CN102323425A - Method for semi-quantitatively diagnosing alpha-fetoprotein by using double-indicatrix immunochromatography - Google Patents
Method for semi-quantitatively diagnosing alpha-fetoprotein by using double-indicatrix immunochromatography Download PDFInfo
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Abstract
The invention discloses a detection method in the technical field of biological engineering, in particular to a method for semi-quantitatively diagnosing an alpha-fetoprotein (AFP) by using a double-indicatrix immunochromatography. The alpha-fetoprotein is a protein generated by embryonic cells and a yolk sac and is peculiar to a fetus. The content of the AFP in blood serum of a normal adult person is extremely low (less than 20ng/ml), and when hepatic cells cancerize, the content is greatly increased (larger than 400ng/ml), therefore the detection of abnormal increase of the content of the AFP in the blood serum is a very effective basis for clinical judgment of early discovery and diagnosis of primary liver cancer. At present, the clinically used AFP detection methods are ELISA (Enzyme Linked Immunosorbent Assay), CLIA (Chemiluminescent Immunoassay) and other methods, which are time-consuming and labor-wasting, are not favorable for popularization and have expensive costs. An immune colloidal gold technique develops fast in recent years and is widely applied to the detection of communicable diseases, early pregnancy, cancer and the like. In the invention, a colloidal gold immunochromatography is adopted, and the method for quickly detecting the AFP is established, and an AFP gray zone range of 20ng/mL-400ng/mL can be semi-quantitatively detected.
Description
Technical field
The present invention relates to a kind of detection method of technical field of bioengineering, specifically is the method for the two index line immunochromatography sxemiquantitative diagnosis of a kind of usefulness alpha-fetoprotein.
Background technology
(hepatocellular carcinoma is one of China's common malignancy HCC) to hepatocellular carcinoma, is the third-largest common cancer that mortality ratio is only second to cancer of the stomach, cancer of the esophagus.Liver cancer grade malignancy height, development is rapid, and its average sicken age is 44 years old, if treatment is untimely or the therapeutic scheme selection is improper, the mean survival time is half a year.China dies from about 110,000 people of liver cancer every year, accounts for 45% of whole world PLC mortality number, but along with the widespread use of AFP and other detection methods, the incidence of disease and the recall rate of liver cancer increase gradually.This shows that following China is for high-quality diagnosing cancer of liver product, and especially the demand of AFP diagnostic products is with increasing.
For liver cancer, generally self is if manifest symptom, and the words of going again to check just reach an advanced stage, and have missed best treatment time.Hepatocarcinoma patient has basically no symptom in early days, in case when occurring such as symptoms such as hepatalgia, abdominal distension, abdominal mass, jaundice, ascites, how to have belonged to late period.Liver cancer patient is difficult to early detection, and the hepatocarcinoma patient of receiving treatment at present about 3/4 just belongs to late period, brings great difficulty to treatment., the patient can feel liver pain, especially after meal for very, and apocleisis, hepatomegaly, upper right abdomen has lump, the becoming thin of unknown cause, abdominal distension, diarrhoea, discontinuity heating, weak, anorexia etc.Therefore, early detection is also in time gone to a doctor and is become the focal issue that improves the liver cancer cure rate, and at present, concentrates on this specific proteins of alpha-fetoprotein AFP for the early detection of liver cancer.
Alpha-fetoprotein (alpha-fetoprotein; AFP) be a kind of by the embryo's property glycoprotein about the molecular weight 70kDa of fetal liver cells generation; Its content of birth back in case canceration takes place liver cell, has then recovered to produce the function of this protein below 20ng/mL; AFP content obviously increases in the blood, so can be used as one of index of diagnosing primary liver cancer.It is generally acknowledged when AFP content is lower than 20ng/mL there is not clinical meaning, during less than 400ng/mL, it is unusual to can be considered liver cell greater than 20ng/mL, when 400ng/mL is above, as primary carcinoma of liver examining finger veins mark really.Therefore, detecting increasing unusually of AFP content in the serum, as early detection and diagnosing primary liver cancer, is very effective judgment basis clinically.AFP content not only can be used as the early diagnosis index of HCC in the serum, also can be used as the index that observation of curative effect and prognosis are judged.At present, the method that AFP commonly used clinically detects is ELISA, methods such as CLIA.Said method is time-consuming, require great effort, be unfavorable for to popularize and expensive.Exploitation simply, the hepatocarcinoma early diagnosis method has crucial economic worth and social effect fast.
