CN1281978A - Anti-alpha-fetoprotein antibody labelled by fluorescein and its preparation method - Google Patents
Anti-alpha-fetoprotein antibody labelled by fluorescein and its preparation method Download PDFInfo
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- CN1281978A CN1281978A CN 00119376 CN00119376A CN1281978A CN 1281978 A CN1281978 A CN 1281978A CN 00119376 CN00119376 CN 00119376 CN 00119376 A CN00119376 A CN 00119376A CN 1281978 A CN1281978 A CN 1281978A
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Abstract
The present invention relates to an anti-alpha-fetoprotein (AFP) antibody labelled by fluorescein, its molecular weight is 16 ten thousand, and at 260, 280 and 490 nm wavelength is possesses absorption peak, under the condition of full-wave light irradiation it produces yellowing-green colour and possesses ability of making specific antigen-antibody reaction with AFP. When it is prepared, firstly the fluorescein is prepared, and dropped into the anti-AFP antibody solution, then the reactant is placed in refrigerator, the label can be formed at low temp., then the label solution is dialyzed, engrafted into phosphoric acid buffer solution, and the glucar gel scphader equilibrium is used to make equilibrium and elution, then the product can be collected. It can be used as indicator, and can impement alloplasmatic body detection within 1 hr., and can prevent radioactive hazard.
Description
The present invention relates to a kind of anti-alpha-fetoprotein antibody.
(Fetoprotein, α-FP) find the sixties alpha-fetoprotein, in the blood of most of hepatocarcinoma patient, can find alpha-fetoprotein.The seventies, China just generally investigated by large-scale, found that alpha-fetoprotein can be used for the early diagnosis of liver cancer.Find again later on, binding ability according to alpha-fetoprotein and lectins (Lectin), the alpha-fetoprotein that alpha-fetoprotein and hepatocarcinoma patient produced that normal fetus and other hepatopathy patients can be produced is distinguished, wherein the alpha-fetoprotein that hepatocarcinoma patient produced is called as alpha-fetoprotein variant, and it can combine with the various plants lectin.And the alpha-fetoprotein that normal fetus and other hepatopathy patients are produced can not combine with lectins, therefore, according to this and difference lectins binding ability, now is used for the specific diagnosis of liver cancer.
Used technology is earlier lectins to be joined in the agarose that has dissolved at present, is prepared into offset plate, and increase serum carries out electrophoresis again; Pour the agarose gel of the anti-alpha-fetoprotein antibody that mixes the 125-iodine labeling behind the electrophoresis in its vertical direction into, carry out the second phase electrophoresis,, show whether alpha-fetoprotein variant exists then by radioautograph.This method needs could obtain the result in about three days, had isotopic contamination simultaneously and produced, and the experimenter is had injury.
The history of the existing many decades of the generation of fluorescence labeling technology, it combines the pinpoint accuracy of immunochemistry and serological high degree of specificity and susceptibility and microscopy.The technology in past need observe or measure with fluorescent microscope or fluorospectrophotometer.
The object of the present invention is to provide a kind of anti-alpha-fetoprotein antibody that replaces the 125-iodine labeling with fluorescein and preparation method thereof.
Fluorescein-labeled anti-alpha-fetoprotein antibody is a kind of antibody with following characteristic:
1) molecular weight: 160,000; 2) extinction characteristic: 260,280, the 490nm wavelength has absorption peak, under the all-wave rayed, can send green-yellow light; 3) biological nature: have with alpha-fetoprotein and carry out specific antigen-antibody response ability.
The preparation method of fluorescein-labeled anti-alpha-fetoprotein antibody is:
1) preparation fluorescein is in an amount equivalent to 1/ (50～150) of anti-alpha-fetoprotein antibody protein content;
2) fluorescein for preparing is added dropwise in the anti-alpha-fetoprotein antibody solution goes;
3) reactant is moved into 4～8 ℃ of refrigerators, make it form label at low temperatures;
4) label solution is packed in the bag filter, in flowing water, dialyse, move into again in the 0.01mol/L phosphate buffer (PBS) of 1000～2000mL, and in refrigerator, continue dialysis;
5) use sephadex (Sephadex) after 0.01mol/L phosphate buffer balance, add dislysate,, collect the first painted peak, be required product with identical buffer solution elution.
