CN109596823B - Special diluent for detecting streptococcus pneumoniae in urine sample - Google Patents
Special diluent for detecting streptococcus pneumoniae in urine sample Download PDFInfo
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- CN109596823B CN109596823B CN201811503782.5A CN201811503782A CN109596823B CN 109596823 B CN109596823 B CN 109596823B CN 201811503782 A CN201811503782 A CN 201811503782A CN 109596823 B CN109596823 B CN 109596823B
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- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
Abstract
The invention relates to a special diluent for detecting streptococcus pneumoniae in a urine sample, which is prepared from borax,
1307Prill, epsilon-polylysine, a C-reactive protein polyclonal antibody and purified water, wherein the contents of each component are respectively as follows: the content of borax as component I is 50 mM-100 mM,
Description
Technical Field
The invention relates to a special diluent for detecting streptococcus pneumoniae in a urine sample, which can rapidly and effectively dissociate a compound of C-reactive protein (CRP) and streptococcus pneumoniae cell wall C-polysaccharide in the urine sample and inhibit the biological activity of the CRP, belongs to the field of immunological application, and is suitable for immunological experimental methods such as immunochromatography, immunodiafiltration, enzyme-linked immunosorbent assay, immunoturbidimetry, chemiluminescence and the like.
Background
Streptococcus pneumoniae is the major pathogen causing bacterial pneumonia. The mortality rate of pneumococcal pneumonia is as high as 30%, depending on bacteremia, age and underlying disease. Streptococcus pneumoniae infection can lead to bacteremia, meningitis, pericarditis, empyema, purpura fulminans, endocarditis and/or arthritis if not properly diagnosed and treated. People of all ages can be infected, but more commonly are children under 5 years of age, adolescents and the elderly. The course of disease progresses rapidly, and the disease can be transformed from mild disease to coma within hours, so that the diagnosis can be made immediately, and the treatment by using antibiotics is very important.
In developing countries, children under the age of 500 ten thousand 5 years die each year from pneumonia caused by streptococcus pneumoniae infection. The early effective antibacterial treatment can obviously reduce the fatality rate of the streptococcus pneumoniae pneumonia of children. At present, the traditional bacterial culture method is mainly used for separating the streptococcus pneumoniae in a medical laboratory as a basis for diagnosing the streptococcus pneumoniae. However, since streptococcus pneumoniae is a fastidious bacterium, the requirement on culture nutrient conditions is high, the growth time is long, the identification procedure is complicated, the detection positive rate is low, the specificity for diagnosing streptococcus pneumoniae is 55%, the sensitivity is 64%, and the requirement on clinical rapid diagnosis cannot be met.
The colloidal gold method is widely applied to rapid detection of streptococcus pneumoniae pneumonia at present, diagnoses the streptococcus pneumoniae pneumonia by detecting streptococcus pneumoniae antigens (C polysaccharide antigens) in urine, and has higher sensitivity and specificity. The sensitivity of the urine test is 86%, and the specificity is 94%. Although the performance of the method is far better than that of the traditional detection method, the sensitivity of the method is still unsatisfactory in view of the great clinical hazard of streptococcus pneumoniae. Therefore, a great deal of research and analysis is carried out on urine samples missed by the method, and the target detection object C polysaccharide antigen is found to exist in a combined state with C-reactive protein (CRP).
C-reactive protein (CRP) is a protein whose content in blood rises sharply when the body is infected or tissue damaged, and it was found in 1941 to appear in blood of patients with acute inflammation and can bind to Streptococcus pneumoniae cell wall C-polysaccharide, and thus is named C-reactive protein (CRP). CRP normally has a very low blood level and its blood concentration rises sharply in acute wounds and infections. However, the existence and the content of CRP in urine samples are not reported at present.
A great amount of combination of CRP and C-polysaccharide antigen of streptococcus pneumoniae is found in urine of a streptococcus pneumoniae infected person through technical means such as density gradient centrifugation, gel electrophoresis, immunoblotting and the like, so that the combination is suspected to cause interference on detection of the streptococcus pneumoniae, and the detection rate of the streptococcus pneumoniae is further influenced. Therefore, a diluent is designed in a targeted manner and used for rapidly dissociating the CRP and C-polysaccharide complex and inhibiting the biological activity of the CRP, so that the detection rate of the streptococcus pneumoniae in the urine sample is obviously improved, and the diluent has great significance for accurately detecting the streptococcus pneumoniae in the urine sample.
