CA1049403A - Reagent for the detection of syphilis - Google Patents
Reagent for the detection of syphilisInfo
- Publication number
- CA1049403A CA1049403A CA222,900A CA222900A CA1049403A CA 1049403 A CA1049403 A CA 1049403A CA 222900 A CA222900 A CA 222900A CA 1049403 A CA1049403 A CA 1049403A
- Authority
- CA
- Canada
- Prior art keywords
- reagent
- dye
- syphilis
- cardiolipids
- reagent according
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
- G01N31/22—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Endocrinology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
A reagent useful in the detection of syphilis is dis-closed. The reagent consists of an alcoholic solution of cardio-lipids and is characterized by the fact that the lipids are stained in solution by means of a compatible dye. This reagent has the advantage that it can be kept and can be used in automatic blood analysis apparatus.
A reagent useful in the detection of syphilis is dis-closed. The reagent consists of an alcoholic solution of cardio-lipids and is characterized by the fact that the lipids are stained in solution by means of a compatible dye. This reagent has the advantage that it can be kept and can be used in automatic blood analysis apparatus.
Description
~i~4~3 -- 1 The present invention relates to a reagent useful in the detection oE syphilis.
It is known that syphilis disease can in fact be detected by a flocculation reaction between cardiolipids and blood serum or blood plasma. A number of reagents are known, which basically consist of an alcoholic solution of cardiolipids such as Kahnis antigen or Kline's antigen, and by means of which syphilis can be detected by performing laboratory reactions manually. For example, it is a conventional practice to place a few drops of serum on a glass plate and then mix them with the antigen by stirring.
If the reaction is positive the presence of a flocculate is de-tected visibly.
On account of the development of automatic methods for performing various blood analyses, it became useful to include the detection of syphilis among the various systematic analyses. The automatic devices currently commercially available perform in parallel and simultaneously five to seven analyses on serum depend- -ing on the situation~ The detection of syphilis hy automatic methods has raised some difficult problems for the investigators since the known antigens already mentioned for the reactions car-ried out by hand are reagents which have a very short shelf-life and which have to be prepared shortly before the test; this prep-aration consisting of diluting the alcoholic solution of cardio-' lipids in water. -~
Of the apparatus known at the present time for carrying out these reactions automatically, those which work according to the "continuous flow" principle are mentioned, and these consist ~f introducing a sufficient amount of reagent into small quantities of serum or plasma from different sources, which are separated by air bubbles. All the samples of serum move continuously along flexible tubes of narrow cross-section with the aid ofa~pump. The number of tubes will be equal to the number of systematic analyses to be performed on the blood, for example blood group ancl syphilis tests. After the reagen~s have been added to the samples the 9~3 reaction products are poured on absorbent paper, thereby enabling the flocculation and thus the "positive" result to be observed a posteriori.
Such an apparatus, available commercially under the trade mark TECHNI~AN is described by McGrew in "Automation of a Flocculation Test for Syphilis" published by "The American Journal of Clinical Pathology", Vol. 50, No.1.
In order to detect syphilis automatically it was thus necessary to have a reagent whose life is at least several weeks and which also produces very visible reactions, such that there will be no doubt about the "sharpness" or "distinctness" of the re-actions when they are automated, for example when the reaction products are added to the paper. Accordingly, a reagent which involves fixing the cardiolipids on particles of carbon has been developed instead of using an alcoholic solution of cardiolipids.
The blackish colour of these particles enables the test to be read very well.
Details regarding the preparation and use of this reagent are given in U.S. Patent 3,074,853. This reagent, which has been used by McGrew in a "continuous flow" automatic apparatus such as the TECHNICAN apparatus, has given good results.
However, this reagent has a serious disadvantage in that it is extremely expensive. In addition, in certain cases it is ; found that the reaction is no longer visible on account of the fact that the antigen has been removed from the carbon.
One important object of the reagent according to the invention is thus to detect syphilis automatically, since the life of tha present reagent enables this to be possible and the reaction is made very ~isible when the flocculate is deposited on a sheet of absorbent paper.
This reagent is much less expensive and is also extrem-; ely sensitive to the reaction, and it is possible to "read" the reaction very clearly and distinctly on account of the fact that . .
~494~)3 the size of the lipid micelles in solution is homogeneous.
