Process for preparing purified syphilis antigen from Treponema palljdum

A process for preparing a purified syphilis antigen from Treponema pallidum is presented. The process comprises the steps of obtaining an extract from Treponema pallidum, adsorbing the extract onto hydroxyapatite gel and eluting the antigen in the presence of a surfactant. The preferred surfactant is octylglucopyranoside, A diagnostic agent is prepared which comprises the purified syphilis antigen adsorbed on an inert carrier, which carrier at least partially a hydrophobic carrier to which the antigen is adsorbed.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a process for preparing an antigen of 
Treponema (Treponema pallidum, hereinafter sometimes abbreviated to TP) 
which is used as a reagent for diagnosing syphilis. More particularly, the 
present invention relates to a method for preparing an antigen which 
enables to prepare a diagnostic agent for syphilis, exhibiting high 
specificity and being able to detect primary syphilis. Further, this 
invention relates to a diagnostic reagent for syphilis and a method for 
preparing the same. 
2. Prior Arts 
Diagnostic methods have been performed which utilize the antigen-antibody 
reaction of TP antigens and anti-treponemal antibodies (hereinafter 
abbreviated to TP antibody) in sera from syphilitic patients. Among such 
methods, TPHA (Treponema pallidum hemaggultination assay test) has been 
widely used in recent years because of the advantages in its sensitivity, 
specificity and convenience in operation. Therefore, the TPHA has been a 
typical diagnostic method for syphilis. 
The antigen solution originated from TP and used in the above-mentioned 
method is prepared as follows: First, TP is inoculated and cultivated in 
rabbit testes. The treponemes are extracted and suspended in a suitable 
buffer and then disrupted by homogenizer, sonicator and so forth. Thus 
disrupted treponemes with or without solubilization was used as the 
antigen solution for sensitization. 
However, the prior art has the following drawbacks. Specifically, primary 
syphilis can not sufficiently be detected by the diagnostic agent for 
syphilis made from the conventional TP antigen solution. In other words, 
the conventional TPHA test or the like does not show a positive result in 
most cases until 2 to 3 months after syphilitic infection. Accordingly, 
there is a great problem that, in order to accomplish reliable diagnosis 
for primary syphilis, a diagnostic reagent using a lipoidal antigen 
(cardiolipin) should be used together with the TPHA method. Although the 
reagent using lipoidal antigens is sensitive to primary syphilis, 
nonspecific reactions are often observed. 
In a syphilitic antibody detection test such as TPHA, sensitivity of 
reagent to a primary antibody (Ig-M) is lower than an advanced antibody. 
This was caused by impurities in the antigen solution used for the 
reagent. Namely, in the conventional TPHA, the TP antigen solution used 
for sensitization of animal erythrocytes inevitably includes impurities 
due to the preparation method. 90% or more of impurities are proteins 
originated from rabbit testes in which TP is cultivated, or from TP 
components having no antigenicity. Consequently, a significant quantity of 
impurities is incorporated in the TP antigen solution, so that the primary 
antibody (Ig-M) cannot be detected. 
The antigen solution used for the conventional TPHA inevitably includes 
components originated from rabbit tissue due to its preparation method, 
which cause nonspecific reaction. Therefore, in order to reduce the 
nonspecific reaction, some components originated from rabbit tissue were 
added to the buffer of TPHA for absorbing heterophil antibodies in serum 
to be tested. 
In order to solve the above-mentioned problems, Japanese Unexamined Patent 
Application No. SHO 58(1983)-71457 discloses a technique using an antigen 
fraction which is obtained by removing fractions having specific gravity 
of 1.01 or less from the extracted treponemal suspension. 
However, a significant quantity of impurities is still mixed in the antigen 
fraction, because the fractionation method by the difference of specific 
gravity does not provide a strict separation of the antigen fraction from 
rabbit tissue. The aforesaid Japanese Application states that the above 
method is applicable even after the disruption of treponemes. However, 
sodium diatrizoate or the like used for density gradient reagents is 
inevitably incorporated in the antigen solution in this method, and hence, 
a process for removing the density gradient reagents should be required. 
Accordingly, this method would not be applied after the disruption of 
treponemes. 
An immunological diagnostic reagent is generally prepared by immobilizing 
an antigen or antibody on a hydrophobic carrier (e.g., plastic particles 
such as latex particles, cellulose powder, polystyrene, polypropylene or 
nylon particles; membrane of nitro-cellulose or nylon; erythrocytes 
treated with tannic acid; or agarose gel). Known immobilization methods 
include a method by physical adsorption wherein an antigen or antibody is 
in contact with a hydrophobic carrier in an aqueous medium and a method 
wherein an antigen or antibody is covalently bonded to a carrier having an 
amino group or a carboxylic group on its surface. The former method 
utilizing physical adsorption is widely used in view of manufacturing 
efficiency, convenience and being easy to reproduce the product of the 
same quality. 
In the case of immobilizing an antigen or antibody by physical adsorption, 
an approximately neutral buffer comprising a salt and a buffering agent is 
usually used as the aqueous medium. A surfactant is not employed as the 
aqueous medium, since the surfactant is considered to interfere with the 
immobilization of antigen or antibody on carrier. Specifically, the 
surfactant is considered to decrease the hydrophobic interaction in 
immobilization by physical adsorption. 
The surfactant is also considered to interfere with the immobilization of 
TP antigens on carriers. In other words, the efficiency of immobilization 
is substantially decreased in the presence of the surfactant, with the 
result that it is difficult to prepare an excellent diagnostic reagent. 
SUMMARY OF THE INVENTION 
The present invention is accomplished to solve the problem of impurities in 
the antigen immobilized on the carrier in the method for preparing the 
antigen originated from T. pallidum. A main object of the present 
invention is to provide a process for preparing a treponemal antigen used 
for a diagnostic agent of syphilis which can detect a primary syphilis as 
well as an advanced syphilis and does not exhibit a nonspecific reaction. 
Thus, the present invention provides a process for preparing a treponemal 
antigen which comprises adsorbing an extract originated from T. pallidum 
on a hydroxyapatite gel, followed by elution, while an aqueous medium is 
used. 
Further, the present invention provides a diagnostic agent employing a 
treponemal antigen, preferably the above-mentioned one and a method for 
preparing the same. 
The diagnostic agent of syphilis in the present invention comprises a 
treponemal antigen and a carrier. Preferably, the agent is prepared by 
treating a carrier with a treponemal antigen in an aqueous medium having a 
pH from about 4.5 to about 7.7 and containing a surfactant in an amount of 
from about 0.01 wt. % to about 2.5 wt. % and removing the remaining 
surfactant if any. 
PREFERRED EMBODIMENT OF THE PRESENT INVENTION 
The extract originated from T. pallidum can be prepared in accordance with 
the present invention as follows. 
(1) Selection, cultivation and collection of T. pallidum 
Suitable seed strain of T. pallidum is, for example, WHO's pathogenic 
standard Nichols strain or T. pallidum strain used for diagnosing syphilis 
in various tests. The WHO's pathogenic standard Nichols strain is easily 
available from, for example, CDC (Center for Disease Control, Public 
Health Service, U.S. Department of Health, Education and Welfare, Atlanta 
Ga.). The cultivation, collection and treatment methods can optionally be 
selected among any known methods. 
(2) Disruption and solubilization of TP 
Subsequently, the collected treponemes are suspended in a buffer solution. 
After cooling and disruption, the resultant is solubilized to obtain the 
extract originated from TP. The method for disruption or solubilization is 
optionally selected among any known methods. The disruption can be 
conducted by a homogenizer, ultrasonication or freeze-thawing method. The 
solubilization can be performed with a surfactant for solubilization of 
slightly soluble protein, chaotropic ion (e.g., SCN.sup.-, Cl.sup.- or 
I.sup.-), urea or an alkaline treatment, an enzymatic treatment, an 
autolysis method or the like. Particularly, the solubilization method is 
more suitable which employs a now ionic strength buffer containing a 
non-ionic or amphoteric surfactant. 
Besides, the present method is applicable to an extract containing TP 
antigen which is obtained from Escherichia coli or the like in accordance 
with a recombinant DNA method, as far as said antigen has the 
physicochemical characteristics existing within the range of those of the 
antigen of the present invention. 
(3) Pretreatment 
It is preferable that any pretreatment is carried out for removing 
impurities beforehand as much as possible from the extract originated from 
TP in order to effectively perform the present invention. of course, the 
present invention can be carried out without the pretreatment. 
Examples of the pretreatment are as follows: 
(a) The fractions other than the antigen fraction are removed beforehand by 
an ion-exchange chromatography (utilizing, for example, a cation 
exchanger). 
(b) A partial purification is conducted with a sodium sulfate or 
polyethylene glycol fractination. 
The above pretreatment (a) is more desirable since it is easier to perform 
compared with (b). 
