CN101614740B - Hepatitis A virus antigen saliva fast detection test paper strip - Google Patents
Hepatitis A virus antigen saliva fast detection test paper strip Download PDFInfo
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Abstract
The invention discloses a hepatitis A virus antigen saliva fast detection test paper strip which comprises a bottom plate that is sequentially stuck with a sample pad, an aurosol pad, a nitrocellulose membrane and a sample absorbing pad; wherein the aurosol pad is attached with polyclonal antibody or first monoclonal antibody of anti-hepatitis A virus antigen marked by aurosol; the nitrocellulose membrane is coated with a detection line formed by second monoclonal antibody of the anti-hepatitis A virus antigen, and a quality control line formed by double anti-antibody resisting the polyclonal antibody or the first monoclonal antibody; the polyclonal antibody or the first monoclonal antibody is matched with the second monoclonal antibody in pairs. The test paper strip adopts aurosol labeling technique to detect the hepatitis A virus antigen ingredient in saliva, and can be used for judging whether the patient is infected by hepatitis B virus; furthermore, the test paper strip has simple operation, rapid reaction speed, high sensibility, strong specificity, being economical and practical and the like, so as to be suitable for field test and self-test.
Description
Technical field
The present invention relates to the biologic applications technical field, particularly relate to a kind of hepatitis A virus antigen saliva fast detection test paper strip.
Background technology
Hepatitis A (abbreviation hepatitis A) is a kind of virus hepatitis that is caused by hepatitis A virus (HAV), mainly be to broadcast approach through the excrement oral instructions to infect, namely enter intestines and stomach by hepatitis A virus polluted source, food, apparatus and life close contact per os in latent period of patient or acute stage ight soil, the blood and propagate.Hepatitis A virus has stronger resistibility to various extraneous factors and can survive in external environment for a long time, can by various contaminated articles (hand, articles for daily use, clothing, coverlet etc.) and water and food transmission, also can carry and propagate through fly.
The hepatitis A infection sources is acute patient and subclinical infection person normally, patient is from hiding latter stage to the rear 10 days infectiousness maximums of falling ill, fecal oral route is its main route of transmission, and water, food are that fulminant master looks into mode, and the daily life contact is the main route of transmission of Sporadic cases.There is the report hepatitis A also can propagate and vertical transmission (foreign medical science spread fascicle 1994) by blood, waits further research.
Hepatitis A is mainly via unclean diet and drink the approach such as unboiled water and infect, hepatitis A virus is mainly take primates such as human body, macaque, apes as the host, be approximately 2-6 week latent period: infecting in the week, can also in ight soil, find the particle of virus; And infected individual just looks like like the flu, and high fever may appear in some individuality, perhaps poor appetite, nonspecific symptom such as general malaise; Minority dark brown urine may occur or be apprised of the phenomenon of jaundice.
At present, detecting the most frequently used method of above infectious disease is immune detection, and the method is take the specific recognition of antigen one antibody as the basis, comprises two kinds of the colloidal gold methods of traditional enzyme linked immunosorbent assay (ELISA) and recently rise.Colloidal gold method is divided into immunochromatography and immunity percolation dual mode, and is wherein convenient, fast with immunochromatographic method.
The immunochromatography colloidal gold technique is novel diagnostic techniques, obtained using comparatively widely, ultimate principle is as follows: utilize a kind of antigen of colloid gold label or antibody, at the coated corresponding pairing antigen of the nitrocellulose filter of test strips or antibody, during detection when containing corresponding specific antibody or antigen in the sample, the part formation compound that combines in colloid gold label particle and the sample, then chromatography on nitrocellulose filter, again coated antigen or antibody capture, form macroscopic detection line (T line), by the judgement that realizes the result that has or not of detection line.Existing test strips all is that blood is sample, and complicated operation can not self check, and testing cost is higher.
Summary of the invention
The invention provides a kind of hepatitis A virus antigen saliva fast detection test paper strip, this test strips can with saliva as sample, solve existing test strips and be not suitable for Site Detection, the problem that testing cost is higher.
A kind of hepatitis A virus antigen saliva fast detection test paper strip, comprise base plate, be pasted with successively sample pad, collaurum pad, nitrocellulose filter on the base plate and inhale the sample pad, be attached with polyclonal antibody or first monoclonal antibody of the anti-hepatitis A viral antigen of colloid gold label on the described collaurum pad; The nature controlling line that is coated with detection line that the second monoclonal antibody by anti-hepatitis A viral antigen consists of on the described nitrocellulose filter and is made of two antiantibodys of anti-described polyclonal antibody or the first monoclonal antibody, described polyclonal antibody or the first monoclonal antibody are matched mutually with described second monoclonal antibody.