The reagent according to the invention consists of analcoholic solution of cardiolipids, characterized by the fact that the lipids are stained in solution by means of a dye which is compatible with and soluble in alcohol.
The invention also relates to a process for preparing this reagent, and which comprises heating an alcoholic solution of cardiolipids and a dye for the cardiolipids to a temperature between about 50 and 60 C., and diluting the mixture with physio- ~
10 logical saline at a temperature of about 30 to 50C. ~ -The invention will be more fully understood from the following description of the reagent and also a process for the preparation thereof.
A dye for lipids which dissolves in alcohol and becomes fixed on the lipids is added to an alcoholic solution of cardio-lipids which may possibly comprise egg yolk, lecithin and may be . other adjuvants; or, more simply, is added to commercially avail- ~:
able Kahn's or Rline's antigens. Various dyes have been tested .-and it has been found that black dyes give the best results, at least as regards the subsequent reading of the flocculation on the paper. Thus, Amido black, Cerol black or Sudan black are mentioned among the conventional dyes for lipids. Scarlet-red, Congo or ruthenium dyes are also satisfactory. The amount of dye used .
with respect to the alcoholic solution of cardiolipids is a .
' function of the nature of the dye and its solubility. An amount . of dye is used which enables a saturated solu-tion to be obtained, :
while avoiding an excess which might precipitate without dissolv- .
ing. Too small an amount of dye will, among other things, tend to reduce the sharpness of the reaction.
. 30 Heating the mixture to 50 - 60 C. for ten to forty :
minutes while shaking will stain the lipids by causing the dyes ~ : :
to be fixed, but will not denature the lipids.
The mixture can then be diluted while hot with physio-.. .. . . . . . .
.. : ~ . , . . . . , , : . : - ~
::IL0~9403 logical saline. In order to obtain a homogeneous formation of micelles during this dilution it is important not to cause any discontinuity in temperature. Physiological saline at the same temperature as the mixture, i.e. between 30 and 50C., will therefore be used.
A preserving agent and various adjuvants for improving ~0 : ~
'" ', .
" .
- .:
~94~3 the sensitivity of the reaction can be added to the solution.
The automatic apparatus employed with a "TEC~INICAN"
apparatus. The syphilis detection was carried out by the Blood Transfusion Centre in order to determine whether the donors' blood is free from disease and can therefore be used in transfusions.
Details of the apparatus and its use in this field are given in the ", , AMERICAN JOURNAL OF CLINIC~L PATHOLOGY, Vol 50, No. 1, and in the bibliography mentioned. The apparatus for employing the re-agent in accordance with the invention was not modified.
A non-limiting example of the preparation which has been found to be completely satisfactory in the automatic detection of syphilis, both as regards the preservation of the product and the sharpness of the reactions, is given hereinafter:
145 mg of Sudan black were added to 45 ml of a solution of Kahn's antigen. The mixture was heated for twenty minutes at , about 60C. while shaking. The mixture was allowed to stand for ', half-an~hour and one cc. of Sumatra benzoin was added.
,, The stained antigen, kept at 50C., is then poured drop by drop into 300 ml of physiological saline (NaCl 15/1000), also ' 20 heated to 50C. The micelles of the stained lipids which are ob-', tained are homogeneous in size.
3.5 ml of (N~I4)2SO4 (saturated solution) are then addedO
The ammonium sulphate increases the sensitivity of the reaction.
'I The mixture is left to stand at ambient temperature and ' the reagent is then kept at 4C. for a week.
~, l g of choline chloride per l ml of solution is added to reduce the percentage of false positives (false positive-, reactions). ,, The reagent may be kept for six weeks at 4C.
Reagents of the same quality have also been prepared from Kline's antigen or the dyes already mentioned.
;~, A reagent and a process for the preparation thereof, ~ which is particularly suitable for detecting syphilis automatically, `' has been described and the mixtu~e~descrihéd above has been found .. ~ ,, ~ . :................ .
.. ...
. `.`
1~9~
to be satisfactory when used in a continuous flow apparatus.
It will of course be possible for a person skilled in the art to produce minor modifications, depending on the desired characteristics of the reagent, functioning of the apparatus used, etc., by adding adjuvants in order to obtain these characteristics.
For example, it would be possible to add tà the reagent products for increasing the density of the micelles and their granulation, with a view to using the reagent in other types of apparatus.
