CN1262440A - Semi-quantitative quick-diagnosing reagent for detecting alpha-fetoglobulin and its preparing process - Google Patents
Semi-quantitative quick-diagnosing reagent for detecting alpha-fetoglobulin and its preparing process Download PDFInfo
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- CN1262440A CN1262440A CN 00111436 CN00111436A CN1262440A CN 1262440 A CN1262440 A CN 1262440A CN 00111436 CN00111436 CN 00111436 CN 00111436 A CN00111436 A CN 00111436A CN 1262440 A CN1262440 A CN 1262440A
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Abstract
A semi-quantitative quick diagnosing reagent for detecting alpha-fetoprotein is composed of micropore membrane as solid-phase carrier, colour particles as marker, and two antibodies with certain concentration are coated on the membrane to form detecting region and quality control region. As the detection result, the colour variation intwo regions, the colour contrast and the content range of the substance to be detected have obvious associated relation. Its advantages are simple operation, high speed, and no need of instrument.
Description
The invention belongs to the biotechnology diagnostic reagent, be specifically related to a kind of diagnostic reagent and preparation method who detects alpha-fetoprotein.
(α-fetoprotein, AFP) measure is the main laboratory diagnostic method of hepatocellular carcinoma to alpha-fetoprotein.Normal human serum also contains the AFP of low concentration.During clinical detection AFP, often with 20-50 μ g/L as the normal reference value upper limit.
The common method of measuring AFP in clinical detection has enzyme immunoassay, radioimmunology, chemiluminescence immunoassay method etc.But these quantitative determination process need be not suitable for extensively promoting the use of, and report time be longer as a result with special instrument and reagent.
Existing AFP fast diagnosis reagent bar is qualitative reagent, prescribes a time limit when AFP content in the sample surpasses on the normal reference value, only reports positive findings.
Because clinically except that liver cancer, Serum AFP such as other hepatopathy such as hepatitis also can significantly raise, but the rising degree is not as good as liver cancer, because of China is high incidence of hepatitis, to surpass the normal positive content range of reporting AFP, lack the reference of antidiastole as only clinically.
The objective of the invention is to overcome above-mentioned shortcoming, develop a kind of testing tool that do not need, easy, sxemiquantitative diagnostic method fast.
The invention provides a kind of detection alpha-fetoprotein semi-quantitative quick-diagnosing reagent, it is characterized in that this reagent is by sample pad 1, is fixed with the absorbent material 2 of the anti-AFP antibody I of coloured particle mark, microporous barrier 3, adsorptive pads 4, base plate 5, anti-AFP antibody I I A, the reagent that antiantibody B forms.As shown in Figure 1 and Figure 2.
Fig. 1 is for detecting alpha-fetoprotein semi-quantitative quick-diagnosing reagent side view
Fig. 2 is for detecting alpha-fetoprotein semi-quantitative quick-diagnosing reagent vertical view
Detection alpha-fetoprotein semi-quantitative quick-diagnosing reagent of the present invention adopts immunity in use Chromatography (immunochromatographic assay, ICA), this is a kind of with microporous membrane Tachysynthesis associated methods for carrier. The sample solution that drips at film one end among the ICA is subjected to film Capillarity move to the other end. In the moving process analyte be fixed on the film certain The acceptor in one zone (antigen or antibody) in conjunction with and be fixed, irrelevant thing is then crossed this zone And separated, the colour developing by label comes the judgment experiment result then.
The sxemiquantitative of detection alpha-fetoprotein semi-quantitative quick-diagnosing reagent of the present invention is under using The row principle reaches.
Whether when the common immunochromatographic method reagent of preparation, people establish detection zone and Quality Control district usually on microporous barrier, lost efficacy but the Quality Control district here only is used to refer to this reagent, do not have definite relation with the content of measured object.In experimenting, we find out that, and along with the increase of measured object content, the color of detection zone is deepened gradually, and that the color in Quality Control district subtracts gradually is light.We are label with the coloured particle, by regulating the antibody sandwich concentration in detection zone and Quality Control district, make the change color in detection zone and Quality Control district and the color contrast in two districts and the content range of measured object be clear and definite corresponding relation, thereby reach semiquantitative purpose.
During detection, reagent sample pad one end is immersed sample to be measured or sample to be measured is dripped on sample pad, wait liquid to move forward to the forward position when reaching microporous barrier, take out reagent and keep flat 15 minutes observationss.
