CN111363037B - Disease detection kit containing antibody specifically binding AFP protein - Google Patents

Disease detection kit containing antibody specifically binding AFP protein Download PDF

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Publication number
CN111363037B
CN111363037B CN202010451891.8A CN202010451891A CN111363037B CN 111363037 B CN111363037 B CN 111363037B CN 202010451891 A CN202010451891 A CN 202010451891A CN 111363037 B CN111363037 B CN 111363037B
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hafp
variable region
afp
chain variable
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CN111363037A (en
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史辛艺
王阳
金鑫
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Shenzhen Taihua Sail Biotechnology Co ltd
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Shenzhen Michael Biotech Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]

Abstract

The invention relates to a disease detection kit containing an antibody specifically binding AFP protein, which contains an antibody capable of specifically binding with AFP and not cross-reacting with HAS, and the kit HAS higher sensitivity and is used for early screening diagnosis, curative effect detection and prognosis judgment of liver cancer with abnormal AFP and fetal congenital diseases.

Description

Disease detection kit containing antibody specifically binding AFP protein
Technical Field
The invention relates to the field of biomedicine, in particular to a disease detection kit containing an antibody specifically binding AFP protein.
Background
Primary liver cancer is one of malignant tumors with extremely poor prognosis, and liver cancer is in the 2 nd position of the fatality rate of malignant tumors in China at present. Although liver cancer can be found at an early stage at present and the treatment methods are more, the treatment effect is still unsatisfactory, most liver cancer patients have no significant effect on chemical drug treatment, radiotherapy and the like, and the number of patients capable of being treated radically by operation is only 15%. Alpha-fetoprotein (AFP) is an important tumor-associated antigen. Currently, AFP is an important target antigen for diagnosis and biological treatment of primary liver cancer. AFP is a fetal specific protein, is mainly synthesized in the yolk sac and liver of a fetus, is a single-chain polypeptide, belongs to alpha globulin, has the molecular weight of about 64-72 KD, has a peptide chain amino acid sequence similar to that of albumin, and consists of 590 amino acids in part. The AFP of the serum is more than 400 ng/mL, which is an important auxiliary index for diagnosing the primary liver cancer. Research shows that AFP has strong effect of independently promoting liver cancer cell proliferation, and high expression of AFP in liver cancer may be an important autocrine phenomenon of endogenous swelling and pain cell proliferation. The detection of AFP becomes an important index for diagnosing and monitoring prognosis of primary liver cancer, and also becomes a target antigen for recognized biological treatment of liver cancer. Except primary liver cancer, some patients with gastric cancer, malignant teratoma, pancreatic acinar cell cancer, colon cancer and renal cell carcinoma all find that the AFP level in serum is obviously increased. In addition, the AFP has high concentration in fetal blood circulation, and the AFP content is low 2-3 months after birth, so that the AFP is difficult to detect in blood. If congenital spina bifida, neural tube defects, Down syndrome and the like of a fetus can cause changes of alpha-fetoprotein content, screening and diagnosis of the diseases can be carried out through AFP monitoring in the gestational period. Therefore, the detection of AFP is helpful for the diagnosis, early intervention and treatment of liver cancer and fetal congenital diseases with AFP abnormality.
There are also several patents in the prior art that disclose AFP antibodies, such as: CN105037546A, CN10331959B, and CN105037544A, but the inventors found that these antibodies still have to be improved in sensitivity after the study. Meanwhile, Human AFP has 50% homology with Human Serum Albumin (HSA), so that the monoclonal antibody prepared by immunizing with AFP as an antigen has higher cross reaction probability with the Human Serum Albumin.
Disclosure of Invention
Based on the above findings, the primary object of the present invention is to provide a novel AFP antibody having higher sensitivity while not reacting with HSA, in order to solve the problems of the prior AFP antibody that the sensitivity is low and non-specific binding with HSA is likely to occur.
The invention provides the following technical scheme:
an anti-AFP antibody, designated hAFP-2, consisting of a heavy chain and a light chain comprising a variable region and a constant region, respectively, wherein the heavy chain variable region is SEQ ID NO 1 and the light chain variable region is SEQ ID NO 2.
An anti-AFP antibody, designated hAFP-6, consisting of a heavy chain and a light chain comprising a variable region and a constant region, respectively, wherein the heavy chain variable region is SEQ ID NO 9 and the light chain variable region is SEQ ID NO 10.
