemed difficult
to believe that the very same antigen could give positive reactions with all
serums tested. The possibility that these reactions are based on an unexplained
artefact was also considered. Continued studies, however, led us to abandon
these views in favor of a concept of antigen-antibody reactions.
As indicated above, each human being consistently gives an individual serologic pattern under conditions of health. That this pattern undergoes changes
in certain diseases will be seen in the present series of articles. It would thus
appear that we are dealing with a biologic reaction of a basic nature. Briefly,
as a working hypothesis of the nature of universal serologic reactions in normal
human beings, it is believed that, in the course of normal tissue wear and tear,
lipids are liberated which act as antigens in stimulating the formation of specific
antibodies and these react with tissue lipids in vitro.
Many explanations have been suggested as to the nature of serologic reactions
in syphilis with lipid antigen. Eagle 3 believes that these reactions are not due to
a general "state" of the serum proteins but to an actual substance which can be
absorbed from the serum. Mackie and Watson,21 in an extensive study on the
nature of the Wassermann and Sachs-Georgi reactions, concluded that they are
due to antibody-like substances homologous with antibodies natural to certain
species of animals. The diagnostic reaction in syphilis they believed to be due
to the nonspecific augmentation of this natural antibody-like substance. Ultramicroscopic precipitation studies carried out in this laboratory in 193122 led us
to conclude that both syphilitic and nonsyphilitic human serums possess the
property of precipitation when mixed with tissue extract antigen, syphJlitic
serums possessing this property to a marked degree and nonsyphilitic serums to
a slight degree. More recently Neurath and associates26 made comprehensive
studies of protein fractions of serums giving specific serologic reactions in syphilis
in relation to those giving false positive reactions. These workers believe that
the antibodies in syphilitic human serum possess chemical and/or serologic
properties which differ from the antibodies occurring in serum of false-positive
reactors.
Sachs, Klopstock and Weil27 injected in rabbits tissue lipids, the same as those
used as antigen in tests for syphilis, and the animals gave negative complementfixation and precipitation reactions for syphilis, but when the tissue lipids were
first mixed with pig serum and the mixture injected in rabbits, the animals gave
positive complement-fixation and precipitation reactions for syphilis. Tin's
358
KAHN
finding is in line with Landsteiner and Simm's observation19 that alcoholic extracts
of horse kidney (Forssman antigen) become antigenic when mixed with protein
solutions, such as human or pig serum. Sachs, Klopstock and Weil suggested
that tissue lipids, liberated as a result of tissue destructive processes in syphilis,
in combination with spirochetal protein, serve as the antigen which stimulates
antibody formation to lipids in syphilis.
However, theories which attempt to explain serologic reactions in syphilis
ought to explain also the occurrence of these same reactions under normal conditions in animals and in a number of diseases other than syphilis in human
beings. Actually, tests for syphilis give as high as 90 per cent of positive reactions with certain animal serums, such as horse and dog serums and perhaps
50 per cent with cow, chicken and rabbit serums. This, aside from the fact that
serologic reactions are obtained also in various diseases in human beings in the
absence of syphilis, and that in lepromatous leprosy these reactions are often
stronger than those obtained in syphilis.
Recalling our lipid antigen-antibody hypothesis presented above, the universal serologic reaction given by normal human beings is the result of the presence of natural antibodies to tissue lipids in human serum. According to this
hypothesis, the universal serologic reaction in different animals, considered in
the following article of this series, is also the result of natural antibodies to tissue
lipids. It follows that the positive reactions given by normal animals with
serodiagnostic tests for syphilis are also the result of the same natural antibodies, since the serodiagnostic technic is embodied in the universal technic.
Insofar as the nature of serologic reactions in syphilis is concerned, the antibody theories which attempt to explain this reaction may be grouped in two categories. One theory assumes the existence of antibodies in syphilis which are
distinctive from those of false-positive reactors, while the other assumes that the
syphilis reaction represents an augmentation of naturally existing antibodies.
The universal serologic studies suggest a theory which comprises both of these
theories. The fact that normal human beings give universal serologic reactions
would indicate the existence of a natural antibody to tissue lipids. In syphilis,
as will be seen in article III of this series, (1) the universal reaction becomes intensified over the normal response, indicating a quantitative increase in antibody
and (2) the same reaction also shows a distinctive serologic pattern, suggesting
the presence of an antibody qualitatively different from the normal.
Let us now apply the same lipid antigen-antibody theory to universal serologic
reactions in leprosy, malaria and tuberculosis, to be reported in the several
articles of this series. In these diseases, acquired antibodies to lipids appear in
the blood stream as a result of increased liberation of tissue lipids due to tissue
destructive processes. These acquired antibodies cause the universal reaction
to become quantitatively intensified over the reaction given by natural antibodies. This intensified reaction in some diseases may be so pronounced that
precipitation "overflows" into the zone of the serodiagnostic reaction for syphilis.
In lepromatous leprosy, as in syphilis, the universal reaction is not only intensi-
SEROLOGIC REACTIONS IN NORMAL PERSONS
359
fied quantitatively but it exhibits also some qualitative changes, as is evident
from the distinctive serologic pattern obtained in this disease. This distinctive
pattern suggests the liberation of particular lipids associated with this disease.
If the universal serologic reaction represents an antigen-antibody reaction,
it would be expected that it conform to basic requirements of antigen-antibody
reactions. Thus, any intensification of the normal universal reaction resulting
from disease should be preceded by an incubation period. Actually, in malaria,
for example, as will be seen later, a change in the normal universal reaction is not
noted until about two to three weeks after the first attack of chills and fever.
Other factors affecting antigen-antibody reactions might be mentioned. Thus,
it is well known that low resistance reduces the production of antibodies, also,
excess of antigen interferes with such production. An illustration of both
situations was observed in tuberculosis. While marked intensification of the
universal reaction was noted in acute tuberculosis, no intensification was noted
in prolonged chronic tuberculosis, and in terminal and miliary forms of this
disease.
Has the universal reaction in normal human beings any clinical value? The
answer is that the reaction was herewith presented primarily to demonstrate what
appears to be a new serologic manifestation, and any clinical value it may have
is yet to be established. It may be mentioned that, as indicated, the serologic
patterns of the universal reaction exhibited in health undergo changes in the
several diseases studied. Hence, in the investigation of these changes, the determination of normal patterns will obviously be essential as controls. Then
again, if it be true that the universal reaction in health represents a reaction of
natural antibodies to lipids, and the production of these antibodies is stimulated
by lipids liberated in normal tissue wear and tear, then this reaction might prove
to be of value as a serologic indicator of normal tissue wear and tear.
SUMMARY
A serologic reaction with lipid antigen is described which has been manifested
by all normal human beings and animals tested thus far and which is referred
to as "universal serologic reaction". The technic employed in eliciting this
reaction is based on precipitation and combines serum dilutions, NaCl concentrations and incubation as variables. The reactions given by different normal
persons are distinguishable within limits by individual serologic patterns.
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