CN115716874A - Monoclonal antibody aiming at AFP-L3 and lectin compound, kit and application thereof - Google Patents
Monoclonal antibody aiming at AFP-L3 and lectin compound, kit and application thereof Download PDFInfo
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- CN115716874A CN115716874A CN202211378127.8A CN202211378127A CN115716874A CN 115716874 A CN115716874 A CN 115716874A CN 202211378127 A CN202211378127 A CN 202211378127A CN 115716874 A CN115716874 A CN 115716874A
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Abstract
The application provides a monoclonal antibody aiming at an AFP-L3 and lectin compound, a kit and application thereof, wherein the monoclonal antibody comprises a heavy chain variable region CDR1, CDR2 and CDR3 with amino acid sequences shown as SEQ ID No.1, 2 and 3 respectively, and a light chain variable region CDR1, CDR2 and CDR3 with amino acid sequences shown as SEQ ID No.4, 5 and 6 respectively. The antibody can identify a space epitope formed after AFP-L3 protein is combined with lectin, so that the specificity and the affinity are improved, and the space epitope is combined into a detection kit of an alpha fetoprotein heteroplasmon, and the kit is good in accuracy and high in sensitivity.
Description
Technical Field
The application relates to the field of medical equipment and in-vitro diagnosis, in particular to a preparation method of a monoclonal antibody and a deglycosylated alpha-fetoprotein antibody aiming at an AFP-L3 and lectin compound, and a kit and application thereof.
Background
The number of people died from liver cancer in 2020 is nearly 83 thousands, liver cancer is the third most common malignant tumor in the world, 90 thousands of liver cancer patients are increased in 2020 in the world, and the rank is 6 on the number of all the newly increased cancer patients, so the severe situation of controlling the occurrence and development of liver cancer is not moderate. The primary liver cancer is divided into hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC) and HCC-ICC mixed type, wherein HCC accounts for 85% -90%. Liver hepatitis B, liver hepatitis C, excessive drinking, smoking and the like can cause hepatocellular carcinoma, but the pathogenesis of hepatocellular carcinoma is not clear so far, and the hepatocellular carcinoma is high in clinical malignancy, so that early screening and diagnosis are the key for improving the survival rate of patients, most of patients lose the best treatment opportunity when in the middle and late stages of treatment, and effective treatment is required, and the clinical efficiency of screening and general survey depends on early diagnosis.
Alpha Fetoprotein (AFP) is a glycoprotein with a molecular weight of about 70KD and is synthesized in mammals primarily by fetal liver cells and yolk sac cells. When hepatic cells become cancerous, part of sugar chains of AFP are changed. According to the difference of affinity between alpha-fetoprotein (AFP) and lentil Lectin (LCA), AFP can be divided into AFP-L1, AFP-L2 and AFP-L3, wherein AFP-L1 is mainly found in benign liver diseases, AFP-L2 is mainly produced by yolk sac and is mostly found in pregnant women, and AFP-L3 is mainly derived from liver cancer cells and is also called alpha-fetoprotein heteroplasms. AFP-L3 has alpha-1-6-fucoidan, which binds specifically to LCA. AFP-L3 in serum of HCC patients is obviously higher than that of other benign liver disease patients and is closely related to the occurrence of HCC, so the AFP-L3 is an important index for diagnosing hepatocellular carcinoma. The national society of clinical oncology (CSCO) Primary liver cancer diagnosis and treatment guidelines 2020 recommend detection of alpha-fetoprotein heteroplasmons in liver cancer patients, and the U.S. Food and Drug Administration (FDA) has approved the detection reagent and method for alpha-fetoprotein heteroplasmons for clinical liver cancer early warning in 2005.
At present, methods for detecting an alpha-fetoprotein heteroplasmon include lectin affinity chromatography, polyacrylamide gel electrophoresis, affinity blotting, immunoelectrophoresis, affinity centrifugation column, chemiluminescence, and the like. The methods such as the lectin affinity chromatography, the polyacrylamide gel electrophoresis, the affinity blotting and the like are complicated to operate, time-consuming, complex in steps, high in requirements for the quality of operators, and incapable of realizing high-throughput detection.
Currently, there are methods for AFP-L3 detection using F (ab ') 2 fragments, fab' fragments, fab fragments, fv fragments, and deglycosylated modified alpha-fetoprotein antibodies as capture antibodies in combination with acridine-labeled lentil lectin. In the method, the alpha fetoprotein antibody needs to be treated in advance, the Fc segment is cut to obtain F (ab ') 2, fab and Fab', the steric hindrance of the antibody after being coupled with solid phase carriers such as magnetic beads is not easy to combine the antibody with the antigen, the process is complex and difficult to operate, and the reagent cost is increased. In addition, there is a method for preparing a monoclonal antibody that recognizes a glycopeptide, the glycopeptide comprising a core fucosylation motif and a glycosylated asparagine motif having an amino acid sequence at the C-terminal portion of the motif. Although the monoclonal antibody has strong specificity, the operation is complex, the preparation difficulty is high, the sensitivity is not high, and samples near the critical value are difficult to distinguish negative and positive.
Disclosure of Invention
Based on the above, the application needs to provide a monoclonal antibody against a compound of AFP-L3 and lectin and a detection kit thereof, and is used for preparing an alpha-fetoprotein heteroplasmon detection product, which has the advantages of good accuracy, high sensitivity, simple operation and short detection time.
The specific technical scheme is as follows:
in a first aspect of the application, a monoclonal antibody against a complex of AFP-L3 and lectin is provided, the monoclonal antibody comprising a heavy chain variable region CDR1 with an amino acid sequence as shown in SEQ ID No.1, a heavy chain variable region CDR2 with an amino acid sequence as shown in SEQ ID No.2, a heavy chain variable region CDR3 with an amino acid sequence as shown in SEQ ID No.3, a light chain variable region CDR1 with an amino acid sequence as shown in SEQ ID No.4, a light chain variable region CDR2 with an amino acid sequence as shown in SEQ ID No.5, and a light chain variable region CDR3 with an amino acid sequence as shown in SEQ ID No. 6.
In one embodiment, the monoclonal antibody comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID No.7 and a light chain variable region having an amino acid sequence as set forth in SEQ ID No. 8.
In a second aspect of the present application, there is provided a nucleic acid molecule comprising a nucleotide sequence encoding the monoclonal antibody of the first aspect.
In a third aspect of the application, there is provided a biological material comprising a nucleic acid molecule according to the second aspect, said biological material comprising a vector or a host cell.
In a fourth aspect of the present application, there is provided a deglycosylated antibody, which has a crosslinking agent or lectin attached to a sugar chain of an Fc region of the antibody. Optionally, the antibody comprises an alpha-fetoprotein antibody.
In one embodiment, the cross-linking agent reacts with hydroxyl groups on the sugar chains of the Fc fragment of the antibody, and is linked by a chemical bond; the lectin is linked to the sugar chain of the Fc fragment of the antibody by intermolecular forces.
In one embodiment, the cross-linking agent contains an epoxy group; optionally, the cross-linking agent comprises at least two epoxy groups.
In one embodiment, the cross-linking agent is a homobifunctional cross-linking agent.
In one embodiment, the homobifunctional crosslinking agent comprises 1,4-butanediol diglycidyl ether, polyethylene glycol diglycidyl ether, trimethylolpropane triglycidyl ether.
In a fifth aspect of the present application, there is provided a method for producing the deglycosylated antibody of any one of the above, comprising treating the sugar chains of the antibody with a crosslinking agent or a lectin to reduce the reaction of the antibody Fc fragment sugar chains with the lectin.
In one embodiment, the step of treating the sugar chain of the antibody with the crosslinking agent comprises subjecting the antibody and the crosslinking agent to a treatment in a molar ratio of 1: (5-50) carrying out the reaction.
In one embodiment, the step of treating the sugar chain of the antibody with the crosslinking agent comprises mixing the antibody, the modifying agent and the crosslinking agent in a molar ratio of 1: (20-100): (5-50) carrying out the reaction.
Optionally, the modifying agent comprises at least one of a PEG series, a small molecule polypeptide, and a hydroxyl-containing amino acid.
In one embodiment, the step of treating the sugar chain of the antibody with the lectin includes mixing the antibody and the lectin in a molar ratio of 1: (1-200) carrying out reaction.
In one embodiment, the lectin comprises one or more of an animal lectin and a plant lectin; optionally, the plant lectin comprises one or more of lentil lectin, orange Huang Wangbao discodermin lectin, sword bean lectin, wheat germ lectin, peanut lectin, soybean lectin, and lotus root lectin.
In a sixth aspect of the present application, there is provided a test kit comprising the monoclonal antibody of the first aspect.
