CN1405565A - Diagnosis reagent for syphilis spirochete antibody - Google Patents
Diagnosis reagent for syphilis spirochete antibody Download PDFInfo
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- CN1405565A CN1405565A CN 02137626 CN02137626A CN1405565A CN 1405565 A CN1405565 A CN 1405565A CN 02137626 CN02137626 CN 02137626 CN 02137626 A CN02137626 A CN 02137626A CN 1405565 A CN1405565 A CN 1405565A
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Abstract
This invention discloses a diagnostic reagent of syphilis spirochete antibody enzyme labeling for diagnosing syphilis spirochete infection, containing synthetic peptide antigen or genetic engineering expressed antigen: antigen P15, P17, P47, 0.1-10 mg/ml coated on the enzyme labeling, the enzyme labeling two antigens (IgG, IgM): containing 1:20-1:100K their diluents solution and color-developing agent combination and solubilizer in which the said color-developing agent in the combination is TMB. The said combination can include combination of TMB with hydrogen peroxide cumon ir its derivatives or TMB with hydroxyurea. On the bases of applied synthetic peptide antigen and genetic engineering expressing antigen, a TMB saline is used in the diagnostic reagent with excellent water solubility and stable performance, matched with proper solubilizers, stabilizers to get much better stable performance.
Description
Technical field
The present invention relates to a kind of syphilis spirochete antibody labeled diagnosis reagent that is used to diagnose infection by Treponema pallidum.
Technical background
Syphilis is a kind of sexually transmitted disease that is caused by microspironema pallidum (spirillum) of complexity, its pathogen is a spirochaeta pallida, mucous membrane and skin to human body have very strong penetration power, and its circulation way is contagion, sexual intercourse, propagate main path through the blood vertical transmission.The course of disease development of syphilis generally includes first phase, second phase and latent period.The tertiary syphilis symptom can occur during the 1-30 after first phase infects, and comprises the gumme damage, cardiovascular syphilis and nervous system syphilis.Because microspironema pallidum can not be in vitro culture, so diagnosis can only rely on the direct sight of clinical sign, damage location bacterium to look into and serological method.Except utmost point early syphilis, serological method is suitable for the controller used in syphilis diagnosis in other all stages.The serodiagnosis of syphilis method can be divided into two classes, promptly non-helical body (antigen) serological method and conveyor screw (antigen) serological method at present.Two methods and case history all are essential for finally making a definite diagnosis.
Syphilis detects common method at present has: RPR, TPHA, ELISA etc.What RPR (being quick seroreaction prime ring shape card test method) detected is the microspironema pallidum non-specific antibody, the susceptibility of method is poor, poor specificity, false negative and false positive more than 30% are arranged, because syphilis is very big to the mankind's harmfulness, and transmissibility is strong, therefore, improves the specificity and the susceptibility no less important of its detection.TPHA (microspironema pallidum oppositely indirectly hemagglutination test) though method specificity height but susceptibility not as ELISA (diagnosis of syphilis spirochete antibody mark) method, and the reagent stability operation must rely on manual, can not realize robotization, can't carry out batch detection, therefore its practical performance is nothing like the ELISA method, so easy to use, sensitive, the stable ELISA method syphilis detection kit of development is used for replacing RPR and TPHA, is the task of top priority as primary dcreening operation and the confirmed diagnosis test of diagnosing syphilis.
In recent years, the application of syphilis ELISA reagent is popularized gradually, common problem of syphilis ELISA reagent ubiquity.Because its Color Appearance System instability, usually chromogenic substrate TMB, hydrogen peroxide are separately preserved to use and drip, this makes blood in enormous quantities source when screening workload to increase and is twice, more dangerous is in thickly dotted enzyme mark micropore just in case leak when adding one of them chromogenic substrate, causing testing result to be false negative, is to well imagine if the syphilis positive serum causes blood transfusion to infect its harm because of false negative.
TMB is an ELISA reagent developer commonly used, because 3,3 ', 5,5 '-tetramethyl benzidine (being called for short TMB) is insoluble in water, and need add a large amount of solvents usually increases its solubleness; Mix when using with another chromogenic substrate hydrogen peroxide in the reaction system and to become blue look easily, cause clinical judgment error (exceeding CutOff value), patient, hospital are all caused burden and waste.
Summary of the invention
The objective of the invention is to provide for the deficiency that solves above-mentioned technology a kind of in syphilis ELISA detects performance more stable, avoid the syphilis positive serum because the test error causes false negative phenomenon, and can alleviate the syphilis spirochete antibody labeled diagnosis reagent of workload.