Disclosure of Invention
The invention aims to provide a diluent which can rapidly and effectively dissociate CRP and streptococcus pneumoniae cell wall C-polysaccharide complexes in a urine sample and inhibit the biological activity of CRP, so that the detection rate of streptococcus pneumoniae in the urine sample is improved, and the subsequent normal immunoassay of streptococcus pneumoniae is not influenced.
The above purpose of the invention is realized by the following technical scheme:
a special diluent for detecting streptococcus pneumoniae in a urine sample comprises the following raw materials: borax,
Prill, epsilon-polylysine, a C-reactive protein polyclonal antibody and purified water, wherein the contents of the components are respectively as follows: the content of borax as component I is 50 mM-100 mM,
prill as component II in 0.5-1 wt%, epsilon-polylysine as component III in 0.5-1 wt%, C-reactive protein polyclonal antibody as component IV in 10-20 ug/ml, and purified water for the rest.
1. The epsilon-polylysine is rich in positive charges, can form stronger repulsion between CRP and C-polysaccharide, further leads the compound to be rapidly dissociated, and simultaneously has the function of antisepsis.
The CRP polyclonal antibody can be combined with CRP to form an immune complex, thereby inhibiting the biological activity of CRP.
3.
Prill is a nonionic surfactant, and has promotion effect on the immunological combination of CRP polyclonal antibody and CRP.
4. The borax provides an alkaline environment, ensures the normal operation of each step of reaction and stabilizes the solubility state of each component.
5. The substances have synergistic effect, so that the CRP and streptococcus pneumoniae cell wall C-polysaccharide complex in a urine sample can be quickly and effectively dissociated, the biological activity of the CRP is inhibited, and the subsequent normal immunoassay of streptococcus pneumoniae is not influenced.
Compared with the prior art, the invention has the following beneficial effects:
1. although the diluent is researched and designed based on a colloidal gold immunochromatographic assay, the diluent can be theoretically applied to any immunological method for detecting streptococcus pneumoniae in urine, such as immunodiafiltration, enzyme-linked immunosorbent assay, immunoturbidimetry, chemiluminescence and the like.
2. The diluent does not contain sodium azide, is safe to use, does not harm the body surface of a human body, and does not have operation risks.
3. Based on the application of the diluent, the detection rate of streptococcus pneumoniae in a urine sample can be obviously improved without influencing the specificity of subsequent streptococcus pneumoniae immunodetection.
Detailed Description
In order to make the technical means, the creation features, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
Example 1
The contents of the components of the special diluent for detecting streptococcus pneumoniae in a urine sample are respectively as follows: the content of borax as component I is 50mM,
prill as an ingredientThe content of II is 1%, the content of epsilon-polylysine as a component III is 0.5%, the content of C-reactive protein polyclonal antibody (CRP polyclonal antibody) as a component IV is 20ug/ml, and the balance is purified water.
A process for preparing the diluent used to detect streptococcus pneumoniae in urine specimen includes such steps as proportionally mixing borax with the diluent,
And sequentially dissolving Prill, epsilon-polylysine and C-reactive protein polyclonal antibody (CRP polyclonal antibody) in purified water, uniformly stirring, and continuously adding the purified water to a constant volume to a required volume. The diluent is used after being mixed with a urine sample for 1 minute according to the volume ratio of 1: 9.
Example 2
The contents of the components of the special diluent for detecting streptococcus pneumoniae in a urine sample are respectively as follows: the content of borax as component I is 75mM,
prill as component II in an amount of 0.75%, epsilon-polylysine as component III in an amount of 0.75%, C-reactive protein polyclonal antibody (CRP polyclonal antibody) as component IV in an amount of 15ug/ml, and the balance purified water.
A process for preparing the diluent used to detect streptococcus pneumoniae in urine specimen includes such steps as proportionally mixing borax with the diluent,
And sequentially dissolving Prill, epsilon-polylysine and C-reactive protein polyclonal antibody (CRP polyclonal antibody) in purified water, uniformly stirring, and continuously adding the purified water to a constant volume to a required volume. The diluent is used after being mixed with a urine sample for 1 minute according to the volume ratio of 1: 9.
Example 3
The contents of the components of the special diluent for detecting streptococcus pneumoniae in a urine sample are respectively as follows: the content of borax as a component I is 100mM,
prill as component II in 0.5%, epsilon-polylysine as component III in 1%, C-reactive protein polyclonal antibody (CRP polyclonal antibody) as component IV in 10ug/ml, and purified water in balance.