If the colour developing of Quality Control district is only arranged, shows that AFP content is less than normal reference value (for example 30ug/L) in the determinand;
If test section and Quality Control district all develop the color, but Quality Control district color is deeper than the test section.Show that AFP content is between normal reference value and positive diagnosis value (for example 200 μ g/L) in the determinand;
If test section and Quality Control district all develop the color, but the test section color is identical with Quality Control district color or it is dark to omit.Show that AFP content is greater than the positive diagnosis value in the determinand;
If test section and Quality Control district all do not develop the color, illustrate that reagent lost efficacy.
Another object of the present invention is the preparation method who discloses above-mentioned detection alpha-fetoprotein semi-quantitative quick-diagnosing reagent, and this method comprises the following steps:
(1) purifying of monoclonal antibody: 1 part of the ascites that contains monoclonal antibody, behind the centrifugal 10min of 12000r/min, get supernatant and add 2 parts of 0.06mol/L pH 4.0 acetate buffer solutions, add caprylic acid in 33.3 μ l/ml ascites ratios, 4 ℃ are stirred 30min, centrifugal 12000r/min, 30min gets supernatant liquid filtering, with the NaCl dialysis 48h of 0.002mol/L pH7.4; On ultraviolet spectrophotometer, measure A
280And A
260: according to formula: (1.45 * A
280-0.74 * A
260) * extension rate calculates protein concentration; Add 0.2%NaN
3, packing ,-20 ℃ are frozen;
(2) many anti-purifying: the antiserum of anti-AFP adds 4 parts of 0.06M pH4.0 acetate buffer solutions for 1 part, adds 0.125 part caprylic acid again, and 4 ℃ are stirred 30min, and the centrifugal 30min of 10000r/min gets supernatant and filters, and adds the PBS of 1/10 volume, transfers pH to 7.0; Be cooled to 4 ℃, add the 0.277g ratio in every ml volume and add ammonium sulfate, 4 ℃ are stirred 1h, and the centrifugal 15min of 4000r/min precipitates with 1 part of physiological saline solution, and mixing adds 1 part of physiological saline again, and protein concentration is calculated in dialysis, and store method is with the monoclonal antibody purifying;
(3) preparation of collaurum: with the preparation of citric acid reducing process, the sodium citrate solution of preparation 1%, the ratio in 1.5% joins in the distilled water, after boiling again the ratio in 1% add 1% chlorauric acid solution, continue to boil 15min, cool off standby;
(4) preparation of golden labeling antibody: as above Zhi Bei colloidal gold solution 0.1mol/LK
2CO
3Regulate pH to neutral, add the anti-AFP antibody I solution of the debita spissitudo of 1/10 (V/V), behind the room temperature reaction 15min, add 1% polyglycol (PEG); The centrifugal 30min of 12000r/min inhales and removes supernatant; With the phosphate buffer washing that contains 1%BSA 2-4 time; Precipitation is suspended from the 1%BSA solution that is equivalent to original volume 1/5 again at last; 4 ℃ of preservations;
(5) golden labeling antibody is fixing: get golden labeling antibody liquid and 1: 1 mixing of 1%BSA solution, add on the absorbent material drying in proportion;
Parallel bag is by two kinds of antibody on microporous barrier, and a kind of is anti-AFP antibody I I, and bag is 2mg/ml by concentration; Another kind is a rabbit anti-mouse igg, and bag is 1.5mg/ml by concentration; Dry;
(6) be combined into reagent strip: the absorbent material 2 (5mm that said fixing the anti-AFP antibody I of coloured particle mark
*6mm), bag is by the microporous barrier 3 (5mm of anti-AFP antibody I I and rabbit anti-mouse igg
*20mm), sample pad 1 (5mm
*23mm) with adsorptive pads 4 (5mm
*31mm) stick on base plate 5 (5mm by accompanying drawing
*Be combined into reagent strip (seeing Fig. 1, Fig. 2) 77mm);
At first, microporous barrier 3 is sticked on the base plate 5, apart from the terminal 30mm of the nearly B of base plate 5, then adsorptive pads 4 is sticked on the nearly B end of base plate 5, adsorptive pads 4 is 1mm with microporous barrier 3 overlappings, absorbent material 2 is sticked on the nearly A end of base plate 5 again, and absorbent material 2 is 1mm with microporous barrier 3 overlappings; At last sample pad 1 is also sticked on the nearly A end of base plate 5, sample pad 1 is 1mm with absorbent material 2 overlappings.