A preferable embodiment will be explained hereinbelow for performing the 
method for preparing the treponemal antigen according to the present 
invention. It is to be noted that the present invention should not be 
limited to the embodiment described below. 
(1) Hydroxyapatite gel 
The hydroxyapatite used in the present invention can be represented by the 
chemical formula of [Ca.sub.5 (PO.sub.4).sub.3 (OH)].sub.2. Usable gels 
are those on market, for example, Bio-Gel.RTM. HTP (Bio-Rad Laboratories) 
or HCA-200 L (Mitsui Toatsu Chemicals, Inc.). There is no limitation in 
the shape and the particle size of the gel. Generally, gels for column 
chromatography can be used. Preferably, the specific surface area of the 
gel is about 1 to 100 m.sup.2 /g and the particle size thereof is about 1 
to 100 .mu.m. 
(2) Kind of the aqueous medium 
Any buffer used for general biochemical experiment such as phosphate 
buffer, Tris buffer, glycine buffer or the like can be used as the aqueous 
medium in the present invention. Phosphate buffer is preferable. 
(3) pH of the aqueous medium 
(a) Adsorption of the antigen 
The pH of the aqueous medium is preferably within the range from about 5.0 
to 8.0, more preferably within the range from about 5.5 to 7.0. When the 
pH is less than 5.0, antigenicity is likely to be lost, thereby decreasing 
recovery of the TP antigen. On the other hand, the aqueous medium having 
pH 8.0 or more causes the reduction in the adsorption efficiency of the 
antigen to the hydroxyapatite gel, thereby decreasing recovery of the TP 
antigen. 
(b) Elution of the antigen 
The pH is preferably within the range from about 5.0 to 11.0, preferably 
about 5.5 to 10.0 for eluting the antigen. When the buffer has pH 8.0 or 
more, its ionic strength is preferably to be in a low ionic strength such 
as 10 to 60 mM to reduce the affinity of antigen for the column. When the 
pH is less than 5.0, antigenicity is likely to be lost, thus undesirable. 
On the other hand, an aqueous medium having a pH more than 11.0 tends to 
elute the impurities together with the antigen, thus undesirable. 
(4) Ionic strength of the aqueous medium 
(a) Adsorption of the antigen 
An aqueous medium having an ionic strength of preferably about 2 to 30 mM, 
more preferably about 10 to 20 mM is used to adsorb the extract originated 
from TP on the hydroxyapatite gel. 
When the ionic strength is less than 2 mM, it is not practical because 
impurities tend to be adsorbed on the hydroxyapatite gel and the capacity 
of the buffer used is reduced. On the other hand, when the ionic strength 
is more than 30 mM, it causes less adsorption of the antigen on the 
hydroxyapatite gel, thereby decreasing the recovery. 
(b) Elution of the antigen 
The ionic strength in the elution is preferably about 10 to about 360 mM, 
more preferably about 50 to about 120 mM. When the ionic strength is less 
than 10 mM, the antigen is hard to be eluted at an acidic pH. The 
impurities are also eluted with an ionic strength of more than 360 mM, 
thus undesirable. In case where the elution is conducted with an ionic 
strength below that in the adsorption, the elution is preferably carried 
out at a pH higher than that in the adsorption. On the other hand, the 
elution is preferably carried out with an ionic strength higher than that 
in the adsorption in case where the elution is conducted with a pH below 
that in the adsorption. 
(5) Additive or the like to the aqueous medium 
Any materials to which the hydroxyapatite gel is resistant such as the 
surfactant, chaotropic ions or urea which can be used for solubilization 
of treponemal antigen can be used as the additive to the aqueous medium 
for the adsorption and elution. A buffer of low ionic strength which 
contains a non-ionic or amphoteric surfactant is preferably used for the 
method of the present invention in consideration of easiness of operation. 
Besides, the use of chelating agent such as EDTA is not recommended since 
it interferes with the adsorption of the antigen protein to the 
hydroxyapatite gel. 
(6) Gradient elution 
The elution can be carried out with stepwise or linear increase of the 
ionic strength or pH, although the increasing pattern is not particularly 
limited. 
The stepwise increasing method would be industrially effective, because the 
antigen can be once eluted under a predetermined suitable condition. 
However, in such method, it is noted that the perfect separation of the 
antigen from impurities may not be attained in some cases. Accordingly, 
the linear gradient elution method which linearly increases the ionic 
strength and/or pH is preferable. By this method, the optimal fraction can 
be obtained by examining the elution profile on chromatogram, or dividing 
the fractions as detailedly as possible and measuring the antigen activity 
thereof. 
By the present method, the substantially pure antigen can be obtained. 
When the extract originated from treponemes is in contact with the 
hydroxyapatite gel in the buffer having a low ionic strength (salt 
concentration), the antigens are adsorbed on the gel. Then, the antigens 
are eluted from the gel at the ionic strength within the predetermined 
range, thereby obtaining the antigen of high-purity. 
Explained next is the application of the antigen obtained by the present 
invention. 
(1) Diagnostic agent and a method for preparing the same 
The treponemal antigens of the present invention are immobilized on a 
carrier by any known method to make a reagent for diagnosing syphilis. 
Any carrier used in this field can be used. Preferable carriers are inert 
carriers in which the surface is at least partially hydrophobic. Examples 
of the carriers are synthetic polymer particles having a particle size of 
about 0.05 to 50 .mu.m prepared by performing polymerization or 
copolymerization with a monomer such as styrene, acrylic acid, methyl 
methacrylate, acrylonitrile or butadiene and particularly microparticles 
having an uniform particle size of 0.1 to 2 .mu.m, i.e., latex particles 
called in the field of an immunological diagnostic reagent, prepared by 
emulsion-polymerizing the aforesaid polymer or its derivatives in an 
aqueous medium; synthetic polymer materials such as polystyrene, 
polyethylene, polypropylene, nylon or cellulose acetate and their molded 
product; membrane of nylon, nitrocellulose or the like; materials of 
living organism such as sheep or hen erythrocytes treated with tannic 
acid; and inorganic materials such as silica powder or glass particles, or 
the like. The latex particles and erythrocytes treated with tannic acid 
are more preferable. 
"The surface is at least partially hydrophobic" means the property for 
immobilizing the antigen via any physical adsorption. The surface may 
entirely be hydrophobic. Some materials may be used by activating the 
surface thereof. 
According to the present invention, it is found that the immobilization of 
the antigen on a carrier is conducted in contacting them in an aqueous 
medium containing about 0.01 to 2.5% by weight of a surfactant and having 
pH about 4.5 to 7.7, thereby providing a reagent with excellent 
sensitivity and improved specificity in which the antigens are much stably 
immobilized. 
Besides, in case where the surfactant is used at a high concentration, it 
is preferable to remove the possibly remaining surfactant after the 
immobilization, in order to avoid the interference of the surfactant with 
the antigen-antibody reaction. 
Usable surfactants for the above immobilization are those which can be used 
for extraction and stabilization of the surface antigen and membrane 
protein of TP, are capable of extracting and solubilizing the object 
constituents, have high specificity of extractability, and are stable at 
pH about 4.5 to 7.7 without separation. Preferable examples of the 
surfactants are non-ionic surfactants such as octylglucopyranoside 
(1-O-n-octyl-.beta.-D-glucopyranoside), Triton X-100.RTM., Tween 20.RTM., 
Tween 80.RTM., octylthioglucoside or the like or ampholytic surfactants 
such as CHAPS (3-[(3-Cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate) 
or the like. Cationic surfactants such as dodecylamine or anionic 
surfactants such as sodium dodecyl sulfate can be used. 
The effective concentration of the surfactant in the aqueous medium for 
immobilizing the TP antigen on the carrier is about 0.01 to 2.5 wt. %, 
preferably about 0.02 to 2.10 wt. %. If the concentration of the 
surfactant is higher, the TP antigen cannot be immobilized on the carrier. 
The activity of the TP antigen is likely to be lost with the surfactant of 
lower concentration. 
Preferable examples of the aqueous media used for immobilizing the TP 
antigen on the carrier are buffers used for general biochemical 
experiments, e.g., phosphate buffer, Tris buffer or the like. The ionic 
strength is adjusted by the addition of salt to the aqueous medium. The 
effective pH is about 4.5 to 7.7, preferably about 4.9 to 7.1, more 
preferably about 5.4 to 6.5. If the pH is less than 5.0, antigenicity is 
likely to be lost, but the adsorption is performed instantaneously. Thus, 
the pH may be more than 4.5. The quantity of the TP antigen immobilized on 
the carrier becomes less with the aqueous medium of pH more than 7.7. In 
order to increase the storage stability of the obtained diagnostic 
reagent, a preservative can be added thereto if necessary. 
Further, choline chloride, EDTA, saccharides (polysaccharide, dextran or 
the like), polyethylene glycol and the like can be added in order to 
improve the sensitivity in measurement. 
Explained subsequently is a specific method for treating the carrier with 
the antigen solution. 