In one embodiment, the kit further comprises an alpha-fetoprotein antibody or a deglycosylated alpha-fetoprotein antibody. Alternatively, the deglycosylated alpha-fetoprotein antibody is obtained by a method comprising the preparation method of the fifth aspect.
In one embodiment, the kit comprises a solid support coated with the alpha-fetoprotein antibody or the deglycosylated alpha-fetoprotein antibody and the monoclonal antibody of the first aspect labeled with a label.
Optionally, the label comprises biotin.
In one embodiment, the kit further comprises an acridinium ester-labeled avidin and/or lectin.
In a seventh aspect of the present application, there is provided a detection kit comprising the deglycosylated antibody of any one of the above, wherein the antibody is a deglycosylated alpha-fetoprotein antibody.
In one embodiment, the kit further comprises a label labeled lectin. Optionally, the label comprises biotin or an acridinium ester.
In one embodiment, the kit comprises a solid support coated with the deglycosylated alpha-fetoprotein antibody, a biotin-labeled lectin, and an acridinium ester-labeled avidin.
In an eighth aspect of the present application, there is provided the use of the monoclonal antibody of the first aspect, the deglycosylated antibody of the fourth aspect, or the deglycosylated alpha-fetoprotein antibody obtained by the preparation method of the fifth aspect, or the kit of the sixth aspect or the seventh aspect, in the preparation of a product for detecting an alpha-fetoprotein variant, or in the preparation of a liver cancer diagnostic product.
Compared with the prior art, the method has the following beneficial effects:
the monoclonal antibody developed by the application, aiming at the AFP-L3 and lectin compound, has the advantages of high specificity and sensitivity and good affinity when detecting alpha-fetoprotein heteroplasmon.
Further, the application provides a method for preparing the deglycosylated alpha-fetoprotein antibody, which comprises the steps of carrying out site-directed modification on a sugar chain of the alpha-fetoprotein antibody by using a cross-linking agent or lectin, further changing the glycosylation of an Fc segment of the antibody, and blocking the interference of the glycosylation on detection. The method has the advantages of simple operation, low cost, high yield of modified antibody, and small influence on affinity of antibody.
The detection kit for the alpha-fetoprotein heteroplasmon assembled by the monoclonal antibody aiming at the AFP-L3 and lectin compound developed by the application or the detection kit for the alpha-fetoprotein heteroplasmon assembled by the cross-linking agent or lectin modified alpha-fetoprotein antibody and the lectin has the advantages of short detection time, low cost, good accuracy and high sensitivity.
Drawings
FIG. 1 is a kinetic curve of Ab-01 antibody;
FIG. 2 is an Ab-02 antibody kinetic curve;
FIG. 3 is an Ab-03 antibody kinetic curve;
FIG. 4 is a schematic view showing a structure in which an antibody sugar chain in example two is rich in hydroxyl groups;
FIG. 5 is the reaction process of the antibody Fc fragment modified PEG molecule in example two;
FIG. 6 is a reaction sequence of the antibody Fc fragment sugar chain to effect multimerization production of antibody molecules in example two;
FIG. 7 shows the results of molecular sieves of the multimeric alpha-fetoprotein antibody;
FIG. 8 shows the reaction process of blocking the glycosylation site of the Fc fragment of the alpha fetoprotein antibody by the lectin in example II.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present application more comprehensible, specific embodiments thereof are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. This application is capable of embodiments in many different forms than those described herein and that modifications may be made by one skilled in the art without departing from the spirit and scope of the application and it is therefore not intended to be limited to the specific embodiments disclosed below.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present application, "plurality" means at least two, e.g., two, three, etc., unless explicitly specified otherwise.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Terms and abbreviations
Hepatocellular carcinoma (HCC);
intrahepatic Cholangiocellular Carcinoma (ICC);
alpha-fetoprotein (AFP);
lentil lectin (Lens culiaris aglutinin, LCA);
enzyme linked immunosorbent assay (ELISA);
1,4-Butanediol Diglycidyl Ether (1,4-Butanediol Diglycidyl Ether, BDDE);
orange Huang Wangbao discodermin Lectin (AAL);
concanavalina (conconnalina, conA);
wheat germ element (Wheat germ aglutinin, WGA);
peanut agglutinin (PNA);
soybean agglutinin (SBA);
lotus root lectin (LTL).
One embodiment of the present application provides a monoclonal antibody (i.e., ab-03 antibody strain in example 1 of the present application) against a complex of AFP-L3 and lectin, comprising a heavy chain variable region CDR1 having an amino acid sequence shown in SEQ ID No.1, a heavy chain variable region CDR2 having an amino acid sequence shown in SEQ ID No.2, a heavy chain variable region CDR3 having an amino acid sequence shown in SEQ ID No.3, a light chain variable region CDR1 having an amino acid sequence shown in SEQ ID No.4, a light chain variable region CDR1 having an amino acid sequence shown in SEQ ID No.5, and a light chain variable region CDR3 having an amino acid sequence shown in SEQ ID No. 6. The specific amino acid sequence is shown as follows:
CDR1 of the heavy chain variable region of SEQ ID NO. 1: DYAMQ;
CDR2 of the heavy chain variable region of SEQ ID NO. 2: VISTYNGNTNYNQKFKG;
CDR3 of the heavy chain variable region of SEQ ID NO. 3: SLYYGSSPYAMDY;
CDR1 of the light chain variable region of SEQ ID NO. 4: KSSQSLLNSRTRKNYLA;
SEQ ID NO.5 light chain variable region CDR2: the WASTRES;
CDR3 of the light chain variable region of SEQ ID NO. 6: KQSYNLFT.
In one particular example, the monoclonal antibody comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID No.7 and a light chain variable region having an amino acid sequence as set forth in SEQ ID No. 8. The specific amino acid sequence is shown as follows:
the heavy chain sequence of SEQ ID No. 7:
MGWSCIIFFLVATATGVHSQVQLQQSGPEVVRPGVSVKISCKGSGYTFTDYAMQWVKQSHAKSLEWIGVISTYNGNTNYNQKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYCARSLYYGSSPYAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK；
the light chain sequence of SEQ ID No. 8:
MDSQAQVLILLLLWVSGTCGDIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。
the above monoclonal antibodies can be prepared by those skilled in the art according to conventional techniques.
Furthermore, the application provides a preparation method of the monoclonal antibody, which comprises the steps of combining the lectin with the alpha-fetoprotein to form an AFP-L3 and lectin compound serving as an immunogen to immunize a mouse to obtain hybridoma cells, injecting the hybridoma cells into the abdomen of the mouse to prepare ascites, and purifying to obtain the monoclonal antibody aiming at the AFP-L3 and lectin compound. The method is simple to operate. Optionally, in the AFP-L3 and lectin complex, the mass ratio of AFP-L3 to lectin is 1:5.
in one particular example, the lectin may be an animal lectin or a plant lectin.
In one particular example, the plant lectins include one or more of lentil lectin, orange Huang Wangbao discodermus lectin, sword bean lectin, wheat germ lectin, peanut lectin, and soybean lectin, and lotus root lectin. Optionally, the lectin is lentil lectin.
One embodiment of the present application also provides a nucleic acid molecule comprising a nucleotide sequence encoding the monoclonal antibody described above.
Further, an embodiment of the present application provides a biological material comprising the nucleic acid molecule as described above, the biological material comprising a vector or a host cell.
In this application, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection such that the genetic material element it carries is expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids, phagemids, cosmids, artificial chromosomes, viral vectors, and the like.
In the present application, the term "host cell" is a eukaryotic cell. For example, the host cell may be a mammalian cell. Suitable host cells for expressing the antibodies of the present application include, but are not limited to: mammalian cells such as NS0, sp2/0, CHO, COS, HEK, fibroblasts, and myeloma cells.
In the detection of alpha fetoprotein heteroplasmon, an Fc section of an alpha fetoprotein antibody structure is glycosylated to a certain extent, lentil agglutinin can be bound to a sugar chain part of the Fc section of the antibody in a non-specific manner, and if the alpha fetoprotein antibody is used as a capture antibody combined marker to mark the lentil agglutinin for detection, the problems of false positive or high background can be generated, and the detection result of AFP-L3 is interfered. The traditional method for modifying the antibody Fc segment glycoprotein is to oxidize hydroxyl in a sugar chain by NaIO4 to produce an aldehyde intermediate product containing carbonyl, and the intermediate product further reacts with a molecule with amino to produce Schiff base so as to finish the modification of the glycoprotein. The technology belongs to the traditional conventional route, and has more defects and more byproducts.