Used fluorescein mainly contains fluorescein isothiocynate (FITC), dichlorotrazinylaminofluorescein (DTAF), RB 200 (RB 200), TRITC (TMRITC) etc.
The present invention replaces 125-iodine labeling anti-alpha-fetoprotein antibody with fluorescein, as indicator, the detection of alpha-fetoprotein variant can be finished in 1 hour.Because the present invention and immuno-electrophoresis combine, fluorescent marker is concentrated, reach the effect that strengthens fluorescence intensity, only need common cash inspecting machine just can observe, and need not use expensive fluorescent microscope or fluorospectrophotometer to observe or measure testing result.In addition, use this fluorescent marker, replace radioactivity 125-iodine labeling anti-alpha-fetoprotein antibody, can avoid radioactive harm and the equipment that develops photographic film when not needing to use radioautograph, testing cost is greatly reduced, fluorescence labeling The Application of Technology scope is increased greatly.Main application of the present invention is 1) detection of liver cancer; 2) early detection of hepatitis B patient canceration (optimum hepatopathy can not produce heteroplasmon); 3) detection of abnormal pregnancy.
The authentication method of fluorescein-labeled anti-alpha-fetoprotein antibody label:
The fair of label determined with fluorescein amount (F) and total protein quality (P) ratio.Concerning FITC, F/P between 1～2 for well.
F/P=0.41 * (fluorescein mcg/ml ÷ albumen milligram/microlitre), the content spectrophotometry of P and F.Protein content is with 260, and the absorbance of 280nm (OD value) is calculated protein concentration (mg/ml)=1.45OD280-0.74OD260.
The mensuration of fluorescein is made typical curve (being concentration and OD value curve) with the fluorescein of known quantity, and wherein, absorption peak is respectively FITC 490nm, RB200 570nm, TMRITC550nm, DTAF 495nm, molecular weight is respectively FITC389, RB200 580, and TMRITC 443, and DTAF 466.
Below provide fluorescein-labeled anti-alpha-fetoprotein antibody preparation method's example.
Reagent: alpha-fetoprotein antibody (anti-fetoprotein) can be selected purifying horse anti-alpha-fetoprotein antibody or anti-alpha-fetoprotein monoclonal antibody for use.Select fluorescein FITC for use.
Preparation:
1) 0.025mol/L PH9.0 carbonate buffer solution (CB): NaHCO
32.10g, Na
2CO
30.16g, add distilled water to 100mL.
2) 0.5mol/L PH9.5 carbonate buffer solution (CB): NaHCO
33.7g, Na
2CO
30.6g, add distilled water to 100mL.
Wherein 1) carbonate concentration 0.02～0.5mol/L 2), PH8.5～9.5 all can, also can use trishydroxymethylaminomethane (Tris) damping fluid replacement carbonate buffer solution.
3) 0.01mol/L PH7.2 phosphate buffer (PBS): Na
2HPO
412H
2O 2.58g, NaH
2PO
42H
2O0.44g, NaCl 8.50g, add distilled water to 1000mL (concentration 0.005-0.1mol/L wherein, PH 6.8-8.0 all can or with this phosphate buffer of physiologic saline for substitute.
4) anti-alpha-fetoprotein antibody is made into 30mg/mL solution with 0.025mol/L PH9.0 carbonate buffer solution (CB).
Preparation:
1) gets the anti-alpha-fetoprotein antibody solution of 30mL.
2) the fluorescein FITC of preparation 3mg (being equivalent to alpha-fetoprotein antibody protein content 1/100), (FITC is dissolved in earlier and is equivalent in the 0.5mol/L PH9.5 carbonate buffer solution (CB) that antibody-solutions amount 1/10 is 3mL.)
