CN107022030A

CN107022030A - Detect monoclonal antibody and kit and the application of alpha-fetoprotein

Info

Publication number: CN107022030A
Application number: CN201710179458.1A
Authority: CN
Inventors: 向军俭; 黄建芳; 孙帆; 孙一帆; 杨少彬
Original assignee: Guangdong Valley Bio Tech Industry Research Institute Co Ltd
Current assignee: Guangdong Valley Bio Tech Industry Research Institute Co Ltd
Priority date: 2017-03-23
Filing date: 2017-03-23
Publication date: 2017-08-08
Anticipated expiration: 2037-03-23
Also published as: CN107022030B

Abstract

The invention discloses the monoclonal antibody of detection alpha-fetoprotein and kit and application.Monoclonal antibody Ab1, Ab3 and Ab4 of the present invention this 3 plants of monoclonal antibodies can in human liver cancer cell and liver cancer tissue specific recognition people source natural hAFP, with good specificity.The invention discloses the mouse monoclonal antibody that can specifically bind human a-fetoprotein, the antibody can combine the different epitopes of human a-fetoprotein, form double-antibody sandwich pattern, realize the quantitative detection of the alpha-fetoprotein in human serum, there is certain effect to pre-natal diagnosis and hepatocarcinoma early diagnosis, with good application value.

Description

Detect monoclonal antibody and kit and the application of alpha-fetoprotein

Technical field

The invention belongs to biological technical field, and in particular to detect the monoclonal antibody and kit of alpha-fetoprotein and answer With.

Background technology

Human a-fetoprotein (human alpha fetoprotein, hAFP) is a kind of by fetal liver cells and yolk bag conjunction Into the Embryo glycoprotein gone out, its function is similar with human serum albumins (human serum albumin, HSA), is considered as The HSA substitutes in development of fetus period.Under normal circumstances, hAFP is existed only in fetal serum, fetal birth hAFP after 1 year Concentration can drop back to normal person's level (<5ng/ml), the concentration in normal human serum is generally not more than 20ng/ml.When body occurs During primary carcinoma of liver, liver cancer cells can largely secrete hAFP in peripheral blood, trigger the abnormal rise of blood hAFP concentration.

HAFP is one of tumor markers for finding earliest, and it is used as liver cancer, Fetal Defects and embryo for a long time The important serum molecules label of related cancer, is the important evaluation index molecule in blood test.Except applied to pre-natal diagnosis and Outside diagnosis of hepatoma, hAFP blood testing to hepatic injury, hepatic sclerosis, hepatitis, stomach cancer, nerve channel damage, endodermal sinus tumor, The diagnosis of the diseases such as reproductive system cancers and state of an illness monitoring are also significant.

China is a populous nation, and pre-natal diagnosis and each family are closely bound up；Meanwhile, China is again a liver cancer height Hair area, the number that liver cancer is died from every year accounts for global more than 40%, because mid and late liver cancer is substantially without effective treatment method, So the whole people's generaI investigation and early diagnosis to primary carcinoma of liver specific tumor antigen hAFP haemoconcentrations are to solve liver cancer height extremely Die the most effective measure of rate problem.Therefore it provides sensitive, special, accurate, easy, inexpensive serum hAFP detection methods are met Closed society in the urgent need to.

Human a-fetoprotein (hAFP) monitors molecule as primary carcinoma of liver most special tumor markers and pre-natal diagnosis, its Monoclonal antibody has extremely important practical application meaning.Although being successfully prepared anti-hAFP monoclonal antibodies both at home and abroad, its It is most of not have clinical value, it is impossible to research and develop and produce for kit.The hAFP detections at home and abroad now sold In ELISA kit product, linear detection range is within 20-400ng/ml, and the overall quality of wherein import reagent box is bright It is aobvious to be better than homemade goodses, but its range of linearity and sensitivity still need further raising.Further, since complicated component is easy in serum There is false positive, hAFP change in concentration scope greatly (5ng/ml-10mg/ml), therefore is used dual anti-in actually detected application Body sandwich ELISA (DAS-ELISA) two-step method is to improve accuracy in detection and stability, and this method requires the capture for pairing Antibody and detection antibody all have the specificity that very high affinity is become reconciled, and are formed " during antibody 1- 2 " compounds of Ag-Ab Locus distance it is more remote pairing effect it is better, therefore continually develop with more high sensitivity, more preferable linear detection range it is dual anti- The sandwich pairing antibody of body is the striving direction of kit research and development, the need for being also clinical detection.

The content of the invention

Resist the technical problem to be solved in the present invention is to provide the monoclonal that can specifically bind human a-fetoprotein different epitopes Body, and set up double-antibody sandwich Elisa methods the alpha-fetoprotein in human serum is detected.

It is an object of the invention to provide one group of targeting human a-fetoprotein (human alpha fetoprotein, hAFP) no With the mouse monoclonal antibody of epitope, it is desirable to antibody energy high-affinity, the combination human a-fetoprotein of high specific, and form double Antibody sandwich pattern, so as to quantitatively be detected to human a-fetoprotein.

It is an object of the invention to provide a kind of monoclonal antibody for detecting alpha-fetoprotein.

Another object of the present invention is to provide a kind of kit for detecting alpha-fetoprotein.

The technical solution used in the present invention is：

A kind of monoclonal antibody of high specific high-affinity combination human a-fetoprotein, the monoclonal antibody is Ab1, Ab3 Or Ab4, monoclonal antibody Ab1, Ab3 or Ab4 variable region gene include weight chain variable district and light chain variable district；

The amino acid sequence of the monoclonal antibody Ab1 weight chain variable districts as shown in SEQ ID NO.7, light chain variable district Amino acid sequence is as shown in SEQ ID NO.8；

The amino acid sequence of the weight chain variable district of the monoclonal antibody Ab3 is as shown in SEQ ID NO.9, light chain variable district Amino acid sequence as shown in SEQ ID NO.10；

The amino acid sequence of the monoclonal antibody Ab4 weight chain variable districts is as shown in SEQ ID NO.11, light chain variable district Amino acid sequence as shown in SEQ ID NO.12.

Further, the gene order of said monoclonal antibody Ab1 weight chain variable districts is as shown in SEQ ID NO.1, light chain The gene order of variable region is as shown in SEQ ID NO.2；

The gene order of the monoclonal antibody Ab3 weight chain variable districts is as shown in SEQ ID NO.3, the base of light chain variable district Because sequence is as shown in SEQ ID NO.4；

The gene order of the monoclonal antibody Ab4 weight chain variable districts is as shown in SEQ ID NO.5, the base of light chain variable district Because sequence is as shown in SEQ ID NO.6.

