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Method for detecting bioactivity of humanized anti-CD52 antibody

A CD52 and antibody biological technology, applied in the biological field, can solve the problems of poor detection method sensitivity, poor detection sensitivity, and difficulty in obtaining a large batch of uniform and sufficient quantities.

Inactive Publication Date: 2008-06-18
上海国健生物技术研究院
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] There are many problems in the above method, such as: using synthetic CD52 antigen, the synthetic antigen has lower affinity compared with natural antigen, so the sensitivity of the detection method is poor; using natural CD52 antigen, the source is difficult (human spleen), and antigens from different sources There is a big difference, and the purified antigen has a tendency to aggregate; using anti-CD52 idiotype antibody instead of CD52 antigen, the detection sensitivity is low, or there is an inevitable cross-reaction with serum Ig; using fresh peripheral blood mononuclear cells, different donors Differences in CD52 expression between patients, virus contamination, it is difficult to obtain a large number of uniform and sufficient quantities; non-specific binding caused by differences in other membrane antigens; use of CD52-positive cell lines, false positives caused by Fc receptors, poor detection sensitivity ; ADCC or CDC method, the use of isotope makes the operability decrease, the detection sensitivity and precision are poor, etc.

Method used

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  • Method for detecting bioactivity of humanized anti-CD52 antibody
  • Method for detecting bioactivity of humanized anti-CD52 antibody
  • Method for detecting bioactivity of humanized anti-CD52 antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1: Construction of CHO-C cells

[0064] 1. Acquisition of human CD52 cDNA and construction of expression vector:

[0065] We designed primer sequences:

[0066] Sense primer: 5′-C CACCATGAAGCGCTTCCTCTTCCTC-3' (SEQ ID NO. 1)

[0067] Antisense primer: 5′-C TCAACTGAAGCAGAAGAGGTG-3' (SEQ ID NO.2)

[0068] This pair of primers has added restriction sites on both sides of the complementary region for loading into the expression vector

[0069] The gene sequence of human CD52 cDNA was obtained from the RNA extract of human peripheral blood by conventional RT-PCR method: 10 ml of normal human peripheral blood was extracted, and mononuclear cells were separated by Ficoll (Beijing Dingguo Biotechnology Co., Ltd.) density gradient centrifugation. Trizol kit (GIBCO company) was used to extract total RNA, and RT-PCR kit (INVITROGEN company) was used for reverse transcription PCR to obtain a specific fragment of about 200 bp. The fragment was recovered with a gel rec...

Embodiment 2

[0076] Embodiment 2: biological activity assay experiment

[0077] 1. Take an aliquoted reference product (Campath-1, product of Genzyme Company), dilute it to 2000 μg / ml with PBSS, and add an equal volume of 20 μg / ml FITC-anti-CD52MAb to each dilution factor of no more than 10 times (PBSS dilution).

[0078] 2. According to the protein quantification of the sample to be tested (that is, rhCD52mAb prepared according to the content disclosed in the Chinese invention patent application number 02155174.X), dilute to 2000 μg / ml with PBSS, and the dilution factor does not exceed 10 times each time, and add an equal volume of 20 μg / ml of FITC-anti-CD52MAb (diluted in PBSS).

[0079] 3. Use 10 μg / ml FITC-anti-CD52MAb (diluted in PBSS) in a centrifuge tube to serially dilute the dilution of the working reference product or the sample by 2 times.

[0080] 4. Take the CHO-C cells in the logarithmic growth phase, count them, and take about 10 for each assay (1 sample and working refer...

experiment example 1

[0091] Experimental Example 1 Identification experiment of CHO-C cells: Refer to "Modern Immunology Experimental Technology" for fluorescent staining

[0092] CHO-K1 cells (ATCC CCL-61) are compared with CHO-cells of the present invention. After labeling, the peak of the staining fluorescence intensity of CHO-C cells can be obviously shifted to the right, that is, FITC-rhCD52mAb can stain CHO-C, that is It shows that CD52 is expressed on the surface of CHO-C cells, but not expressed on the surface of untransfected CHO-K1 cells. The results are detailed in the attached image 3

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Abstract

The invention discloses a method to detect the biological activity of humanized antibody CD52, which comprises dilution of specimens and a reference product having given activity, making use of CD52 positive cells for competitive binding assay and detection with a flow cytometry, and calculation of the results. The CD52 positive cells used in the competitive binding assay steps are CHO-C cells. The CHO-C cells used by the invention have the advantages of high sensitivity and high stability.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and relates to a method for detecting the biological activity of an antibody. Background technique: [0002] CD52, also known as CAMPATH-1 antigen, is expressed on the surface of normal and malignant B and T lymphocytes, NK cells, monocytes, macrophages and male reproductive system tissues. Antigens on the surface of lymphocytes. The monoclonal antibody against the CD52 antigen can temporarily clear the lymphocytes in the blood in the body, so that the immune system is in a state of failure, so that the organ transplanted into the patient will not be rejected by the patient's immune system. Can cause antibody-dependent cytolytic or cytocidal effects, thereby clearing the blood, bone marrow, and other affected cells of malignant lymphocytes. Anti-CD52 monoclonal antibody (CAMPATH-1 series) has been widely used in clinical treatment. This antibody is a humanized anti-CD52 antibody. It was approved b...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/554
Inventor 侯盛王皓寇庚李博华郭亚军
Owner 上海国健生物技术研究院
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