In order to check the effectiveness and reliability of the reagent prepared in the example above, the following procedure ; is adopted:
; 389 samples of fresh serum were used, all of which ga~e four positive reactions using the following known methods:
Treponeme: Reiter and Kolmer method.
Cardiolipids; Kline and Kahn's reagents.
If the reaction was doubtful the sample was discarded.
These samples were analysed with a TECHNICAN apparatus using the prepared reagent:
384 reactions were positive 5 reactions were doubtful The results are thus found to be accurate in 98.7~ of the cases analysed.
.1 . .
:` :
It is known that syphilis disease can in fact be detected by a flocculation reaction between cardiolipids and blood serum or blood plasma. A number of reagents are known, which basically consist of an alcoholic solution of cardiolipids such as Kahnis antigen or Kline's antigen, and by means of which syphilis can be detected by performing laboratory reactions manually. For example, it is a conventional practice to place a few drops of serum on a glass plate and then mix them with the antigen by stirring.
If the reaction is positive the presence of a flocculate is de-tected visibly.
On account of the development of automatic methods for performing various blood analyses, it became useful to include the detection of syphilis among the various systematic analyses. The automatic devices currently commercially available perform in parallel and simultaneously five to seven analyses on serum depend- -ing on the situation~ The detection of syphilis hy automatic methods has raised some difficult problems for the investigators since the known antigens already mentioned for the reactions car-ried out by hand are reagents which have a very short shelf-life and which have to be prepared shortly before the test; this prep-aration consisting of diluting the alcoholic solution of cardio-' lipids in water. -~
Of the apparatus known at the present time for carrying out these reactions automatically, those which work according to the "continuous flow" principle are mentioned, and these consist ~f introducing a sufficient amount of reagent into small quantities of serum or plasma from different sources, which are separated by air bubbles. All the samples of serum move continuously along flexible tubes of narrow cross-section with the aid ofa~pump. The number of tubes will be equal to the number of systematic analyses to be performed on the blood, for example blood group ancl syphilis tests. After the reagen~s have been added to the samples the 9~3 reaction products are poured on absorbent paper, thereby enabling the flocculation and thus the "positive" result to be observed a posteriori.
Such an apparatus, available commercially under the trade mark TECHNI~AN is described by McGrew in "Automation of a Flocculation Test for Syphilis" published by "The American Journal of Clinical Pathology", Vol. 50, No.1.
In order to detect syphilis automatically it was thus necessary to have a reagent whose life is at least several weeks and which also produces very visible reactions, such that there will be no doubt about the "sharpness" or "distinctness" of the re-actions when they are automated, for example when the reaction products are added to the paper. Accordingly, a reagent which involves fixing the cardiolipids on particles of carbon has been developed instead of using an alcoholic solution of cardiolipids.
The blackish colour of these particles enables the test to be read very well.
Details regarding the preparation and use of this reagent are given in U.S. Patent 3,074,853. This reagent, which has been used by McGrew in a "continuous flow" automatic apparatus such as the TECHNICAN apparatus, has given good results.
However, this reagent has a serious disadvantage in that it is extremely expensive. In addition, in certain cases it is ; found that the reaction is no longer visible on account of the fact that the antigen has been removed from the carbon.
One important object of the reagent according to the invention is thus to detect syphilis automatically, since the life of tha present reagent enables this to be possible and the reaction is made very ~isible when the flocculate is deposited on a sheet of absorbent paper.
This reagent is much less expensive and is also extrem-; ely sensitive to the reaction, and it is possible to "read" the reaction very clearly and distinctly on account of the fact that . .
~494~)3 the size of the lipid micelles in solution is homogeneous.
The reagent according to the invention consists of analcoholic solution of cardiolipids, characterized by the fact that the lipids are stained in solution by means of a dye which is compatible with and soluble in alcohol.
The invention also relates to a process for preparing this reagent, and which comprises heating an alcoholic solution of cardiolipids and a dye for the cardiolipids to a temperature between about 50 and 60 C., and diluting the mixture with physio- ~
10 logical saline at a temperature of about 30 to 50C. ~ -The invention will be more fully understood from the following description of the reagent and also a process for the preparation thereof.