Immune colloidal gold technique is to be firstly appeared by Faulk and Taylor phase early 1970s, is used for immunoelectronmicroscopy at first.Colloidal gold-labeled method is as tracer label thing or developer with collaurum; In a kind of novel immunolabelling technique of antigen-antibody reaction, successfully be used for the neck q territories such as manufacturing of Electronic Speculum, flow cytometer, Western blotting, protein staining, in-vitro diagnosis preparation now.Present golden labelling technique often cooperates with membrane carrier, forms specific immune detection mode, like immunity percolation test and immunity-chromatography test etc.Immuno-chromatographic test paper strip is exactly the important development direction that this technology is used for external quick diagnosis, is the novel detection technique that grows up on monoclonal antibody technique, colloidal gold immunochromatographimethod technology and the new material technology basis in modern times.This technical development in recent years is rapid, has particularly obtained widespread use in the bedside detection (POCT) in clinical diagnosis, like the detection of infectious disease, early pregnancy, cancer etc.
At present; There have been a lot of companies to develop the kit that detects alpha-fetoprotein; For example the alpha-fetoprotein (AFP) of the biological medicine company (Beijing) of Blue-Cross company limited detects test paper (colloidal gold method), the alpha-fetoprotein AFP colloidal gold diagnosis reagent of Chongqing Tai Kehua friendship medicine company limited etc.People such as Hou Huiren only need a small amount of serum is dripped on test-strips through experiment development AFP immune chromatograph testing strip, approximately behind the 10min, can learn the yin and yang attribute of AFP through range estimation.But can not distinguish for hepatocellular unusual and liver cancer clinically; Using this standard of 20ng/mL can not make a definite diagnosis liver cancer directly, fast separately, also is very necessary so exploitation can detect the sxemiquantitative colloid gold test paper of this AFP diagnosis gray area of AFP 20～400ng/mL.All not having relevant test strips at present both at home and abroad sells.
Summary of the invention
The object of the invention: exploitation simply, the alpha-fetoprotein diagnostic method has crucial economic worth and social value fast.Colloidal gold immunity chromatography is adopted in this experiment, sets up the method for fast detecting AFP.And can this AFP gray area scope of half-quantitative detection 20ng/mL～400ng/mL.
The present invention realizes through following technical scheme: adopt trisodium citrate reduction method to prepare colloid gold particle, the anti-AFP monoclonal antibody 1 of mark is also evenly coated on the glue gold pad, as the trace labelling thing.On nitrocellulose filter, the AFP monoclonal antibody 2 of two variable concentrations is fixed in two detection lines (being the T line), the sheep anti mouse two anti-nature controlling lines (being the C line) that are fixed in.Successively with sample pad, glue gold pad, nitrocellulose filter and absorbent filter assembling, be cut into colloid gold test paper and the test card of packing in.The gold labeling antibody combines to be trapped with corresponding antigen, antibody generation specificity and develops the color, and whether develops the color according to T line, C line and judges the yin and yang attribute result.
The inventive method comprises the steps:
One, colloidal gold solution preparation
1. prepare 1% chlorauric acid solution;
2. prepare 2% citric acid three sodium solution;
3. 0.02% chlorauric acid solution is heated to boiling, adds 2% trisodium citrate 2mL rapidly;
4. solution is by light blue, and dark blue, claret appears in heating again, continues to boil 10min;
Stop heating, continue to be stirred to room temperature.
Two, the preparation of colloid gold label thing
1. get two 1.5mL test tubes, add the 1mL colloidal gold solution respectively;
2. add an amount of borate buffer solution and be adjusted into 8.8 to pH;
3. add 20 μ g/mL AFP monoclonal antibodies 1, make it reach minimum protein concentration, mixing, vibration 30min;
4. the 10%BSA that adds 20 μ L, mixing, vibration 30min;
5.12000rpm centrifugal 5min absorbs supernatant gently;
6. the collaurum deposition that suspends loose with 1mL wash buffer solution weight;
7. repeat the operation of 5. steps;
8. repeat the operation of 6. steps;
9. repeat the operation of 5. steps;
10. being formulated in the OD of 540nm place (optical density) value with fix buffer is 0.5～1.0 colloid gold immune compound;
11. the plain film spot appearance of spun glass forms glue gold pad, vacuum freeze-drying 3 hours.