A process for preparing the diluent used to detect streptococcus pneumoniae in urine specimen includes such steps as proportionally mixing borax with the diluent,
And sequentially dissolving Prill, epsilon-polylysine and C-reactive protein polyclonal antibody (CRP polyclonal antibody) in purified water, uniformly stirring, and continuously adding the purified water to a constant volume to a required volume. The diluent is used after being mixed with a urine sample for 1 minute according to the volume ratio of 1: 9.
Comparative example
The matched diluent of the existing commercial Binax NOW colloidal gold method product contains sodium citrate/phosphate buffer solution with sodium dodecyl sulfate, Tween 20 and sodium azide.
The test effect of the diluents in the examples and comparative examples:
urine samples of 100 streptococcus pneumoniae infectors and urine samples of 100 healthy people are respectively subjected to same-ratio treatment detection by using the special diluent of the embodiment and the Binax NOW matched diluent of the comparative example, the detection result can be visually distinguished through the color development condition of the test paper, and the detection effects of the diluents in the embodiment and the comparative example are shown in table 1.
Table 1: test effect of Diluent in examples and comparative examples
| Performance index | The invention is special for diluting liquid | Binax NOW matched diluent |
| Sensitivity of the probe | 98％ | 86％ |
| Specificity of | 94％ | 94％ |
As shown in table 1, the special diluent for detecting streptococcus pneumoniae in a urine sample in the present application significantly improves the sensitivity of streptococcus pneumoniae in a urine sample without affecting the normal immunoassay of subsequent streptococcus pneumoniae, thereby significantly improving the detection rate of streptococcus pneumoniae in a urine sample.
Claims (6)
1. The special diluent for detecting streptococcus pneumoniae in a urine sample is characterized by mainly comprising the following raw materials: borax,
1307Prill, epsilon-polylysine, C-reactive protein polyclonal antibody (CRP polyclonal antibody), purified water.
2. The special diluent for detecting streptococcus pneumoniae in a urine sample according to claim 1, wherein the special diluent mainly comprises the following raw materials: the content of borax as component I is 50 mM-100 mM,
1307Prill as component II content of 0.5% -1%, epsilon-polylysine as component III content of 0.5% -1%, C-reactive protein polyclonal antibody as component IV content of 10 ug/ml-20 ug/ml, the rest is purified water.
3. A kind ofThe preparation method of the special diluent for detecting the streptococcus pneumoniae in the urine sample in claim 1 is characterized in that borax with required content is added,
1307Prill, epsilon-polylysine and C-reactive protein polyclonal antibody are sequentially dissolved in purified water, stirred and mixed evenly, and then purified water is continuously added to the mixture until the volume is constant to the required volume.
4. The use method of the special diluent for detecting streptococcus pneumoniae in the urine sample according to any one of claims 1-3, wherein the special diluent is mixed with the urine sample according to a volume ratio of 1:9 for 1 minute.
5. The special diluent for detecting the streptococcus pneumoniae in the urine sample as claimed in any one of claims 1-3 is applied to any immunological method for detecting the streptococcus pneumoniae in the urine.
6. The special diluent for detecting streptococcus pneumoniae in urine samples according to claim 5, which is applied to immunochromatography, immunodiafiltration, enzyme-linked immunosorbent assay, immunoturbidimetry and chemiluminescence.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1324247A (en) * | 1998-09-18 | 2001-11-28 | 比南股份有限公司 | Process and materials for the repid detection of i(streptococcus pneumoniae) employing purified antigen-specific antibodies |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1324247A (en) * | 1998-09-18 | 2001-11-28 | 比南股份有限公司 | Process and materials for the repid detection of i(streptococcus pneumoniae) employing purified antigen-specific antibodies |
Non-Patent Citations (4)
| Title |
|---|
| Detection of C-polysaccharide in serum of patients with Streptococcus pneumonia bacteraemia;S H Gillespie et al.;《Journal of Clinical Pathology》;19951231;第48卷;第803-806页 * |
| Detection of Streptococcus pneumoniae Antigen by a Rapid Immunochromatographic Assay in Urine Samples;José Domínguez et al.;《Chest》;20010131;第119卷(第1期);第243-249页 * |
| 肺炎链球菌C多糖的研究现状;吴元元 等;《微生物学免疫学进展》;20110228;第39卷(第1期);第66-70页 * |
| 肺炎链球菌检测方法及耐药研究;俞蕙;《儿科药学杂志》;20091231;第15卷(第3期);第1-3页 * |
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