Accordingly, one embodiment of the present application also provides a method of making a deglycosylated antibody, which can be an alpha-fetoprotein antibody. In other embodiments, the antibody may be other antibodies with glycosylated Fc region. The method comprises site-directed modification of the sugar chains of the alpha-fetoprotein antibody with a cross-linking agent or lectin to alter glycosylation of the Fc region of the antibody to reduce reaction of the antibody Fc region sugar chains with the lectin. Specifically, the crosslinking agent performs site-directed reaction on the sugar chain structure of the Fc segment of the antibody, changes the sugar chain structure, and blocks the binding of the sugar chain to the lectin. After the agglutinin blocks the Fc segment sugar chain region of the alpha fetoprotein antibody, the non-specific binding of the agglutinin marked by the marker and the antibody can be avoided.
In one specific example, the cross-linking agent reacts with hydroxyl groups on the sugar chains of the Fc fragment of the antibody, and is attached by chemical bonding. Alternatively, the chemical bond may comprise
The lectin is attached to the sugar chain of the Fc fragment of the antibody by intermolecular forces. Alternatively, intermolecular forces include van der waals forces and/or hydrogen bonding, among others.
In one specific example, the cross-linking agent has a group that reacts with a hydroxyl group on a sugar chain of the Fc fragment of the antibody, the group including an epoxy group. In other examples, other groups that react with hydroxyl groups may also be included. Optionally, the cross-linking agent comprises at least two epoxy groups. Optionally, the crosslinking agent is a homobifunctional crosslinking agent.
In one specific example, homobifunctional crosslinkers containing epoxy groups include, but are not limited to, 1,4-butanediol diglycidyl ether, polyethylene glycol diglycidyl ether, and trimethylolpropane triglycidyl ether.
In one specific example, the step of treating the sugar chain of the antibody with the crosslinking agent comprises subjecting the antibody and the crosslinking agent to a treatment in a molar ratio of 1: (5-50) carrying out the reaction. Alternatively, the molar ratio is 1: (20-50); alternatively, the molar ratio is 1: (20-40); alternatively, the molar ratio is 1: (40-50). The method realizes multimerization of the antibody by reacting the hydroxyl of the antibody sugar chain, and is favorable for improving the binding affinity with the antigen.
In one specific example, the step of treating the sugar chain of the antibody with the crosslinking agent includes a step of treating the antibody, the modifying agent and the crosslinking agent in a molar ratio of 1: (20-100): (5-50) carrying out the reaction. Alternatively, the molar ratio is 1: (50-100): (30-50). The method adopts cross-linking agent, which greatly increases the modification direction. In addition, according to the difference of the modifying agent, the modified antibody can show different properties, and the appropriate modifying agent is selected, so that the hydrophilicity and hydrophobicity and the charge distribution of the antibody can be further changed, the actual application requirement is met, and the more excellent reagent performance is met.
In a particular example, the modifying agent includes at least one of a PEG-based series, a small molecule polypeptide, and a hydroxyl-containing amino acid.
The term "PEG series" is meant to include PEGs of different molecular weights, such as PEG200, 300, 400, 600, and the like.
In a more specific example, the modifying agent may be a PEG molecule. Through PEG modification of the Fc segment sugar chain part of the antibody, the sugar chain structure can be effectively changed, and simultaneously, due to the self-characteristics of PEG, the hydrophilicity and hydrophobicity and the charge distribution of the modified antibody molecule are further improved. In other specific examples, other modifying agents can be selected as the modifying agent, and modified antibodies with different properties can be obtained by using different modifying agents, so that the application range is wide.
In one specific example, the step of treating the sugar chain of the antibody with the lectin includes mixing the antibody with the lectin in a molar ratio of 1: (1-200) carrying out reaction. Alternatively, the molar ratio is 1: (10-100). The method has simple operation and low cost, and does not affect the activity of the antibody.
In a particular example, the lectin comprises one or more of an animal lectin or a plant lectin. In one embodiment, the plant lectins include lentil lectin, orange Huang Wangbao discodermin lectin, sword bean lectin, wheat germ lectin, peanut lectin, soybean lectin, and lotus root lectin. Optionally, lentil lectin.
An embodiment of the present application also provides a deglycosylated antibody having a crosslinker or lectin attached to the sugar chain of the Fc region of the antibody.
In one specific example, the crosslinking agent reacts with hydroxyl groups on the sugar chains of the Fc fragment of the antibody, and is linked by chemical bonds. Alternatively, the chemical bond comprises
Of lectins with antibodies by intermolecular forcesThe sugar chain of the Fc segment is connected. Alternatively, intermolecular forces include van der waals forces and/or hydrogen bonding, among others.
In one specific example, the crosslinking agent contains an epoxy group. Optionally, the cross-linking agent comprises at least two epoxy groups. Alternatively, the crosslinking agent is a homobifunctional crosslinking agent including, but not limited to, 1,4-butanediol diglycidyl ether, polyethylene glycol diglycidyl ether, and trimethylolpropane triglycidyl ether.
One embodiment of the application provides a detection kit, which comprises the monoclonal antibody.
In a particular example, the kit further comprises an alpha-fetoprotein antibody or a deglycosylated alpha-fetoprotein antibody. Alternatively, deglycosylated alpha-fetoprotein antibodies are prepared using methods that include the above-described deglycosylated antibodies.
In one particular example, the kit includes a solid support coated with an alpha-fetoprotein antibody or a deglycosylated alpha-fetoprotein antibody and a label labeled with the monoclonal antibody described above. Alternatively, the label comprises at least one of biotin and other labels conventional in the art. Optionally, the label is biotin. Alternatively, the solid phase carrier may be magnetic beads, latex microspheres, enzyme-free plates, nitrocellulose membranes, or the like.
In one particular example, the kit further comprises a lectin and/or an acridinium ester-labeled avidin.
In one specific example, the kit is used in a method comprising combining AFP-L3 in a sample with magnetic beads coated with alpha fetoprotein antibodies, incubating for a period of time, washing, adding LCA and a biotin-labeled monoclonal antibody component of a complex of AFP-L3 and LCA, incubating for a period of time, washing, adding a pre-excitation solution and an excitation solution, and detecting a light signal. Alternatively, LCA and biotin-labeled AFP-L3 can be premixed with monoclonal antibodies to the LCA complex.
An embodiment of the present application further provides another detection kit, which comprises the deglycosylated antibody, wherein the antibody is a deglycosylated alpha-fetoprotein antibody.
In one particular example, the kit further comprises a label labeled lectin. Alternatively, the label may be selected from labels conventional in the art, such as acridinium esters, fluorescein, biotin, and the like. Alternatively, the lectin may be selected from lentil lectins.
In one particular example, the kit includes a solid support coated with deglycosylated alpha-fetoprotein antibody, biotin-labeled lectin, and acridinium ester-labeled avidin.
In one specific example, the method for labeling a lectin or antibody with biotin is to mix the lectin and biotin NHS ester in phosphate or carbonate at a molar ratio of 1: (5-50) carrying out reaction.
In one specific example, the method for labeling avidin with acridine ester is to label avidin and acridine NHS ester in a phosphate or carbonate at a molar ratio of 1: (5-50) carrying out reaction.
The kit is prepared by combining a crosslinking agent or a lectin-fixed-point modified alpha fetoprotein antibody and a lectin, or combining a monoclonal antibody of an AFP-L3 and lectin compound and the alpha fetoprotein antibody, and combining a biotin labeling technology and an acridine labeled avidin, and in the AFP-L3 detection, the kit not only can amplify a detection signal and improve the specificity and sensitivity of the kit, but also can achieve the effects of low production cost and short detection time.
The monoclonal antibody or the deglycosylated alpha-fetoprotein antibody obtained by the preparation method of the deglycosylated antibody is applied to preparation of products for detecting alpha-fetoprotein heteroplasms or liver cancer diagnosis products. Alternatively, the product may be a reagent, test strip, kit or system, or the like. Optionally, the diagnosis includes early diagnosis or assisted diagnosis. The kit for detecting AFP-L3 has high sensitivity and specificity in detecting liver cancer or early liver cancer.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
Embodiments of the present application will be described in detail with reference to examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present application. The experimental methods in the following examples, in which specific conditions are not indicated, can be performed according to the instructions given in the present application, according to the experimental manual or the conventional conditions in the art, according to the conditions suggested by the manufacturer, or according to the experimental methods known in the art.
In the following specific examples, the measurement parameters relating to the components of the raw materials, if not specified otherwise, may be subject to slight deviations within the accuracy of the weighing. Temperature and time parameters are involved to allow for acceptable deviation of the instrument test accuracy or operational accuracy.