Contain the monoclonal antibody described in any of the above-described in a kind of kit for detecting human a-fetoprotein, the kit.

Containing any of the above-described described in a kind of double-antibody sandwich elisa kit for detecting human a-fetoprotein, the kit Monoclonal antibody Ab1 and Ab4, either Ab3 and Ab4 or Ab1, Ab3 and Ab4.

Further, above-mentioned monoclonal antibody Ab1 or/and Ab3 are capture antibody, and described monoclonal antibody Ab4 is Detect antibody.

A kind of double-antibodies sandwich ELISA for detecting human a-fetoprotein, this method is with any of the above-described described monoclonal Antibody A b1 or/and Ab3 is used as detection antibody as capture antibody using any of the above-described described monoclonal antibody Ab4；This method Diagnosis or treatment for non-diseases.

Further, the above method specifically includes following steps：

S1. it is coated with：Coating plate is carried out to be coated with overnight with Ab1 containing monoclonal antibody or/and Ab3 solution；

S2. close：Ab1 or/and Ab3 coating plate PBST washes cleans will be coated with, then entered with skimmed milk power or BSA Row closing；

Plus antigen S3.：By the coating plate PBST washes cleans closed, plasma sample to be detected is added, it is incubated 50~ 70min；

Plus secondary antibody S4.：Will be incubated sample after coating plate PBST washes cleans, add secondary antibody HRP-Ab4, be incubated 30~ 50min；

S5. PBST washes cleans are used, TMB nitrite ions are added after patting dry, color development stopping after room temperature lucifuge is incubated, ELIASA is read Take OD₄₅₀Value.

Application of the monoclonal antibody in hepatocarcinoma early diagnosis kit or reagent is prepared described in any of the above-described.

Application of the monoclonal antibody in the kit or reagent of detection human a-fetoprotein is prepared described in any of the above-described.

The beneficial effects of the invention are as follows：

1) the invention discloses the mouse monoclonal antibody that can specifically bind human a-fetoprotein, the antibody can combine people The different epitopes of alpha-fetoprotein, form double-antibody sandwich pattern, the quantitative detection of the alpha-fetoprotein in human serum are realized, to antenatal Diagnosis and hepatocarcinoma early diagnosis have certain effect.

2) this 3 plants of monoclonal antibodies of Ab 1 of the present invention, Ab 3, Ab 4 can in human liver cancer cell and liver cancer tissue specific recognition The natural hAFP in people source, and Ab 1, Ab 3, Ab 4 and high concentration to intersect albumen HSA reactionless, with good specificity.

3) Ab 1/HRP-Ab4 of the present invention, the detection sensitivity of this 2 couple of Ab3/HRP-Ab 4 pairing Antibody Combination are 5ng/ml, 10~200ng/ml of linear detection range, detect the 400ng/ml that reaches the standard grade.The line of Ab 1+Ab3/HRP-Ab4 combinations of pairs Property detection range is about 5-250ng/ml, and Monitoring lower-cut is up to 2ng/ml, upper limit of detection 400ng/ml.

4) present invention discover that antibody A b 1, Ab3 and Ab 4 there are good Detection results to hAFP, especially with anti- When body combinations of pairs Ab 1+Ab3/HRP-Ab 4 carry out double-antibody sandwich elisa detection to sample, its linear detection range is 5 ~250ng/ml, minimum detection limit 2ng/ml, upper limit of detection 400ng/ml, better than import reagent box (ABCam ELISA Kit) 5~200ng/ml of the range of linearity and minimum detection limit 5ng/ml；Clinical sample testing result shows detection method Positive serum Detection accuracy 100%, negative serum accuracy rate 100%.Illustrate the double-antibody sandwich elisa side that the present invention is set up Method has the bigger range of linearity, with more preferable application value.

Brief description of the drawings

Fig. 1 determines for 1~Ab of monoclonal antibody Ab 4 relative affinity

Fig. 2 is that monoclonal antibody Ab1-Ab 4 carries out immunofluorescence dyeing detection to HepG2 human liver cancer cells；

Fig. 3 is the carry out SABC detection that monoclonal antibody Ab1-Ab 4 is organized to human primary liver cancer；

Fig. 4 is CDR (complementary determining region) area of Ab1 heavy chain of antibody variable region 3；

Fig. 5 is CDR (complementary determining region) area of Ab1 antibody light chains variable region 3；

Fig. 6 is CDR (complementary determining region) area of Ab3 heavy chain of antibody variable region 3；

Fig. 7 is CDR (complementary determining region) area of Ab3 antibody light chains variable region 3；

Fig. 8 is CDR (complementary determining region) area of Ab4 heavy chain of antibody variable region 3；

Fig. 9 is CDR (complementary determining region) area of Ab4 antibody light chains variable region 3；

Figure 10 is the detection curve of the double-antibody sandwich elisas of different pairing antibody；

The linear standard curve that Figure 11 detects for pairing antibody A b 1/HRP-Ab4 double-antibody sandwich elisas；

The linear standard curve that Figure 12 detects for the double-antibody sandwich elisas of pairing antibody A b 3/HRP-Ab 4；

The linear standard curve that Figure 13 detects for the double-antibody sandwich elisas of pairing antibody A b 1+Ab 3/HRP-Ab 4.

Embodiment

With reference to specific embodiment, the present invention is further illustrated, but is not limited thereto.

The preparation of the monoclonal antibody of embodiment 1

Antigen human a-fetoprotein (Human Myoglobin) is purchased from Guilin Immunetech Co, Ltd., separates pure Change from human cord blood, its concentration is 7,6mg/ml, is natural human a-fetoprotein molecule, molecular weight is 69kDa, by 609 ammonia Base acid residue composition.HAFP is a kind of multi-functional transport protein of the expression silencing after the expression of fetal period height, birth.Serum HAFP concentration is the direct or indirect evaluation index of a variety of diseases, is especially common in pre-natal diagnosis and the diagnosis of liver cancer and the state of an illness Monitoring.

