U.S. patent number 3,882,224 [Application Number 05/396,169] was granted by the patent office on 1975-05-06 for reagents and tests for syphilis.
This patent grant is currently assigned to American Cyanamid Company. Invention is credited to Peter Salvatore Forgione.
United States Patent |
3,882,224 |
Forgione |
May 6, 1975 |
Reagents and tests for syphilis
Abstract
An antigen preparation for use in the detection of syphilis and
other treponemal diseases comprising an antigen reagent and a class
of dyes and a method of using said antigen preparation, are
disclosed.
Inventors: |
Forgione; Peter Salvatore
(Stamford, CT) |
Assignee: |
American Cyanamid Company
(Stamford, CT)
|
Family
ID: |
23566148 |
Appl.
No.: |
05/396,169 |
Filed: |
September 11, 1973 |
Current U.S.
Class: |
435/7.36;
436/543; 436/536; 436/805 |
Current CPC
Class: |
G01N
33/532 (20130101); G01N 33/571 (20130101); Y10S
436/805 (20130101) |
Current International
Class: |
G01N
33/571 (20060101); G01N 33/532 (20060101); G01n
031/02 (); G01n 031/22 (); G01n 033/16 () |
Field of
Search: |
;424/3,7,8,11,12,13 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Berger, Chem. Abs., Vol. 38, 1944, pp. 3996-3998. .
Berger, Brit. Exptl. Path., Vol. 24, 1943, pp. 252-261..
|
Primary Examiner: Meyers; Albert T.
Assistant Examiner: Fagelson; A. P.
Attorney, Agent or Firm: Van Riet; Frank M.
Claims
I claim:
1. An antigen preparation for use in carrying out an agglutination
test for syphilis which comprises
a. 0.1-25 percent by weight of a syphilis antigen reagent,
b. 0.0001-0.2 percent by weight of a dye compound comprising
##SPC8##
and
c. a flocculating amount of polyvinylimidazoline.
2. The antigen preparation of claim 1 wherein said dye compound has
the formula ##SPC9##
3. The antigen preparation of claim 1 wherein said dye compound has
the formula ##SPC10##
4. The antigen preparation of claim 1 wherein said dye compound has
the formula ##SPC11##
5. A method for carrying out an agglutination test for syphilis on
a test card having dried thereon a test spot containing a
flocculating amount of polyvinylimidazoline said method comprising
contacting said test spot with
1. a test serum or plasma and
2. an antigen preparation comprising
a. 0.1-25 percent by weight of a syphilis antigen reagent and
b. 0.0001-0.2 percent by weight of a dye compound comprising
##SPC12##
6. The method of claim 5 wherein said dye compound has the formula
##SPC13##
7. The method of claim 5 wherein said dye compound has the formula
##SPC14##
8. The method of claim 5 wherein said dye compound has the formula
##SPC15##
9. A method for carrying out an agglutination test for syphilis
which comprises mixing a test serum or plasma with the antigen
preparation of claim 1.
Description
BACKGROUND OF THE INVENTION
The use of serological tests for the detection of syphilis and
other treponemal diseases has become more and more commonly
practiced in recent years. These tests are usually based on an
agglutination reaction and are conducted in clinics and doctors'
offices preparatory to more extensive diagnosis of the patient.
The most commonly practiced test constitutes the use of a finely
divided solid such as charcoal in conjunction with a card having a
surface color contrasting to that of the solid material, see U.S.
Pat. No. 3,074,853. In practice, the test utilizes a common antigen
liquid which is buffered and to which is added the charcoal. A drop
of the resultant antigen solution-charcoal suspension is then
placed on a test card in admixture with one drop of serum. The card
is then shaken and the results are visually interpreted. Additional
diagnostic reagents have also recently been patented, see U.S. Pat.
Nos. 3,564,089 and 3,600,494, while Lockyer, Brit. J. vener. Dis.
(1970), Vol. 46, pages 290-294 sets out a modified Laughlin
flocculation test using a Kahn antigen coupled with a scarlet red
stain and a Wassermann complement-fixation technique, using a
cardiolysin antigen.
The known test methods, while resulting in adequate syphilis
detection in most cases, are by no means perfect and each possesses
its own difficulties. The charcoal test, for example, is very
difficult to interpret due to the tendency of the positive and
negative results to be similar in many instances. Where a
definitely positive test is encountered, no difficulty arises,
however, in cases where the results are in doubt, further extensive
tests must be conducted before a definite conclusion can be drawn.
With regard to the scarlet red stain test, the results tend to be
nebulous because of the faint pink color of the aggregated
particles and the concomitant difficulty in analyzing and
interpreting the test.