Example 1 preparation of monoclonal antibodies against AFP-L3 and lectin Complex and affinity testing thereof
1. Preparation of AFP-L3 and agglutinin compound monoclonal antibody
(1) Preparation of immunogens
Firstly, synthesizing a full-length sequence of AFP alpha fetoprotein, constructing into a pCMV expression vector to obtain a plasmid, transforming the plasmid into Escherichia coli TOP10, extracting the plasmid by using a kit after the TOP10 is amplified, wherein the concentration of the plasmid is 800 ng/mu L. The plasmid uses PEI to transfect 293F mammal cells, collects cell supernatant after expressing for 4 days, purifies by nickel column after centrifugal filtration, and then obtains AFP protein by purifying by ion exchange column. The eluted sample was 90% pure AFP by SDS-PAGE and was used to form complexes with lectins. The AFP-L3 and lectin complex was obtained by utilizing the ability of the lectin (Latin Dolichos lablab lectin (LCA/LcH) cat number: L140096-10 mg) to specifically bind to alpha-fetoprotein sugar chains, and the mass ratio of AFP-L3 to lectin in the AFP-L3 and lectin complex was 1:5.
Wherein: the alpha-fetoprotein amino acid sequence consists of (SEQ ID No. 9):
MKWVESIFLIFLLNFTESRTLHRNEYGIASILDSYQCTAEISLADLATIFFAQFVQEATYKEVSKMVKDALTAIEKPTGDEQSSGCLENQLPAFLEELCHEKEILEKYGHSDCCSQSEEGRHNCFLAHKKPTPASIPLFQVPEPVTSCEAYEEDRETFMNKFIYEIARRHPFLYAPTILLWAARYDKIIPSCCKAENAVECFQTKAATVTKELRESSLLNQHACAVMKNFGTRTFQAITVTKLSQKFTKVNFTEIQKLVLDVAHVHEHCCRGDVLDCLQDGEKIMSYICSQQDTLSNKITECCKLTTLERGQCIIHAENDEKPEGLSPNLNRFLGDRDFNQFSSGEKNIFLASFVHEYSRRHPQLAVSVILRVAKGYQELLEKCFQTENPLECQDKGEEELQKYIQESQALAKRSCGLFQKLGEYYLQNAFLVAYTKKAPQLTSSELMAITRKMAATAATCCQLSEDKLLACGEGAADIIIGHLCIRHEMTPVNPGVGQCCTSSYANRRPCFSSLVVDETYVPPAFSDDKFIFHKDLCQAQGVALQTMKQEFLINLVKQKPQITEEQLEAVIADFSGLLEKCCQGQEQEVCFAEEGQKLISKTRAALGVHHHHHH。
(2) Mouse immunization and acquisition of Positive hybridoma cells
Using the obtained AFP-L3 and agglutinin compound to immunize Balb/c mice for 8-12 weeks, and detecting the tail blood of the mice by an ELISA indirect method until the titer reaches 10 5 The immunization was stopped and the mouse spleen was removed. Fusing with mouse myeloma cell SP2/0 after treatment, and obtaining hybridoma secreting monoclonal antibody by a limiting dilution method; and respectively coated with AFP-L3 protein, agglutinin and AFP-L3 and agglutinin compound to screen positive hybridoma. Injecting the obtained positive hybridoma cells into the abdomen of a mouse to prepare ascites, and carrying out affinity detection on the monoclonal antibody obtained after Protein A purification.
2. Affinity assay
3 monoclonal antibodies Ab-01, ab-02 and Ab-03 which have high ELISA detection OD values and aim at the AFP-L3 and lectin compound are selected for carrying out affinity detection, the affinity of the monoclonal antibodies is detected by using Biacore T200, the AFP-L3 and lectin compound is fixed on a CM5 chip, the monoclonal antibodies to be detected with different concentrations and the immobilized compound are used for carrying out reaction, and a kinetic curve obtained by the reaction is fitted by software to obtain an affinity constant. The results of affinity detection are shown in FIGS. 1, 2 and 3. The affinity KD of the obtained Ab-01 antibody is 4.191 multiplied by 10 --9 Wherein kd is 8.265 × 10 -4 Ka is 1.972X 10 5 (ii) a The affinity KD of the obtained Ab-02 antibody is 6.269X 10 -9 Wherein kd is 1.037X 10 -3 Ka is 1.6X 10 5 (ii) a The affinity KD of the obtained Ab-03 antibody is 2.656X 10 -9 Wherein kd is 2.893 × 10 -4 Ka is 1.089X 10 5 . Therefore, the Ab-03 monoclonal antibody with the highest affinity is selected for labeling.
The present example develops that monoclonal antibodies against the AFP-L3 and lectin complex can recognize the spatial epitope formed by binding AFP-L3 protein and lectin, and improve specificity and affinity.
Example 2 method for modifying Fc fragment sugar chain of alpha-fetoprotein antibody
The alpha fetoprotein antibody structure has certain glycosylation in the Fc segment, the lentil agglutinin can be non-specifically combined with the sugar chain part of the Fc segment of the antibody, if the alpha fetoprotein antibody is used as a capture antibody combined marker to mark the lentil agglutinin for detection, the problems of false positive or high background can be generated, and the detection result of AFP-L3 is interfered. In order to solve the problem that lentil lectin is non-specifically bound to the core fucosylation part of the Fc segment of an antibody, the application provides two processing methods aiming at glycoprotein from the characteristics of antibody structure and sugar chain:
1. based on the site-directed reaction of the antibody Fc segment sugar chain structure by the cross-linking agent, thereby changing the sugar chain structure and blocking the binding of the sugar chain to the lectin
According to the structural characteristics of the antibody sugar chain with rich hydroxyl groups (figure 4), a cross-linking agent capable of specifically reacting the hydroxyl groups is selected to realize site-directed modification of the sugar chain. The epoxy group has high reactivity due to the special structural characteristics, and can perform nucleophilic reaction with a nucleophilic reagent (with primary amino group, hydroxyl group and sulfhydryl group) and perform ring-opening addition reaction with an electrophilic reagent (with carboxyl group) in actual reaction according to different reaction conditions, as shown in the following:
based on the reaction characteristics, the epoxy group can modify the Fc fragment sugar chain of the alpha fetoprotein antibody.
Further, this example takes 1,4-Butanediol Diglycidyl Ether (1,4-butandediol Diglycidyl Ether, BDDE) as an example of a cross-linking agent, which is a homobifunctional cross-linking agent having one epoxy group at each end. 1,4 butanediol diglycidyl ether the structural formula is as follows:
the reaction mode comprises the following two modes:
one is to modify the Fc part of the antibody by PEG, which can effectively change glycosylation sites, and further improve the properties of the modified antibody molecule, such as hydrophilicity and hydrophobicity, charge distribution, etc. due to the self-property of PEG. The other is multimerization of antibody molecules, based on coupling of the Fc-fragment sugar chain portion of the antibody, which alters the conformation of the sugar chain and prevents it from performing its biological role in binding to lectins.
First, alpha fetoprotein antibody Fc segment modified PEG molecule
Under a certain condition (pH is more than 11, and the reaction temperature is more than or equal to 37 ℃), adding the alpha fetoprotein antibody, PEG molecules (with the molecular weight of 400-600) and 1,4-butanediol diglycidyl ether (BDDE) according to a certain proportion, activating sugar chain hydroxyl of an Fc segment of the alpha fetoprotein antibody by the BDDE in the reaction process to generate an intermediate product with an epoxy group, and further reacting the intermediate product with PEG or antibody self-hydroxyl, but because the antibody self-reaction has larger steric hindrance, the PEG molecules with smaller molecular weight preferentially participate in the reaction. And after the modification reaction is finished, purifying and removing redundant PEG molecules and a small amount of polymerized antibody to obtain the modified alpha-fetoprotein antibody molecule. Please refer to fig. 5.
Preparing an alpha fetoprotein antibody of Fc segment sugar chain modified PEG molecules, comprising the following steps a-c:
a, step a: selecting alpha-fetoprotein antibody (manufacturer: meridian cat # M86641M), with molecular weight of about 150KD and concentration of 5mg/mL;1,4-butanediol diglycidyl ether (manufacturer: sigma cat # 220892-50G) diluted with ultrapure water to a concentration of 5mg/mL; PEG molecules (manufacturer: sigma cat # 202398-250G), average molecular weight 400, were diluted with ultrapure water to a concentration of 5mg/mL.
Step b: taking 0.1M carbonate buffer solution with pH of more than 11 as a reaction system, and under the environment of 37 ℃, the content of alpha fetoprotein antibody: modifying agent: the cross-linking agent is added into the mixture according to the molar ratio of 1: (20/50/100): 30, the reaction is carried out. Specifically, three reaction tubes were taken, 0.2ml of the alpha-fetoprotein antibody at 5mg/mL and 0.8ml of 0.1m carbonate buffer (pH = 11.2) were mixed, 10.67 μ L,26.67 μ L, and 53.33 μ L of the modifier (PEG molecule) were added, and finally 8.09 μ L of the crosslinking agent (1,4-butanediol diglycidyl ether) was added to each tube, mixed, and reacted at 37 ℃ for 3 hours.