The preparation method of anti-human alpha-fetoprotein mouse monoclonal antibody is as follows：

(1) animal immune

1) initial immunity：30 μ g antigens carry out subcutaneous multiple spot after adding isometric Freund's complete adjuvant fully emulsified to mouse Injection；

2) secondary immunity：After 2 weeks, after the amount of antigen same with initial immunity adds equivalent incomplete Freund's adjuvant fully emulsified Subcutaneous multi-point injection；

3) three times be immunized：After 2 weeks, after the amount of antigen same with initial immunity adds equivalent incomplete Freund's adjuvant fully emulsified Subcutaneous multi-point injection (tail vein blood surveys its potency after 10 days)；

4) booster immunization：Three immunizing potencies are reached after cell fusion requirement, are not added with the same amount of antigen of initial immunity Adjuvant is injected intraperitoneally；

5) spleen is taken to merge after 72h.

(2) cell fusion

1) preparation of feeder cells：A healthy Balb/c mouse is taken, eyeball blood sampling, after blood is put totally, neck is plucked Dislocation is put to death, after body surface sterilizes and be fixed, and skin, exposure peritonaeum, cotton ball soaked in alcohol sterilization peritonaeum are cut off from thigh.Noted with 5mL Emitter injection 10ml RPMI1640 basal mediums (RPMI Medium 1640basic, gibco purchases, article No. 8114056, Using preceding addition penicillin and streptomysin, addition is double per penicillin of the 100ml culture mediums addition 1ml containing 100U and streptomysin It is anti-) abdominal cavity is arrived, the right hand fixes syringe, and left hand holds cotton ball soaked in alcohol gently abdomen massage, draws back intraperitoneal liquid, and injection is ready for In good sterile 10ml centrifuge tubes, 1000rmp is centrifuged 7 minutes, with 10% hyclone RPMI 1640 (containing HAT) culture completely Base is resuspended, dilution about 2 × 10⁵Individual/ml, is subsequently added in 96 orifice plates, per the μ l of hole 100, be placed in cell culture incubator (37 DEG C, 5%CO₂) in it is standby.

2) take the logarithm the murine myeloma cell SP2/0 of growth, is washed with RPMI1640 basal mediums, blows after outstanding dilution Count；

3) mouse spleen is taken, RPMI1640 basal mediums are washed, milling prepares single splenocyte suspension, counted；

4) myeloma cell and splenocyte are pressed 1：10 ratio is mixed, 1000rpm centrifugations 7min；

5) supernatant is abandoned, residual liquid is exhausted with dropper, 1mL poly- second two is added dropwise in 1min under 37 DEG C of water bath conditions Alcohol (PEG), stands 90 seconds, the basal medium terminating reactions of 15mL RPMI 1640 is added dropwise in 2~4min；

6) 1000rpm centrifuges 7min, abandons supernatant, is gently mixed with the hyclone RPMI 1640 (containing HAT) of 100mL 10% It is outstanding；It is added dropwise in being covered with advance in 96 orifice plates of feeder cells, 100 μ l/ holes；37 DEG C, 5%CO₂Cultivated in incubator.

(3) screening and cloning of fused cell

1) cells and supernatant was taken in the 7th day or so after cell fusion, with being coated with 30ng/ holes human a-fetoprotein Elisa plate carries out indirect ELISA detection, the positive hole of screening；With the serum taken before immune mouse fusion as positive control, use SP2/0 supernatants are used as negative control.It is final to filter out 14 plants of positive cell strains.

2) 14 plants of cell lines are found into the potency of wherein 3 plants cell line supernatants after 3~4 continuous cloning experiments It is unstable, it is undesirable.Finally, screened altogether by cloning and obtain 12 plants and be capable of the anti-hAFP monoclonal antibodies of stably excreting Positive hybridoma cell strain (is named as 1~AB of AB 12), and further antibody conjugates requirement of experiment can be met substantially.

(4) preparation of ascitic type monoclonal antibody

1) 10 week old female Balb/c mouse peritoneal injection 0.5mL incomplete Freund's adjuvants are taken；

2) it is inoculated with about 5*10 after mouse peritoneal within 1 week⁵Individual hybridoma, cell can induce ascites after being inoculated with 7~12 days；

3) ascites is extracted when ascites is as more as possible；

4) it is spaced 1~2 day, after after ascites regeneration accumulation, is taken out again with method, ascites 3000rpm centrifuges 10min after extraction, takes Supernatant is placed in -20 DEG C of preservations.

(5) purifying of monoclonal antibody and bioactivity

1) 4 DEG C of 12000rpm centrifugation 30min of ascites are taken, supernatant is taken；

2) isometric saturated ammonium sulfate solution is added dropwise under lasting stirring, 4 DEG C stand overnight；

3) liquid after staying overnight abandons supernatant, precipitation 0.1M PBS redissolve in 7500rpm, 4 DEG C of centrifugation 30min；

4) liquid will be redissolved and desalting processing is carried out with desalting column, concrete operation step is as follows：

1. pillar is balanced：With 0.1M PBS (5~10 times of column volumes), pillar is balanced, 20% ethanol in post is gone out, by nucleic acid Albumen instrument returns to zero；

2. loading：Before loading, first constant flow pump is closed, then slow loading, loading is continually fed into 0.1M PBS after finishing, when When A values are begun to ramp up, liquid (destination protein) is collected, stops collecting when A values drop to below 10.The sample of collection continue into The purifying of row next step.

3. pillar is balanced：When A values are " 0 ", post is crossed with 0.1M PBS (more than 5 times column volumes)；

4. preserve：Pillar is crossed with 20% ethanol (5 times of column volumes), pillar is preserved.

5) albumen after Protein G affinity columns purifying desalination.

Purification step is as follows：

1. column equilibration pillar is filled：With 0.1M PBS (5~10 times of column volumes), pillar is balanced, 20% ethanol in post is gone out, will Nucleic acid-protein instrument returns to zero；

2. loading：Before loading, first constant flow pump is closed, then slow loading, when A values are begun to ramp up, collect liquid (miscellaneous egg The uncombined upper pillar of protein is prevented in vain), and 0.1M PBS dilutions are added when sample finishes loading, make protein almost complete Full upper prop；

3. elute：When A values are down to " 0 ", use elution destination protein, when A values are begun to ramp up collect liquid (by In protein belt negative electricity, in alkalescent, a certain amount of neutralizer is previously added in collecting pipe, make collection liquid pH be maintained at 7.0 with On)；

4. pillar is balanced：When A values are " 0 ", post is crossed with 0.1M PBS (more than 5 times column volumes)；

5. preserve：Pillar is crossed with 20% ethanol (5 times of column volumes), pillar is preserved.