Three, on nitrocellulose filter, rule
1.T1 line uses AFP monoclonal antibody 2 line of concentration as 0.2mg/mL, is used to detect the AFP of 20ng/mL;
2.T2 line uses AFP monoclonal antibody 2 line of concentration as 0.1mg/mL, is used to detect the AFP of 400ng/mL;
3.C line is rule with 3.5mg/mL sheep anti mouse polyclonal antibody;
4. nitrocellulose filter was placed 40 ℃ of oven dryings 3 days.
Four, the assembling of colloidal gold strip
1. sample pad, glue gold pad, NC film and absorption pad 4 parts are assembled (seeing accompanying drawing 1) in order;
2. it is assembled and detect after the back is cut into the 4mm/ bar with scissors.
Five, detect
Test paper keeps flat, and sample is added on the sample pad of detector bar, because capillary effect, the chromatography direction of liquid combines to be trapped and develops the color with material on being fixed on T line, C line forward.Behind the 5min, judge the yin and yang attribute result according to the colour developing situation of T line, C line.The C line with near the detection of C line take out of existing redness be in the sample AFP level greater than 20ng/mL (seeing accompanying drawing 2); C line and two detect band all occurs red be in the sample AFP level greater than 400ng/mL (seeing accompanying drawing 3); Have only the C line red negative (seeing accompanying drawing 4) to occur, T line and C line all do not develop the color, and then test strips is invalid.
Detected object of the present invention is single and with strong points, and accuracy rate is high.Detection speed is fast, and required time is short, only needs 5min, does not need just can use the inventive method to detect through the professional of training, satisfies department's requirements of diagnosing liver cancer quickly and accurately such as hospital, outpatient service, and is convenient to basic unit's popularization and utilization.
Description of drawings
Fig. 1 is the diagram of embodiment of the invention test strips
Fig. 2 is the positive findings (the AFP level is greater than 20ng/mL) of embodiment of the invention test strips
Fig. 3 is the positive findings (the AFP level is greater than 400ng/mL) of embodiment of the invention test strips
Fig. 4 is the negative findings of embodiment of the invention test strips
Embodiment
Below in conjunction with accompanying drawing embodiments of the invention are elaborated: present embodiment provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment being to implement under the prerequisite with technical scheme of the present invention.
Among the following embodiment, the experimental technique of unreceipted actual conditions, usually according to normal condition, or the condition of advising according to manufacturer.
Jinsui River that this instance prepares 20nm with 2% citric acid three sodium solution earlier; And mark AFP monoclonal antibody 1; Down payment mark monoclonal antibody, AFP monoclonal antibody 2, sheep anti mouse polyclonal antibody metal spraying mark pad, detection line (T line), nature controlling line (C line); Be assembled into test strips then, put 37 ℃ of oven for drying and be cut into the 4mm/ bar, drying at room temperature is preserved subsequent use.
Embodiment
1. prepare AFP monoclonal antibody 1 and 2.And the required glassware of cleaning experiment.
2. the preparation of collaurum
With 0.02% chlorauric acid solution solution heated and boiled, add 2% trisodium citrate 2mL rapidly earlier;
Solution colour becomes dark bluely by light blue, continues heating and claret occurs, continues to boil 10min; Transparent claret occurs, stop heating, continue to be stirred to room temperature.The colloidal gold solution that has so just prepared 20nm.Use the Electronic Speculum microscopy then, guarantee that the gold grain for preparing makes its big or small consistent and uniform as far as possible, particle diameter is about 20nm, otherwise making again.
3. the preparation of colloid gold label thing
Get two 1.5mL test tubes, add the 1mL colloidal gold solution respectively; Be adjusted into 8.8 to pH to wherein adding an amount of borate buffer solution; Add 20 μ g/mLAFP monoclonal antibodies 1, make it reach minimum protein concentration, mixing, quick oscillation 30min on shaker; The 10%BSA that adds 20 μ L, mixing, vibration 30min; 12000rpm in hydro-extractor, centrifugal 5min absorbs supernatant gently; With the loose collaurum deposition of 1mL wash buffer solution weight suspension; 12000rpm in hydro-extractor, centrifugal 5min absorbs supernatant gently; With the loose collaurum deposition of 1mL wash buffer solution weight suspension; 12000rpm in hydro-extractor, centrifugal 5min absorbs supernatant gently; Being formulated in the OD of 540nm place (optical density) value with fix buffer is 0.5～1.0 colloid gold immune compound; Point sample on the plain film of spun glass forms glue gold pad, vacuum freeze-drying 3 hours.