The hAFP-2 heavy and light chain variable region shares 6 complementarity determining regions. 3 CDR sequences of the heavy chain variable region of the antibody 1 are respectively SEQ ID NO 3, SEQ ID NO 4 and SEQ ID NO 5; the variable region of light chain has 3 CDR sequences of SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8.
The 3 CDR sequences of the heavy chain variable region of the hAFP-6 are respectively SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 13; the variable region of light chain has 3 CDR sequences of SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16.
The heavy and light chains of the AFP antibodies of the invention further comprise constant regions, and the antibody light chain constant regions further comprise murine kappa and lambda chain sequences. The antibody heavy chain constant region further comprises a murine IgG1, IgG2a, IgG2b, or IgG3, or IgA or IgM sequence.
The invention aims to provide two novel AFP antibodies, which do not have cross reaction with HSA, recognize different AFP epitopes and are used for detecting the antibodies of the existence and the content of AFP protein in a sample.
It is another object of the present invention to provide a method for determining AFP using two or more different AFP antibodies that do not cross-react with HAS and recognize different AFP epitopes. The method is a double antibody sandwich ELISA detection method.
Still another objective of the present invention is to provide a more sensitive AFP antibody kit, which contains the above two antibodies recognizing different AFP epitopes, and is used for early screening diagnosis, therapeutic effect detection and prognosis judgment of AFP-abnormal liver cancer, fetal congenital diseases, etc.
Advantageous effects
The invention has the following beneficial effects: the antibody does not cross-react with human serum albumin; the AFP antibodies recognize different AFP epitopes; the AFP detection kit established by the antibody has higher sensitivity, and provides help for early diagnosis of AFP abnormal diseases, such as liver cancer and fetal congenital diseases.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.
FIG. 1 is a diagram of the sensitivity detection of a human AFP double antibody sandwich ELISA kit.
Detailed Description
The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.
Example 1 anti-AFP antibody preparation and purification
Mixing AFP antigen (with the concentration of 1 mug/mul) withAfter the equal volume of Complete Freund's Adjuvant (CFA) is fully and uniformly mixed to form a water-like oil bag, a BALB/C mouse is primarily immunized by intraperitoneal injection, and 200 mul is injected into each mouse. And (3) performing first reinforcement after 3 weeks, uniformly mixing and emulsifying an AFP antigen (with the concentration of 1 mug/mul) and an Incomplete Freund's Adjuvant (IFA) in an equal volume, and performing back multi-point injection on the mixture, wherein 200 mul is obtained for each mouse. The subsequent booster immunization was performed every 2 weeks, and tail blood was collected 7 days after each immunization to detect the antibody titer by ELISA. When the antibody titer reaches 10-4And (3) mixing and emulsifying the AFP antigen (with the concentration of 1 mug/mul) and an Incomplete Freund's Adjuvant (IFA) in an equal volume, and performing back multi-point injection to enhance immunity once, wherein 200 mul is obtained for each mouse. Spleens were harvested on day four and subjected to cell fusion. Taking splenocytes of immunized Balb/c mice, fusing the splenocytes with myeloma Sp2/0 cell strain, adding HAT into fused cells for plating, changing the liquid for half after 3 days, changing the liquid for one week, and changing HAT culture medium for culture. After 12 days, cell supernatants were removed and primary cultures showing a positive reaction with AFP protein in the supernatants were examined by high throughput ELISA. The hybridoma cells in the wells were diluted and subcloned, and then screened by ELISA method, and finally 6 positive hybridoma cell lines were selected and named hAFP1-6, respectively. Respectively culturing the hybridoma cell lines, collecting the hybridoma cells, resuspending with PBS, and configuring to 107Hybridoma cell suspension in ml. Freund's incomplete adjuvant was used as an inducer. Injecting Freund's incomplete adjuvant into Balb/c abdominal cavity for about 8 weeks, and injecting 500ul of 10-concentration adjuvant into Balb/c mouse abdominal cavity after 3 days7Hybridoma cell suspension in ml. When the abdomen of the mouse swells, ascites purified antibodies are collected. And (3) precipitating by saturated ammonium sulfate (PH = 7.8) to obtain crude AFP monoclonal antibody, and performing protein G column chromatography to obtain the AFP antibody with higher purity.