Step c: after the reaction is finished, the product is purified by a Zeba desalting centrifugal column (7K MWCO, 2mL), unreacted modifying agent and cross-linking agent are removed, and a carbonate buffer solution is replaced by a phosphate buffer solution, so that the alpha-fetoprotein antibody of the Fc segment sugar chain modified PEG molecule is obtained.
Performance testing
Step d: and (3) carrying out magnetic bead coating on the obtained alpha fetoprotein antibody of the Fc segment sugar chain modified PEG molecule. The specific process is as follows: 10mg of magnetic beads (JSR, MS 160/Carboxyl) were washed 3 times with coating buffer (15mM MES, pH 6.0), 0.4mL of coating buffer (15mM MES, pH 6.0) was added to resuspend the magnetic beads, 0.2mL of activator (1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide sulfonic acid sodium salt each 0.1mL, DMSO was dissolved to 10 mg/mL) was added to react at 25 ℃ for 30min, after the reaction was completed, magnetic separation was performed, 1 time washing was performed with coating buffer (15mM MES, pH 6.0), 1.0mL of coating buffer (1mM MES, pH 6.0) was added to resuspend, 0.5mg of the above antibody was added to react at 25 ℃ for 3h, after the reaction was completed, 2 times washing was performed with washing buffer (150 mM phosphate containing 1 Twenn 20) after the completion of washing, the coated magnetic beads were treated with blocking buffer (150 mM phosphate containing BSA) for 2h, finally the Fc fragment of the blocking Fc-modified immunoglobulin was treated with PEG as a control molecule, and the intact albumin.
Step e: combining the magnetic bead coating of the coupled alpha fetoprotein antibody with an LCA acridine marker, detecting AFP-L3 in a sample by an indirect method, and reading a luminescent value, wherein the result is shown in Table 1.
TABLE 1
Sample preparation: PBS buffer solution is used as a blank sample, serum samples 1-4 are positive samples with different concentrations, and each data is an average value after three times of repeated tests;
experimental group 1: the molar ratio of the alpha fetoprotein antibody to the modifier to the cross-linking agent is 1:20:30, of a nitrogen-containing gas;
experimental group 2: the molar ratio of the alpha fetoprotein antibody to the modifier to the cross-linking agent is 1:50:30;
experimental group 3: the molar ratio of the alpha fetoprotein antibody to the modifier to the cross-linking agent is 1:100:30.
the test results show that the control group has low cost and low signal-to-noise ratio. Compared with the experimental groups 1 to 3, the background is obviously reduced, and the signal-to-noise ratio is obviously increased. Wherein the background of the experimental group 2 is lowest and the signal-to-noise ratio is highest, and the experimental group 2 is preferred for the experimental parameters.
(II) multimerization of antibody molecules by reaction of Fc-fragment sugar chains of antibodies
1,4-butanediol diglycidyl ether is a homotype bifunctional cross-linking agent, and can simultaneously react sugar chain hydroxyl of an Fc segment of an alpha fetoprotein antibody under certain conditions (pH is more than 11, and the reaction temperature is more than or equal to 37 ℃), so that self-crosslinking of two or more antibody molecules is realized, and multimerization of the antibody molecules through the sugar chains of the Fc segment of the antibody is realized. After the reaction is completed, the multimerized antibody is purified according to molecular weight. Please refer to fig. 6.
The preparation of the polymerized alpha fetoprotein antibody comprises the following steps of a to c:
step a: selecting alpha-fetoprotein antibody (manufacturer: meridian cat # M86641M), with molecular weight of about 150KD and concentration of 5mg/mL;1,4-butanediol diglycidyl ether (manufacturer: sigma cat # 220892-50G) was diluted with ultrapure water to a concentration of 5mg/mL.
Step b: using 0.1M carbonate buffer solution, pH >11, as a reaction system, and under the environment of 37 ℃, the ratio of alpha-fetoprotein antibody: the cross-linking agent is added into the mixture according to the molar ratio of 1: (20/40/50) the reaction was carried out. Specifically, three reaction tubes were used, 0.4ml of the alpha-fetoprotein antibody at 5mg/mL and 1.6mL of the 0.1m carbonate buffer (pH = 11.2) were mixed, 10.79 μ L,21.57 μ L, and 26.97 μ L of the crosslinking agent (1,4-butanediol diglycidyl ether) were added thereto, and the mixture was mixed and reacted at 37 ℃ for 3 hours.
Step c: after the reaction, the antibody which has undergone the reaction is purified by using an AKTA protein purification system (pure 150M) and using a phosphate buffer as a flow phase, and the polymer component is collected according to the difference in molecular weight, thereby obtaining an alpha-fetoprotein antibody which is multimerized by an Fc fragment sugar chain.
Performance testing
Step d: the thus obtained alpha-fetoprotein antibody multimerized by the Fc fragment sugar chain is coated with magnetic beads to obtain a multimerized alpha-fetoprotein antibody coated magnetic beads, and at the same time, an untreated alpha-fetoprotein antibody is coated for control, with reference to step d in the above-mentioned method (one).
Step e: the magnetic bead coating coupled with the alpha fetoprotein antibody is combined with an LCA acridine marker, an AFP-L3 in a sample is detected by an indirect method, a luminescent value is read, and the result is shown in Table 2.
TABLE 2
Sample preparation: PBS buffer solution is used as a blank sample, serum samples 1-4 are positive samples with different concentrations, and each data is an average value after three times of repeated tests;
experimental group 1: the molar ratio of the alpha fetoprotein antibody to the cross-linking agent is 1:20;
experimental group 2: the molar ratio of the alpha fetoprotein antibody to the cross-linking agent is 1:40;
experimental group 3: the molar ratio of the alpha fetoprotein antibody to the cross-linking agent is 1:50.
the results show that compared with the control group, the background of the experimental group is generally lower, and the signal-to-noise ratio is obviously improved. Wherein the experimental group 2 has a lower background and a highest signal-to-noise ratio, and the experimental parameters are preferably selected from the experimental group 2.
Further, the alpha-fetoprotein antibody obtained as described above, which is multimerized by an Fc fragment sugar chain, is subjected to molecular sieve analysis. The multimerized molecular sieve takes the ratio of the peak area of a polymer peak and the peak area of a monomer peak of the alpha fetoprotein antibody as an index of the quality of a multimerization result, and the larger the ratio is, the better the multimerization effect is. The results in fig. 7 show that in a 1: at 40, the multimerization effect was best. In the condition that the mole ratio of the alpha fetoprotein antibody to the cross-linking agent is 1: at 20, the effect is the worst. Therefore, the reaction molar ratio based on the multimerization of the Fc fragment sugar chain of the alpha-fetoprotein antibody is preferably 1:40.
2. the lectin is adopted to block the Fc segment sugar chain of the alpha-fetoprotein antibody, thereby avoiding the nonspecific binding of the lectin and the antibody
The glycosylation sites of the alpha fetoprotein antibodies can be noncovalently, specifically and reversibly bound to lectins. Based on this property, the method uses lectins to site-specifically block the sugar chain region of the Fc fragment of AFP antibody, see FIG. 8.
The preparation of the agglutinin-blocked alpha fetoprotein antibody comprises the following steps A-C:
step A: selecting alpha-fetoprotein antibody (manufacturer: meridian cat number: M86641M), with molecular weight of about 150KD and concentration of 5mg/mL; lentil lectin (manufacturer: allantin cat # L140096-10 mg) has a molecular weight of about 50KD and is diluted with ultrapure water to a concentration of 5mg/mL.
And B: taking 0.1M phosphate buffer solution with pH of more than 7.4 as a reaction system, and under the environment of 25 ℃, mixing alpha fetoprotein antibody and lentil agglutinin according to the molar ratio of 1: (1-200) carrying out reaction. The specific operation is as follows:
(1) 0.2ml of the alpha-fetoprotein antibody at 5mg/mL and 0.8ml of 0.1m phosphate buffer (pH = 7.4) were mixed, 667 μ L of a lentil lectin solution (wherein the molar ratio of antibody to lectin is 1.
(2) Taking 3mL of the coated alpha-fetoprotein antibody magnetic bead coating material (10 mg/mL, each mg of the magnetic bead is coated with 20 mu g of the alpha-fetoprotein antibody, the preservation solution is phosphate buffer solution), adding 0.4mL of lentil agglutinin solution (wherein the molar ratio of the antibody to the agglutinin is 1 100), mixing, and reacting at 25 ℃ overnight (16-18 h).