First, the antigen solution for immobilization is prepared by any known 
method. For example, the treponemes are extracted from rabbit testicular 
materials containing treponemes. Then, the treponemes are washed, to which 
a surfactant is added. The resultant mixture is incubated to disrupt the 
treponemes and extract the TP antigen. The extract is centrifuged to 
collect the supernatant which is then diluted with a buffer containing a 
surfactant to obtain treponemal antigen solution. The obtained solution is 
adjusted to have a predetermined surfactant concentration and pH, thus 
making the sensitizing solution, i.e., the antigen solution for 
immobilization. 
Subsequently, the sensitizing solution is in contact with the carrier 
described above in an aqueous medium containing about 0.01 to 2.5 wt. % of 
surfactant and having pH about 4.5 to 7.7. The resultant mixture was 
incubated for a predetermined period to immobilize the TP antigen on the 
carrier. 
Other methods for immobilizing the TP antigen on the carrier include 
various ones, e.g., a method in which an antigen solution containing a 
suitable concentration of surfactant is added to a carrier suspension 
containing or not containing surfactant to adjust the concentration of 
surfactant and pH to a predetermined value, and then results in 
immobilization of the antigens, or a method in which a carrier is 
contacted with an antigen solution and then a diluent comprising a 
surfactant or buffer is added to adjust the concentration of surfactant 
and pH to a predetermined value thereby immobilizing the TP antigen. 
After the immobilization, the carrier is separated from the aqueous medium 
and then washed with a buffer containing bovine serum albumin, saline 
solution or the like to remove the surfactant. 
The antigen sensitizing solution described above is prepared from a 
so-called partially purified antigen fraction. An excellent diagnostic 
reagent can be obtained as mentioned below by using the treponemal antigen 
purified according to the present invention with the carrier of latex 
particles. 
The surfactant used for immobilizing the antigen on the carrier in the 
present invention is considered to stabilize the antigens in the antigen 
solution and prevent unnecessary components in the material from being 
immobilized on the carrier. In general, the surfactant interferes with the 
immobilization of antigens on carriers. The antigens of the present 
invention are slightly soluble in water and hence low isoelectric points, 
whereby the solubility of the antigen is decreased by lowering pH so that 
the immobilization on carrier occurs even in the presence of the 
surfactant. As a result, the dignostic reagent of the present invention 
has an increased amount of the antigen carried on the carrier and more 
excellent sensitivity compared with that prepared by the conventional 
method. Further, amount of other components immobilized the carrier 
decreases, thereby improving specificity. 
(2) Application of the diagnostic reagent 
The disgnostic agent of the present invention is used for detecting 
anti-treponemal antibodies in the subject serum. The detection methods 
include radio-immunoassay (RIA), fluorescent immunoassay (FIA), enzyme 
immunoassay (EIA or ELISA), latex agglutination assay, TPHA method 
(Treponema pallidum hemaggultination assay test) or the like. The 
diagnostic reagent of the present invention can be formed to suit the 
above-mentioned detection methods. 
The immunoassay utilizing the antigen-antibody reaction is preferably 
conducted in the presence of a water-soluble polymer and/or water-soluble 
copolymer containing at least one kind of glycoside derivatives 
represented by the following formula (I) as a monomer: 
##STR1## 
wherein G--O-- represents a saccharic residue not having a protective 
group. R is a hydrogen atom, a methyl group or an ethyl group, m is an 
integer of 1 to 3 and n is an integer of 1 to 4. 
The saccharic residue in the above glycoside derivative is a group in which 
a hydrogen atom is removed from a hydroxyl group bonded to the glycosidic 
carbon atom of the reduced end of a saccharide. Specifically, the 
saccharic residue means a residue of a monosaccharide comprising 1 to 3 
sugar units or an oligosaccharide. 
Examples of the monosaccharides are hexoses such as glucose, mannose, 
galactose, glucosamine, mannosamine or galactosamine, or pentoses such as 
arabinose, xylose or ribose. 
Examples of the oligosaccharides are disaccharides such as maltose, 
lactose, trehalose, cellobiose, isomaltose, gentiobiose, melibiose, 
laminaribiose, chitobiose, mannobiose or sophorose; or maltotriose, 
isomaltotriose, taltotetraose, maltopentaose, mannotriose or 
manninotriose. 
The polymers or copolymers containing glycoside derivatives disclosed in 
PCT Application No. 90/04598 can be used for the above method. 
There is no limitation in the molecular weight of the polymer or copolymer 
containing glycoside derivative if said polymer or copolymer is soluble in 
the reaction medium. A law molecular weight requires much amount of 
polymers or copolymers, thus taking much time to dissolve them in the 
reaction medium. Therefore, the molecular weight is preferably more than 
3,000. 
The concentration of the polymer or copolymer containing glycoside 
derivatives in the reaction system of the antigen-antibody reaction is 
suitably determined depending upon the molecular weight of the polymer or 
copolymer, coexisting additives such as salts, proteins, or saccharides. 
Generally, the polymer or copolymer is adjusted to be contained in the 
reaction system in an amount such that the final concentration at the time 
of reaction is 0.01 to 10.0% (W/V), preferably about 0.1 to 5.0% (W/V), 
more preferably about 0.5 to 2.0% (w/V). When the concentration of the 
above polymer or copolymer is less than 0.01% (W/V), the above polymer or 
copolymer is less effect for accelerating the antigen-antibody reaction. 
On the other hand, the polymer or copolymer having more than 10.0% (w/v) 
of the concentration increases in nonspecific reaction with the materials 
other than the object constituents.

The present invention will be explained in detail hereinbelow with 
reference to Examples, by which no limitation shall not be given. 
EXAMPLE 1 
Method for purifying antigen 
TP antigens were purified according to a method of the present invention. 
1. Reagents and ethers 
(1) Buffer solution 
(1-1) Phosphate buffered saline (pH:6.5) (hereinafter abbreviated to PBS) 
A buffer was prepared from potassium dihydrogen phosphate, sodium 
dihydrogen phosphate (12 hydrate) and sodium chloride to make phosphate 
concentration of 0.036M, NaCl concentration of 0.156M and pH 6.5. To this 
buffer was added NaN.sub.3 at a concentration of 0.1% (w/v). 
(1-2) 1% BSA/PBS 
Bovine serum albumin (hereinafter abbreviated to BSA, manufactured by Miles 
Laboratories Co.) was dissolved in PBS to make 1% (W/V) BSA solution. 
(1-3) 10 mM potassium phosphate buffer 
(pH:6.0 and pH:7.0) (hereinafter abbreviated to KPB) 
10 mM potassium dihydrogen phosphate solution was mixed with 10 mM 
dipotassium hydrogen phosphate to obtain potassium phosphate buffers 
having pH 6.0 and 7.0. 
(1-4) 350 mM KPB (pH:6.0) 
350 mM potassium dihydrogen phosphate was mixed with 350 mM dipotassium 
hydrogen phosphate to obtain mM potassium phosphate buffer having pH 6.0. 
(2) Surfactant 
Octylglucopyranoside (1-O-n-octyl-.beta.-D-glucopyranoside (hereinafter 
abbreviated as OG) used for a study of slightly soluble proteins 
(manufactured by Nacalai Tesque, Inc.) was employed as a surfactant. 
(3) Chromatographic gel for purifying protein 
(3-1) Cation exchanger 
Sepharose Fast Flow (Pharmacia LKB Biotechnology), which is cation 
exchanger wherein a sulfonic acid group was introduced to the surface of 
agarose gel, was used as cation exchanger. 
(3-2) Hydroxyapatite gel 
Bio-Gel.RTM. HTP (Bio-Rad Laboratories) and HCA-200L (Mitsui Toatsu 
Chemicals, Inc.) were used as hydroxyapatite gel. 
(4) TP 
Treponemes were used was cultivated and isolated by the following method. 
A suspension of a pathogenic standard Nichols strain of Treponema pallidum 
(6.0.times.10.sup.7 /ml) was inoculated in rabbit testes in an amount of 1 
ml per testis. After the cultivation for 10 days, testes were taken out 
from 10 rabbits, sliced and then shaked for 30 minutes at 37.degree. C. in 
2.2% sodium citrate solution (500 ml). Thereafter, proliferated treponemes 
were extracted. The extract was centrifuged for 5 minutes at 200.times.g 
to remove the precipitate of rabbit tissue. The supernatant was 
centrifuged for 30 minutes at 3000.times.g to precipitate treponemes. Thus 
obtained treponemes were well washed with PBS and suspended in PBS to 
adjust the number of the treponemes to 1.times.10.sup.9 after the counting 
with a dark-field microscope. Thus, a suspension of TP mycelia was 
obtained. This suspension was confirmed with a dark-field microscope that 
no sperms and tissues of rabbit were included. 
(5) Reagent for measuring concentration of protein 
BCA.RTM. Protein Assay Reagent (Pierce Co.) was used as a reagent for 
measuring concentration of protein. 
