A kind of anti-pla2r recombinant rabbit monoclonal antibody and its application

A monoclonal antibody, PLA2R technology, applied in the field of immunochemistry, can solve the problems of immunofluorescence method that cannot be stored for a long time, and achieve the effect of easy scoring, strong positive signal, and accurate type of kidney disease

Active Publication Date: 2022-07-05
苏州百道医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the domestic diagnosis of IMN is mainly based on the semi-quantitative method of serum protein and immunofluorescence method, but the literature shows that the se

Method used

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  • A kind of anti-pla2r recombinant rabbit monoclonal antibody and its application
  • A kind of anti-pla2r recombinant rabbit monoclonal antibody and its application
  • A kind of anti-pla2r recombinant rabbit monoclonal antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] This embodiment is the preparation and screening of anti-PLA2R recombinant rabbit monoclonal antibody, and the steps include:

[0035] (1) Antigen preparation

[0036] The specific sequences of the PLA2R antigens are shown in SEQ ID NOs: 1-3 below.

[0037] SEQ ID No. 1: IGLQEERANDEFRWRDGTPVIYQNWD

[0038] SEQ ID No. 2: SNWGIRKPDTDYFKP

[0039]SEQ ID No. 3: DTGEYTWKPVGQKPE.

[0040] The above-mentioned polypeptide sequence is selected by analyzing the PLA2R molecular sequence according to the structure, antigenicity, hydrophobicity and secondary structure of the PLA2R protein molecule on the cell membrane. The polypeptides of the sequences shown in SEQ ID NOs: 1-3 were artificially synthesized, and the synthesized polypeptides were used as antigens for immunizing rabbits. During immunization, the polypeptides of the sequences shown in SEQ ID NOs: 1-3 were coupled via KLH or OVA as immunogens to immunize rabbits.

[0041] (2) Immunity

[0042] The polypeptide seque...

Embodiment 2

[0067] This example is the immunohistochemical detection of anti-PLA2R recombinant rabbit monoclonal antibody as the primary antibody, and the method is as follows:

[0068] (1) Preparation of sample slices: The formalin-fixed, paraffin-embedded renal cancer tissue slices were baked in a 60°C incubator for 1-2 hours, and stored for later use.

[0069] (2) Dewaxing of sections: paraffin sections were first placed in fresh xylene for dewaxing, and soaked for 2 times for 10 min each time.

[0070] (3) Slice hydration: soak in absolute ethanol, absolute ethanol, 95% ethanol, 85% ethanol, and 70% ethanol for 5 minutes in order for hydration, and then rinse with purified water twice, 3 minutes each time.

[0071] (4) Antigen retrieval: It is recommended to use the high-temperature thermal repair method for 3 minutes (if using an automatic repair instrument, set a high temperature of 98 °C for 20 minutes), and then cool the slices to room temperature naturally. Rinse 2 times, 3 min ...

Embodiment 3

[0081] This example is for the determination of the affinity of the 54D11 anti-PLA2R recombinant rabbit monoclonal antibody. The determination method is as follows:

[0082] (1) Take out the purified PLA2R protein from 4°C and return to room temperature. Dilute to a concentration of 1 μg / ml, add 100 μL / well to a 96-well microtiter plate and incubate at 4°C overnight, followed by blocking with 2% BSA at 4°C overnight.

[0083] (2) The PLA2R recombinant rabbit monoclonal antibody cloned from 54D11 and the commercially available anti-PLA2R antibody were respectively diluted to an initial concentration of 0.5 μg / mL, and then subjected to 2-fold gradient dilution, and a total of 7 concentration gradients were set up for comparison.

[0084] (3) Add 100 μL / well of the diluted anti-PLA2R recombinant rabbit monoclonal antibody to a 96-well ELISA plate with peptides, cover with a sealing film, and incubate at 37°C for 1 h to allow the reaction to reach equilibrium.

[0085] (4) After ...

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Abstract

The present invention relates to an anti-PLA2R recombinant rabbit monoclonal antibody, comprising a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 6; the light chain can be The amino acid sequence of the variable region is shown in SEQ ID No.7. Compared with the commercially available anti-PLA2R recombinant rabbit monoclonal antibody, the anti-PLA2R recombinant rabbit monoclonal antibody provided by the present invention has higher affinity with the PLA2R protein, and can recognize and detect tumor cells or immune cells with high specificity and sensitivity. The expression of PLA2R protein can be used in immunohistochemistry (IHC), indirect ELISA, Western blotting, antibody chip preparation, flow cytometry and other detection and screening processes at lower concentrations, which is conducive to obtaining more accurate results. Detect and evaluate results, reducing detection costs and interference from background signals.

Description

technical field [0001] The invention belongs to the technical field of immunochemistry, and in particular relates to an anti-PLA2R antibody and application thereof, especially the application in immunohistochemical detection. Background technique [0002] Idiopathic membranous nephropathy (IMN) is the main type of primary glomerular disease, and its incidence is gradually increasing and younger in recent years. As an autoimmune disease, the search and detection of IMN target antigens has always been the focus of the disease treatment. Relevant foreign literature points out that 70% of IMN patients are related to the target antigen PLA2R (M-type phospholipase A2 receptor, M-type phospholipase A2 receptor), which is higher in China, and another antigen is type 1 thrombospondin 7A Domain (thrombospondin type1 domain-containing 7A, THSD7A), only 2.5% -7.5% of IMN. [0003] PLA2R is expressed by glomerular podocytes, and its mediated production of anti-PLA2R antibodies is consi...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K1/22C12N15/13C12N15/85G01N33/68G01N33/577
CPCC07K16/2851C12N15/85G01N33/6872G01N33/6893G01N33/577C07K2317/56C07K2317/92C07K2317/20C12N2800/107G01N2333/705G01N2800/347
Inventor 李静孟凡华程育苗刘杨张东旭
Owner 苏州百道医疗科技有限公司
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