Example 2 Cross-reactivity of antibodies with human serum Albumin experiment
The AFP protein HAS high homology with human serum albumin, and is easy to generate cross reaction, and the specificity of the AFP antibody is influenced, so that the antibody which does not have cross reaction with HAS is screened out by adopting a cross reaction experiment. Human serum albumin was added to 96-well plates at 100ng per well and incubated overnight at 4 ℃. Removing supernatant, adding 200 μ l PBST lotion into each well, standing for 2min, removing lotion, and washing repeatedly for 3 times, wherein the elisa plate is dried as much as possible. Bovine serum albumin was added and blocked at 37 ℃ for 1 h. Removing supernatant, adding 200 μ l PBST lotion into each well, standing for 2min, removing lotion, and washing for 3 times. The antibodies prepared in example 1 were added to the detection wells, respectively, and incubated at 37 ℃ for 2 h. Removing supernatant, adding 200 μ l PBST lotion into each well, standing for 2min, removing lotion, and washing repeatedly for 3 times, wherein the elisa plate is dried as much as possible. Goat anti-mouse IgG-HRP labeled antibody was added and incubated at 37 ℃ for 1 h. Removing supernatant, adding 200 μ l PBST lotion into each well, standing for 2min, removing lotion, and washing repeatedly for 3 times, wherein the elisa plate is dried as much as possible. Adding a chromogenic substrate, and reacting for 15 min. Adding a stop solution to stop the color reaction. And (3) placing the ELISA plate into an ELISA reader, and detecting the light absorption value at the wavelength of 450 nm. From the OD450 values of each sample, the P/N values were calculated. P/N value = = (sample OD-blank OD)/(negative control OD control-blank OD), if P/N is greater than 2.1 positive, it indicates that the AFP sample cross-reacts with human serum albumin; if P/N is less than 2.1, it is negative, indicating that the AFP sample does not cross-react with human serum albumin, and only specifically binds the AFP protein. The experimental results are shown in table one: hAFP-1 and hAFP-4 were positive, while hAFP-2, hAFP-3, hAFP-5 and hAFP-6 were negative.
epi-AFP antibody Cross-reactive with human serum Albumin
Figure 484875DEST_PATH_IMAGE001
Example 3 competitive inhibition ELISA assay
Because two AFP antibodies binding to different epitopes are required for establishing a double sandwich ELISA detection system, the AFP antibodies binding to different epitopes are screened through a competitive inhibition ELISA experiment. The hAFP-6 antibody was labeled with horseradish peroxidase (HRP) and used as an enzyme-labeled control antibody. Wherein the HRP labeling technique is well known in the art. hAFP-2, hAFP-3, and hAFP-5 were used as unlabeled antibodies to be detected. Adding an AFP enzyme-labeled antibody into 96-hole enzyme coated with AFP antigenIn the standard plate, antibodies to be detected (antibodies to be detected: enzyme-labeled antibody =1, 2, 4, 8, 16, 32) with different proportions are respectively added into the experimental group, and meanwhile, AFP enzyme-labeled antibody positive control added with PBS is set up; incubating at 37 ℃ for 1 h; removing supernatant, adding 200 μ l PBST lotion into each well, standing for 2min, removing lotion, and washing repeatedly for 3 times, wherein the elisa plate is dried as much as possible. Adding a chromogenic substrate, and reacting for 15 min. Adding a stop solution to stop the color reaction. And (3) placing the ELISA plate into an ELISA reader, and detecting the light absorption value at the wavelength of 450 nm. The inhibition was calculated from the value of OD450 of each sample. Inhibition (%) = [ (OD)Enzyme-labeled antibody–ODBlank hole)-(ODAntibodies to be tested–ODBlank hole)]/ （ODEnzyme-labeled antibody–ODBlank hole) X 100%. The final results are shown in table two: when the concentration of the AFP enzyme-labeled antibody hAFP-6 is fixed, the inhibition rate of the mixed reaction with the AFP antigen is gradually increased along with the increase of the concentration of the hAFP-5 antibody; and when hAFP-5: when hAFP-6 was 32, the competitive inhibition rate reached 72.3%, indicating that hAFP-5 and hAFP-6 bind to the same epitope. The inhibition rates of hAFP-2 and hAFP-3 are determined in the following steps: enzyme-labeled antibody =32, only 8.7% and 11.6%. The results show that hAFP-2, hAFP-3 and hAFP-6 bind different epitopes and can establish a double sandwich ELISA detection system with hAFP-6.