(6) Microtiter plate 
A microplate having 96 wells (Nunc Co., U-bottom) was used. 
(7) TP antigen sensitized erythrocyte 
Used sensitized erythrocyte was the one used for Seroclit TP (The Chemo- 
Sero-Therapeutic Research Institute), which is a TPHA kit on market. 
(8) Syphilis-positive serum of rabbit 
Used serum was the one taken out from the rabbit which was subjected to the 
cultivation of TP in its testes for 45 days. Antibody titer was measured 
by the commercially available TPHA kit, obtaining a value of 102,400. This 
serum was diluted with 1% BSA/PBS for use. 
2. Experimental Method 
(2-1) Solubilization and extraction of antigen from TP 
The suspension of treponemes (10 ml) was washed three times with PBS (50 
ml), followed by suspending in PBS (20 ml). The resultant suspension was 
sonicated for disruption. The suspension was subjected to centrifugation 
for 30 minutes at 12,600.times.g to take out the precipitate. 
Thus obtained precipitate was washed twice with KPB (10 mM, pH 7.0) using 
centrifugation for 30 minutes at 12,600.times.g. Thereafter, KPB (10 mM, 
pH 7.0) containing OG in 1% (W/V) was added in an amount of 25 ml to the 
precipitate. The suspension was then slightly sonicated to be solubilized. 
After being left at 4.degree. C. for 16 hours or more, the mixture was 
centrifuged for 1 hour at 50,000.times.g. The resulting supernatant was 
filtered through a filter of 0.22 .mu.m (Millex-GS produced by Millipore 
Corporation). The extract obtained from the treponemes in this way was 
referred to as the extracted antigen hereinbelow. 
(2-2) Pretreatment 
(1) Dialysis of antigen solution 
The extracted antigen which was dissolved in a buffer having pH 7.0 was 
dialyzed against a buffer having pH 6.0 and containing 1% OG. The dialysis 
was carried out with the volume ratio of the dialyzing solution to the 
extracted antigen solution being 4 to 1. The dialyzing solution was 
exchanged three times. After the final dialysis, the pH value of the 
dialyzing solution (external solution for dialysis) was confirmed to be in 
the range of from 6.0.+-.0.1. 
(2) The antigen obtained in (1) was passed through a column (Pharmacia LKB 
Biotechnology, SR 25/45) of S Sepharose Gel (30 ml) to collect 50 ml of 
the passed-through fraction as the antigen fraction (hereinbelow referred 
to as the partially purified antigen). 
(2-3) Purification of antigen by hydroxyapatite gel 
(1) Washing of hydroxyapatite gel 
A column (Pharmacia LKB Biotechnology, HR 10/10) was filled with the 
hydroxyapatite gel (8 ml), followed by equilibrated with 10 mM KPB 
containing 1% OG (pH 6.0). The optical density (hereinafter abbreviated to 
O.D.) of the washing solution at 280 nm was measured. Washing was 
continued until the absorbance of the eluate decreases to 0.010. 
(2) Addition of the partially purified antigen 
The partially purified antigen was added to a hydroxyapatite column. 
Thereafter, 10 mM KPB containing 1% OG (pH 6.0) was passed through the 
column. The column was washed until the absorbance of the eluate solution 
becomes 0.010 or less at 280 nm. The fraction thus obtained was defined as 
the passed-through fraction. 
(3) Elution of antigen 
A linear gradient elution was conducted by gradually increasing the ratio 
of 350 mM KPB containing 1% OG to 10 mM KPB containing 1% OG (pH 6.0) from 
0 to 40%, and finally increasing it to 100% The fractions of the ratios of 
0-8%, 8-16%, 16-24% 24-32% and 32-40% were collected. The fraction at the 
ratio of 100% is defined as the 40% or more fraction. 
(4) Assay of each fraction 
The antigen activity and protein concentration of each of the extracted 
antigen, the passed-through fraction, 0-8% fraction, 8-16% fraction, 
16-24% fraction, 24-32% fraction, 32-40% fraction and 40% or more fraction 
were measured by the following methods. Specific activitiy was calculated 
from the antigen activity and protein concentration. 
(2-4) Antigen assay method 
(1) Protein concentration 
The protein concentration was measured by BCA.RTM. protein Assay Reagent 
(Pierce Co.) in which a measuring method [Smith, P. K., Krohn, R. I. etc., 
(1985) Anal. Biochme. 150, 76-85] was used as its principle. Said 
measuring method uses bicinchoninic acid and is one of the modified Lawry 
method. The used standard was a solution of BSA in 10 mM KPB containing 1% 
OG. The unit was expressed by .mu.g/ml. 
(2) Antigen activity 
(a) 25 .mu.l of 1% BSA/PBS was dispersed in each well of the microtiter 
plate. 
(b) 25 .mu.l of each antigen fraction was dispersed in the well of the 
plate and 25 .mu.l of it was transferred in the above next well repeatedly 
to be diluted serially 2.sup.1 to 2.sup.n fold with 1% BSA/PBS on the 
plate. 
(c) Syphilis-positive rabbit serum, in which the antibody titer was diluted 
to 50 fold, was added to and mixed with the antigen fractions diluted 
2.sup.1 to 2.sup.n fold in (b). 
(d) The mixture was incubated for 30 minutes or more at room temperature. 
The antibody in the well having a high concentration of antigen was 
consumed by antigen-antibody reaction, while the antibody on the well 
having a low concentration of antigen remained thereon. 
(e) Subsequently, TP antigen sensitized erythrocyte in the commercially 
available TPHA kit (Serodia-TP) was added to each well. A final dilution 
ratio of the rabbit serum which caused the hemagglutination was defined as 
the antigen activity. This antigen activity was represented by titer 
(titer/ml). 
(g) Among the fractions exhibiting the antigen activity, those exhibiting a 
specified activity of 12 titer/.mu.g or more were collected. Thus 
collected fractions were concentrated under reduced pressure by using 
cellophane tube (Wako Pure Chemical Industries, Ltd.) until the protein 
concentration became to 50 .mu.g/ml or more. The obtained fractions were 
defined as the HAp purified antigen. 
3. Other experiments 
Antigens were purified with Bio-Gel.RTM. HTP and HCA-200 L by the same 
manner as described above. Besides the antigen activity and protein 
concentration of each of the fractions, those of extracted antigen and 
partially purified antigen were measured, whereby total antigen activity, 
total amount of protein and total specific activity of antigen were 
calculated. Table 1 shows the results. 
4. Conclusion 
As is apparent from Table 1, when the TP extract was eluted after adsorbing 
on the hydroxyapatite gel, the antigens were eluted from Bio-Gel.RTM. HTP 
at a salt concentration of 8% (37.2 mM) to 40% (146.0 mM) and from HCA-200 
L at a salt concentration of 8% (37.2 mM) to 32% (118.8 mM). Thus obtained 
fractions were combined, thereby obtaining the TP antigen of high purity 
having specific activities of 26.6 and 36.7 (titer/pg) respectively. 
TABLE 1-(1) 
______________________________________ 
Purification from Bio-Gel .RTM. HTP 
Specified 
Total Antigen 
Total amount 
Activity 
Activity of Protein of Antigen 
Fractions (titer) (.mu.g) (titer/.mu.g) 
______________________________________ 
passed-through 
0 850 -- 
0-8% 0 90 -- 
8-16% 4610 170 27.1 
16-24% 9220 240 38.4 
24-32% 4610 180 25.6 
32-40% 2300 190 12.1 
40% or more 
0 430 -- 
Total of 8-40% 
20740 780 26.6 
Extracted antigen 
24000 4250 5.6 
Partially purified 
24000 2370 10.1 
antigen 
______________________________________ 
TABLE 1-(2) 
______________________________________ 
Purification from HCA-200L 
Specified 
Total Antigen 
Total amount 
Activity 
Activity of Protein of Antigen 
Fractions (titer) (.mu.g) (titer/.mu.g) 
______________________________________ 
passed-through 
0 760 -- 
0-8% 0 110 -- 
8-16% 2300 120 12.1 
16-24% 9220 140 65.0 
24-32% 4610 180 25.0 
32-40% 580 360 1.6 
40% or more 
0 700 -- 
Total of 8-40% 
16130 440 36.7 
Extracted antigen 
24000 4250 5.6 
Partially purified 
24000 2370 10.1 
antigen 
______________________________________ 
Reference Example 1 
Confirmation of protein purity by SDS-PAGE 
1. Materials 
(1) Electrophoretic apparatus 
Phastsystem (Pharmacia) was used in accordance with its instruction. 
(2) Molecular weight marker 
Used molecular weight marker was the LMW kit E manufactured by Pharmacia 
LKB Biotechnology. 
(3) Buffer for treating sample 
The buffer was prepared by adding sodium dodecyl sulfate (5%) and 
mercaptoethanol (10%) to a solution of 10 mM Tris-HCl and 2 mM EDTA (pH 
8.0). 
