Download ADVENTITIOUS SUBSTANCES REMOVED FROM SLIDE TEST

ADVENTITIOUS SUBSTANCES REMOVED FROM SLIDE TEST
ANTIGEN BY EXTRACTION WITH WATER
MIRIAM S. LEVY
From the Laboratory Department, Mt. Sinai Hospital, Cleveland, Ohio
Most present day antigens for tests for syphilis are composed of alcoholic
solutions of acetone insoluble phospholipids, especially diaminomonophosphatide, and more or less adventitious substances that may cause fajse positive
reactions. Much of the undesirable material is removed by preliminary treatment of the tissues used for antigen preparation with ether or acetone, or by
treatment of the residue of a primary alcoholic extract with acetone. A new
method of removing impurities and further increasing the specificity of an antigen by extraction with water has recently been reported 1 . Analyses of the
impurities removed from slide test antigen by extraction with water show small
quantities of all the important constituents of heart muscle with the exception
of proteins, creatine and glycogen.
In 1911 Noguchi and Bronfenbrenner2 made a careful chemical study of tissue
extracts using alcohol, ether and acetone in various procedures and determined
the value of the fractions isolated as antigen in the complement fixation test for
syphilis. They found that the acetone insoluble portion was the most potent
and specific fraction, and that such substances as neutral fats, fatty acids, soaps,
cholesterol and other unidentified lipoidal substances; proteins, cleavage products
of proteins, bile salts and other salts especially sodium chloride, were undesirable
in the antigen solution. Some of these latter substances (acetone soluble)
although having some antigenic power and increasing the activity of the antigen
extract also possess undesirable hemolytic and anticomplementary properties.
In 1917, Neymann and Gager3, using the more precise chemical methods of
Erlandsen 4 with alcohol, ether and acetone, made similar observations and
demonstrated the high antigenic potency and specificity of the acetone insoluble
diaminomonophosphatide and the undesirable properties (hemolytic and anticomplementary) of the other fractions isolated.
Wellman and Lankelma 5 in 1938 determined the antigenic value of the various
Erlandsen fractions in a flocculation test for syphilis (Kline test) and corroborated the fact that the diaminomonophosphatide fraction was the most potent
and specific portion and that the other fractions were less potent and less specific
or had no antigenic value whatever.
EXPERIMENTAL
In the purification of standard slide test antigen with water, described elsewhere in this
journal, a 10 per cent alcoholic solution of antigen wax, which has been chilled and filtered,
and contains approximately 8.7 per cent purified wax, is treated with half its volume of
62
PTJEIFIED SLIDE TEST ANTIGEN
63
distilled water. On agitation of the mixture, a wax precipitates out, leaving an alcohol
water solution containing impurities. This solution was analyzed for all the substances
originally present in beef heart muscle, the source of the antigen wax. Because of the complexity of the mixture, and the large proportion of lipoidal constituents, separation was
extremely difficult and the figures given are necessarily approximations.
The alcohol water solutions decanted from six purified antigen waxes were analyzed
separately. All these antigens had been satisfactory for the slide test prior to purification,
but showed increased specificity after the treatment. The method described below for the
separation and analysis was followed with minor modifications in all cases.
The pale yellow solution discarded from the water treatment of the antigen was evaporated to dryness on a 60C C water bath. The brown wax remaining averaged 16.7 per cent by
weight of the antigen before water purification. The wax was dissolved in ether and filtered
through a weighed filter paper. The portion on the filter paper was washed with small
portions of ether, then dried, weighed, and dissolved in water to a known volume. This
ether insoluble fraction which contained only a trace of phospholipid and no other lipoidal
constituents, was analyzed for the other constituents of beef heart muscle.
The ether filtrate and washings were combined, evaporated to a small volume in the
60CC. oven, and treated with ten volumes of acetone to precipitate the phospholipids. The
supernatant solution was decanted through a filter paper. The insoluble portion was
washed three times with small portions of acetone. The combined filtrate and washings
were evaporated to dryness, dissolved in ether and treated as was the primary ether solution. In three of the six analyses, a third extraction and phospholipid precipitation was
employed, but the yield of ether and acetone insoluble substances was negligible.
The combined acetone insoluble phospholipid fractions represent the antigen wax lost in
the purification process. The acetone solution of lipids (ether soluble fraction) was evaporated until no solvent remained, leaving a pale yellow, oily substance, which was weighed.
This was then dissolved in chloroform to a known volume. Aliquots were analyzed for the
lipoidal constituents of beef heart muscle.
The methods used for the analysis of the impurities in the aqueous and acetone solutions
were in most cases those employed in blood chemical determinations, although some modifications were necessary. An attempt was made to use an aliquot which would bring the
value of each constituent within the range of greatest accuracy for the method.
The non-lipoidal organic constituents of the aqueous solution (ether insoluble fraction)
were classified as nitrogenous and non-nitrogenous. The nitrogenous fraction represented
about 13 per cent of the total impurities (0.5 per cent of the weight of the antigen wax before
water purification). Protein, which constitutes 75-80 per cent of the dry weight of beef
heart muscle was not detected. Moreover, all the nitrogen in the phosphotungstic acid
precipitate was accounted for by extractives. About a third of the total nitrogen (microKjeldahl) was creatinine nitrogen. A determination of the organic phosphorus gave an
approximation of the amount to be calculated as phosphatide nitrogen. There was a small
amount of organic sulfur, which was assumed to be combined with nitrogen in either bile
salts or sulfur-containing amino acids. Qualitative tests indicated the presence of small
quantities of the purine bases, xanthine, guanine, hypoxanthine, and uric acid. The other
nitrogen-containing substances detected in small amounts were urea, ammonia, amino
acids, and polypeptides. An accurate quantitative distribution of this group was impossible, because methods were not available for accurate estimation of bile salts and purine
bases in the presence of phospholipids.