It has also been well recognized that other commonly used tests
have been known to give completely erroneous results in 2.0 percent
of the tests run and false positive results in 20 percent of the
test cases.
SUMMARY OF THE INVENTION
I have now found a novel visual method for the detection of
syphilis which has material advantages over other techniques such
as the use of charcoal or latex emulsions. Since my system does not
utilize solid particles, no light scattering is caused and
therefore a more easily visible particle is produced if a positive
serum or plasma is used. Therefore, the instant test is more
accurate and reliable.
According to my novel test, when my novel antigen reagent is
contacted with an antibody-containing serum or plasma, a complex is
formed which forms a coaccervate with one component of the reagent.
A positive test is indicated by colored flocs which are especially
noticeable on a light background, e.g., a white card.
DESCRIPTION OF THE INVENTION INCLUDING PREFERRED EMBODIMENTS
As mentioned briefly above, I have discovered a novel antigen
preparation for use in carrying out an agglutination test for
syphilis and other treponemal diseases. The antigen preparation
consists of two basic ingredients, the first being a common
V.D.R.L., U.S.R. or Reiter antigen reagent, known to those skilled
in the art and more completely identified in U.S. Pat. No.
3,564,089, mentioned above.
The second component of my novel antigen preparation is a dye
compound having the formula ##SPC1##
wherein R and R.sup.1 are, individually, hydrogen, lower alkoxy,
lower alkyl, amino, monoalkylamino, dialkylamino or monoarylamino
groups, no more than one of R and R.sup.1 being hydrogen and
R.sup.2 is hydrogen, amino or monoarylamino groups, R.sup.3 is an
SO.sub.3 Na group, Z is SO.sub.3 or Cl, X is oxygen, sulfur or
##SPC2##
R.sup.4 and R.sup.5 are, individually, hydrogen, lower alkoxy or
lower alkyl groups and AR.sup.1 is ##SPC3##
wherein R.sup.6 is hydrogen or lower alkyl, R.sup.7 and R.sup.8
are, individually, hydrogen, amino, monoarylamino, lower alkyl,
dialkylamino, monoalkylamino, hydroxy or nitro groups, no more than
one of R.sup.7 and R.sup.8 being hydrogen and R.sup.9 and R.sup.10
are, individually, hydrogen, amino, monoarylamino,
monodialkylaminoarylamino, or hydroxy groups, and the zinc double
chlorides thereof.
The dye compounds and mixtures are present in my novel antigen
preparations in conjunction with solvents, buffers, etc., in a
manner as is known in the art. These solvents, buffers etc. per se
form no part of the instant invention and are used as described in
known antigen systems such as those discussed in the
above-identified U.S. patents, hereby incorporated herein by
reference.
The components of the novel antigen preparation of my invention are
employed in concentrations ranging from about 0.1 percent to about
25.0 percent, by weight, of the standard V.D.R.L., U.S.R. or Reiter
antigen and from about 0.0001 percent to about 0.2 percent, by
weight, of the dye compounds, said weights being based on the total
weight of the resultant mixture including solvents, e.g., water,
alcohol, etc., and buffer.
By "standard V.D.R.L. and U.S.R. antigen," as used herein, is meant
the compositions set forth and prescribed by the Manual of Tests
for Syphilis (1969), U.S. Dept. of Health, Education and Welfare,
Public Health Service, National Communicable Disease Center.
A third component may also be present in my novel antigen
preparations. This component is preferred but not critical and
comprises a flocculating agent. The flocculating agent can be
employed in amounts ranging from about 0.0001 to about 0.05
percent, by weight, based on the total weight of the novel antigen
preparation of the instant invention, i.e., standard antigen and
dye compound, and includes such known flocculants as
vinylminidazoline, acrylamide, acrylic acids, acrylonitrile,
styrene, maleic anhydride, etc., polymers and copolymers thereof
with each other and other known copolymerizable monomers.
In practice, the flocculating agent aids in the precipitation and
coaccervation of the antigen-antibody complex and thereby aids in
the visual detection of the positive test.
In preparing the antigen formulations of the present invention,
standard V.D.R.L. antigen can be used. The dye compound may be
incorporated into an alcoholic solution of the V.D.R.L. antigen or
added after the reagent antigen has been prepared in buffer. If a
flocculating agent is to be incorporated into the basic
preparation, it is also added with slight agitation at this
time.