And step C, after the reaction is finished, purifying the alpha fetoprotein antibody treated in the step (1) by using a Millipore ultrafiltration tube (50K MWCO, 2mL), and removing unreacted lentil lectin molecules. And (3) directly carrying out magnetic separation on the alpha fetoprotein antibody magnetic bead coating substance in the step (2) to remove redundant unreacted lentil lectin molecules, washing the magnetic bead coating substance after sealing for 3 times by using a phosphate buffer solution, and finally re-suspending the magnetic bead coating substance according to the concentration of 10 mg/mL.
Performance testing
Step D, the obtained antibody in the step (1) is the alpha fetoprotein antibody which is obtained by adopting lentil agglutinin to seal Fc segment sugar chain molecules, and then the magnetic bead coating is further carried out, and the step D in the method (I) is referred to in the concrete step, so that the magnetic bead coating object of the alpha fetoprotein antibody is obtained; and (3) in the magnetic bead coating obtained by magnetic separation and cleaning in the step (2), the lentil lectin molecules are reacted on the sugar chains of the Fc segment of the alpha fetoprotein antibody. At the same time, control was performed by coating untreated alpha-fetoprotein antibody.
Step E: combining the magnetic bead coating of the alpha fetoprotein antibody treated by the agglutinin molecules with an LCA acridine marker, detecting AFP-L3 in a sample by adopting an indirect method, reading a luminous value, and obtaining the result shown in the table 3.
TABLE 3
Sample preparation: PBS buffer solution is used as a blank sample, serum samples 1-4 are positive samples with different concentrations, and each data is an average value after three times of repeated tests;
experimental group 1: the molar ratio of the alpha fetoprotein antibody to the lentil agglutination molecules is 1:10;
experimental group 2: the molar ratio of the antibody on the alpha fetoprotein antibody magnetic bead coating material to the lentil agglutinin is 1:100.
the results show that: compared with a control group, the experimental group 1 and the experimental group 2 can reduce the background of a blank sample and improve the signal-to-noise ratio of a positive sample. The experimental conditions of experimental group 1 and experimental group 2 are superior to those of the control group in which the antibody is not treated.
Example 3 Signal amplification Using Biotin-labeled antibodies, acridine-labeled avidin
The embodiment provides a detection kit based on a signal amplification mode of a biotin-labeled antibody or lectin and acridine-labeled avidin, which comprises an AFP antibody modified by a cross-linking agent or lectin in a site-specific manner and/or a monoclonal antibody aiming at a compound of AFP-L3 and the lectin. In particular to an AFP-L3 detection kit with the following two forms:
1. kit consisting of alpha fetoprotein antibody coated magnetic bead component, biotin-labeled LCA biotin component and acridinium ester-labeled avidin marker component after cross-linking agent treatment or LCA blocking
Taking the crosslinking agent treated alpha-fetoprotein antibody coated magnetic beads as an example:
(I) kit of experiment group
The preparation method comprises the following steps of preparing a crosslinking agent-treated alpha-fetoprotein coated magnetic bead, a biotin-labeled LCA and an acridinium ester-labeled avidin component:
1. preparing crosslinking agent treated alpha-fetoprotein coated magnetic beads
The polymerized alpha-fetoprotein antibody-coated magnetic beads after the treatment with the crosslinking agent were obtained with reference to the procedure of method (two) in example 2.
2. Biotin marker LCA
1mg of LCA was added to a phosphate buffer solution (pH 7.4) at a concentration of 1mg/mL. Add DMSO solution of biotin NHS ester (molar ratio of LCA to biotin NHS ester 1: 20) and react at 25 ℃ for 3h. The reaction was quenched by addition of lysine solution. Desalting was performed with a 3mL dialysis card (Thermo fish) with a molecular weight cut-off of 10KD. The recovered solution was diluted to 10mL with biotin-preserving solution to obtain biotin-labeled LCA stock solution, which was diluted to 0.2. Mu.g/mL in 10mM Tris-HCl buffer containing 0.1% Triton X-405.
3. Acridinium ester labeled avidin
1mg of avidin was added to a phosphate buffer (pH 7.4) at a concentration of 1mg/mL. A DMSO solution of acridine NHS ester (the molar ratio of avidin to acridine NHS ester is 1. The reaction was quenched by addition of lysine solution. Desalting was performed with 3mL of a 10KD molecular weight cut-off dialysis card (Thermo fish Co.). The volume of the recovered solution was adjusted to 10mL by adding an acridine preservative solution to obtain an acridine ester-labeled avidin mother solution, which was diluted to concentrations of 0.1. Mu.g/mL, 0.2. Mu.g/mL and 0.3. Mu.g/mL in a 10mM Tris-HCl buffer containing 0.1% Triton X-405. The avidin used includes SA, SA4 and SA8, optionally SA4.
(II) control group kit
Comprises crosslinking agent treated alpha-fetoprotein coated magnetic beads and acridinium ester labeled LCA component.
1. Preparing crosslinking agent treated alpha-fetoprotein coated magnetic beads
The polymerized alpha-fetoprotein antibody-coated magnetic beads after the treatment with the crosslinking agent were obtained with reference to the procedure of method (two) in example 2.
2. Acridinium ester labeled LCA
1mg of LCA was added to a phosphate buffer solution (pH 7.4) at a concentration of 1mg/mL. A DMSO solution of acridine NHS ester (the molar ratio of LCA to acridine NHS ester is 1. The reaction was quenched by addition of lysine solution. Desalting was performed with a 3mL dialysis card (Thermo fish) with a molecular weight cut-off of 10KD. The volume of the recovered solution was adjusted to 10mL by adding an acridine preservative solution to obtain an acridine ester-labeled LCA mother liquor, which was diluted to a concentration of 0.2. Mu.g/mL with 0.1% Triton X-405 in 10mM Tris-HCl buffer.
Kit performance testing
AFP-L3 was measured in the experimental group and the control group, and the results of the measurement of luminescence values were shown in Table 4.
The specific detection method comprises the following steps:
and (3) taking 50 mu L of calibrator, quality control material or sample by using a full-automatic chemiluminescence determinator, adding 50 mu L of magnetic bead component suspension, incubating for 10min at 37 ℃, adding 50 mu L of biotin component and 50 mu L of marker component after magnetic separation and cleaning, incubating for 10min at 37 ℃, adding excitation liquid and pre-excitation liquid after magnetic separation and cleaning, detecting a luminescence value, and calculating the concentration of AFP-L3 according to a calibrator curve.
TABLE 4
The concentrations of the biotin-labeled LCA and the acridinium ester-labeled avidin in the experimental group 1 are 0.2 mu g/ml and 0.1 mu g/ml respectively;
the concentrations of the biotin-labeled LCA and the acridinium ester-labeled avidin of the experimental group 2 are 0.2 mug/ml and 0.2 mug/ml respectively;
the concentrations of the biotin-labeled LCA and the acridinium ester-labeled avidin in the experimental group 3 were 0.2. Mu.g/ml and 0.3. Mu.g/ml.
From the results, the signal-to-noise ratios were higher in the experimental group than in the control group without the biotin system, with the highest signal-to-noise ratio in experimental group 2.
2. Kit consisting of magnetic bead components coated by alpha fetoprotein antibodies, biotin components containing LCA and antibodies of biotin-labeled AFP-L3 and LCA complexes, and marker components of acridinium ester-labeled avidin
(I) kit of experiment group
The preparation method comprises the following steps of coating magnetic beads with alpha fetoprotein antibodies, LCA, an AFP-L3 and LCA compound antibody labeled by biotin and an acridinium ester labeled avidin component:
1. preparation of alpha-fetoprotein antibody coated magnetic beads
An alpha-fetoprotein antibody (manufacturer: meridian cat # M86641M) was selected with a molecular weight of about 150KD and a concentration of 5mg/mL. After completion of the reaction, the magnetic beads were washed 3 times with a coating buffer (15mM MES, pH 6.0), 0.4mL of a coating buffer (15mM MES, pH 6.0) was added to resuspend the magnetic beads, 0.2mL of an activator (1-ethyl- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide sulfonic acid sodium salt each 0.1mL, DMSO-dissolved) was added and reacted at 25 ℃ for 30min, magnetic separation was performed, the coating buffer (15mM MES, pH 6.0) was washed 1 time, 1.0mL of a coating buffer (15mM MES, pH 6.0) was added to resuspend the coated magnetic beads, 0.5mg of the above antibody was added and reacted at 25 ℃ for 3h, after completion of the reaction, the coated magnetic beads were washed 2 times with a washing buffer (150 mM phosphate containing 1 Twen20), the coated blocking buffer (150 mM phosphate) was used to block the magnetic beads after completion of the washing for 2h, and finally, the coating of the alpha-fetoprotein antibody magnetic beads was obtained, and the concentration of the magnetic beads was adjusted to 50.5 mM PBS containing the working buffer containing the BSA solution of 0.5 mM PBS was adjusted to 2mL of the BSA.