6. protein takes a small amount of progress PAGE gel electroresis appraisal purity after being concentrated by ultrafiltration, and uses ELISA method Detect antibody titer.

Success is screened and obtains 12 plants of hybridoma cell strains for being capable of the anti-hAFP monoclonal antibodies of stably excreting, wherein there is 5 plants The Monoclonal Antibody Against (1~Ab of Ab 5) of secretion has high titer of ascites, up to up to 30,000,000.

(6) the relative affinity detection of monoclonal antibody

According to IgG antibody concentration in ascites, the ascites or supernatant of the above-mentioned 12 plants of monoclonal antibodies filtered out are diluted to IgG respectively Antibody concentration 10⁵Ng/ml, 10 multiple proportions gradient dilutions simultaneously draw (such as Fig. 1).

When Antibody dilution curves reach OD₄₅₀During the half of maximum, corresponding IgG antibody concentration (P50 values, 50%of Maximal binding) it is lower, represent its correspondence affinity higher.As shown in figure 1, in the 12 plants of monoclonal antibodies prepared, having The affinity of 4 strain antibodies (1~Ab of Ab 4) is higher than or close to import standard antibody AFP-01 (with arrow mark in mark 01, figure Go out) and AFP-11 (being marked in mark 11, figure with arrow), show that this 5 strain antibody has high affinity, be potential optimal pairing The selection of antibody.

Determined through above-mentioned relative affinity, successfully screening obtains 12 plants and is capable of the miscellaneous of the anti-hAFP monoclonal antibodies of stably excreting Tumor cell strain is handed over, wherein the affinity for having the monoclonal antibody (1~Ab of Ab 4) of 4 plants of secretions is higher than or be similar to ABCam pairings Antibody A FP-01 and AFP-11 affinity, therefore show that this 4 plants of monoclonal antibodies have reached the affinity requirement of commercial pairing antibody.

The specificity analysis of the monoclonal antibody of embodiment 2

Above-mentioned 4 plant weights filtered out want monoclonal antibody Ab 1-A b 4 to have very high affinity to immunogene hAFP, to enter One step identifies its specificity, verifies whether it can specifically bind with naturally occurring hAFP, then respectively in cell aspect Following two experiments are devised with tissue aspect.

1) HepG2 human liver cancer cells immunofluorescence dyeing

Known human liver cancer cell HepG2 can largely synthesize hAFP in intracellular, and using Ab1-Ab 4, this 4 plants of monoclonal antibodies carry out cell Fluorescent staining is tested.

Experimental method：1. the previous day is tested, takes HepG2 cells in good condition that cell suspension is made, through cell count, with Cell concentration 5 × 10⁵The amount in individual/hole is spread in six orifice bores for being placed with cover glass；2. culture one day in incubator, treats thin Born of the same parents' degree of converging starts staining procedure when reaching 70% or so；3. PBS rinses cover glass one time, abandons after most liquid, with 4% poly first Aldehyde fixes cell 10min, rear PBS and washes 3 times, 5min/ times；4. with the solution of triton-X 100 of PBS preparations 3%, to thin Born of the same parents carry out film permeabilization, handle 10min, and PBS washes 3 times, 5min/ times；5. 5% skimmed milk power closes 30min, and PBS is washed 3 times；6. plus Enter suitably dilute prepare monoclonal antibody on cover glass, negative control group is done with irrelevant antibody, 37 DEG C of incubation 1h, PBS is washed 3 times；⑦ Add 1:The FITC mark donkey anti-mouse IgG secondary antibodies of 100 times of dilutions, 37 DEG C are incubated 40min, and PBS is washed 2 times；8. 0.5ug/ is added Ml DAPI dyeing liquors dye 10min, and PBS is washed 3 times；9. add and (400 are observed under 20ul mountant mountings, Laser Scanning Confocal Microscope ×) and store results.

Experimental result：Observation result shows (Fig. 2, multiplication factor：400 ×), in the corresponding experimental group of 4 plants of monoclonal antibodies, HepG2 is intracellular to be presented obvious green fluorescence, and irrelevant antibody cellular control unit does not observe green fluorescence, illustrates Ab1-Ab 4 monoclonal antibodies are specific must to identify the intracytoplasmic hAFP molecules of human liver cancer cell, it was demonstrated that 4 plants of monoclonal antibodies can specific recognition it is natural The people source AFP molecules of generation.

2) human liver cancer tissue SABC：

When primary carcinoma of liver occurs for human body, liver cancer cells can secrete a large amount of AFP in peripheral blood.Primary carcinoma of liver is suffered from The hepatic tissue of person does immunohistochemical analysis.

Experimental method：1. fixed and embedding：4% paraformaldehyde fixing organization blocks, through 70% ethanol 30min, 80% ethanol 30min, 90% ethanol 30min 2 times, 95% ethanol 30min 2 times, 100% ethanol 30min 2 times, the transparent 30min of dimethylbenzene 2 times, 30min 2 times, mould Zhong La investing tissues block in 55 DEG C of paraffin；2. cut into slices：Thickness 3-5um histotomy is placed in many On the slide of polylysine activation, 60 DEG C overnight；3. dewax, enter water：Section is dipped in dimethylbenzene 5min 2 times, 100% second Alcohol 5min 2 times, 95% ethanol 5min 2 times, 90% ethanol 5min 2 times, 85% ethanol 5min 2 times, 75% ethanol 5min 2 Secondary, PBS is washed 2 times；4. 1% methanol hydrogen peroxide room temperature 10min, distillation washing 1 time, PBS is washed 3 times；5. 0.01M lemons are inserted into section Repaired in lemon phthalate buffer (pH 6.0), 10min in micro-wave oven, PBS is washed 3 times；6. close：5% skimmed milk power is closed, room temperature 20min, is not washed；7. enzyme labelled antibody (Ab1-HRP, Ab2-HRP, Ab3-HRP, Ab4- of appropriateness dilution is added dropwise in section respectively HRP), stay overnight for 4 DEG C, PBS is washed 5 times；8. horseradish enzyme mark strepto- avidin working solution (S-A/HRP), 37 DEG C are added dropwise in section 20min, PBS are washed 3 times；9. DAB develops the color：Add each drop of developer A, B, C using DAB colour reagent box 1ml distilled water, mix, Add on sample, develop the color 6min, washing is terminated；10. haematoxylin redyes nucleus 1min, washing, the differentiation of 1% hydrochloride alcohol, 1% Aqueous amine is anti-blue, washing, through 70% ethanol 5min, 80% ethanol 5min, 90% ethanol 5min 2 times, 95% ethanol 5min 2 times, 100% ethanol 5min 2 times dehydrations, the transparent 5min of dimethylbenzene 2 times, resinene mounting.