Microporous barrier of the present invention is the hybrid films of nylon membrane, pvdf membrane, polyester film, nitrocellulose filter, cellulose acetate membrane or cellulose nitrate and cellulose acetate; Described coloured particle is collaurum, electroselenium or colored latex; Described antibody is monoclonal antibody or polyclonal antibody.
Reagent of the present invention does not need to use testing tool, has easy, quick, semiquantitative advantage, can judge the content range of AFP in the measured body, for the antidiastole of hepatitis and liver cancer clinically provides better detection method.
Embodiment 1
The purifying of monoclonal antibody: the ascites 1ml that contains monoclonal antibody is through 12000r/min, after 10min is centrifugal supernatant is moved in the small beaker, add 2ml 0.06mol/L pH 4.0 acetate buffer solutions, slowly drip 33.3 μ l caprylic acids again, 4 ℃ are stirred 30min, centrifugal 12000r/min, 30min, supernatant is filtered into bag filter through absorbent cotton, to the NaCl dialysis 48h of 0.002mol/L pH 7.4.Take out the monoclonal antibody liquid of purifying and after dilution in 1: 20, survey A
280, A
260According to formula: (1.45 * A
280-0.74 * A
260) * extension rate calculates protein concentration.Add 0.2%NaN
3, packing ,-20 ℃ are frozen.
The preparation of collaurum; Use the citric acid reducing process.In the 100ml distilled water, add 1% sodium citrate solution 1.5ml, add 1% chlorauric acid solution 1ml after boiling again, continue to boil 15min, cool off standby.
The preparation of gold labeling antibody: as above Zhi Bei colloidal gold solution 0.1mol/L K
2CO
3Regulate pH to neutral, add the anti-AFP monoclonal antibody I solution of the debita spissitudo of 1/10 (V/V), behind the room temperature reaction 15min, add 1%PEG.The centrifugal 30min of 12000r/min inhales and removes supernatant.With the phosphate buffer washing of going into 1%BSA 2-4 time.Precipitation is suspended from the 1%BSA solution of 20ml again at last.4 ℃ of preservations.
Fixing of gold labeling antibody: get golden labeling antibody liquid 400 μ l, add 1%BSA solution 400 μ l, mixing adds on the glass fibre membrane of 0.6 * 20cm drying.
Parallel bag is by two kinds of antibody on nitrocellulose filter, and a kind of is anti-AFP monoclonal antibody II, and bag is 2mg/ml by concentration; Another kind is a rabbit anti-mouse igg, and bag is 1.5mg/ml by concentration.Dry.
Above-mentioned glass fibre membrane, nitrocellulose filter, sample pad and adsorptive pads are assembled into reagent strip by accompanying drawing on the PVC base plate.
Embodiment 2
Many anti-purifying: the antiserum 1ml of the anti-AFP of horse adds 4ml 0.06M pH 4.0 acetate buffer solutions, adds the caprylic acid of 125 μ l again, and 4 ℃ are stirred 30min, the centrifugal 30min of 10000r/min gets supernatant and filters, and volume is 4.5ml, add 450 μ l PBS, transfer pH to 7.0.Be cooled to 4 ℃, add 1.37 gram ammonium sulfate, 4 ℃ are stirred 1h, the centrifugal 15min of 4000r/min, and precipitation is used the 1ml physiological saline solution, and mixing adds 1ml physiological saline again, and protein concentration is calculated in dialysis, and store method is with the monoclonal antibody purifying.
The preparation of gold body antibody: golden labelled antibody is the anti-AFP of horse, and the preparation method is with embodiment 1.
All the other steps such as fixing of the preparation of monoclonal antibody purifying, collaurum, golden labeling antibody are all with embodiment 1.
Embodiment 3:
Parallel bag is by two kinds of antibody on nitrocellulose filter, and a kind of is the anti-AFP of horse, and bag is 2mg/ml by concentration; Another kind is a rabbit anti-mouse igg, and bag is 1.5mg/ml by concentration, drying.
All the other steps are all with embodiment 1.