A dye for lipids which dissolves in alcohol and becomes fixed on the lipids is added to an alcoholic solution of cardio-lipids which may possibly comprise egg yolk, lecithin and may be . other adjuvants; or, more simply, is added to commercially avail- ~:
able Kahn's or Rline's antigens. Various dyes have been tested .-and it has been found that black dyes give the best results, at least as regards the subsequent reading of the flocculation on the paper. Thus, Amido black, Cerol black or Sudan black are mentioned among the conventional dyes for lipids. Scarlet-red, Congo or ruthenium dyes are also satisfactory. The amount of dye used .
with respect to the alcoholic solution of cardiolipids is a .
' function of the nature of the dye and its solubility. An amount . of dye is used which enables a saturated solu-tion to be obtained, :
while avoiding an excess which might precipitate without dissolv- .
ing. Too small an amount of dye will, among other things, tend to reduce the sharpness of the reaction.
. 30 Heating the mixture to 50 - 60 C. for ten to forty :
minutes while shaking will stain the lipids by causing the dyes ~ : :
to be fixed, but will not denature the lipids.
The mixture can then be diluted while hot with physio-.. .. . . . . . .
.. : ~ . , . . . . , , : . : - ~
::IL0~9403 logical saline. In order to obtain a homogeneous formation of micelles during this dilution it is important not to cause any discontinuity in temperature. Physiological saline at the same temperature as the mixture, i.e. between 30 and 50C., will therefore be used.
A preserving agent and various adjuvants for improving ~0 : ~
'" ', .
" .
- .:
~94~3 the sensitivity of the reaction can be added to the solution.
The automatic apparatus employed with a "TEC~INICAN"
apparatus. The syphilis detection was carried out by the Blood Transfusion Centre in order to determine whether the donors' blood is free from disease and can therefore be used in transfusions.
Details of the apparatus and its use in this field are given in the ", , AMERICAN JOURNAL OF CLINIC~L PATHOLOGY, Vol 50, No. 1, and in the bibliography mentioned. The apparatus for employing the re-agent in accordance with the invention was not modified.
A non-limiting example of the preparation which has been found to be completely satisfactory in the automatic detection of syphilis, both as regards the preservation of the product and the sharpness of the reactions, is given hereinafter:
145 mg of Sudan black were added to 45 ml of a solution of Kahn's antigen. The mixture was heated for twenty minutes at , about 60C. while shaking. The mixture was allowed to stand for ', half-an~hour and one cc. of Sumatra benzoin was added.
,, The stained antigen, kept at 50C., is then poured drop by drop into 300 ml of physiological saline (NaCl 15/1000), also ' 20 heated to 50C. The micelles of the stained lipids which are ob-', tained are homogeneous in size.
3.5 ml of (N~I4)2SO4 (saturated solution) are then addedO
The ammonium sulphate increases the sensitivity of the reaction.
'I The mixture is left to stand at ambient temperature and ' the reagent is then kept at 4C. for a week.
~, l g of choline chloride per l ml of solution is added to reduce the percentage of false positives (false positive-, reactions). ,, The reagent may be kept for six weeks at 4C.
Reagents of the same quality have also been prepared from Kline's antigen or the dyes already mentioned.
;~, A reagent and a process for the preparation thereof, ~ which is particularly suitable for detecting syphilis automatically, `' has been described and the mixtu~e~descrihéd above has been found .. ~ ,, ~ . :................ .
.. ...
. `.`
1~9~
to be satisfactory when used in a continuous flow apparatus.
It will of course be possible for a person skilled in the art to produce minor modifications, depending on the desired characteristics of the reagent, functioning of the apparatus used, etc., by adding adjuvants in order to obtain these characteristics.
For example, it would be possible to add tà the reagent products for increasing the density of the micelles and their granulation, with a view to using the reagent in other types of apparatus.
In order to check the effectiveness and reliability of the reagent prepared in the example above, the following procedure ; is adopted:
; 389 samples of fresh serum were used, all of which ga~e four positive reactions using the following known methods:
Treponeme: Reiter and Kolmer method.
Cardiolipids; Kline and Kahn's reagents.
If the reaction was doubtful the sample was discarded.
These samples were analysed with a TECHNICAN apparatus using the prepared reagent:
384 reactions were positive 5 reactions were doubtful The results are thus found to be accurate in 98.7~ of the cases analysed.
.1 . .