(4) Polyacrylamide gel 
PhastGel Gradient 10-15 (Phast System) was used. 
(5) Staining solution 
A high-sensitive argentation solution for electrophoresis "Sil-Best Stain 
for Protein/PAGE" (Nacalai Tesque, Inc.) was used in accordance with the 
instruction. 
2. Operation Method 
(1) Preparation of samples 
The extracted antigen, partially purified antigen or HAp purified antigen 
as obtained in Example 1 were mixed with the sample buffer in an equal 
amount and incubated for 5 minutes at 100.degree. C. 
(2) Preparation of the molecular weight marker 
The molecular weight marker was dissolved in the sample buffer which was 
diluted in two fold with purified water, and then the solution was 
incubated for 5 minutes at 100.degree. C. 
(3) The above-mentioned samples were loaded on the polyacrylamide gel in an 
amount of 1 .mu.l for electrophoresis. 
(4) After electrophoresis, the polyacrylamide gel was stained and the 
molecular weight of protein was calculated from its position in the gel. 
3. Result 
About 20 bands were observed in the extracted antigen, while about 10 and 3 
bands (at molecular weights of about 31,000, 41,000 and 47,000) were 
observed in the partially purified antigen and HAp purified antigen 
respectively. 
4. Conclusion 
As is apparent from the result, an extremely high-purified TP antigen 
fraction was obtained by using the hydroxyapatite gel. 
EXAMPLE 2 
TPHA 
The purified antigen obtained in accordance with the present invention was 
carried on sheep erythrocyte for confirming the effect of the invention by 
TPHA. 
1. Materials 
The same materials as used in Example 1 were used if unspecified. The 
buffer was prepared by the same manner as in Example 1. 
(1) Buffer 
(a) 0.15M sodium phosphate buffer (pH:7.4): 
The buffer was prepared by mixing 0.15M sodium dihydrogen phosphate (2 
hydrate) with 0.15M disodium hydrogen phosphate (12 hydrate) so as to show 
pH 7.4. 
(b) Physiological saline solution 
The saline solution was prepared by dissolving sodium chloride (9.0 g) in 
purified water (1000 g). 
(c) McIlvaine buffer (hereinafter abbreviated to McI) 
McI was prepared by mixing 0.10M citric acid with 0.20M disodium hydrogen 
phosphate (12 hydrate) so as to show pH 6.5. 
(d) 1% OG/McI 
The above-identified solution was obtained by dissolving OG (1% W/V) in 
McI. 
(e) PHA buffer 
The above-identified buffer was prepared by mixing the solutions and 
reagents described as follows (The amount is expressed per 1000 ml of 
buffer.) 
______________________________________ 
Rabbit normal serum 30 ml 
Sheep erythrocyte stroma 
10 ml 
Sodium azide 1 g 
0.15M sodium phophate buffer 
100 ml 
(pH 7.4) 
Physiological saline 860 ml 
______________________________________ 
(2) Reagents 
(a) Tannic acid was bought from Nacalai Tesque, Inc. 
(b) Fixed sheep erythrocyte was the one immobilized with glutaraldehyde. 
(c) TPHA kits commercially available 
Serodia TP (Fuji Rebio Inc.) and Seroclit TP (The Chemo- Sero-Therapeutic 
Research Institute) were used. 
(3) Serum Samples 
(a) Syphilis-positive control 
Three control sera (G.sub.1, G.sub.2 and G.sub.3) were used, those of which 
were collected from fully cured advanced syphilitic patients. These 
control sera were supposed to contain a lot of IgG antibodies. Further, 
three control sera (M.sub.1, M.sub.2 and M.sub.3) were used, those of 
which were collected from primary syphilitic patients, i.e., three to five 
weeks after the infection. The latter controls were supposed to contain 
few Ig-G antibodies, but Ig-M antibodies. 
(b) Normal control (Syphilis-negative control) 
Three control sera (N.sub.1, N.sub.2 and N.sub.3) were used, those of which 
were observed to show nonspecific reaction with the TPHA kit commercially 
available and further found not to be syphilic by FTA-ABS. 
(c) Anti-rabbit tissue antiserum 
A normal rabbit testis sliced and solubilized with 1% OG was immunized to a 
goat for obtaining the above-identified serum. 
(d) Anti-Reiter strain antiserum 
Nonphthogenic treponemes, Treponema phagedenis (biotype Reiter), were 
solubilized with 1% OG. This solution was immunized to a goat for 
obtaining the above-identified serum. 
(4) TP antigen 
The extracted antigen, partially purified antigen and HAp purified antigen 
as obtained in Example 1 were used. Table 2 shows the antigen activity and 
protein concentration of each antigen solution. 
TABLE 2 
______________________________________ 
Specified 
Antigen Activity 
Protein cocn. 
Activity 
Antigen (titer/ml) (.mu.g/ml) (titer/.mu.g) 
______________________________________ 
Extracted Antigen 
2048 365 5.6 
Partially purified 
1024 101 10.1 
antigen 
(HAp Purification) 
Bio-Gel .RTM. HTP 
2048 77.0 26.6 
HCA-200L 1536 41.9 36.7 
______________________________________ 
2. Method 
(1) Blood cell processing method 
(a) The fixed sheep blood cells were washed four times with a physiological 
saline by using centrifugation for 5 minutes at 700.times.g, which was 
suspended in a physiological saline to have solid content of 6%. A 
solution (tannic acid in physiological saline, 120 .mu.g/ml) was added to 
the blood cell suspension, followed by stirring. 
(b) After stirring, the resultant solution was washed twice with a 
physiological saline and once with McI, and then suspended in McI to have 
solid content of 6%. Immediately, the resultant solution was used for 
antigen sensitization. 
(c) TP antigen solution (the extracted antigen solution, partially purified 
antigen solution or HAp purified solution) was in advance dialyzed against 
1% OG/McI. The obtained solution was adjusted as described in Table 3 to 
serve as the sensitizing solution. The sensitizing solution A was adjusted 
to have antigen activity of 100 (titer/ml). The sensitizing solution B was 
adjusted to have protein concentration of 10 .mu.g/ml. One volume of the 
sensitizing solution was added to one volume of the blood cell suspension 
as described at (b) and the mixture was stirred for 1 hour at 25.degree. 
C. 
TABLE 3 
______________________________________ 
Sensitizing Solution A 
Sensitizing Solution B 
(Sensitization with 
(Sensitization with 
a predetermined 
a predetermined 
antigen amount) 
protein amount) 
Antigen 1% OG/ Antigen 
1% OG/ 
Solution McI Solution 
McI 
Antigen (ml) (ml) (ml) (ml) 
______________________________________ 
Extracted 0.049 0.951 0.027 0.973 
Partially purified 
0.098 0.902 0.099 0.901 
(HAp 
purification) 
Bio-Gel .RTM. HTP 
0.049 0.951 0.130 0.870 
HCA-200L 0.065 0.935 0.239 0.761 
______________________________________ 
(d) The sensitized blood cells were washed twice with a physiological 
saline and suspended in PHA buffer to have blood cell solid content of 
0.2%. After standing for 3 hours at room temperature, the resultant 
suspension was used for assay. 
(2) TPHA assay method 
(a) 100 .mu.l of PHA buffer was dispensed in each well of the microtiter 
plate, and 25 .mu.l in other wells. 
(b) 25 .mu.l of each control was dispensed in each well of the microtiter 
plate, which was serially diluted 2.sup.1 to 2.sup.n on the plate. 
(c) The blood cell suspension prepared in (1) was shaken to obtain a 
homogeneous suspension. 75 .mu.l of the suspension was dispensed in each 
well. 
(4) The plate was vibrated to mix sufficiently. Thereafter, the plate was 
covered with an empty plate in order to prevent evaporation and then 
incubated at room temperature. The determination was conducted after 2 
hours. 
(5) The plate was placed on white paper, observing hemagglutination by 
visual observation. The maximum dilution (20, 40, 80, . . . ) exhibiting 
agglutination was defined as the antibody titer. 
3. Result 
Table 4 shows the results of determination of hemagglutination on each 
control and of measurement of antibody titer by using the sensitized blood 
cell obtained by the above method. 
As shown in Table 4, three primary syphilitic sera which were all negative 
with the sensitized blood cell using the extracted antigen were positive 
with the sensitized blood cell using the HAp purified antigen of the 
present invention.. Normal control sera exhibiting nonspecific reaction by 
the TPHA kit showed no agglutination. Further, the sensitized blood cell 
using the present invention exhibited no agglutination with the 
anti-rabbit tissue antiserum and anti-Reiter strain-antiserum. 
4. Conclusion 
As is apparent from the result, even a primary syphilis is detectable by 
using the antigen purified according to the present invention, without 
giving a false positive result due to the nonspecific reaction. 