In order to achieve the above object, a kind of syphilis spirochete antibody labeled diagnosis reagent provided by the present invention, comprise that the antigen with antigenic synthetic peptide or gene engineering expression: P15, P17, P47 antigen 0.1～10 μ g/ml are coated on the ELISA Plate, ELIAS secondary antibody (IgG, IgM): contain 1: 20～1: 100K ELIAS secondary antibody dilute solution and developer combination and solubilizer, the developer in the described developer combination is a TMB propane sulfonic acid sodium; Described developer combination comprises the combination of TMB propane sulfonic acid sodium and hydrogen phosphide cumene or derivatives thereof, also can be the combination of TMB propane sulfonic acid sodium and hydroxycarbamide.Described solubilizer can be N-N-methyl-2-2-pyrrolidone N-, ethanol, methyl alcohol, acetone, dimethyl sulfoxide (DMSO).Described developer combined content can be 0.01～0.5 part in a TMB propane sulfonic acid sodium, all the other cooperate composition to be: 0.01～0.5 part of ProClin300 (2-methyl-4-isothiazoline-3-ketone), piperazine-N, two (2-ethanesulfonic acid) damping fluid 5-200mmol/L of N`-, pH of buffer 3～9,0.01～0.5 part of hydrogen phosphide cumene or derivatives thereof, 0.1～50 part of N-N-methyl-2-2-pyrrolidone N-, ethanol 1-50 part.Described developer TMB propane sulfonic acid sodium content is 0.01～0.5 part, all the other cooperate composition to be: 0.01～0.5 part of ProClin300 (2-methyl-4-isothiazoline-3-ketone), piperazine-N, two (2-ethanesulfonic acid) damping fluid 5-200mmol/L of N`-, 1～50 part of pH of buffer 3～9,0.01～0.5 part of hydroxycarbamide, hydroxypropyl cyclodextrin 0.1～50mmol/L, dimethyl sulfoxide (DMSO).
Syphilis spirochete antibody labeled diagnosis reagent provided by the invention, on the antigen basis of using antigenic synthetic peptide, gene engineering expression, used that a kind of new type water-solubility is fabulous, the TMB salt of stable performance, cooperate an amount of solubilizer, stabilizing agent, make the syphilis spirochete antibody labeled diagnosis reagent obtain better stability.Its high specific, high sensitivity, easy and simple to handle, stable reagent make that screen in batches in the robotization of syphilis detection energy, blood source, standardized management is guaranteed.
Embodiment
The present invention is further described by the following embodiment.
Embodiment 1:
Reagent is formed:
1, with the antigen of antigenic synthetic peptide or gene engineering expression: P15, P17, P47 antigen 1 μ g/ml are coated on the ELISA Plate.
2, ELIAS secondary antibody (IgG, IgM): contain 1: 35K ELIAS secondary antibody dilute solution
3, two-in-one developer:
0.05 part in TMB propane sulfonic acid sodium;
0.05 part of hydrogen phosphide cumene;
10 parts of N-N-methyl-2-2-pyrrolidone N-s;
10 parts of ethanol;
0.05 part of ProClin300 (2-methyl-4-isothiazoline-3-ketone);
Piperazine-N, two (2-ethanesulfonic acid) damping fluid 20mmol/L pH7.0 of N`-.
Embodiment 2:
Reagent is formed:
1, with the antigen of antigenic synthetic peptide or gene engineering expression: P15, P17, P47 antigen 1 g/ml wrap quilt
On ELISA Plate.
2, ELIAS secondary antibody (IgG, IgM): contain 1: 35K ELIAS secondary antibody dilute solution
3, two-in-one developer:
0.05 part in TMB propane sulfonic acid sodium;
0.05 part of hydroxycarbamide;
Hydroxypropyl cyclodextrin-5mmol/L;
10 parts of dimethyl sulfoxide (DMSO)s;
0.05 part of ProClin300 (2-methyl-4-isothiazoline-3-ketone);
Piperazine-N, two (2-ethanesulfonic acid) damping fluid 20mmol/L pH7.0 of N`-.
Reference examples:
Reagent is mainly formed:
1, with the antigen of antigenic synthetic peptide or gene engineering expression: P15, P17, P47 antigen 1 g/ml are coated on the ELISA Plate.