3) under the stirring of magnetic stirrer, FITC slowly is added dropwise in the antibody-solutions goes.In dripping fluorescein (FITC) process, should often measure the PH of antibody-solutions, reduce available 0.5mol/K Na as PH
2CO
3Adjust PH to 9.0-9.5.PH is on the low side, and mark is slow, and PH is higher, and fluorescein easily decomposes, 2～24 hours mark time, 2～30 ℃ of temperature.
4) fluorescein finishes, and reactant is moved into 4 ℃ of refrigerators, continues to stir 12 hours, or moves into 8 ℃ of refrigerators, continues to stir 6 hours.
5) above-mentioned label solution is packed in the bag filter, dialysis is 5 minutes in flowing water, moves in the 0.01mol/LPH7.2 phosphate buffer (PBS) of 2000mL again, continues dialysis 4 hours in 4 ℃ of refrigerators; Or move among the PBS of 1000mL, in 8 ℃ of refrigerators, continue dialysis 3.5 hours.
6) (1.2 * 30cm), after 0.01mol/L PH7.2 phosphate buffer (PBS) balance, the adding dislysate with identical PBS eluant solution, is collected the first painted peak, is required product with sephadex (Sephadex) G-25 (or G-50) post.
Among this preparation method, the calculating of FITC amount: generally with FITC: protein content=1: 100 is for well, can be from 1: 50-150, the preceding protein concentration 5-50 mg/ml of reaction; And the temperature during mark, can be from 2-30 ℃, the time, wherein temperature was low from 2 hours-24 hours, and the reaction time is long, the temperature height, the reaction time is short; Can drip FITC gradually during mark, the bag filter of also can directly packing into was by stirring reaction 10-24 hour.If the protein concentration of anti-alpha-fetoprotein antibody is low, the bag filter of then packing into is labeled as.
In preparation process, the fluorescein of preparation can be selected DTAF for use, and at this moment available sodium borate buffer liquid 0.1mol/L PH9.0 replaces sodium carbonate buffer for well.The amount of preparation of fluorescein DTAF can be equivalent to DTAF: alpha-fetoprotein antibody protein content=1～2.0mg: 100mg, antibody protein solution concentration 10～30mg/mL.Reaction time is identical with FITC with temperature.
The fluorescein of preparation can be selected TMRITC for use, and amount of preparation is TMRITC: antibody protein=1: 20～40mg, and the association reaction time is 12～24 hours, all the other are identical with FITC.
If select RB200 for use, then RB200 must change into sulfonic acid chloride (SO earlier
2Cl) base, could combine with antibody protein, the phosphorus pentachloride of RB200 and doubled amount (1: 2) grinds rapidly, the anhydrous propanone that after 3～10 minutes, adds 5 times of amounts, the extractive reaction product, with filter paper filtering or centrifugal removal insolubles, solution can be used for mark immediately, or with ampoule encapsulation, freezing (≤-15 ℃) are preserved.Antibody-solutions, carbonate buffer solution and physiological saline are mixed antibody protein mg: RB200=100 in 1: 1: 1 ratio: 0.1～0.5, be preferably in below 10 ℃, carry out association reaction under the condition of PH＞8.5, reaction time＞15 hour.
Claims (6)
1. fluorescein-labeled anti-alpha-fetoprotein antibody, its molecular weight is 160,000,260,280, the 490nm wavelength has absorption peak, under the long illumination of all-wave, sends green-yellow light, has with alpha-fetoprotein and carries out specific antigen-antibody response ability.
2. the preparation method of fluorescein-labeled anti-alpha-fetoprotein antibody is characterized in that
1) preparation fluorescein is in an amount equivalent to 1/ (50～150) of anti-alpha-fetoprotein antibody protein content;
2) fluorescein for preparing is added dropwise in the anti-alpha-fetoprotein antibody solution goes;
3) in the refrigerator with 4～8 ℃ of reactant immigrations, make it form label at low temperatures;
4) label solution is packed in the bag filter, in flowing water, dialyse, move into again in the 0.01mol/L phosphate buffer of 1000～2000mL, and in refrigerator, continue dialysis;
5) use sephadex after 0.01mol/L phosphate buffer balance, add dislysate,, collect the first painted peak, be required product with identical buffer solution elution.