The U.S.R. antigen-dye compound formulation is made by first
preparing the V.D.R.L. antigen suspension in buffer. This system is
then centrifuged in a stainless steel vessel at approximately 2,000
g for 15 minutes. The fluid is decanted and the sides of the vessel
wiped dry. The sediment is suspended in U.S.R. suspending medium
which consists of a known and appropriate mixture of monopotassium
phosphate, disodium phosphate, merthiolate, choline chloride and
ethylenedinitrilotetraacetic acid. The dye compound or compounds
may be incorporated into the alcoholic solution of V.D.R.L. antigen
or the U.S.R. antigen after its suspension of U.S.R. suspending
medium.
When standard Reiter antigen is used, the dye compound is merely
added thereto and the resultant antigen preparation is used as such
in the test.
The tests utilizing the antigen preparations of the instant
invention entail the use of a test card which may or may not have a
flocculating agent deposited thereon. The test card should be
smooth in order to render the test results easily discernible.
Although it has been previously indicated in the prior art that the
surface must be wettable, I have found that such is not the case
and a wettable surface is merely preferred. The card may be
composed of well calendered paper or cardboard and may be
absorbable, however, only small degrees of absorbability are
preferred. One feature of the use of my novel antigen preparations
is that the test may be read for the test card whether the spot is
wet or dry and therefore results can be ascertained more rapidly
than when using other techniques. The card may also be a laminate
of a paper or cardboard base having a water-permeable or
water-impermeable material coated thereon such as polyethylene. The
paper itself or the coating, however, should be of a color which is
contrasting with regard to the color of the dye employed in the
test. Basically, any card similar in construction and composition
to that disclosed in U.S. Pat. No. 3,074,853 may be used. The
flocculating agent may be deposited on the card, if desired, by
merely evaporating it from a solution after having placed a
solution thereof thereon. The solvent may be either water or an
organic material such as methylene chloride, etc. An adhesive may
also be added to the card beforehand to insure adequate adhesion of
the flocculating agent thereto. A suitable adhesive for this
purpose is methyl cellulose. The same flocculating agents as
mentioned above with regard to direct addition thereof to the
antigen preparation may be used. The flocculating agent is usually
deposited in selected areas only of the test card, these areas
usually being designated as a circle, etc., the area within which
the test is actually conducted. When the flocculating agent is
present on the card per se, it is not necessary to add further
flocculating agent to the antigen preparation being used to conduct
the test.
Tests are usually carried out by shaking the cards after the
antigen preparation and serum have been added dropwise thereto. The
shaking may be carried out on, for example, a horizontal disc about
10 inches square at about 80-240 strokes per minute with a slight
eccentric motion. However, hand shaking is also effective.
The following examples are set forth for purposes of illustration
only and are not to be construed as limitations on the instant
invention except as set forth in the appended claims. All parts and
percentages are by weight unless otherwise indicated.
EXAMPLE 1
U.S.R. Test
The antigen used in this test is an alcoholic solution of 0.03
percent cardiolipin, 0.9 percent cholesterol and 0.21 .+-. 0.01
percent lecithin. This standard V.D.R.L. antigen and the standard
buffered saline diluent for the preparation of the reagent V.D.R.L.
antigen are commercially available.
Unheated Serum Reagin (U.S.R.) Test
Preparation of U.S.R. Antigen-Dye Compound Reagent
A. EDTA (0.1 M)
Dissolve 3.72 g [(ethylenedinitrilo)tetraacetic acid disodium
salt](EDTA) to a volume of 100 ml.
B. Choline Chloride Solution (40 percent)
Dissolve the contents of a 250 g bottle of choline chloride in
distilled water to a final volume of 625 ml. Filter and store at
room temperature.
C. Phosphate (0.02 M), Merthiolate (0.2 percent) Solution
Dissolve 1.42 g Na.sub.2 HPO.sub.4 , 1.36 g KH.sub.2 PO.sub.4, 1.00
g merthiolate in distilled water to a final volume of 500 ml. at a
pH of 6.9.
______________________________________ Suspending Solution
______________________________________ Solution A 1 part Solution B
2 parts Solution C 4 parts Distilled Water 1 part
______________________________________
Preparation of the Antigen Suspension
1.6 Ml of commercially available V.D.R.L. buffered saline are
pipetted to the bottom of a 30 ml flat-bottom, glass-stoppered
vessel. Two (2.0) ml of antigen is added drop by drop so that it is
added in about 6 seconds directly onto the saline solution. The
vessel is then continuously but gently rotated on a flat surface
while the antigen is being added. The last drop is blown out
without touching the pipette to the saline solution. The vessel is
rotated for an additional 10 seconds. Sixteen and four tenths
(16.4) ml of standard buffered saline is then added and the vessel
shaken (with the top on) bottom to top and back thirty (30) times
in about 10 seconds. The resultant product is the V.D.R.L. antigen
suspension used below.