TABLE II competitive inhibition ELISA experiments
Figure 57808DEST_PATH_IMAGE002
Example 4 detection of sensitivity of double antibody Sandwich ELISA kit
And finally, selecting a hAFP-2 and hAFP-6 pair to establish a double sandwich ELISA system by screening, wherein hAFP-2 is used as a coating antibody, and hAFP-6-HRP is used as a detection antibody. The anti-AFP antibody hAFP-2 was diluted to 0.5. mu.g/ml with the coating solution, incubated at 100. mu.l/well overnight at 4 ℃ and immobilized on an ELISA plate. Removing supernatant, adding 200 μ l PBST lotion into each well, standing for 2min, removing lotion, and washing repeatedly for 3 times, wherein the elisa plate is dried as much as possible. 3% skimmed milk was used as the blocking solution, 100. mu.l of the blocking solution was added to each well, and the wells were blocked at room temperature for 1 hour. Removing supernatant, adding 200 μ l PBST lotion into each well, standing for 2min, removing lotion, and washing repeatedly for 3 times, wherein the elisa plate is dried as much as possible. AFP antigen (diluted according to a gradient of 600ng/ml, 150ng/ml, 37.5ng/ml, 9.37ng/ml, 2.34ng/ml, 0.59ng/ml, 0.15 ng/ml) is added to an ELISA plate in sequence, 100 mul of the diluted antigen is added to the ELISA plate at 37 ℃, the incubation is carried out for 30min at 37 ℃, supernatant is removed, 200 mul of PBST washing solution is added to each well, the mixture is kept stand for 2min, washing solution is removed, washing is repeated for 3 times, the ELISA plate is washed to be as dry as possible each time, anti-AFP antibody hAFP-6 marked by Horse Radish Peroxidase (HRP), namely hAFP-6-HRP, is used as detection antibody, the antibody is diluted to be 0.5 mul/ml, 100 mul of the washing solution is added to each well, the incubation is carried out for 1h at room temperature, supernatant is removed, 200 mul of PBST washing solution is added to each well, the standing is carried out for 2min, washing solution is removed, washing is repeated for 3 times, 100 mul of the ELISA plate is washed to, incubating for 15-30min at room temperature in dark. The chromogenic reaction was stopped by adding 50. mu.l of stop solution to each well. And (3) placing the ELISA plate into an ELISA reader, and detecting the light absorption value at the wavelength of 450 nm. The resulting data are shown in FIG. 1 with a lowest detection line of 0.15ng/ml, a linear range of 0.59ng/ml to 150ng/ml, and a linear relationship of 0.986. Whereas the lowest concentration detected according to the prior art, e.g. the results of the double-sandwich standard curve in CN105037544A, was 1.21 ng/ml. Therefore, the sensitivity of the double-sandwich ELISA detection kit prepared by the AFP antibody is obviously higher than that of the kit in the prior art CN 105037544A.
EXAMPLE 5 monoclonal antibody sequencing
Cell preparation: recovering hybridoma cell lines corresponding to hAFP-2 and hAFP-6, and culturing to total amount of 107 The cells were centrifuged at 1000rpm for 5min to collect the cells, and RNA was extracted. TRNzol-A + was added to the cell pellet for lysis and allowed to stand at room temperature for 15 min. Mu.l chloroform was added to each ml of TRNzol-A +, vortexed for 15 seconds, and allowed to stand for 3 minutes. 13000 rpm, 4 ℃ for 10 minutes, Trizol-A + cell solution is divided into three layers: transferring the water phase dissolved with the RNA into a centrifuge tube, adding isopropanol with the same volume into the water phase, uniformly mixing, and standing at room temperature for 25 minutes. 13000 rpm, 4 ℃ for 10 minutes, discard the waste liquid to get the bottom RNA precipitation.After washing the RNA pellet twice with 75% ethanol, the RNA was dissolved in PEDC water and stored at-80 ℃. Synthesizing first chain cDNA by reverse transcription kit SMARTERRACE, and amplifying antibody variable region DNA sequence corresponding to hybridoma cell by using the first chain cDNA as subsequent template. Designing specific nested PCR primer, the primer sequence used in the amplification reaction is complementary with the first frame region and the constant region of the antibody variable region, and amplifying the target gene by adopting a conventional PCR method. After sequencing the amplified product, the heavy chain variable region sequence of anti-AFP antibody hAFP-2 secreted by hybridoma clone hAFP-2 is SEQ ID NO:1 and light chain variable region sequences SEQ ID NO: 2; the 3 CDR sequences of the heavy chain variable region of the antibody are respectively SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5; the variable region of light chain has 3 CDR sequences of SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8. Hybridoma clone hAFP-6 secreted heavy chain variable region sequence of anti-AFP antibody hAFP-6 SEQ ID NO: 9 and the light chain variable region sequence SEQ ID NO: 10; the 3 CDR sequences of the heavy chain variable region of the antibody are respectively SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 13; the variable region of light chain has 3 CDR sequences of SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16.