2, ELIAS secondary antibody (IgG, IgM): contain 1: 35K ELIAS secondary antibody dilute solution
3, two-in-one developer:
TMB0.03 part
0.05 part of hydroxycarbamide
20 parts in acetone
Citrate buffer solution 20mmol/L pH4.5
Detection method:
Sample-10 μ l is with 100 μ l sample dilutions mixing in the ELISA Plate hole of above-mentioned envelope antigen, putting 37 ℃ takes out after 30 minutes and washes plate 5 times with washing lotion, add above-mentioned enzyme labelled antibody 100 μ l/ holes, put 37 ℃ of taking-ups in 20 minutes and wash plate 5 times with washing lotion, add the two-in-one developer 100 μ l/ holes in above-mentioned each example, 37 ℃ 10 minutes, take out to add stop buffer 100 μ l/ holes, read the A value at microplate reader 450nm/650nm place.
Native system CutOff value is between 0.18-0.20
1, detects the absorbance of positive sample and the stability of reagent blank (developer)
Table one
| Positive sample OD value | Reagent blank | ||
| Newly join | Embodiment 1 | ????1.878 | ????0.020 |
| Embodiment 2 | ????1.638 | ????0.020 | |
| Reference examples | ????1.727 | ????0.020 | |
| Seven days | Embodiment 1 | ????1.872 | ????0.022 |
| Embodiment 2 | ????1.636 | ????0.023 | |
| Reference examples | ????1.700 | ????0.256 * | |
| Half a year | Embodiment 1 | ????1.833 | ????0.023 |
| Embodiment 2 | ????1.588 | ????0.022 | |
| Reference examples | ?????- | ????** | |
| 1 year | Embodiment 1 | ????1.782 | ????0.025 |
| Embodiment 2 | ????1.539 | ????0.026 | |
| Reference examples | ??????- | ????** | |
*It is blue that the reagent color becomes
*The reagent blueness that darkens
As can be seen from Table I, implementing 1,2 reaction sensitivities and reference examples does not have too big-difference, still has higher sensitivity, and the TMB Color Appearance System of enforcement 1,2 is highly stable, still kept blank very low in 1 year, and reference examples seven days Lan Seyi just obviously occur and exceeds the CutOff value and can't use.
2, clinical control
Assay method is the same
105 routine syphilis serum results of comparison
Table two
| Stadium/be subordinate to | ??????RPR | Embodiment 1 | Embodiment 2 | Reference examples | ||||
| Number positive | Positive rate | Number positive | Positive rate | Number positive | Positive rate | Number positive | Positive rate | |
| 1 phases 48 example | 29 examples | ?60.4％ | 47 examples | 97.9％ | 47 examples | 97.9％ | 47 examples | 97.9％ |
| 2 phases 57 examples | 55 examples | ?96.5％ | 57 examples | 100％ | 57 examples | 100％ | 57 examples | 100％ |
| Add up to 105 | 84 examples | ?80％ | 104 examples | 99％ | 104 examples | 99％ | 104 examples | 99％ |
Embodiments of the invention, reference examples are compared with domestic common RPR method as can be seen from Table II higher specificity, is a kind of new method that is worthy to be popularized.
For selected different solubilizer and stabilizing agent, also can provide different syphilis spirochete antibody labeled diagnosis reagent embodiment:
Embodiment 3:
Reagent is formed:
1, with the antigen of antigenic synthetic peptide or gene engineering expression: P15, P17, P47 antigen 1 μ g/ml are coated on the ELISA Plate.
2, ELIAS secondary antibody (IgG, IgM): contain 1: 35K ELIAS secondary antibody dilute solution
3, two-in-one developer:
0.5 part in TMB propane sulfonic acid sodium;
0.01 part of hydroxycarbamide;
50% part of N-N-methyl-2-2-pyrrolidone N-;
0.5 part of ProClin300 (2-methyl-4-isothiazoline-3-ketone);
Piperazine-N, two (2-ethanesulfonic acid) damping fluid 100mmol/L pH8.0 of N`-.
Embodiment 4:
Reagent is formed:
1, with the antigen of antigenic synthetic peptide or gene engineering expression: P15, P17, P47 antigen 1 g/ml are coated on the ELISA Plate.
2, ELIAS secondary antibody (IgG, IgM): contain 1: 35K ELIAS secondary antibody dilute solution
3, two-in-one developer:
0.01 part in TMB propane sulfonic acid sodium;
0.5 part of hydroxycarbamide;
Hydroxypropyl cyclodextrin 50mmol/L; 20 parts of dimethyl sulfoxide (DMSO)s;
0.01 part of ProClin300 (2-methyl-4-isothiazoline-3-ketone);
Piperazine-N, two (2-ethanesulfonic acid) damping fluid 5mmol/L pH4.0 of N`-.