The invention is not restricted to above-mentioned embodiment, also can put into plastic casing to the reagent of making and form kit or agent plate, therefore everyly utilize kit that the principle of the invention makes or agent plate to think to drop within protection scope of the present invention.
Claims (3)
1, a kind of detection alpha-fetoprotein semi-quantitative quick-diagnosing reagent is characterized in that this reagent is by sample pad (1), is fixed with the absorbent material (2) of the anti-AFP antibody I of coloured particle mark, microporous barrier (3), adsorptive pads (4), base plate (5), anti-AFP antibody I IA, the reagent that antiantibody B forms.
2, a kind of preparation method of detection alpha-fetoprotein semi-quantitative quick-diagnosing reagent as claimed in claim 1 is characterized in that this method comprises the following steps:
1. the purifying of monoclonal antibody: 1 part of the ascites that contains monoclonal antibody, behind the centrifugal 10min of 12000r/min, get supernatant and add 2 parts of 0.06mol/L pH 4.0 acetate buffer solutions, add caprylic acid in 33.3 μ l/ml ascites ratios, 4 ℃ are stirred 30min, centrifugal 12000r/min, 30min gets supernatant liquid filtering, with the NaCl dialysis 48h of 0.002mol/L pH7.4; On ultraviolet spectrophotometer, measure A
280And A
260According to formula: [1.45 * A
280-0.74 * A
260] * extension rate calculates protein concentration; Add 0.2%NaN
3, packing ,-20 ℃ are frozen;
2. many anti-purifying: the antiserum of anti-AFP adds 4 parts of 0.06M pH4.0 acetate buffer solutions for 1 part, adds 0.125 part caprylic acid again, and 4 ℃ are stirred 30min, and the centrifugal 30min of 10000r/min gets supernatant and filters, and adds the PBS of 1/10 volume, transfers pH to 7.0; Be cooled to 4 ℃, add the 0.277g ratio in every ml volume and add ammonium sulfate, 4 ℃ are stirred 1h, and the centrifugal 15min of 4000r/min precipitates with 1 part of physiological saline solution, and mixing adds 1 part of physiological saline again, and protein concentration is calculated in dialysis, and store method is with the monoclonal antibody purifying;
3. the preparation of collaurum: with the preparation of citric acid reducing process, the sodium citrate solution of preparation 1%, the ratio in 1.5% joins in the distilled water, after boiling again the ratio in 1% add 1% chlorauric acid solution, continue to boil 15min, cool off standby;
4. the preparation of golden labeling antibody: as above Zhi Bei colloidal gold solution 0.1mol/L K
2CO
3Regulate pH to neutral, add the anti-AFP antibody I solution of the debita spissitudo of 1/10 (V/V), behind the room temperature reaction 15min, add 1% polyglycol; The centrifugal 30min of 12000r/min inhales and removes supernatant; With the phosphate buffer washing that contains 1%BSA 2-4 time; Precipitation is suspended from the 1%BSA solution that is equivalent to original volume 1/5 again at last; 4 ℃ of preservations;
5. golden labeling antibody is fixing: get golden labeling antibody liquid and 1: 1 mixing of 1%BSA solution, add on the absorbent material drying in proportion;
Parallel bag is by two kinds of antibody on microporous barrier, and a kind of is anti-AFP antibody I I, and bag is 2mg/ml by concentration; Another kind is a rabbit anti-mouse igg, and bag is 1.5mg/ml by concentration; Dry;
6. be combined into reagent strip: absorbent material (2), bag that said fixing the anti-AFP antibody I of coloured particle mark are sticked on the base plate (5) by microporous barrier (3), sample pad (1) and the adsorptive pads (4) of anti-AFP antibody I I and rabbit anti-mouse igg and are combined into reagent strip;
At first, microporous barrier (3) is sticked on the base plate (5), apart from the terminal 30mm of the nearly B of base plate (5), then adsorptive pads (4) is sticked on the nearly B end of base plate (5), adsorptive pads (4) is 1mm with microporous barrier (3) overlapping, absorbent material (2) is sticked on the nearly A end of base plate (5), absorbent material (2) is 1mm with microporous barrier (3) overlapping again; At last sample pad (1) is also sticked on the nearly A end of base plate (5), sample pad (1) is 1mm with absorbent material (2) overlapping.