The above prepared V.D.R.L. antigen suspension is centrifuged in an
angle centrifuge at room temperature at a relative centrifugal
force of approximately 2,000 .times. g for 15 minutes. The
supernatant fluid is decanted by inverting the tube away from the
side containing the sediment. The inside of the vessel is wiped
with a cotton gauze without disturbing the sediment while the tube
is held in an inverted position. The sediment is then resuspended
in the above suspending solution with a volume equal to that of the
original portion of antigen suspension that was centrifuged. If
more than one container is used for centrifuging, the contents are
pooled and mixed gently. The result is U.S.R. antigen. Seven
hundredths (0.07) ml of a 1 percent solution of "Amethyst Violet",
having the formula ##SPC4##
and identified as Basic Dye C.I. No. 50225, is slowly added to the
U.S.R. antigen while the antigen is gently agitated. The result is
an antigen preparation ready for testing.
Procedure for the Detection of Reagin Antibody (U.S.R. Test)
The test may be conducted on serum or plasma samples which have not
been heated, although heat inactivated samples are
satisfactory.
Fifty .mu.1 of serum and 1/60 ml of the above antigen preparation
are added to a commercially available white test card. The mixture
is briefly mixed with a mixing stick or other device. The card is
then put on a clinical rotator and rotated for 6 minutes at 130
r.p.m. Dark violet aggregated antigen-dye particles are visible
under an incandescent, incident beam of light, denoting a positive
syphilis test. A negative reaction is one in which the reagents do
not aggregate into discrete particles but remain as a homogenous
suspension.
EXAMPLE 2
Antigen Preparation
To the V.D.R.L. antigen suspension (see above) is added 0.04 ml of
0.1 percent alcoholic solutions of "Nile Blue A", having the
formula ##SPC5##
and designated as Basic Blue C.I. No. 51180. The antigen
preparation is now ready for use.
Procedure for the Detection of Reagin Antibody (V.D.R.L. Test)
Plasma is removed from the blood cells of a suspect patient and
heated at 56.degree.C. for 30 minutes. Fifty .mu.1 of the sample
and 1/60 ml of the above V.D.R.L. antigen preparation are placed on
a commercially available white test card. After mixing, the card is
placed on a clinical rotator and rotated as in Example 1.
Aggregated dark blue particles visible under an incandescent,
incident beam of light denote a positive syphilis test. A negative
reaction is one in which the reagents do not aggregate into
discrete particles but remain as a homogenous dispersion.
EXAMPLE 3
An antigen preparation is again produced as in Example 1. The test
is conducted in the same manner as the U.S.R. test except that 1
.mu.1 of a 0.1 percent aqueous solution of polyvinylimidazoline is
added to the test card and dried at room temperature before
rotating. The aggregated particles are such that visibility thereof
is further enhanced over the card wherein no imidazoline polymer is
used.
EXAMPLE 4
An antigen preparation is produced as in Example 2. The test is
conducted in the same manner as the V.D.R.L. test except 1 .mu.1 of
a 0.1 percent aqueous solution of polyvinylimidazoline is added to
the test card and dried as in Example 3. Visibility of the
aggregated particles is again amplified.
EXAMPLE 5
An aqueous solution of 0.1 percent of "Azocarmine G", having the
formula ##SPC6##
and designated as Acid Red-C.I. No. 50085, is prepared and 0.5 ml.
of it are added to 1.0 ml of Reiter antigen. This constitutes the
Reiter-dye compound antigen preparation. One drop of this
preparation is placed on a glass slide and one drop of suspect
serum is added, followed by brief mixing with a wooden device. The
resultant mixture is rotated for 4 minutes at 160 r.p.m. and read
under a microscope at 2X power. Fluffy, translucent particles of
red intensity appear after standing 1 minute indicating a positive
syphilis reaction.
By "Reiter antigen," as used herein is meant that such as disclosed
by De Alessandro et al., Isolation and Purification of a Protein
Antigen of the Reiter Treponema, Amer. Jour. of Syphilis and
Gonorrhea, Vol. 37, page 135 et seq. 1953.
A typical testing procedure is set forth by Cammefax et al., Reiter
Protein Complement Fixation Test for Syphilis, Public Health
Reports, Vol. 72, page 335 et seq., 1957.
Following the procedures of Examples 1-5, various other dye
compounds are substituted for the compounds used therein. The dye
compounds correspond to Formula I, above, and, in each instance,
show an easily detected, positive syphilis reaction via the
presence of aggregated particles. The dye compounds used are set
forth in Table I, below. ##SPC7##
* * * * *