4. the assembling of colloidal gold strip
On 2 strokes of detection lines of AFP monoclonal antibody (T line) with 0.2mg/mL and 0.1mg/mL, 3.5mg/mL sheep anti mouse polyclonal antibody is drawn on nature controlling line (C line) respectively at nitrocellulose filter.Then nitrocellulose filter was placed 40 ℃ of dryings of baking oven 3 days.Then various piece is assembled into test strips by accompanying drawing 1.
5. the use of colloidal gold strip and interpretation as a result
Before the detection, extract examinee's serum sample earlier.
Keep flat the test strips test paper then, sample is added on the sample pad of detector bar, because capillary effect, the chromatography direction of liquid combines to be trapped and develops the color with material on being fixed on T line, C line forward.Behind the 5min, judge the yin and yang attribute result according to the colour developing situation of T line, C line.The C line with near the detection of C line take out of existing redness be in the sample AFP level greater than 20ng/mL (seeing accompanying drawing 2); C line and two detect band all occurs red be in the sample AFP level greater than 400ng/mL (seeing accompanying drawing 3); Have only the C line red negative (seeing accompanying drawing 4) to occur, T line and C line all do not develop the color, and then test strips is invalid.
Embodiment is the AFP in the test sample directly, and method of application does not need professional training, and is easy to operate, quick, and 5min can obtain the result, can reach quick, easy, detect the purpose of AFP in time.
Claims (3)
1. the method with two index line immunochromatography sxemiquantitative diagnosis alpha-fetoproteins is characterized in that, adopts trisodium citrate reduction method to prepare colloid gold particle, and the anti-AFP monoclonal antibody 1 of mark is also evenly coated on the collaurum pad, as the trace labelling thing.On nitrocellulose filter, the AFP monoclonal antibody 2 of two variable concentrations is fixed in two detection lines (being the T line), the sheep anti mouse two anti-nature controlling lines (being the C line) that are fixed in.Successively with sample pad, glue gold pad, nitrocellulose filter and absorbent filter assembling, be cut into colloid gold test paper and the test card of packing in.The gold labeling antibody combines to be trapped with corresponding antigen, antibody generation specificity and develops the color, and whether develops the color according to T line, C line and judges the yin and yang attribute result.
2. the method for the two index line immunochromatography sxemiquantitative diagnosis of a kind of usefulness according to claim 1 alpha-fetoprotein is characterized in that antibody line concentration is:
The T1 line uses AFP monoclonal antibody 2 line of concentration as 0.2mg/mL, is used to detect the AFP of 20ng/mL;
The T2 line uses AFP monoclonal antibody 2 line of concentration as 0.1mg/mL, is used to detect the AFP of 400ng/mL;
The C line is rule with 3.5mg/mL sheep anti mouse polyclone carrier;
Nitrocellulose filter was placed 40 ℃ of oven dryings 3 days.
3. the method for the two index line immunochromatography sxemiquantitative diagnosis of a kind of usefulness according to claim 1 alpha-fetoprotein; It is characterized in that; The antibody-solutions of utilization variable concentrations is rule on identical matrix, detects the method for the horizontal material of a plurality of variable concentrations with the collaurum method.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114560936A (en) * | 2022-04-08 | 2022-05-31 | 北京科跃中楷生物技术有限公司 | Fluorescent microsphere marking method and detection kit |
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| CN1262440A (en) * | 2000-01-12 | 2000-08-09 | 上海本草生物医学工程研究所 | Semi-quantitative quick-diagnosing reagent for detecting alpha-fetoglobulin and its preparing process |
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| CN114560936A (en) * | 2022-04-08 | 2022-05-31 | 北京科跃中楷生物技术有限公司 | Fluorescent microsphere marking method and detection kit |
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Application publication date: 20120118 |