3, a kind of detection alpha-fetoprotein semi-quantitative quick-diagnosing reagent according to claim 1 is characterized in that wherein said microporous barrier is the hybrid films of nylon membrane, pvdf membrane, polyester film, nitrocellulose filter, cellulose acetate membrane or cellulose nitrate and cellulose acetate; Described coloured particle is collaurum, electroselenium or colored latex; Described antibody is monoclonal antibody or polyclonal antibody.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00111436 CN1262440A (en) | 2000-01-12 | 2000-01-12 | Semi-quantitative quick-diagnosing reagent for detecting alpha-fetoglobulin and its preparing process |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00111436 CN1262440A (en) | 2000-01-12 | 2000-01-12 | Semi-quantitative quick-diagnosing reagent for detecting alpha-fetoglobulin and its preparing process |
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| CN1262440A true CN1262440A (en) | 2000-08-09 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100360684C (en) * | 2005-02-01 | 2008-01-09 | 合肥中科大生物技术有限公司 | Fluorescent quantitative RT-PCR detecting kit of 2-f(o)etoprotein (AFP)mRNA |
| CN102253094A (en) * | 2011-05-06 | 2011-11-23 | 中国人民解放军第三军医大学 | Electromagnetic control electrode and preparation method thereof, and detection method of biological sample |
| CN102323425A (en) * | 2011-06-03 | 2012-01-18 | 正元盛邦(天津)生物科技有限公司 | Method for semi-quantitatively diagnosing alpha-fetoprotein by using double-indicatrix immunochromatography |
| CN102662061A (en) * | 2012-04-17 | 2012-09-12 | 北京九强生物技术股份有限公司 | Kit for determination of human alpha-fetoprotein content by latex-enhanced immunoturbidimetry |
| CN102879576A (en) * | 2012-10-23 | 2013-01-16 | 广州万孚生物技术股份有限公司 | Early pregnancy detection test strip and detection method thereof |
| CN103954773A (en) * | 2014-05-08 | 2014-07-30 | 北京玖佳宜科技有限公司 | Kit for detecting alpha fetal protein and preparation method of kit |
| CN115616228A (en) * | 2022-12-20 | 2023-01-17 | 协和生物制药（天津）有限公司 | Detection method and detection kit for alpha-fetoprotein antigen |
-
2000
- 2000-01-12 CN CN 00111436 patent/CN1262440A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100360684C (en) * | 2005-02-01 | 2008-01-09 | 合肥中科大生物技术有限公司 | Fluorescent quantitative RT-PCR detecting kit of 2-f(o)etoprotein (AFP)mRNA |
| CN102253094A (en) * | 2011-05-06 | 2011-11-23 | 中国人民解放军第三军医大学 | Electromagnetic control electrode and preparation method thereof, and detection method of biological sample |
| CN102253094B (en) * | 2011-05-06 | 2013-12-18 | 中国人民解放军第三军医大学 | Electromagnetic control electrode and preparation method thereof, and detection method of biological sample |
| CN102323425A (en) * | 2011-06-03 | 2012-01-18 | 正元盛邦(天津)生物科技有限公司 | Method for semi-quantitatively diagnosing alpha-fetoprotein by using double-indicatrix immunochromatography |
| CN102662061A (en) * | 2012-04-17 | 2012-09-12 | 北京九强生物技术股份有限公司 | Kit for determination of human alpha-fetoprotein content by latex-enhanced immunoturbidimetry |
| CN102662061B (en) * | 2012-04-17 | 2014-06-18 | 北京九强生物技术股份有限公司 | Kit for determination of human alpha-fetoprotein content by latex-enhanced immunoturbidimetry |
| CN102879576A (en) * | 2012-10-23 | 2013-01-16 | 广州万孚生物技术股份有限公司 | Early pregnancy detection test strip and detection method thereof |
| CN103954773A (en) * | 2014-05-08 | 2014-07-30 | 北京玖佳宜科技有限公司 | Kit for detecting alpha fetal protein and preparation method of kit |
| CN103954773B (en) * | 2014-05-08 | 2016-02-24 | 北京玖佳宜科技有限公司 | Alpha-fetoprotein detection kit and preparation thereof |
| CN115616228A (en) * | 2022-12-20 | 2023-01-17 | 协和生物制药（天津）有限公司 | Detection method and detection kit for alpha-fetoprotein antigen |
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