3. the preparation method of fluorescein-labeled anti-alpha-fetoprotein antibody as claimed in claim 2 is characterized in that said fluorescein is fluorescein isothiocynate, dichlorotrazinylaminofluorescein, RB 200 or TRITC.
4. as the preparation method of claim 1 and 3 described fluorescein-labeled anti-alpha-fetoprotein antibodies, it is characterized in that said fluorescein is a fluorescein isothiocynate, in an amount equivalent to 1/100 of anti-alpha-fetoprotein antibody protein content.
5. the preparation method of fluorescein-labeled anti-alpha-fetoprotein antibody as claimed in claim 2 is characterized in that the fluorescein for preparing is added dropwise in the anti-alpha-fetoprotein antibody solution under condition of stirring, and the pH value of solution is 9.0～9.5.
6. the preparation method of fluorescein-labeled anti-alpha-fetoprotein antibody as claimed in claim 2 is characterized in that and will continue to stir 6～12 hours in 4～8 ℃ of refrigerators of reactant immigration.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00119376 CN1281978A (en) | 2000-07-13 | 2000-07-13 | Anti-alpha-fetoprotein antibody labelled by fluorescein and its preparation method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00119376 CN1281978A (en) | 2000-07-13 | 2000-07-13 | Anti-alpha-fetoprotein antibody labelled by fluorescein and its preparation method |
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| CN1281978A true CN1281978A (en) | 2001-01-31 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EA008925B1 (en) * | 2004-12-14 | 2007-08-31 | Товарищество С Ограниченной Ответственностью "Реал Мед Компани" | Method of protection immune state in an organism suffering from diabetes mellitus |
| CN102321167A (en) * | 2011-09-02 | 2012-01-18 | 北京利德曼生化股份有限公司 | Antigen epitope peptide of alpha fetoprotein, nucleic acid, preparation method of nucleic acid, recombinant vector, host cell, hybridoma cell, monoclonal antibody and kit |
| CN103063845A (en) * | 2012-12-18 | 2013-04-24 | 苏州浩欧博生物医药有限公司 | Nanometer magnetic particle chemiluminiscence kit, preparation method and detection method of hepatitis B virus surface-antibody |
| CN104311651A (en) * | 2014-10-22 | 2015-01-28 | 浙江中医药大学 | Method for preparing fluorescent single-labeled cobra neurotoxin |
| CN106146642A (en) * | 2016-06-27 | 2016-11-23 | 浙江星博生物科技股份有限公司 | A kind of for protein microsphere conjugate detecting sperm acrosin activities in assessing and its preparation method and application |
-
2000
- 2000-07-13 CN CN 00119376 patent/CN1281978A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EA008925B1 (en) * | 2004-12-14 | 2007-08-31 | Товарищество С Ограниченной Ответственностью "Реал Мед Компани" | Method of protection immune state in an organism suffering from diabetes mellitus |
| CN102321167A (en) * | 2011-09-02 | 2012-01-18 | 北京利德曼生化股份有限公司 | Antigen epitope peptide of alpha fetoprotein, nucleic acid, preparation method of nucleic acid, recombinant vector, host cell, hybridoma cell, monoclonal antibody and kit |
| CN103063845A (en) * | 2012-12-18 | 2013-04-24 | 苏州浩欧博生物医药有限公司 | Nanometer magnetic particle chemiluminiscence kit, preparation method and detection method of hepatitis B virus surface-antibody |
| CN104311651A (en) * | 2014-10-22 | 2015-01-28 | 浙江中医药大学 | Method for preparing fluorescent single-labeled cobra neurotoxin |
| CN106146642A (en) * | 2016-06-27 | 2016-11-23 | 浙江星博生物科技股份有限公司 | A kind of for protein microsphere conjugate detecting sperm acrosin activities in assessing and its preparation method and application |
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