LCA and biotin-labeled monoclonal antibody against AFP-L3 and LCA Complex (hereinafter may be abbreviated as AFP-L3-LCA antibody)
(1) Dolichos lablab lectin (manufacturer: allantin cat # L140096-10 mg), molecular weight approximately 50kD, was diluted to a concentration of 5mg/mL with 10mM Tris-HCl buffer containing 0.1% Triton X-405.
(2) Biotin-labeled AFP-L3-LCA antibodies: 1mg of AFP-L3-LCA antibody was added to a carbonate buffer (pH 9.0) at a concentration of 1mg/mL. Adding a DMSO solution of biotin NHS ester (the molar ratio of AFP-L3-LCA antibody to biotin NHS ester is 1. The reaction was quenched by addition of lysine solution. Desalting was performed with 3mL of a 10KD molecular weight cut-off dialysis card (Thermo fish Co.). Adding biotin preservation solution into the recovered solution to a constant volume of 10mL to obtain a biotin-labeled AFP-L3-LCA antibody mother solution.
(3) A biotin-labeled working solution comprising two components was prepared by adjusting 5mg/mL of LCA and a biotin-labeled AFP-L3-LCA antibody stock solution to 0.4. Mu.g/mL and 0.2. Mu.g/mL, respectively, with a 10mM Tris-HCl buffer containing 0.1% Triton X-405, and mixing 0.4. Mu.g/mL of LCA and 0.2. Mu.g/mL of biotin-labeled AFP-L3-LCA antibody.
3. Acridinium ester labeled avidin
Reference is made in particular to the above-mentioned method for labeling avidin with acridinium esters.
(II) control group kit
The preparation method comprises the following steps of:
1. preparation of alpha-fetoprotein antibody coated magnetic beads
The magnetic beads coated with the alpha-fetoprotein antibody were obtained according to step 1 in the test kit of this example.
Preparation of Mixed Components of LCA and acridinium ester-labeled AFP-L3-LCA antibody
(1) The preparation of LCA at a concentration of 5mg/mL was performed according to the procedure for preparing LCA in the test kit of this example.
(2) 1mg of AFP-L3-LCA antibody was added to a phosphate buffer (pH 7.4) at a concentration of 1mg/mL. A DMSO solution of acridine NHS ester (the molar ratio of SA4 to acridine NHS ester is 1. The reaction was quenched by addition of lysine solution. Desalting was performed with a 3mL dialysis card (Thermo fish) with a molecular weight cut-off of 10KD. And adding acridine preservation solution into the recovered solution to a constant volume of 10mL to obtain an AFP-L3-LCA antibody mother solution marked by acridine ester.
(3) A5 mg/mL stock solution of LCA and acridinium ester-labeled AFP-L3-LCA complex antibody was adjusted to 0.4. Mu.g/mL and 0.2. Mu.g/mL with 0.1% Triton X-405 in 10mM Tris-HCl buffer, respectively, and 0.4. Mu.g/mL LCA and 0.2. Mu.g/mL biotin-labeled AFP-L3-LCA antibody were mixed to make up an acridine working solution containing both components.
Kit performance testing
AFP-L3 was measured in the experimental group and the control group, and the results of the measurement of luminescence values were shown in Table 5.
TABLE 5
From the results, it is clear that the signal-to-noise ratio of the biotin system in the experimental group is significantly improved compared to the control group of the non-biotin system.
In summary, in this embodiment, the avidin-biotin system is applied to acridine labeling, and mainly adopts a method of labeling biotin with an antibody or LAC, and labeling acridine with avidin for signal amplification. The method can obviously improve detection signals and sensitivity in AFP-L3 detection.
Example 4 preparation and detection of the kit
This example provides two forms of AFP-L3 detection kits, kit A and kit B, respectively.
1. Kit A
Comprises an alpha fetoprotein antibody coated magnetic bead component treated by a cross-linking agent or sealed by LCA, a biotin component for labeling the LCA by biotin and a marker component for labeling avidin by acridinium ester.
Preparation of kit A, comprising the following steps:
1. magnetic bead particles for preparing coupling cross-linking agent or LCA blocking treated alpha-fetoprotein antibody
Step a: selecting alpha-fetoprotein antibody (manufacturer: meridian cat number: M86641M), with molecular weight of about 150KD and concentration of 5mg/mL;1,4-butanediol diglycidyl ether (manufacturer: sigma cat # 220892-50G) diluted with ultrapure water to a concentration of 5mg/mL; PEG molecules (manufacturer: sigma cat # 202398-250G), average molecular weight 400, were diluted with ultrapure water to a concentration of 5mg/mL. Lenticular lens agglutinin (manufacturer: allantin cat # L140096-10 mg) has a molecular weight of about 50KD, and is diluted with ultra pure water to a concentration of 5mg/mL.
Step b: the coating material of alpha-fetoprotein antibody magnetic beads having Fc-fragment sugar chain-modified PEG molecules obtained according to the procedure of the method (I) of example 2 was stored in 50mM PBS buffer containing 0.5% BSA for further use.
Step c: the LCA-blocked coating of the alpha-fetoprotein antibody magnetic beads obtained in steps B, C and D (1) of the method of example 2, which was used to block the Fc fragment sugar chains of the alpha-fetoprotein antibody, with lectins, was stored in 50mM PBS buffer containing 0.5% BSA until use.
2. Biotin-labeled lectin
1mg of LCA was added to a carbonate buffer (pH 8.5) at a concentration of 1mg/mL. Add DMSO solution of biotin NHS ester (molar ratio of LCA to biotin NHS ester 1: 20) and react at 25 ℃ for 3h. The reaction was quenched by addition of lysine solution. Desalting was performed with a 3mL dialysis card (Thermo fish) with a molecular weight cut-off of 10KD. The volume of the recovered solution is adjusted to 10mL by adding biotin preservation solution.
3. Acridinium ester labeled avidin
1mg of SA8 was added to a carbonate buffer solution (pH 8.5) at a concentration of 1mg/mL. A DMSO solution of acridine NHS ester (molar ratio of SA8 to acridine NHS ester 1. The reaction was quenched by addition of lysine solution. Desalting was performed with a 3mL dialysis card (Thermo fish) with a molecular weight cut-off of 10KD. The volume of the recovered solution is adjusted to 10mL by adding acridine preservation solution.
4. Assembling the kit:
adjusting the concentration of the coated beads obtained in step 1B to 0.2mg/ml with a magnetic bead working solution containing 0.5% BSA in 50mM PBS buffer; biotin-labeled lectin was adjusted to 0.2. Mu.g/ml with 10mM Tris-HCl buffer containing 0.1% Triton X-405; the acridinium ester-labeled avidin was adjusted to 0.2. Mu.g/ml of acridine working solution with a 10mM Tris-HCl buffer containing 0.1% Triton X-405, and combined into kit A1.
Adjusting the concentration of the coated beads obtained in step 1 to 0.2mg/ml with 50mM PBS buffer containing 0.5% BSA; biotin-labeled lectin was adjusted to 0.2. Mu.g/ml with 10mM Tris-HCl buffer containing 0.1% Triton X-405; the acridinium ester-labeled avidin was adjusted to 0.2. Mu.g/ml of acridine working solution with a 10mM Tris-HCl buffer containing 0.1% Triton X-405, and combined into kit A2.
(II) minimum detection limit
The kit A1 and the kit A2 adopt an iFlash 3000 series full-automatic chemiluminescence determinator of Yahuilong biological science and technology Limited company to carry out full-automatic detection. Three different batches of detection kits were used to simultaneously detect 20 calibrator diluent samples or samples with zero concentration values, the Mean (Mean) and Standard Deviation (SD) were calculated, RLU values were calculated from the fit equation Mean +2SD, and the concentration values were calculated from RLU, with the results for kit A1 and kit A2 shown in tables 6 and 7, respectively.
TABLE 6 results of kit A1
TABLE 7 results of kit A2
According to the analysis of the detection results of the three batches of reagents of the kit A1 and the kit A2, the highest detection result of the blank detection limit of the kit A1 is 0.0746ng/ml which is less than 0.1ng/ml; the highest detection result of the blank detection limit of the kit A2 is 0.086ng/ml and is less than 0.1ng/ml.
(III) accuracy of detection result
A total of 632 clinical samples were collected, 274 primary liver cancer (HCC) samples, 143 chronic hepatitis samples, 117 cirrhosis samples, and 98 healthy human serum samples. The combined kit A1 and kit A2 of this example were used for detection, and the detection results are shown in Table 8.