Claims (4)
1, a kind of syphilis spirochete antibody labeled diagnosis reagent, comprise that the antigen with antigenic synthetic peptide or gene engineering expression: P15, P17, P47 antigen 0.1～10 μ gg/ml are coated on the ELISA Plate, ELIAS secondary antibody (IgG, IgM): contain 1: 20～1: 100K ELIAS secondary antibody dilute solution and developer combination is characterized in that the developer in the described developer combination is a TMB propane sulfonic acid sodium.
2, a kind of syphilis spirochete antibody labeled diagnosis reagent according to claim 1 is characterized in that described developer combination comprises the combination of TMB propane sulfonic acid sodium and hydrogen phosphide cumene or derivatives thereof or the combination of TMB propane sulfonic acid sodium and hydroxycarbamide.
3, a kind of syphilis spirochete antibody labeled diagnosis reagent according to claim 2, it is characterized in that described developer combined content is 0.01～0.5 part in a TMB propane sulfonic acid sodium, all the other cooperate composition to be: 0.01～0.5 part of ProClin300 (2-methyl-4-isothiazoline-3-ketone), piperazine-N, two (2-ethanesulfonic acid) damping fluid 5-200mmol/L of N`-, pH of buffer 3～9,0.01～0.5 part of hydrogen phosphide cumene or derivatives thereof, 0.1～50 part of N-N-methyl-2-2-pyrrolidone N-, ethanol 1-50 part.
4, a kind of syphilis spirochete antibody labeled diagnosis reagent according to claim 2, it is characterized in that described developer is 0.01～0.5 part in a TMB propane sulfonic acid sodium, all the other cooperate composition to be: 0.01～0.5 part of ProClin300 (2-methyl-4-isothiazoline-3-ketone), piperazine-N, two (2-ethanesulfonic acid) damping fluid 5-200mmol/L of N`-, 1～50 part of pH of buffer 3～9,0.01～0.5 part of hydroxycarbamide, hydroxypropyl cyclodextrin 0.1～50mmol/L, dimethyl sulfoxide (DMSO).
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02137626 CN1405565A (en) | 2002-10-24 | 2002-10-24 | Diagnosis reagent for syphilis spirochete antibody |
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|---|---|---|---|
| CN 02137626 CN1405565A (en) | 2002-10-24 | 2002-10-24 | Diagnosis reagent for syphilis spirochete antibody |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101738473B (en) * | 2008-11-13 | 2013-06-05 | 威海威高生物科技有限公司 | Treponema pallidum antibody diagnostic kit and preparation method thereof |
| CN104155450A (en) * | 2014-08-20 | 2014-11-19 | 厦门大学附属中山医院 | Treponema pallidum IgG antibody biotin avidin enzyme-linked immune detection kit and preparation method thereof |
| CN104316681A (en) * | 2014-11-10 | 2015-01-28 | 厦门大学附属中山医院 | Treponema pallidum specific antibody chemical light emitting detection kit and preparation method thereof |
| CN111896744A (en) * | 2020-07-28 | 2020-11-06 | 重庆澳龙生物制品有限公司 | Goat echinococcus antibody ELISA detection kit and preparation method and application thereof |
-
2002
- 2002-10-24 CN CN 02137626 patent/CN1405565A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101738473B (en) * | 2008-11-13 | 2013-06-05 | 威海威高生物科技有限公司 | Treponema pallidum antibody diagnostic kit and preparation method thereof |
| CN104155450A (en) * | 2014-08-20 | 2014-11-19 | 厦门大学附属中山医院 | Treponema pallidum IgG antibody biotin avidin enzyme-linked immune detection kit and preparation method thereof |
| CN104155450B (en) * | 2014-08-20 | 2016-02-17 | 厦门大学附属中山医院 | Microspironema pallidum IgG antibody biotin-labeled pentylamine enzyme-linked immunologic detecting kit and preparation method thereof |
| CN104316681A (en) * | 2014-11-10 | 2015-01-28 | 厦门大学附属中山医院 | Treponema pallidum specific antibody chemical light emitting detection kit and preparation method thereof |
| CN111896744A (en) * | 2020-07-28 | 2020-11-06 | 重庆澳龙生物制品有限公司 | Goat echinococcus antibody ELISA detection kit and preparation method and application thereof |
| CN111896744B (en) * | 2020-07-28 | 2022-02-22 | 重庆澳龙生物制品有限公司 | Goat echinococcus antibody ELISA detection kit and preparation method and application thereof |
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