TABLE 4 
__________________________________________________________________________ 
Result of TPHA 
Partially 
Antigen HAp purified antigen 
purified 
Extracted 
Commercial 
Control Bio-Gel .RTM. HTP 
HCA-200L 
antigen 
antigen 
TPHA 
__________________________________________________________________________ 
(1) Sensitizing Solution (A) Sensitization with a predetermined antigen 
amount 
Primary 
M1 + + .+-. - - 
syphilis .times.160 
.times.160 
.times.80 
.times.40 
.times.40 
M2 + + + .+-. .+-. 
.times.320 
.times.320 
.times.160 
.times.80 
.times.80 
M3 + + .+-. - - 
.times.160 
.times.160 
.times.80 
.times.40 
.times.40 
Advanced 
G1 + + + + + 
syphilis .times.1280 
.times.1280 
.times.1280 
.times.320 
.times.320 
G2 + + + + + 
.times.640 
.times.640 
.times.640 
.times.320 
.times.320 
G3 + + + .+-. .+-. 
.times.320 
.times.320 
.times.160 
.times.80 
.times.80 
Normal 
N1 - - - - - 
&lt;20 &lt;20 &lt;20 &lt;40 &lt;40 
N2 - - - + + 
&lt;20 &lt;20 .times.40 
.times.160 
.times.160 
N3 - - - .+-. .+-. 
&lt;20 &lt;20 .times.20 
.times.80 
.times.80 
Anti-rabbit 
- - - + - 
tissue antiserum 
&lt;20 &lt;20 .times.40 
.times.160 
.times.20 
Anti-Reiter 
- - - + - 
strain antiserum 
&lt;20 &lt;20 .times.40 
.times.160 
&lt;20 
__________________________________________________________________________ 
(2) Sensitizing solution (B) Sensitization with a predetermined protein 
amount 
Primary 
M1 + + .+-. - - 
syphilis .times.160 
.times.160 
.times.80 
&lt;20 .times.40 
M2 + + + - .+-. 
.times.320 
.times.320 
.times.160 
.times.40 
.times.80 
M3 + + .+-. - - 
.times.160 
.times.160 
.times.80 
.times.40 
.times.40 
Advanced 
G1 + + + + + 
syphilis .times.1280 
.times.1280 
.times.1280 
.times.320 
.times.320 
G2 + + + + + 
.times.640 
.times.640 
.times.640 
.times.160 
.times.320 
G3 + + + .+-. .+-. 
.times.320 
.times.320 
.times.160 
.times.40 
.times.80 
Normal 
N1 - - - - - 
&lt;20 &lt;20 &lt;20 .times.40 
.times.40 
N2 - - - .+-. + 
&lt;20 &lt;20 .times.40 
.times.80 
.times.160 
N3 - - - .+-. .+-. 
&lt;20 &lt;20 .times.20 
.times.80 
.times.80 
Anti-rabbit 
- - - .+-. - 
tissue antiserum 
&lt;20 &lt;20 .times.40 
.times.80 
.times.20 
Anti-Reiter 
- - - .+-. - 
strain antiserum 
&lt;20 &lt;20 .times.40 
.times.80 
&lt;20 
__________________________________________________________________________ 
+: positive (antibody titer more than 80) 
.+-.: false positive (antibody titer of 80) 
-: negative (antibody titer less than 80) 
The value shown below each result represents an antibody titer. 
EXAMPLE 3 
Latex reagent (for measuring method using fully-automatic analyzer) 
In the case of detecting anti-treponemal antibodies, the effect of the 
diagnostic latex reagent as prepared by the present invention was 
confirmed by measuring the agglutination with the use of a fully-automatic 
analyzer. Controls as those used in Example 1 and Example 2 were prepared 
by the same manner as in Example 1 and Example 2 if unspecified. 
1. Preparation of reagent and control 
(1) Latex 
Polystyrene latex (solid:10%) of 0.400 .mu.m (Sekisui Chemical Co., Ltd.) 
was used. 
(2) PBS (pH:7.4) 
A solution of 0.02M phosphate and 0.15M sodium phosphate (2 hydrate), 
disodium phosphate (2 hydrate) and sodium chloride, to which NaN.sub.3 (as 
preservative) was added at 0.1%. 
(3) NaCl--PBS (pH: 6.5) 
A buffer was prepared from sodium phosphate (2 hydrates), disodium 
phosphate (2 hydrates) and sodium chloride to make phosphate concentration 
of 0.02M, NaCl concentration of 1.00M and pH 6.5. 
(4) 100 mM NaPB 
A solution of 100 mM NaPB (pH:7.5) were prepared from disodium hydrogen 
phosphate (anhydrous) and sodium dihydrogen phosphate (12 hydrate), to 
which NaN.sub.3 was added at 0.1%. 
(5) 1% BSA--NaPB 
The above-identified solution was prepared to have BSA in 100 mM NaPB. 
(6) 5% BSA--NaPB 
The above-identified solution was prepared to have 5% BSA in 100 mM NaPB. 
(7) Diluent 
The diluent was prepared by dissolving polyethylene glycol (average 
molecular weight:500,000, Wako Pure Chemical Industries, Ltd.) in 0.25% 
(W/V) in BSA--NaPB. 
(8) Instrument 
Measurement was performed on Hitachi 7050 Type, a fully-automatic analyzer. 
2. Method 
(1) Preparation of antigen sensitizing solution 
To each antigen solution having antigen titer and protein concentration 
shown in Table 2 was added a mixture of 10 mM KPB, of NaCl--PBS and 1% OG 
in the amount shown in Table 5. The obtained solution was defined as the 
sensitizing solution (the antigen solution for immobilization) having pH 
5.4 to 6.5. The sensitizing solution A was prepared to have antigen 
activity of 250 (titer/ml), while the sensitizing solution B was prepared 
to have protein concentration of about 25 .mu.g/ml. 
TABLE 5 
______________________________________ 
Sensitizing solution A 
(Sensitization with a predetermined antigen amount) 
Antigen 1% OG/KPB NaCl/PB 
Antigen Solution (ml) 
pH 6.0 (ml) 
(ml) 
______________________________________ 
Extracted antigen 
0.049 0.251 0.100 
Partially purified 
0.098 0.202 0.100 
antigen 
(HAp purification) 
HCA-200L 0.049 0.251 0.100 
Bio-Gel .RTM. HTP 
0.065 0.235 0.100 
______________________________________ 
______________________________________ 
Sensitizing solution B 
(Sensitization with a predetermined protein amount) 
Antigen 1% OG/KPB NaCl/PB 
Antigen Solution (ml) 
pH 6.0 (ml) 
(ml) 
______________________________________ 
Extracted antigen 
0.027 0.273 0.100 
Partially purified 
0.099 0.201 0.100 
antigen 
(HAp purification) 
HCA-200L 0.130 0.170 0.100 
Bio-Gel .RTM. HTP 
0.239 0.061 0.100 
______________________________________ 
(2) Immobilization of treponemal antigen 
Polystyrene Latex (100 .mu.l) (solid content of 10% by weight) was stirred 
by a magnetic stirrer in an incubator at 4.degree. C., with which the 
treponemal antigen solution (400 .mu.l) prepared in (1) was quickly mixed 
and stirred for 1 hour at 4.degree. C. After the addition of 1% BSA (5 
ml), the resultant mixture was stirred for 1.5 hours at 4.degree. C. and 
then centrifuged for 1 hour at 15,000 rpm. To the obtained pellets was 
added again 1% BSA--NaPB (5 ml). The resultant mixture was centrifuged by 
the same manner as described above and washed. 1% BSA--NaPB (5 ml) was 
added to the final pellets and sufficiently dispersed, thereby affording 
latex reagent having solid content of 0.2%. Thus obtained Latex reagent 
was kept at 4.degree. C. 
(3) Parameters of Hitachi 7050 Type, a fully-automatic analyzer 
______________________________________ 
Sample content 20 .mu.l (serum) 
R1 content 50 .mu.l (Latex reagent) 
R2 content 350 .mu.l (diluent) 
Wavelength 570 nm 
______________________________________ 
(4) Measuring method 
The difference of the absorbance between 80 seconds after the beginning of 
the measurement and 320 seconds after the beginning of the measurement was 
taken. 10.sup.4 of this difference was defined as the variation at O.D. 
570. 
3. Result 
The reaction of each control with the Latex reagent prepared from the TP 
antigen solution by the manner described above was measured as the 
variation of turbidity at O.D. 570. Table 6 shows the results. 
As shown in Table 6, three primary syphilitic sera which were all negative 
with the latex reagents using the extracted antigen and partially purified 
antigen exhibited sensitivity sufficient for determining to be positive 
with the latex reagent using the purified TP antigen of the present 
invention. Three advanced syphilitic sera exhibiting nonspecific reaction 
with the TPHA kit showed no agglutination with the latex reagent using the 
purified TP antigen of the present invention. Further, the latex reagent 
using the present invention did not exhibit a turbidity for determining to 
be positive with the anti-rabbit tissue antiserum and anti-Reiter strain 
antiserum. 