Above-mentioned test strips is identical with the detection principle of traditional immunochromatographydetecting detecting test strip, the pairing of above-mentioned polyclonal antibody and second monoclonal antibody refers to that all antibody and second monoclonal antibody in the polyclonal antibody be combined with the different antigenic determinants of hepatitis A viral antigen, and above-mentioned the first monoclonal antibody and second monoclonal antibody match mutually and refer to that both are combined with the different antigenic determinants of hepatitis A viral antigen.
Be attached with 0.15～0.45 μ g/cm on the described collaurum pad
2Polyclonal antibody or first monoclonal antibody of anti-hepatitis A viral antigen of colloid gold label, in this concentration range, can with Most patients saliva in determined antigen content be complementary.
Described collaurum pad prepares by the following method:
Get the colloidal gold solution that the colloid diameter is 15～35nm, regulate pH value to 8.0～8.2, the polyclonal antibody or the first monoclonal antibody that add the anti-hepatitis A viral antigen of 0.75～1.0mg by every 100ml colloidal gold solution, seal with bovine serum albumin after stirring, the centrifugal supernatant that goes, the colloidal gold solution that adds equal volume redissolves, with every ml soln uniform spreading at 22～50cm
2On the glass fibre membrane, make the collaurum pad after the drying.
The second monoclonal antibody content of anti-hepatitis A viral antigen is 0.05～1.0 μ g/cm in the described detection line; Two antiantibody content of anti-described polyclonal antibody or the first monoclonal antibody are 0.05～1.0 μ g/cm in the described nature controlling line.Under this concentration, just in time can with most of salivas in antigen be complementary.
Described detection line and nature controlling line method for coating are as follows:
Getting concentration is that 0.01mol/L, pH are 8.0 phosphate buffer solution, two antiantibodys of the second monoclonal antibody of anti-hepatitis A viral antigen and anti-described polyclonal antibody or the first monoclonal antibody are dissolved in respectively make two kinds of solution that concentration is 0.05～1.0mg/ml in the phosphate buffer solution, these two kinds of solution are rule at the nitrocellulose filter two ends respectively with the parameter of 1～1.5 μ l/cm, namely get detection line and nature controlling line after the drying.
The width of described test strips is 2.5～6mm.
The present invention also provides a kind of detection method of above-mentioned test strips, may further comprise the steps: get people's saliva, dilute with phosphate buffer solution, the saliva of getting after 50～100 μ l dilute drops on the sample pad, according to the color status of detection line (T line) and nature controlling line (C line), judge testing result.
All occur such as C, T line, judge that sample is positive; The C line occurs, and the T line does not occur, and judges that sample is negative; C, T line do not occur or the appearance of T line does not appear in the C line, judge that all test paper is invalid.
Test strips of the present invention adopts colloidal gold-labeled method, detect the hepatitis A viral antigen composition in the saliva, thereby judge whether to infect hepatitis A virus, have simple to operate, reaction fast, high, the high specificity of susceptibility, be fit to Site Detection and self check, the advantage such as economical and practical.
Embodiment
Embodiment 1
The preparation of collaurum pad
The colloidal gold solution for preparing diameter 15nm with gold chloride-trisodium citrate reduction method is got the 100ml colloidal gold solution in beaker after preparation is finished, and uses 0.1M K
2CO
3Transfer to pH8.0, press the polyclonal antibody (Shanghai Linc-Bio Science Co., Ltd.) that the 100ml colloidal gold solution adds the corresponding anti-hepatitis A viral antigen of 0.75mg, stirring at room 1 hour, added percentage by weight and be 5% bovine serum albumin(BSA) (BSA) sealing 1 hour, 12000rpm, 4 ℃ are centrifugal 30 minutes, abandon supernatant, redissolve to 100ml with colloidal gold solution, press 1ml solution and spread 22cm
2Ratio, solution is layered on the glass fibre membrane equably, 37 ℃ of dryings 30 minutes are made the collaurum pad.
Being coated with of nitrocellulose filter
Two antiantibodys (sheep anti mouse, Shanghai Linc-Bio Science Co., Ltd.) of the second monoclonal antibody (Shanghai Linc-Bio Science Co., Ltd.) of anti-hepatitis A viral antigen and anti-above-mentioned polyclonal antibody are dissolved in the solution that 0.01M, pH8.0 phosphate buffer (PBS) are mixed with 0.05mg/ml, rule with the parameter of 1.5 μ l/cm at the nitrocellulose filter two ends respectively with spray film instrument, coated T line and C line, after the line with nitrocellulose filter drying at room temperature 8 hours.
The polyclonal antibody of above-mentioned anti-hepatitis A viral antigen matches mutually with second monoclonal antibody.