Experimental result：Testing result shows (Fig. 3, multiplication factor：400 ×), in 4 plants of corresponding livers of monoclonal antibody Ab1~Ab 4 In cancerous tissue section, the cytosolic part of most cells is dyed to brown, shows the ability that these cells have a large amount of secretion hAFP, The as liver cell of canceration, may be for the liver cell of canceration occur for the cell that is dyed to brown.Meanwhile, groupization result also table Understand that Ab 1~Ab 4 this 4 plants of monoclonal antibodies specific recognition and can combine in liver cancer tissue the intracytoplasmic hAFP of liver cancer cells points Son.

3) cross reaction is analyzed

Known hAFP primary cross reactive protein is human serum albumin HSA, copies HSA concentration in human blood, with Whether the HSA of 40mg/ml concentration detects monoclonal antibody Ab 1~Ab 4 to human serum as cross antigen by indirect elisa method There is cross reaction in albumin HAS.HSA cross reactive groups and PBS negative control groups are set up in experiment.Experiment is repeated 3 times.(AFP 400ng/ml, HSA 40mg/ml)

The concrete operation method of indirect ELISA determination method is：It is dilute with 0.05M pH9.6 carbonate buffer solution (coating buffer) HAFP antigens are released to 500ng/ml, 100 37 DEG C of μ l/ holes are coated with 3h or 4 DEG C overnight, PBS-T board-washings 3 times, 3min/ times；5% degreasing 1h is closed in 37 DEG C of 200 μ l/ holes of milk powder, and PBS-T is washed 3 times；By mouse polyvalent antibody to be measured and negative control sera, with 2 multiple proportions Gradient doubling dilution, adds 100ul/ holes in hole, and 37 DEG C are incubated 1h, and PBS-T is washed 3 times；Plus HRP mark goat anti-mouse igg antibodies (1:8000 dilutions) 100ul, 37 DEG C are incubated 40min, and PBS-T is washed 5 times；Tmb substrate develops the color, 37 DEG C of lucifuges colour developing 10min；2M H₂SO₄50 μ L/ holes terminating reactions, ELIASA wavelength 450nm surveys A values.

Testing result shows, 1~Ab of monoclonal antibody Ab 4 and the complete no cross reactions of HSA.

Above-mentioned monoclonal antibody specificity identification result shows that this 4 plants of monoclonal antibodies of 1~Ab of Ab 4 can be in human liver cancer cell and liver cancer The natural hAFP in specific recognition people source in tissue, and 1~Ab of Ab 4 and high concentration to intersect albumen HSA reactionless.

The monoclonal antibody Ab1 of embodiment 3~Ab4 sequence and structural analysis

Make further sequence and structural analysis to resulting monoclonal antibody Ab 1, Ab 3, Ab 4, find Ab 1, Ab 3, Ab 4 variable region include weight chain variable district and light chain variable district.

(1) from the point of view of gene order aspect：

The gene order of monoclonal antibody Ab1 weight chain variable districts is as shown in SEQ ID NO.1, the gene sequence of light chain variable district Row are as shown in SEQ ID NO.2；

The gene order of monoclonal antibody Ab3 weight chain variable districts is as shown in SEQ ID NO.3, the gene sequence of light chain variable district Row are as shown in SEQ ID NO.4；

The gene order of monoclonal antibody Ab4 weight chain variable districts is as shown in SEQ ID NO.5, the gene sequence of light chain variable district Row are as shown in SEQ ID NO.6.

(2) from the point of view of peptide sequence aspect：

The amino acid sequence of monoclonal antibody Ab1 weight chain variable districts is as shown in SEQ ID NO.7, the amino of light chain variable district Acid sequence is as shown in SEQ ID NO.8；

The amino acid sequence of monoclonal antibody Ab3 weight chain variable district is as shown in SEQ ID NO.9, the ammonia of light chain variable district Base acid sequence is as shown in SEQ ID NO.10；

The amino acid sequence of monoclonal antibody Ab4 weight chain variable districts is as shown in SEQ ID NO.11, the ammonia of light chain variable district Base acid sequence is as shown in SEQ ID NO.12.

(3) architectural feature of variable region

The Ab1 weight chain variable districts encode 119 amino acid by 360 base compositions, and 3 CDR are contained (mutually in variable region Mend determinant) area.CDR1 encodes 5 amino acid, and CDR2 encodes 17 amino acid, and CDR3 encodes 11 amino acid (such as Fig. 4 institutes Show).The framework region of variable region is higher than 91% with the homology of other murine antibodies, and 3 CDR regions are then specific sequences, It is variant with other murine antibody weight chain variable district CDR regions.

Monoclonal antibody Ab1 light chain variable districts encode 104 amino acid, variable region contains 3 by 314 base compositions CDR (complementary determining region) area.CDR1 encodes 11 amino acid, and CDR2 encodes 7 amino acid, and CDR3 encodes 9 amino acid (as schemed Shown in 5).The framework region of variable region is higher than 97.1% with the homology of other murine antibodies, and 3 CDR regions are then specificity Sequence, it is variant with other murine antibody light chain variable district CDR regions.

Monoclonal antibody Ab3 weight chain variable districts encode 119 amino acid, variable region contains 3 by 357 base compositions CDR (complementary determining region) area.CDR1 encodes 5 amino acid, and CDR2 encodes 17 amino acid, and CDR3 encodes 11 amino acid (such as Shown in Fig. 6).The framework region of variable region is higher than 94.8% with the homology of other murine antibodies, and 3 CDR regions are then special Property sequence, it is variant with other murine antibody weight chain variable district CDR regions.

Monoclonal antibody Ab3 light chain variable districts encode 104 amino acid, variable region contains 3 by 314 base compositions CDR (complementary determining region) area.CDR1 encodes 11 amino acid, and CDR2 encodes 7 amino acid, and CDR3 encodes 9 amino acid (as schemed Shown in 7).The framework region of variable region is higher than 97.1% with the homology of other murine antibodies, and 3 CDR regions are then specificity Sequence, it is variant with other murine antibody light chain variable district CDR regions.