4. Conclusion 
As is apparent from the result, the reagent prepared from the purified 
antigen according to the present invention is more reactive, i.e., 
high-sensitive compared to there from the conventional extracted antigen. 
As a result, even primary syphilis, which cannot be detected by the 
conventional extracted antigen can be detected by using the purified 
antigen according to the present invention. 
Further, the use of the purified antigen can detect primary syphilis, which 
cannot be detected by the commercially available TPHA kit, and also does 
not give the false positive result by the nonspecific reaction. 
TABLE 6 
______________________________________ 
Result with a latex reagent 
HAp purified antigen 
Partially 
Ex- 
Antigen Bio-Gel .RTM. purified 
tracted 
Control HTP HCA-200L antigen 
antigen 
______________________________________ 
Sensitizing Solution (A) 
Sensitization with a predetermined antigen amount 
Primary M1 323 639 157 44 
syphilis 
M2 857 1433 184 103 
M3 646 626 108 38 
Advanced 
G1 2101 3289 1624 678 
syphilis 
G2 1655 2273 683 275 
G3 849 1274 154 110 
Normal N1 4 2 19 55 
N2 3 5 16 21 
N3 2 7 19 21 
Anti-rabbit 3 3 40 105 
tissue 
antiserum 
Anti-Reiter 5 9 32 97 
strain 
antiserum 
Sensitizing Solution (B) 
Sensitization with a predetermined protein amount 
Primary M1 1231 1710 161 16 
syphilis 
M2 3144 3882 171 41 
M3 1231 1695 122 21 
Advanced 
G1 7812 7540 1585 291 
syphilis 
G2 6129 6148 652 100 
G3 3102 3225 171 65 
Normal N1 5 3 317 5 
N2 2 2 15 3 
N3 3 7 19 10 
Anti-rabbit 9 6 39 66 
tissue 
antiserum 
Anti-Reiter 5 8 41 35 
strain 
antiserum 
______________________________________ 
EXAMPLE 4 
Assay for syphilis antibody by ELISA method 
The effect of the purified antigen of the present invention was confirmed 
by ELISA method. 
1. Preparation of reagent and control 
The following reagents and controls were prepared for use. The same 
reagents and controls as those used in Examples 1, 2 and 3 were prepared 
by the same manner as in Examples 1, 2 and 3. 
(1) TP antigen solution 
Used antigen solution were the extracted antigen, partially purified 
antigen and HAp purified antigen obtained in Example 1. 
(2) Control 
The controls used in Example 2 were diluted by a hundred fold with 1% 
BSA/PBS. 
(3) Peroxidase labelled anti-goat Ig-G 
Peroxidase labelled anti-goat Ig-G (originated from sheep) (Miles 
Laboratories Co.) was diluted by a thousand fold with 1% BSA/PBS without 
NaN.sub.3. 
(4) Peroxidase labelled anti-human Ig-G and Ig-M 
Peroxidase labelled anti-goat Ig-G and Ig-M (originated from sheep) (Miles 
Laboratories Co.) were diluted by a thousand fold with 1% BSA/PBS without 
NaN.sub.3. 
(5) Microtiter plate 
A microtiter plate having 96 wells (Nunc Co., flat bottom for ELISA) was 
used. 
(6) Peroxidase substrate 
o-Phenylenediamine (dihydrochloride) (2 mg/ml) and aqueous hydrogen 
peroxide (0.03%) were dissolved in a phosphoric acid-citric acid buffer 
(pH 5.0). The substrate was prepared immediately before being used. 
(7) Stop solution 
1N sulfuric acid solution was used as the stop solution of enzyme reaction. 
2. Method 
(1) Preparation of antigen solution 
By the same manner as in Example 3, to each antigen solution was added a 
mixture of 10 mM KPB, NaCl--PB and 1% OG in the amount shown in Table 7. 
TABLE 7 
______________________________________ 
(Composition of sensitizing solution for ELISA) 
Antigen 
Solution 1% OG/KPB NaCl/PBS 
Antigen (ml) (ml) (ml) 
______________________________________ 
Sensitizing solution A 
(Sensitization with a predetermined antigen amount) 
Extracted 0.049 0.351 0.100 
antigen 
Partially 0.098 0.302 0.100 
purified 
antigen 
(HAp 
purification) 
HCA-200L 0.049 0.351 0.100 
Bio-Gel .RTM. HTP 
0.065 0.335 0.100 
Sensitizing solution B 
(Sensitization with a predetermined protein amount) 
Extracted 0.027 0.373 0.100 
antigen 
Partially 0.099 0.301 0.100 
purified 
antigen 
(HAp 
purification) 
HCA-200L 0.130 0.270 0.100 
Bio-Gel .RTM. HTP 
0.239 0.161 0.100 
______________________________________ 
(2) Immobilization of the TP antigen 
The TP antigen solution prepared in (1) was dispersed into the microtiter 
plate in an amount of 50 .mu.l and incubated for 1 hour at room 
temperature. 
After the incubation, the excess TP antigen solution was removed and washed 
three times with 1% BSA/PBS (200 .mu.l) under suction. Thereafter, 1% 
BSA/PBS (200 .mu.l) was added to the resultant and incubated for 1 hour at 
room temperature for effecting blocking. The plate to which blocking was 
completed was used for antigen-antibody reaction. 
(3) Antigen-antibody reaction 
As a first antibody, the control diluted hundredfold with 1% BSA/PBS was 
pipetted into each well in an amount of 100 .mu.l . As a control, the 
control was similarly pipetted into each well to which 1% BSA/PBS was 
added instead of the antigen. After incubated for 1 hour at room 
temperature, the solution was removed under suction and washed three times 
with 1% BSA/PBS (200 .mu.l) under suction. 
Then, the peroxidase labelled anti-human Ig-G and anti-human Ig-M were 
pipetted in an amount of 100 .mu.l into each well into which the primary 
syphilis sera, advanced syphilis sear and syphilis-negative sear were 
pipetted. Further, the anti-goat Ig-G was pipetted in an amount of 100 
.mu.l into each well into which the anti-Reiter strain antiserum and 
anti-rabbit tissue antiserum were pipetted. After the incubation for 1 
hour at room temperature, each well was sucked and washed three times with 
200 .mu.l of 1% BSA/PBS. Immediately after washing, enzyme activity bound 
to each well was measured. 
(4) Enzymatic reaction 
100 .mu.l of peroxidase substrate was added to each well and the plate was 
incubated for 15 minutes at room temperature. As a substrate blank, the 
substrate was pipetted into each well not containing antigen, first 
antibody or second antibody. After the incubation, 1N stop solution (100 
.mu.l) was added to stop the enzymatic reaction. After stopping the 
reaction, the absorbance at 492 nm was measured with a microtiter plate 
reader (MTP-100, Corona Electric Co., Ltd.), in comparison with the 
substrate blank. 
3. Result 
Table 8 shows the results of the absorbance at 492 nm. 
Three primary syphilitic sear increase in detection sensitivity of IgM by 
using the purified antigen of the present invention. Three 
syphilis-negative controls exhibit falsely positive (the value of more 
than O.D. 0.057 is determined to be positive) by using the extracted 
antigen, while do not exhibit positive by using the purified antigen of 
the present invention. 
Moreover, the purified antigen of the present invention did not show a 
value for determining to be positive with the anti-rabbit tissue antiserum 
and anti-Reiter strain antiserum. 
4. Conclusion 
As is apparent from the above result, the purified antigen according to the 
present invention can detect even primary syphilis, which can not be 
detected by ELISA method using the conventional extracted antigen, and 
also does not give a false positive result by the nonspecific reaction. 