The assembling of Test paper
In hothouse, 20～25 ℃ of temperature, humidity is less than 30%, sample pad (glass fibre membrane), collaurum pad, nitrocellulose filter and suction sample pad (suction test paper) are sticked on the base plate, wherein nitrocellulose filter is positioned at the base plate middle part, nitrocellulose filter is coated with an end of T line and 1/3 overlap joint of collaurum pad, is coated with an end and 1/10 overlap joint of inhaling the sample pad of C line, 1/5 overlap joint of sample pad and collaurum pad.Be cut at last the test strips that width is 2.5mm, make test card in the plastic clip of also test strips can being packed into.
Embodiment 2
The preparation of collaurum pad
The colloidal gold solution for preparing diameter 35nm with gold chloride-trisodium citrate reduction method is got the 100ml colloidal gold solution in beaker after preparation is finished, and uses 0.1M K
2CO
3Transfer to pH8.2, the first monoclonal antibody (Shanghai Linc-Bio Science Co., Ltd.) that adds the corresponding anti-hepatitis A viral antigen of 1mg to colloidal gold solution, stirring at room 1 hour, added percentage by weight and be 5% bovine serum albumin(BSA) (BSA) sealing 1 hour, 12000rpm, 4 ℃ are centrifugal 30 minutes, abandon supernatant, redissolve to 100ml with the collaurum dilution, press 1ml solution and spread 50cm
2Ratio, solution is layered on the glass fibre membrane uniformly, 37 ℃ of dryings 30 minutes are made the collaurum pad.
Being coated with of nitrocellulose filter
Two antiantibodys (sheep anti mouse, Shanghai Linc-Bio Science Co., Ltd.) of the second monoclonal antibody (Shanghai Linc-Bio Science Co., Ltd.) of anti-hepatitis A viral antigen and anti-above-mentioned the first monoclonal antibody are dissolved in the solution that 0.01M, pH8.2 phosphate buffer (PBS) are mixed with 0.1mg/ml, rule with the parameter of 1 μ l/cm at the nitrocellulose filter two ends respectively with spray film instrument, coated T line and C line, after the line with nitrocellulose filter drying at room temperature 8 hours.
The first monoclonal antibody and the second monoclonal antibody of above-mentioned anti-hepatitis A viral antigen match mutually.
The assembling of Test paper
In hothouse, 20～25 ℃ of temperature, humidity is less than 30%, stick on successively sample pad (glass fibre membrane), collaurum pad, nitrocellulose filter and suction sample pad (suction test paper) on the base plate, wherein nitrocellulose filter is positioned at the base plate middle part, nitrocellulose filter is coated with an end of T line and 1/3 overlap joint of collaurum pad, is coated with an end and 1/10 overlap joint of inhaling the sample pad of C line, 1/5 overlap joint of sample pad and collaurum pad.Be cut at last the test strips that width is 6mm, make test card in the plastic clip of also test strips can being packed into.
The clinical sample test
People to be checked is gathering saliva sample fasting in front 30 minutes, get every people's saliva 0.5ml to be checked in dixie cup, get in two phosphate buffer solutions that are added drop-wise to concentration 0.02M, pH8.0 with suction pipe and to dilute, the saliva 100 μ l that get dilution are added in the sample pad, naked-eye observation 30 minutes, the color status of record T line and C line.Simultaneously saliva sample is detected with HAV-Ag ELISA kit, if both test result are inconsistent, detect with other two kinds of HAV-Ag ELISA kits again, if two kinds or above ELISA kit are positive, result of determination is positive, two kinds or above ELISA kit are negative, and result of determination is negative.