TABLE 8
Wherein, the content of AFP-L3 refers to the proportion of AFP-L3 in AFP. The AFP-L3 accounts for more than 10 percent of the AFP and is judged to be positive.
Experimental results show that the sensitivity of the reagent kit A1 and the sensitivity of the reagent kit A2 in the application to primary liver cancer respectively reach 98.5% and 96.8%, the specificity to a chronic hepatitis sample in a high risk group of liver cancer respectively reaches 97.5% and 95.2%, and the specificity to a liver cirrhosis sample respectively reaches 95.4% and 93.8%.
2. Kit B
Comprises a magnetic bead component coated by alpha fetoprotein antibody, a biotin component containing LCA and biotin-labeled AFP-L3-LCA complex antibody, and a marker component of acridinium ester-labeled avidin.
Preparation of kit B, comprising the following steps:
1. magnetic bead particles for preparing coupled alpha-fetoprotein antibody
The preparation process is referred to example 3.
2. AFP-L3-LCA antibody mixed component for preparing LCA and labeled biotin
The preparation process is referred to example 3.
3. Acridine-labeled avidin
The preparation process is referred to example 3.
And combining the magnetic bead working solution, an AFP-L3-LCA antibody containing LCA and biotin labeling and the acridine labeled avidin into a kit B.
(II) minimum detection limit
The kit B adopts an iFlash 3000 series full-automatic chemiluminescence determinator of Asia-glow-dragon biological science and technology Limited company to carry out full-automatic detection. Three different batches of test kits were used to simultaneously test 20 calibrator diluent samples or samples with zero concentration values, the Mean (Mean) and Standard Deviation (SD) were calculated, RLU values were calculated from the fit equation Mean +2SD, and the concentration values were calculated from RLU, with the results for kit B shown in table 9.
TABLE 9
According to the analysis of the three reagent detection results of the kit B, the highest detection result of the blank detection limit of the kit 3 is 0.062ng/ml.
(III) accuracy of detection result
A total of 632 clinical samples were collected, 274 primary liver cancer (HCC) samples, 143 chronic hepatitis samples, 117 cirrhosis samples, and 98 healthy human serum samples. The kit B was used for detection, and the detection results are shown in Table 10.
The experimental result shows that the sensitivity of the kit 3 in the invention to primary liver cancer reaches 85.5%, the specificity to chronic hepatitis samples in high risk group of liver cancer reaches 97.7%, and the specificity to cirrhosis samples reaches 96.2% respectively.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the patent is subject to the appended claims, and the description and the drawings can be used for explaining the contents of the claims.
Claims (20)
1. A monoclonal antibody aiming at a compound of AFP-L3 and lectin, which is characterized by comprising a heavy chain variable region CDR1 with an amino acid sequence shown as SEQ ID No.1, a heavy chain variable region CDR2 with an amino acid sequence shown as SEQ ID No.2, a heavy chain variable region CDR3 with an amino acid sequence shown as SEQ ID No.3, a light chain variable region CDR1 with an amino acid sequence shown as SEQ ID No.4, a light chain variable region CDR2 with an amino acid sequence shown as SEQ ID No.5 and a light chain variable region CDR3 with an amino acid sequence shown as SEQ ID No. 6.
2. The monoclonal antibody of claim 1, comprising a heavy chain variable region having an amino acid sequence as set forth in SEQ ID No.7 and a light chain variable region having an amino acid sequence as set forth in SEQ ID No. 8.
3. A nucleic acid molecule comprising a nucleic acid fragment encoding the monoclonal antibody of claim 1 or 2.
4. A biological material comprising a nucleic acid molecule according to claim 3, wherein the biological material comprises a vector or a host cell.
5. A deglycosylated antibody, characterized in that the sugar chain of the Fc fragment of said antibody is linked to a cross-linking agent or a lectin.
6. The antibody of claim 5, wherein said cross-linking agent is reactive with hydroxyl groups on the sugar chains of the Fc fragment of the antibody and is chemically bonded thereto; the lectin is linked to the sugar chain of the Fc fragment of the antibody by intermolecular forces.
7. The antibody of claim 5 or 6, wherein the cross-linking agent comprises an epoxy group;
optionally, the cross-linking agent comprises at least two epoxy groups;
optionally, the cross-linking agent is a homobifunctional cross-linking agent; the homobifunctional crosslinking agent comprises 1,4-butanediol diglycidyl ether, polyethylene glycol diglycidyl ether and at least one of trimethylolpropane triglycidyl ether.
8. The method for producing an antibody according to any one of claims 5 to 7, which comprises treating the sugar chains of the antibody with a crosslinking agent or a lectin to reduce the reaction of the sugar chains of the Fc fragment of the antibody with the lectin.
9. The method according to claim 8, wherein the step of treating the sugar chain of the antibody with the crosslinking agent comprises subjecting the antibody and the crosslinking agent to a treatment in a molar ratio of 1: (5-50) carrying out the reaction.
10. The method according to claim 8, wherein the step of treating the sugar chain of the antibody with the crosslinking agent comprises subjecting the antibody, the modifying agent and the crosslinking agent to a treatment in a molar ratio of 1: (20-100): (5-50) carrying out reaction; optionally, the modifying agent comprises at least one of a PEG series, a small molecule polypeptide, and a hydroxyl-containing amino acid.
11. The method according to claim 8, wherein the step of treating the sugar chain of the antibody with the lectin comprises mixing the antibody with the lectin in a molar ratio of 1: (1-200) carrying out reaction.
12. The method of claim 11, wherein the lectin comprises one or more of an animal lectin and a plant lectin; optionally, the plant lectin comprises one or more of lentil lectin, orange Huang Wangbao discodermin lectin, sword bean lectin, wheat germ lectin, peanut lectin, soybean lectin, and lotus root lectin.
13. A test kit comprising the monoclonal antibody of claim 1 or 2.
14. The kit of claim 13, further comprising an alpha-fetoprotein antibody or a deglycosylated alpha-fetoprotein antibody; alternatively, the deglycosylated alpha-fetoprotein antibody is obtained by a method comprising the preparation of any one of claims 8 to 12.
15. The kit of claim 14, wherein the kit comprises the monoclonal antibody of claim 1 or 2 labeled with a solid support coated with the alpha-fetoprotein antibody or the deglycosylated alpha-fetoprotein antibody and a label; optionally, the label comprises biotin.
16. The kit of claim 15, further comprising one or more of an acridinium ester labeled avidin and a lectin.
17. A test kit comprising an antibody according to any one of claims 5 to 7, wherein the antibody is a deglycosylated alpha-fetoprotein antibody.
18. The kit of claim 17, further comprising a label labeled lectin; optionally, the label comprises biotin or an acridinium ester.
19. The kit of claim 18, wherein said kit comprises a solid support coated with said deglycosylated alpha-fetoprotein antibody, a biotin-labeled lectin, and an acridinium ester-labeled avidin.
20. Use of the monoclonal antibody of claim 1 or 2 or the antibody of any one of claims 5 to 7 or the deglycosylated alpha-fetoprotein antibody obtained by the preparation method of any one of claims 8 to 12 or the kit of any one of claims 13 to 19 for the preparation of a product for detecting alpha-fetoprotein heteroplasmons or for the preparation of a product for diagnosing liver cancer.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2021038980A (en) * | 2019-09-02 | 2021-03-11 | 富士レビオ株式会社 | Afp-l3 measurement method and afp-l3 measurement kit, and blocked labeled lectin used for these |
| CN113433318A (en) * | 2021-06-29 | 2021-09-24 | 山东中鸿特检生物科技有限公司 | Kit for detecting alpha-fetoprotein heteroplasmon AFP-L3 content and detection method and application thereof |
| CN114487385A (en) * | 2021-12-29 | 2022-05-13 | 深圳市国赛生物技术有限公司 | Alpha-fetoprotein heteroplasmon detection composition and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2021038980A (en) * | 2019-09-02 | 2021-03-11 | 富士レビオ株式会社 | Afp-l3 measurement method and afp-l3 measurement kit, and blocked labeled lectin used for these |
| CN113433318A (en) * | 2021-06-29 | 2021-09-24 | 山东中鸿特检生物科技有限公司 | Kit for detecting alpha-fetoprotein heteroplasmon AFP-L3 content and detection method and application thereof |
| CN114487385A (en) * | 2021-12-29 | 2022-05-13 | 深圳市国赛生物技术有限公司 | Alpha-fetoprotein heteroplasmon detection composition and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| YURIKO EGASHIRA等: "establishment and characterization of a fucosylated α-fetoprotein-specific monoclonal antibody: a potential application for clinical research", SCIENTIFIC REPORTS, vol. 9, pages 1 - 12 * |
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