Monoclonal antibody Ab4 weight chain variable districts encode 119 amino acid, variable region contains 3 by 357 base compositions CDR (complementary determining region) area.CDR1 encodes 5 amino acid, and CDR2 encodes 17 amino acid, and CDR3 encodes 11 amino acid (such as Shown in accompanying drawing 8).The framework region of variable region is higher than 95.2% with the homology of other murine antibodies, and 3 CDR regions are then special Different in nature sequence, it is variant with other murine antibody weight chain variable district CDR regions.

Monoclonal antibody Ab4 light chain variable districts encode 108 amino acid, variable region contains 3 by 324 base compositions CDR (complementary determining region) area.CDR1 encodes 15 amino acid, and CDR2 encodes 7 amino acid, and CDR3 encodes 8 amino acid (as schemed Shown in 9).The framework region of variable region is more than 95.6% with the homology of other murine antibodies, and 3 CDR regions are then specificity Sequence, it is variant with other murine antibody light chain variable district CDR regions.

The function for 3 plants of anti-human alpha-fetoprotein antibodies (Ab 1, Ab 3, Ab 4) that the present invention is obtained can by antibody light and heavy chain Become the complementation of area's antigen and determine race (complementarity determing regions CDRs) CDR1, CDR2, CDR3 (function Active region) in specific nucleotide sequences determine, its corresponding amino acid sequence constitutes antibody specificity combination people's first tire Different epitopes on albumen.

The antibody conjugates of the double-antibody sandwich elisa of embodiment 4 detection

Selection purifying, 7 plants of monoclonal antibodies (Ab 1, Ab 2, Ab 3, Ab 4, Ab 5, Ab 6, Ab 10) of HRP marks are used to resist Body pairing experiment.HSA cross reactive groups and PBS negative control groups are set up in experiment.Experiment is repeated 3 times.(AFP 400ng/ml, HSA 40mg/ml)。

Double-antibody sandwich elisa detection method is comprised the following steps that：

S1. it is coated with：Capture monoclonal antibody is diluted to the μ g/ml of working concentration 3~8 with pH9.6 carbonate buffer solution, 100 μ l add coating plate per hole, and 4 DEG C of coatings are stayed overnight；

S2. close：PBST (containing 0.05% tween) washing 3 times, 3min/ times, is sealed with 5% skimmed milk power or 2%BSA Close, per hole 200 μ l, 37 DEG C of incubation 1h；

Plus antigen S3.：PBST wash 3 times, 3min/ times, sequentially added after patting dry human N-terminal plasma pro-brain natriuretic peptide levels antigen (10~ 10000g/ml), per hole 50 μ l, 37 DEG C of incubation 1h；

Plus secondary antibody S4.：PBST is washed 3~5 times, and with 5% skimmed milk power, 8000 times of dilution secondary antibodies, (detection of HRP marks resists Body), per hole 50 μ l, 37 DEG C of incubation 45min；

S5. washed with PBST 3~5 times, each 3min, addition TMB nitrite ions after patting dry, room temperature lucifuge incubation 10~ Terminated after 15min, ELIASA reads OD₄₅₀Value.

The double antibody sandwich ELISA of table 1 screening pairing antibody

Note："+" quantity representative OD₄₅₀The height of value：“+”>1.0, " ++ ">2.0, " +++ ">3.0；"-" represents OD450 values< 0.5；" * ", which is represented, cross reaction.

Match result as shown in table 1, planted in 49 (7 × 7) in antibody conjugates combination, there are 13 kinds of combinations to form double antibody folder The heart is matched, wherein pairing effect preferably has 8 pairs of antibody, being total to the complete no cross reactions of HSA and with preferably pairing effect There are 4 kinds of combinations：Ab 1/HRP-Ab 2、Ab 1/HRP-Ab 4、Ab 3/HRP-Ab 2、Ab 3/HRP-Ab 4.

The sensitivity of the double-antibody sandwich elisa of embodiment 5 detection and linear detection range

Experimental method：With Ab 1, Ab 3, Ab 1+Ab 3, (capture antibody A b1 and Ab 3 is with 1 respectively:1 mixing) it is used as and catches Antibody is obtained, is detection antibody with HRP-Ab 4, is detected using double-antibody sandwich elisa detection method (described in embodiment 4), And to the corresponding detection curve of system and standard curve, each double-antibody sandwich elisa detection under the conditions of the different capture antibody of detection Sensitivity and linear detection range.

Experimental result：

Figure 10 is the detection curve of the double-antibody sandwich elisas of different pairing antibody；Figure 11~13 are respectively to match antibody Ab1/HRP-Ab 4, Ab 3/HRP-Ab 4, the linear criterion song of the double-antibody sandwich elisas of Ab 1+Ab 3/HRP-Ab 4 detection Line.

As can be seen that Ab 1/HRP-Ab4, the inspection of this 2 couple pairing Antibody Combinations of Ab 3/HRP-Ab 4 from Figure 10~13 Survey curve very close, its detection sensitivity is 5ng/ml, 10~200ng/ml of linear detection range, detects the 400ng/ that reaches the standard grade ml。

When the HRP-Ab4 using same concentrations is detection antibody, by capture antibody A b1 and Ab 3 with 1:1 mixing coating When, Ab 1+Ab 3/HRP-Ab4 are compared to Ab 1/HRP-Ab4 and Ab 3/HRP-Ab 4, its sensitivity and linear detection range There is slight raising；In the bottom and top of detection curve, Ab 1+Ab 3/HRP-Ab4 OD values are apparently higher than Ab 1/HRP- Ab4 and Ab 3/HRP-Ab 4 (Figure 10), and the linear detection range of the combinations of pairs of Ab 1+Ab 3/HRP-Ab 4 be about 5~ 250ng/ml, Monitoring lower-cut 2ng/ml, upper limit of detection 400ng/ml (Figure 10 and 13).This shows mixing coating group Ab 1+Ab3/ The ability of HRP-Ab4 capture antigens and the ability of formation double-antibody sandwich compound have slight raising than independent coating group.

Application of the double-antibody sandwich elisa of the present invention of embodiment 6 detection in clinical sample detection

HAFP positive patient seras 120 (pregnant woman 118, primary carcinoma of liver 2), negative normal human serum are have collected altogether 40, respectively with present invention pairing antibody A b 1+Ab 3/HRP-Ab4 double-antibody sandwich elisas and the ABCam ELISA of import Kit is detected, counts the sample inspection accuracy rate (table 2) of the two.