:` :
Claims (7)
PROPERTY OR PRIVILEGS IS CLAIMED ARE DEFINED AS FOLLOWS:
1. Reagent for use in the detection of syphilis, com-prising an alcoholic solution of cardiolipids, the cardiolipids being coloured by means of a dye which is compatible with and is soluble in alcohol.
2. Reagent according to claim 1, wherein the dye is a black dye, selected from Sudan black, Amido black or Cerol.
3. Reagent according to claim 1, including Sumatra benzoin as preserving agent.
4. Reagent according to claim 1, 2, or 3, including a saturated ammonium sulphate solution.
5. Reagent according to claim 1, 2, or 3, including choline chloride.
6. Reagent according to claim 1, 2, or 3, wherein the amount of dye used saturates the alcoholic solution of cardio-lipids.
7. Process for preparing a reagent used in the detect-ion of syphilis, comprising heating an alcoholic solution of cardiolipids and a dye for the cardiolipids to a temperature between about 50 and 60°C. and diluting the mixture with physio-logical saline at a temperature of about 30 to 50°C.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR7411004A FR2266169B1 (en) | 1974-03-26 | 1974-03-26 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1049403A true CA1049403A (en) | 1979-02-27 |
Family
ID=9137018
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA222,900A Expired CA1049403A (en) | 1974-03-26 | 1975-03-24 | Reagent for the detection of syphilis |
Country Status (10)
| Country | Link |
|---|---|
| JP (1) | JPS5315132B2 (en) |
| CA (1) | CA1049403A (en) |
| CH (1) | CH589294A5 (en) |
| DE (1) | DE2511688A1 (en) |
| ES (1) | ES436045A1 (en) |
| FR (1) | FR2266169B1 (en) |
| GB (1) | GB1447470A (en) |
| IL (1) | IL46963A (en) |
| SE (1) | SE7503428L (en) |
| ZA (1) | ZA751873B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2349836A2 (en) * | 1976-04-27 | 1977-11-25 | Lille Transfusion Sanguine | Automatic test for syphilis - using a cardio-lipid soln. satd. with a dye |
| EP0009088A1 (en) * | 1978-09-18 | 1980-04-02 | F. HOFFMANN-LA ROCHE & CO. Aktiengesellschaft | Reagent for the determination of Wassermann antibodies, method for its preparation, its use as such and testkit containing this reagent |
| JPS584778U (en) * | 1981-06-30 | 1983-01-12 | ダイキン工業株式会社 | Safety device for lubrication pump |
| JPH0198976U (en) * | 1987-12-24 | 1989-07-03 | ||
| JPH0711925A (en) * | 1993-06-25 | 1995-01-13 | Kubota Corp | Pressure adjuster for lubrication oil of engine |
-
1974
- 1974-03-26 FR FR7411004A patent/FR2266169B1/fr not_active Expired
- 1974-07-03 CH CH913974A patent/CH589294A5/xx not_active IP Right Cessation
-
1975
- 1975-03-18 DE DE19752511688 patent/DE2511688A1/en active Pending
- 1975-03-24 CA CA222,900A patent/CA1049403A/en not_active Expired
- 1975-03-25 SE SE7503428A patent/SE7503428L/xx unknown
- 1975-03-25 ZA ZA00751873A patent/ZA751873B/en unknown
- 1975-03-25 GB GB1243175A patent/GB1447470A/en not_active Expired
- 1975-03-26 JP JP3563475A patent/JPS5315132B2/ja not_active Expired
- 1975-03-26 ES ES436045A patent/ES436045A1/en not_active Expired
- 1975-03-28 IL IL46963A patent/IL46963A/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| FR2266169A1 (en) | 1975-10-24 |
| IL46963A0 (en) | 1975-08-31 |
| IL46963A (en) | 1977-07-31 |
| JPS517119A (en) | 1976-01-21 |
| ZA751873B (en) | 1976-02-25 |
| GB1447470A (en) | 1976-08-25 |
| ES436045A1 (en) | 1977-01-01 |
| CH589294A5 (en) | 1977-06-30 |
| FR2266169B1 (en) | 1977-06-17 |
| DE2511688A1 (en) | 1975-10-02 |
| SE7503428L (en) | 1975-09-29 |
| JPS5315132B2 (en) | 1978-05-23 |
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