The only non-nitrogenous organic substances found were glucose, inosite and traces of
lactic acid and soaps. This group represented less than 3 per cent of the impurities.
In the estimation of the inorganic substances, the ash weight was corrected for organic
phosphorus and sulfur, and constituted about 2 per cent of the impurities. The relative
quantities of the inorganic elements closely followed that of the original beef heart muscle.
The acetone solution (ether soluble fraction) was analyzed for lipoidal constituents.
Since some of the phosphatide was soluble in acetone, the phosphorus was determined and
Total solids removed
Phospholipids (slide test antigen)
Impurities
1. Acetone-soluble lipids
a. Neutral fats, 65% (Bloor)
b. Cholesterol, 11% (Bloor)
c. Fatty acids, 4% (Bloor)
etc.
2. Nitrogenous extractives
a. Unidentified, 6.5% (Estimated)
b. Creatinine, 3.3% (Folin & Wu)
c. Purine bodies, 1.5% (Estimated)
Xanthine (Fischer)
Guanine (Capranica)
Hypoxanthine (Kossel)
Uric acid (Brown)
d. Urea, 0.8% (Van Slyke & Cullen)
e. Ammonia, 0.6% (Van Slyke & Cullen)
f. Amino-acid, 0.5% (Folin)
etc.
3. Other organic substances
a. Glucose, 1.5% (Folin & Wu)
b. Inosite, 1% (Estimated)
c. Lactic acid, Trace
d. Soaps, Trace
etc.
4. Inorganic substances
a. Chlorine, 0.79% (Van Slyke & Sendroy)
b. Potassium, 0.46% (Kramer & Gittleman)
c. Sodium, 0.37% (Breh & Gaebler)
d. Phosphorus, 0.26% (Bodansky)
e. Calcium, 0.17% (Kramer & Tisdall)
f. Sulfur, 0.09% (Hubbard)
g. Magnesium, 0.04% (Briggs)
etc.
5. Protein
S56
per
cent
Average
S51,
S52
S56
SS9
S60
S61
S63
per
cent
Average
1.7
1.8
4.0
1.1
1.4
2.6
0.09 0.17 0.06 0.08 0.05 0.05 0.08 2.8
1.9
2.2
2.3
0
2.2
2.7
13.2
14.4 16.7
11.2 13.0
3.2 3.7
100
2.60 3.03 77.3 77.7 83.8 86.3 84.5 81.4 81.9
S63
4.0
16.8
12.5
4.3
3.64
S61
0.13 0.18 0.08 0.09 0.08 0.08 0.10 4.2
15.1
10.9
4.2
3.62
S60
9.7 12.5 14.6
16.9
13.5
3.4
2.86
S59
PER CENT IMPURITIES (APPROXIMATE)
0.49 0.63 0.41 0.41 0.54 0.47 0.49 15.7 14.3 12.2
19.3 18.1
16.2 13.7
3.1 4.4
2.40 3.42
8(8'
PBB CENT OF ANTIGEN WAX (APPROXIMATE)
TABLE 1
£
PURIFIED SLIDE TEST ANTIGEN
65
calculated as lecithin. The lipoidal impurities, neutral fat, cholesterol, and fatty acid
were estimated by the methods of Bloor. They represented approximately 3 per cent of
the antigen wax prior to water purification; 82 per cent of the total adventitious substances.
Table 1 gives the complete analysis of the impurities found in the six samples analyzed.
SUMMARY
Analyses of the impurities removed from slide test antigens by extraction with
water show small quantities of almost all the important constituents of beef
heart muscle, the source of the antigens. The principal exceptions are protein,
creatine, and glycogen; probably absent because of their insolubility in absolute
ethyl alcohol, the solvent that was employed in making the original extract.
Lipoidal impurities, chiefly neutral fats, make up the greater proportion of the
adventitious material.
Further study of the antigen impurities may lead to identification of the
substances responsible for many nonspecific reactions in tests for syphilis, and
to methods for completely eliminating them.
REFERENCES
(1) KLINE, B. S.: Further
(2)
purification.of
(3) NEYMANN, C. A., AND GAGER, L. T.: A
new method for making Wassermann
the antigen for the microscopic slide
antigens from normal heart tissue.
precipitation test for syphilis. Am.
Jour. Immunol., 2: 573, 1917.
Jour. Clin. Path., 10: 853 (December)
1940 also Ven. Dis. Infor., 21: 387 (4) ERLANDSEN, A.: Untersuchungen iiber
die lecithinartigen substancen des
(December) 1940.
myocardiums und der quergestreiften
NOGTJCHI, H . , AND B R O N F E N B R E N N E R ,
muskeln. Ztschr. Physiolog. Chem.,
J.: Biochemical studies on so-called
61:71,1907.
syphilis antigen. Jour. Exp. Med., (5) WELLMAN, J. W., AND LANKELMA, H. P .
13: 43, 1911.
Personal communication.