The step of present invention pairing antibody A b 1+Ab 3/HRP-Ab4 double-antibody sandwich elisa detection methods：

S1. it is coated with：Antibody A b1 and Ab3 are captured with 1:1 mixing, working concentration is diluted to pH9.6 carbonate buffer solution 7.5 μ g/ml, 100 μ l add coating plate per hole, and 4 DEG C of coatings are stayed overnight；

Plus antigen S3.：PBST is washed 3 times, and 3min/ time, twice of the use Sample dilution of addition various concentrations is dilute after patting dry The human a-fetoprotein standard antigen and human serum sample released, the μ l of addition 80；37 DEG C of incubation 1h；

Plus secondary antibody S4.：PBST is washed 3~5 times, and with 5% skimmed milk power, 8000 times of dilution secondary antibodies, (detection of HRP marks resists Body HRP-Ab4), per hole 50 μ l, 37 DEG C of incubation 1h；

S5. washed with PBST 5 times, 3min/ times, nitrite ion is added after patting dry, room temperature lucifuge is incubated after 10min and terminated, enzyme Mark instrument and read OD₄₅₀Value.

Meanwhile, with import reagent box (be purchased from ABCam companies, article No. Ab108631) to hAFP positive patient seras 120 (pregnant woman 118, primary carcinoma of liver 2) and negative normal human serum 40 are detected that detection method is in strict accordance with kit Specification is operated.The testing result of the two is recorded, the degree of accuracy of pattern detection is verified.

The distinct methods of table 2 detect the testing result of sample

Statistical result showed (table 2), is examined using the present invention pairing Antibody Combination Ab 1+Ab 3/HRP-Ab4 hAFP set up Survey method and ABCam ELISA Kit can be in effective detection human serum AFP molecules.Wherein, import reagent box is to yin, yang Property blood serum sample Detection accuracy be 100%, detection is stable, accurate；Prove that detection method can be examined well Measure the hAFP antigens in serum.

In summary, present invention discover that antibody A b 1, Ab 3 and Ab 4 there are good Detection results to hAFP, especially It is its linearity test when combining Ab 1+Ab 3/HRP-Ab4 using antibody conjugates to carry out double-antibody sandwich elisa detection to sample Scope is 5~250ng/ml, minimum detection limit 2ng/ml, upper limit of detection 400ng/ml, better than import reagent box (ABCam ELISA Kit) 5~200ng/ml of the range of linearity and minimum detection limit 5ng/ml；The clinical sample testing result display present invention The positive serum Detection accuracy 100% of detection method, negative serum accuracy rate 100%.Illustrate the double antibody that the present invention is set up Sandwich ELISA method has the bigger range of linearity, with more preferable application value.With antibody of the present invention set up it is dual anti- Body sandwich ELISA method can be used for the detection of human a-fetoprotein, pre-natal diagnosis and the early diagnosis of liver cancer be carried out, with good Commercial application value.

Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification, Equivalent substitute mode is should be, is included within protection scope of the present invention.

SEQUENCE LISTING

<110>Guang Donggu is full of Co., Ltd of biotech industry research institute

<120>Detect monoclonal antibody and kit and the application of alpha-fetoprotein

<130>

<160> 12

<170> PatentIn version 3.5

<210> 1

<211> 360

<212> DNA

<213>Artificial sequence

<400> 1

taggtgaaac tgcagcagtc tggggcagaa cttgtgaagc caggggcctc ggtcaagttg 60

tcctgttcag cttctggctt caacattcaa gactccttca tgcactgggt gaaacagagg 120

cctgaacagg gcctggagtg gattggaggg attgatcctg caaatggaaa tattagatat 180

gacccgaaat tccaggacaa ggccactatg acatccgaca catcctccaa tacagcctac 240

ctgaccctca acagcctgac atctgaggac actgccgtct attactgtgt tagaggacaa 300

ctcggaggtc gaggctggtt tgtttactgg ggccaaggga ccacggtcac cgtctcctca 360

<210> 2

<211> 314

<212> DNA

<213>Artificial sequence

<400> 2

gacattcagc tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60

ctcacttgtc gggcaagtca ggaaattagt gattacttaa tctggcttca gcagaaacca 120

gatggaactt ttaaacgcct gatctacgcc gcgtccactt tagattctgg tgtcccaaaa 180

aggttcagtg gcagtaagtc tgggtcagat ttttctctca ccatcagcag ccttgagtct 240

gaagattttg cagactatta ctgtctacaa tatgctagtt ttccgtacac gttcggaggg 300

gggaccaagc tgga 314

<210> 3

<211> 357

<212> DNA

<213>Artificial sequence

<400> 3

gtcaagctgc aggagtctgg ggcagaactt gtgaagccag gggcctcagt caagttgtcc 60

tgcacagctt ctggcttcaa cattaaagac acctatatgc actgggtgaa gcagaggcct 120

gaacagggcc tggagtggat tggagggatt gatcctgcga atggtaagac taaatttgac 180

ccgaagttcc agggcaaggc cactataaca gcagacacat cctccaacac agcctacctg 240

cacctcagcc gcctgacatc tgacgacacg gccgtctatt actgtgttag agggcaagtc 300

ggaggtcgag gctggtttgc ttactggggc caagggacca cggtcaccgt ctcctca 357

<210> 4

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<212> DNA

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gacattcagc tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60

ctcacttgtc gggcaagtca ggaaattagt ggttacttaa gctggcttca gcggaaacca 120

gatggaacta ttaaacgcct gatctacgcc gcatccactt tagattctgg tgtcccaaaa 180

aggttcagtg gcagtaggtc tgggtcagat tattctctca ccatcagcag ccttgagtct 240

gaagattttg cagactatta ctgtctacag tatgctagtt atccgtacac gttcggaggg 300

gggaccaagc tgga 314

<210> 5

<211> 288

<212> DNA

<213>Artificial sequence

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tctggctaca ccttcacaag ctactatata cactgggtga agcagaggcc tggacaggaa 60

cttgaatgga ttggatggat ttatcctgga aatgttaata ctaagtacaa tgagaagttc 120

aagggcaagg ccacactgac tgcagacaaa tcctccagca cagcctacat gcagctcagc 180

agcctgacct ctgaggactc tgcggtctat ttctgtgcta gatgctcggg ccgtctttac 240

tatgctatgg actactgggg ccaagggacc acggtcaccg tctcctca 288

<210> 6

<211> 324

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gacattgtga tgacacagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60