Sequence listing
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<120> a disease detection kit comprising an antibody specifically binding to an AFP protein
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Claims (6)

1. An AFP protein detection kit, which is characterized in that: the kit is a double-antibody sandwich ELISA kit, and comprises two different antibodies hAFP-2 and hAFP-6 which are specifically combined with AFP, wherein the two different antibodies are respectively as follows:
a) 3 CDR sequences of the heavy chain variable region of hAFP-2 are respectively SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5; the variable region of the light chain has 3 CDR sequences of SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8;
b) the 3 CDR sequences of the heavy chain variable region of hAFP-6 are respectively SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 13; the variable region of light chain has 3 CDR sequences of SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16.
2. An AFP protein detection kit, which is characterized in that: the kit is a double-antibody sandwich ELISA kit, and comprises two different antibodies hAFP-2 and hAFP-6 which are specifically combined with AFP, wherein the two different antibodies are respectively as follows:
a) the heavy chain variable region sequence of hAFP-2 is shown in SEQ ID NO. 1, and the light chain variable region sequence is shown in SEQ ID NO. 2;
b) the heavy chain variable region sequence of hAFP-6 is shown in SEQ ID NO 9, and the light chain variable region sequence is shown in SEQ ID NO 10.
3. Use of a combination of two antibodies for the preparation of a kit for the detection of liver cancer, characterized in that: two different antibodies hAFP-2 and hAFP-6 were as follows:
a) the heavy chain variable region sequence of hAFP-2 is shown in SEQ ID NO. 1, and the light chain variable region sequence is shown in SEQ ID NO. 2;
b) the heavy chain variable region sequence of hAFP-6 is shown in SEQ ID NO 9, and the light chain variable region sequence is shown in SEQ ID NO 10.
4. Use of a combination of two antibodies for the preparation of a kit for the detection of an AFP protein, characterized in that: two different antibodies hAFP-2 and hAFP-6 were as follows:
a) the heavy chain variable region sequence of hAFP-2 is shown in SEQ ID NO. 1, and the light chain variable region sequence is shown in SEQ ID NO. 2;
b) the heavy chain variable region sequence of hAFP-6 is shown in SEQ ID NO 9, and the light chain variable region sequence is shown in SEQ ID NO 10.
5. Use of a combination of two antibodies for the preparation of a kit for the detection of liver cancer, characterized in that: two different antibodies hAFP-2 and hAFP-6 were as follows:
a) 3 CDR sequences of the heavy chain variable region of hAFP-2 are respectively SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5; the variable region of the light chain has 3 CDR sequences of SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8;
b) the 3 CDR sequences of the heavy chain variable region of hAFP-6 are respectively SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 13; the variable region of light chain has 3 CDR sequences of SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16.
6. Use of a combination of two antibodies for the preparation of a kit for the detection of an AFP protein, characterized in that: two different antibodies hAFP-2 and hAFP-6 were as follows:
a) 3 CDR sequences of the heavy chain variable region of hAFP-2 are respectively SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5; the variable region of the light chain has 3 CDR sequences of SEQ ID NO 6, SEQ ID NO 7 and SEQ ID NO 8;
b) the 3 CDR sequences of the heavy chain variable region of hAFP-6 are respectively SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 13; the variable region of light chain has 3 CDR sequences of SEQ ID NO 14, SEQ ID NO 15 and SEQ ID NO 16.
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