Collect 145 parts of saliva samples, wherein the ELISA kit detects 33 parts of positive, and test strips of the present invention (embodiment 1) detects 34 parts of positive, and concrete outcome is as shown in table 1 below:
Table 1 test strips of the present invention is to the testing result of clinical sample hepatitis A viral antigen
Sensitivity=33/33=100%; Specificity=111/112=99.1%
Claims (1)
1. hepatitis A virus antigen saliva fast detection test paper strip, comprise base plate, be pasted with successively sample pad, collaurum pad, nitrocellulose filter on the base plate and inhale the sample pad, described nitrocellulose filter is positioned at the base plate middle part, nitrocellulose filter is coated with an end of detection line and 1/3 overlap joint of collaurum pad, be coated with an end and 1/10 overlap joint of inhaling the sample pad of nature controlling line, 1/5 overlap joint of sample pad and collaurum pad;
It is characterized in that: the polyclonal antibody or the first monoclonal antibody that are attached with the anti-hepatitis A viral antigen of colloid gold label on the described collaurum pad; The nature controlling line that is coated with detection line that the second monoclonal antibody by anti-hepatitis A viral antigen consists of on the described nitrocellulose filter and is consisted of by two antiantibodys of anti-described polyclonal antibody or the first monoclonal antibody, described polyclonal antibody or the first monoclonal antibody are matched mutually with described second monoclonal antibody;
When the polyclonal antibody of the anti-hepatitis A viral antigen that adheres to colloid gold label on the collaurum pad,
Described collaurum pad prepares by the following method:
Get the colloidal gold solution that the colloid diameter is 15nm, regulate pH value to 8.0, the polyclonal antibody that adds the anti-hepatitis A viral antigen of 0.75mg by every 100ml colloidal gold solution, seal with bovine serum albumin after stirring, the centrifugal supernatant that goes, the colloidal gold solution that adds equal volume redissolves, with every ml soln uniform spreading at 22cm
2On the glass fibre membrane, make the collaurum pad after the drying;
Described detection line and nature controlling line method for coating are as follows:
Getting concentration is that 0.01mol/L, pH are 8.0 phosphate buffer solution, two antiantibodys of the second monoclonal antibody of anti-hepatitis A viral antigen and anti-described polyclonal antibody are dissolved in respectively make two kinds of solution that concentration is 0.05mg/ml in the phosphate buffer solution, these two kinds of solution are rule at the nitrocellulose filter two ends respectively with the parameter of 1.5 μ l/cm, namely get detection line and nature controlling line after the drying;
The width of test strips is 2.5mm;
When the first monoclonal antibody of the anti-hepatitis A viral antigen that adheres to colloid gold label on the collaurum pad,
Described collaurum pad prepares by the following method:
The colloid diameter is the colloidal gold solution of 35nm, regulate pH value to 8.2, the first monoclonal antibody that adds the anti-hepatitis A viral antigen of 1.0mg by every 100ml colloidal gold solution, seal with bovine serum albumin after stirring, the centrifugal supernatant that goes, the colloidal gold solution that adds equal volume redissolves, with every ml soln uniform spreading at 50cm
2On the glass fibre membrane, make the collaurum pad after the drying;
Described detection line and nature controlling line method for coating are as follows:
Getting concentration is that 0.01mol/L, pH are 8.2 phosphate buffer solution, two antiantibodys of the second monoclonal antibody of anti-hepatitis A viral antigen and anti-described the first monoclonal antibody are dissolved in respectively make two kinds of solution that concentration is 0.1mg/ml in the phosphate buffer solution, these two kinds of solution are rule at the nitrocellulose filter two ends respectively with the parameter of 1 μ l/cm, namely get detection line and nature controlling line after the drying;
The width of test strips is 6mm.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2009101010387A CN101614740B (en) | 2009-07-30 | 2009-07-30 | Hepatitis A virus antigen saliva fast detection test paper strip |
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| CN2009101010387A CN101614740B (en) | 2009-07-30 | 2009-07-30 | Hepatitis A virus antigen saliva fast detection test paper strip |
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| CN101614740B true CN101614740B (en) | 2013-01-02 |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102183633B (en) * | 2011-02-24 | 2013-05-15 | 南京基蛋生物科技有限公司 | Colloidal gold labeling method |
| CN102288758A (en) * | 2011-07-29 | 2011-12-21 | 北京中检安泰诊断科技有限公司 | Hepatitis A virus (HAV) IgM antibody colloidal gold method detection kit and preparation method thereof |
| CN104502590A (en) * | 2014-12-23 | 2015-04-08 | 北京出入境检验检疫局检验检疫技术中心 | Hepatitis A virus chromatography test paper strip based on low-noise excitation fluorescent label |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1508879A (en) * | 1974-12-09 | 1978-04-26 | Merck & Co Inc | Immunological assay |
| CN101183106A (en) * | 2007-09-21 | 2008-05-21 | 马北峰 | Device for detecting hepatitis b virus marker in mouth cavity liquid |
| CN101266246A (en) * | 2008-04-30 | 2008-09-17 | 天津中新科炬生物制药有限公司 | HIV antibody and antigen combined rapid detection reagent kit |
-
2009
- 2009-07-30 CN CN2009101010387A patent/CN101614740B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1508879A (en) * | 1974-12-09 | 1978-04-26 | Merck & Co Inc | Immunological assay |
| CN101183106A (en) * | 2007-09-21 | 2008-05-21 | 马北峰 | Device for detecting hepatitis b virus marker in mouth cavity liquid |
| CN101266246A (en) * | 2008-04-30 | 2008-09-17 | 天津中新科炬生物制药有限公司 | HIV antibody and antigen combined rapid detection reagent kit |
Non-Patent Citations (1)
| Title |
|---|
| 马吉湘 等.草莓携带甲肝病毒ELISA 检测方法的研究.《食品科学》.2008,第29卷(第10期),459-462. * |
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