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caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa cctagaatct 180

ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240

cctgtggagg aggaggatgc tgcaacctat tactgtcagc acattaggga gcttacacgt 300

tcggaggggg gaccaagctg gaaa 324

<210> 7

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<212> PRT

<213>Artificial sequence

<400> 7

Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser

1 5 10 15

Val Lys Leu Ser Cys Ser Ala Ser Gly Phe Asn Ile Gln Asp Ser Phe

20 25 30

Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly

35 40 45

Gly Ile Asp Pro Ala Asn Gly Asn Ile Arg Tyr Asp Pro Lys Phe Gln

50 55 60

Asp Lys Ala Thr Met Thr Ser Asp Thr Ser Ser Asn Thr Ala Tyr Leu

65 70 75 80

Thr Leu Asn Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Val

85 90 95

Arg Gly Gln Leu Gly Gly Arg Gly Trp Phe Val Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 8

<211> 104

<212> PRT

<213>Artificial sequence

<400> 8

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Asp Tyr

20 25 30

Leu Ile Trp Leu Gln Gln Lys Pro Asp Gly Thr Phe Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly

50 55 60

Ser Lys Ser Gly Ser Asp Phe Ser Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Phe Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu

100

<210> 9

<211> 119

<212> PRT

<213>Artificial sequence

<400> 9

Val Lys Leu Gln Glu Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser

1 5 10 15

Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr

20 25 30

Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly

35 40 45

Gly Ile Asp Pro Ala Asn Gly Lys Thr Lys Phe Asp Pro Lys Phe Gln

50 55 60

Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr Leu

65 70 75 80

His Leu Ser Arg Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys Val

85 90 95

Arg Gly Gln Val Gly Gly Arg Gly Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 10

<211> 104

<212> PRT

<213>Artificial sequence

<400> 10

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr

20 25 30

Leu Ser Trp Leu Gln Arg Lys Pro Asp Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu

100

<210> 11

<211> 119

<212> PRT

<213>Artificial sequence

<400> 11

Val Lys Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser

1 5 10 15

Val Thr Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Tyr

20 25 30

Ile His Trp Val Lys Gln Arg Pro Gly Gln Glu Leu Glu Trp Ile Gly

35 40 45

Trp Ile Tyr Pro Gly Asn Val Asn Thr Lys Tyr Asn Glu Lys Phe Lys

50 55 60

Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met

65 70 75 80

Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala

85 90 95

Arg Cys Ser Gly Arg Leu Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 12

<211> 108

<212> PRT

<213>Artificial sequence

<400> 12

Asp Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg

85 90 95

Glu Leu Thr Arg Ser Glu Gly Gly Pro Ser Trp Lys

100 105

Claims

1. a kind of monoclonal antibody of high specific high-affinity combination human a-fetoprotein, the monoclonal antibody be Ab1, Ab3 or Ab4, monoclonal antibody Ab1, Ab3 or Ab4 variable region gene include weight chain variable district and light chain variable district；

The amino acid sequence of the monoclonal antibody Ab1 weight chain variable districts is as shown in SEQ ID NO.7, the amino of light chain variable district Acid sequence is as shown in SEQ ID NO.8；

The amino acid sequence of the weight chain variable district of the monoclonal antibody Ab3 is as shown in SEQ ID NO.9, the ammonia of light chain variable district Base acid sequence is as shown in SEQ ID NO.10；

The amino acid sequence of the monoclonal antibody Ab4 weight chain variable districts is as shown in SEQ ID NO.11, the ammonia of light chain variable district Base acid sequence is as shown in SEQ ID NO.12.

2. a kind of monoclonal antibody of high specific high-affinity combination human a-fetoprotein according to claim 1, it is special Levy and be：The gene order of the monoclonal antibody Ab1 weight chain variable districts is as shown in SEQ ID NO.1, the base of light chain variable district Because sequence is as shown in SEQ ID NO.2；

The gene order of the monoclonal antibody Ab3 weight chain variable districts is as shown in SEQ ID NO.3, the gene sequence of light chain variable district Row are as shown in SEQ ID NO.4；

The gene order of the monoclonal antibody Ab4 weight chain variable districts is as shown in SEQ ID NO.5, the gene sequence of light chain variable district Row are as shown in SEQ ID NO.6.

3. a kind of kit for detecting human a-fetoprotein, it is characterised in that：Contain any one of claim 1 ~ 2 institute in the kit The monoclonal antibody stated.

4. a kind of double-antibody sandwich elisa kit for detecting human a-fetoprotein, it is characterised in that：Contain in the kit and have the right It is required that 1 ~ 2 any described monoclonal antibody Ab1 and Ab4, either Ab3 and Ab4 or Ab1, Ab3 and Ab4.

5. a kind of double-antibody sandwich elisa kit for detecting human a-fetoprotein according to claim 4, its feature exists In：Described monoclonal antibody Ab1 or/and Ab3 is capture antibody, and described monoclonal antibody Ab4 is detection antibody.

6. a kind of double-antibodies sandwich ELISA for detecting human a-fetoprotein, it is characterised in that this method is with claim 1 ~ 2 Any described monoclonal antibody Ab1 or/and Ab3 is as capture antibody, with any described monoclonal antibody of claim 1 ~ 2 Ab4 is used as detection antibody；This method is used for the diagnosis or treatment of non-diseases.

7. method according to claim 6, it is characterised in that：This method specifically includes following steps：

S2. close：Ab1 or/and Ab3 coating plate PBST washes cleans will be coated with, then sealed with skimmed milk power or BSA Close；

Plus antigen S3.：By the coating plate PBST washes cleans closed, plasma sample to be detected is added, 50 ~ 70min is incubated；

Plus secondary antibody S4.：Will be incubated sample after coating plate PBST washes cleans, add secondary antibody HRP-Ab4, be incubated 30 ~ 50min；

S5. PBST washes cleans are used, TMB nitrite ions are added after patting dry, color development stopping after room temperature lucifuge is incubated, ELIASA is read OD₄₅₀Value.

8. the answering in hepatocarcinoma early diagnosis kit or reagent is prepared of the monoclonal antibody described in any one of claim 1 ~ 2 With.

9. the monoclonal antibody described in any one of claim 1 ~ 2 is in the kit or reagent of detection human a-fetoprotein is prepared Using.