Lp(a) (Lipoprotein(a)) resisting monoclonal antibody and latex-enhanced immunonephelometric detection kit for Lp(a)

A monoclonal antibody and detection kit technology, which is applied in anti-animal/human immunoglobulin, biological testing, measuring devices, etc., can solve the problems of increased workload and large differences in kit performance, and achieve high accuracy and reliability , the effect of high coincidence degree of measured value

Active Publication Date: 2014-03-12
BEIJING LEADMAN BIOCHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Take latex-enhanced immunoturbidimetric reagents as an example: in the preparation of reagents, the increase in antibody types will lead to corresponding coupling procedures and concentration adjustment procedures, the workload will double, and the risk of greater differences in kit performance will also arise from this.
[0005] The development of monoclonal antibodies that recognize epitopes other than KringleIV-2 of apo(a) molecules can solve the problem of large batch-to-batch variation of antibodies and apo(a) polymorphism question of sexual influence

Method used

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  • Lp(a) (Lipoprotein(a)) resisting monoclonal antibody and latex-enhanced immunonephelometric detection kit for Lp(a)
  • Lp(a) (Lipoprotein(a)) resisting monoclonal antibody and latex-enhanced immunonephelometric detection kit for Lp(a)
  • Lp(a) (Lipoprotein(a)) resisting monoclonal antibody and latex-enhanced immunonephelometric detection kit for Lp(a)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The construction of embodiment 1 hybridoma cell line

[0040] Four 6-week-old female Balb / c mice were immunized with Lp(a) antigen. For the first immunization, the antigen was emulsified with Freund's complete adjuvant, and each mouse was injected with 40ug of antigen, and then the antigen was emulsified with Freund's incomplete adjuvant , 2-3 immunizations were performed at intervals of 21 days. After 3 times of immunization, blood was collected from the orbit to detect the serum antibody titer, and mice with higher titers were selected for cell fusion. 3 days before the fusion, the selected mice were immunized with Lp(a) antigen without adjuvant. After 3 days, the mice were sacrificed by breaking the cervical spine, and the spleen was aseptically isolated to prepare the spleen cell suspension, and the spleen cells were cultured in vitro. The SP2 / 0 cells were mixed at a ratio of 10:1, centrifuged and washed with PEG1450 for cell fusion, and the fusion cells were cultu...

Embodiment 2

[0041] Example 2 The paired screening of anti-Lp (a) monoclonal antibody

[0042] The Lp(a) monoclonal antibody hybridoma cells that were cloned and established were prepared by the ascites method to produce Lp(a) monoclonal antibody ascites, and the Lp(a) monoclonal antibody was purified with a ProteinG column. The antibodies were labeled with HRP, and then the pairing characteristics of each strain of Lp(a) monoclonal antibodies were analyzed by the checkerboard method. Each strain of Lp(a) monoclonal antibody was diluted to 5ug / ml with 0.05MpH9.6Na2CO3-NaHCO3 buffer solution, 100ul per hole was coated with a microtiter plate, and each monoclonal antibody was coated with 1 row of well strips; Lp(a) was diluted with PBST ) antigen was diluted to 0.1ug / ml, 100ul per well, incubated at 37°C for 30min, and washed twice with PBST; the HRP markers of each monoclonal antibody were diluted to the working concentration with PBST, and each monoclonal antibody marker was added 1 Arran...

Embodiment 3

[0045] Example 3 Lp (a) detection kit prepared with LPa-3 monoclonal antibody

[0046] 1) Reagent 1 is:

[0047] PBS buffer (pH7.4)

[0048] BSA0.5%

[0049] Tween - 200.1%

[0050] NaN30.1%

[0051] 2) Reagent 2 is:

[0052] PBS buffer (pH7.4)

[0053]

[0054] 3) Preparation of LPa-3 monoclonal antibody latex microspheres

[0055] a) Add LPa-3 mAb to 1 mg / ml with 50 mM HEPES buffer (pH 7.4).

[0056] b) Dilute 100nm latex microspheres with MES buffer (10mMpH5.0) to a mass concentration of 2%, 400μl in total.

[0057] c) Prepare 400 μl of EDC (2 mg / ml) and 400 μl of NHS (2 mg / ml) with MES buffer (10 mM pH 5.0).

[0058] d) Add 300 μl EDC solution dropwise to the latex microsphere solution, then add 300 μl NHS solution dropwise, and stir at room temperature for 15 minutes.

[0059] e) The activated microspheres and mAb were mixed and stirred at room temperature for 2 hours.

[0060] f) Centrifuge after the reaction, remove the supernatant, resuspend the pellet wit...

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Abstract

The invention discloses an Lp(a) (Lipoprotein(a)) resisting monoclonal antibody and a latex-enhanced immunonephelometric detection kit for Lp(a). The invention provides a hybridoma cell strain which can stably secrete the Lp(a) monoclonal antibody and has the collection number of CGMCC No. 8509. The hybridoma cell strain provided by the invention can stably produce the Lp(a) monoclonal antibody, and personal pairing, specific Lp(a) combination and no combination with Kringle IV-2 can be realized. Shown by a test that serum samples of patients are detected by an Lp(a) kit prepared from the Lp(a) resisting monoclonal antibody, values detected by the latex-enhanced immunonephelometric detection kit prepared from the Lp(a) resisting monoclonal antibody have high conformity with detected values of commercially available kits, and the accuracy is reliable, so that the latex-enhanced immunonephelometric detection kit can be applied to Lp(a) detection.

Description

technical field [0001] The present invention relates to a hybridoma cell strain and a secreted monoclonal antibody, in particular to a monoclonal antibody against lipoprotein (a) [Lipoprtein(a), Lp(a)] and a hybridoma cell strain secreting the monoclonal antibody , the present invention further Lp(a) latex enhanced immunoturbidimetric detection kit, belongs to the detection field of lipoprotein (a). Background technique [0002] Lipoprotein (a) [Lipoprotein (a), Lp (a)] is a unique lipoprotein in the human body, which is composed of low-density lipoprotein (LDL)-like particles and apolipoprotein (a) [apolipoprotein (a), apo (a)] composition. Apo(a) is highly homologous to the plasminogen gene and belongs to the human plasminogen gene superfamily. Apo(a) consists of multiple copies of plasminogen KringleIV-like repeats and a single copy of KringleV and serine protease domains. Apo(a) has 10 plasminogen KringleIV-like motifs (named KringleIV-1~KringleIV-10), and each Kringl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/18G01N33/92G01N33/577
Inventor 不公告发明人
Owner BEIJING LEADMAN BIOCHEM
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