TABLE 8 
__________________________________________________________________________ 
Result of ELISA 
HAp purified antigen 
Partially 
Bio-Gel .RTM. 
purified 
Extracted 
Antigen HTP HCA-200L 
antigen 
antigen 
Control 
1st anti 
anti 
anti 
anti 
anti 
anti 
anti 
anti 
antibody 
Ig-G 
Ig-M 
Ig-G 
Ig-M 
Ig-G 
Ig-M 
Ig-G 
Ig-M 
__________________________________________________________________________ 
(1) Sensitizing Solution (A) 
Sensitization with a predetermined antigen amount 
Primary 
M1 0.125 
0.452 
0.182 
0.518 
0.123 
0.257 
0.050 
0.030 
syphilis 
M2 0.098 
0.258 
0.082 
0.210 
0.102 
0.220 
0.056 
0.062 
M3 0.058 
0.142 
0.064 
0.264 
0.045 
0.084 
0.057 
0.042 
Advanced 
G1 0.984 
0.245 
0.845 
0.214 
0.789 
0.145 
0.874 
0.168 
syphilis 
G2 1.489 
0.478 
1.378 
0.347 
1.540 
0.210 
1.554 
0.355 
G3 0.258 
0.154 
0.321 
0.148 
0.265 
0.081 
0.291 
0.067 
Negative 
N1 0.015 
0.014 
0.012 
0.016 
0.032 
0.024 
0.064 
0.056 
syphilis 
N2 0.020 
0.016 
0.009 
0.013 
0.051 
0.024 
0.224 
0.095 
N3 0.021 
0.012 
0.013 
0.016 
0.042 
0.032 
0.102 
0.081 
Anti-rabbit 
0.009 
-- 0.015 
-- 0.104 
-- 0.278 
-- 
tissue 
antiserum 
Anti-Reiter 
0.015 
-- 0.012 
-- 0.069 
-- 0.225 
-- 
strain 
antiserum 
(2) Sensitizing solution (B) 
Sensitization with a predetermined protein amount 
Primary 
M1 0.138 
0.513 
0.173 
0.476 
0.138 
0.289 
0.042 
0.046 
syphilis 
M2 0.082 
0.376 
0.099 
0.410 
0.132 
0.253 
0.043 
0.051 
M3 0.043 
0.227 
0.061 
0.232 
0.035 
0.075 
0.045 
0.057 
Advanced 
G1 0.965 
0.233 
0.846 
0.256 
0.822 
0.109 
0.278 
0.078 
syphilis 
G2 1.625 
0.489 
1.478 
0.512 
1.579 
0.224 
0.456 
0.125 
G3 0.289 
0.167 
0.335 
0.154 
0.258 
0.084 
0.085 
0.054 
Negative 
N1 0.013 
0.012 
0.009 
0.006 
0.045 
0.030 
0.102 
0.048 
syphilis 
N2 0.016 
0.013 
0.015 
0.008 
0.051 
0.014 
0.152 
0.054 
N3 0.017 
0.018 
0.016 
0.006 
0.038 
0.031 
0.106 
0.043 
__________________________________________________________________________ 
Anti-rabbit 
0.014 
-- 0.012 
-- 0.067 
-- 0.156 
-- 
tissue 
anti-serum 
Anti-Reiter 
0.009 
-- 0.008 
-- 0.078 
-- 0.115 
-- 
strain 
antiserum 
__________________________________________________________________________ 
Average value at O.D. 492 nm (n = 4) 
Reference Example 2 
Confirmation of antigen protein by immunoplotting assay 
1. Reagent and sample 
The same reagents and samples as those used in Example 1, Reference Example 
1 and Example 4 were prepared by the same manner as these Examples. 
(1) Buffer for blotting 
The buffer was prepared by mixing 25 mM Tris buffer, 192 mM glycine and 20% 
methanol to have pH 8.3. 
(2) Nitrocellulose membrane (hereinafter abbreviated to NC) 
Used membrane was 9.times.12 cm sheet having a pore size of 0.45.mu. 
(Bio-Rad Laboratories). 
(3) Syphilis-positive serum 
Pooled primary syphilitic serum and advanced syphilitic serum were used. 
(4) Peroxidase substrate 
4-Chloro-1-naphtol (Nacalai Tesque Inc.) (10 mg) was dissolved in 
ice-cooled methanol (3.34 ml) and then mixed with citrate phosphate buffer 
(pH:6.0, 16.66 ml). To the resultant mixture was added 30% aqueous 
hydrogen peroxide (10 .mu.l) immediately before being used. The substrate 
was used immediately after preparation. 
2. Operation method 
(1) Phast System.RTM. (Pharmacia LKB Biotechnolgy) was used in accordance 
with its instruction. Electrophoresis of the extracted antigen, partially 
purified antigen and HAp purified antigen was conducted by the same manner 
as in Example 2. The same five gels obtained by the electrophoresis were 
prepared. 
(2) NC were in advance immersed in the buffer for blotting to be 
equilibrated. The NC were pleased on the gel for transferring protein from 
the gel to NC by electrophoresis with blotting. 
(3) After washing three times with PBS, NC were immersed in PBS buffer 
containing 3% BSA for blocking. 
(4) The primary syphilitic serum, advanced syphilitic serum, normal 
(syphilis-negative) serum, anti-rabbit tissue antiserum and anti-Reiter 
strain antiserum were diluted hundred fold with 1% BSA/PBS. Each serum was 
reacted with each antigen transferred onto the above-mentioned NC. 
(5) After washing three times with 1% BSA/PBS, the NC reacted with the 
primary syphilitic serum, advanced syphilitic serum and normal serum were 
immersed in 1% BSA/PBS containing the peroxidase labelled anti-human Ig-G 
and anti-human Ig-M for reaction. Similarly, the NC reacted with the 
anti-Reiter strain antiserum and anti-rabbit tissue antiserum were 
immersed in 1% BSA/PBS containing the anti-goat Ig-G conjugated with 
peroxidese for reaction. After the incubation for 1 hour at room 
temperature, the NC were washed three times with PBS, and immediately 
after that, enzyme activity bounded to each NC was observed. 
(6) Peroxidase substrate was added to the NC and incubated at room 
temperature. When a suitable colored image was observed, the membranes 
were washed with purified water and dried. Thereafter, the position and 
degree of the color development were observed. 
3. Result 
It was confirmed that three bands (at the vicinity of the molecular weights 
31,000, 41,000 and 47,000) of the HAp purified antigen were proteins 
specifically reactive with syphilis-positive serum only. The bands reacted 
with the anti-rabbit tissue-antiserum and anti-Reiter strain antiserum 
were observed in the extracted antigen and partially purified antigen, 
while such bands were not found in the HAp purified antigen. 
4. Conclusion 
As is apparent from the result, the TP purified antigen obtained by using 
the hydroxyapatite gel is a high-pure antigen solution consisting of the 
component specifically reactive with the syphilis-positive serum only. 
EXAMPLE 5 
Conditions for adsorption to and elution by hydroxyapatite gel 
The partially purified antigen solution obtained in Example 1 was used. 
Before adding to a column, the buffer of the partially purified antigen 
solution was exchanged to the buffer for adsorption by dialysis. 
Bio-Gel.RTM. HTP (Bio-Rad) was used as hydroxyapatite gel. 
Other reagents and conditions and methods for chromatography are the same 
as those in Example 1. 
Antigen purity (specific activity) in the obtained fractions were observed 
by varying the conditions for adsorption and elution by use of 
Bio-Gel.RTM.HTP. Table 9 shows the result. 
TABLE 9 
______________________________________ 
Buffer 
Solution A Solution B 
(Adsorption) 
(Elution) Fraction Specific 
phosphate Phosphate 
(ratio of 
Activity 
pH conc. pH conc. B, %) (titer/.mu.g) 
______________________________________ 
5.5 10 mM 5.5 350 mM 15-50 27.5 
6.0 10 mM 6.0 350 mM 8-40 28.4 
6.5 5 mm 6.5 350 mM 2-20 20.3 
7.0 5 mM 7.0 350 mM 1-5 17.2 
6.0 10 mM 8.0 10 mM 10-40 19.7 
______________________________________ 
note: Solution A and B are potassium phosphate buffer containing 1% OG. 
EXAMPLE 6 
Latex reagent 
The partially purified antigen solution obtained in Example 1 was used 
which was dissolved in 10 mM potassium phosphate buffer (pH:6.0) 
containing 1% OG. Sensitizing solution having protein concentration of 
30.mu.g/ml, predetermined OG concentration and pre-determined pH was 
prepared by diluting the antigen solution with buffer or adding OG to the 
antigen solution. 
Latex reagent was prepared by the same manner as in Example 3. 
Used control was obtained by diluting rabbit serum with phosphate buffered 
saline [0.02M phosphate buffer, 0.13M sodium chloride (pH:7.4) and 0.1% 
sodium azide] containing 1% BSA. 
The controls were measured with the obtained latex reagent by the same 
manner as in Example 3. Table 10 shows the result. 
TABLE 10 
______________________________________ 
Sensitizing solution 
% for Variation 
cocn. of dilution syphilis- 
syphilis- 
OG (%) pH (%) negative 
positive 
______________________________________ 
0.5 5.0 400 8.0 205.2 
200 6.0 423.0 
100 7.0 1024.0 
0.5 6.0 400 3.0 237.0 
200 5.0 481.2 
100 13.0 1312.0 
0.5 7.0 400 10.0 225.0 
200 9.0 453.6 
100 7.0 1200.0 
0.5 7.5 400 4.0 186.6 
200 7.0 372.0 
100 12.0 960.0 
0.025 6.0 400 6.0 198.0 
200 11.0 420.0 
100 10.0 1200.0 
1.0 6.0 400 7.0 177.0 
200 8.0 372.0 
100 6.0 1088.0 
2.0 6.0 400 3.0 192.0 
200 6.0 378.0 
100 9.0 1016.0 
______________________________________ 
note: 
Controls are syphilisnegative and syphilispositive (10,000 titer) rabbit 
serum. 
Four hundred fold, two hundred fold and one hundred fold the 
syphilispositive serum respectively correspond to 25, 50 and 100 titer.