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Monoclonal antibody of disodium EDTA

A technology of disodium ethylenediaminetetraacetic acid and monoclonal antibody, which is applied in the biological field, can solve the problems of loss of protein activity, no relevant literature reports, loss of metal cofactors, etc., and achieve highly specific effects

Inactive Publication Date: 2015-03-11
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is currently believed that the mechanism is Na 2 -The metal chelation of EDTA deprives metalloproteins and metalloproteases of metal cofactors, which indirectly leads to the loss of protein activity
Na 2 - In addition to its outstanding chelation effect, it is not known whether there are other mechanisms for the biological effects of EDTA, and there is no relevant literature report

Method used

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  • Monoclonal antibody of disodium EDTA
  • Monoclonal antibody of disodium EDTA
  • Monoclonal antibody of disodium EDTA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1 prepares anti-Na 2 -Monoclonal antibody to EDTA

[0034] Derivatives of EDTA (ethylenediaminetetraacetic acid), ITCBE (1-(4-isothiocyanobenzyl) vinyldiamine-N,N,N',N'-tetraacetic acid, Dojindo Laboratories, Japan) as The antigen was connected to the carrier protein BSA (bovine serum albumin, Ameresco, USA) through the amino group at the end of the isothiocyanate benzyl group to prepare the immunogen ITCBE-BSA. BALB / c mice (purchased from the West China Experimental Animal Center of Sichuan University) were immunized subcutaneously in multiple points according to conventional methods, and the mice with the highest antibody titer were screened by indirect ELISA. The operation of indirect ELISA is as follows: (1) Antigen coating: Dilute ITCBE-OVA to 100ng / mL with coating buffer, add to 96-well microplate plate, 100μL / well, control plate with antigen OVA, the operation is the same as above, 4°C Overnight; (2) Blocking: Wash the plate with PBST for 3 min×3 times...

Embodiment 2 Embodiment 1

[0040] The anti-Na prepared in embodiment 2 embodiment 1 2 - Cross-reactivity of EDTA monoclonal antibody

[0041] In this study, several cations commonly found in the human body, Ca 2+ , Mg 2+ , Fe 2+ , Fe 3+ , Cu 2+ , Zn 2+ , Mn 2+ respectively with Na 2 - Complexation of EDTA, as a cross-reactive species. At a concentration of 0.001-10 μmol / L, the absorbance value was measured by indirect competitive ELISA (the standard procedure of ELISA is as described in Example 1). The method for drawing the ELISA standard curve as described in Example 1 was used to draw the curves respectively, and then calculate the IC50 of the curves respectively. According to IC50, the cross-reactivity rate was calculated respectively. The calculation method is as follows: cross-reactivity rate CR (%)=[IC50 of EDTA] / [cross-reaction ion-IC50 of EDTA]. The cross-reaction results showed that the IC50 of K2-EDTA, Ca-EDTA and Mg-EDTA were 0.185, 0.171 and 0.181 μmol / L, which were very close to...

Embodiment 4

[0047] Example 4 Preliminary Exploration Using the Anti-Na2-EDTA Monoclonal Antibody Prepared in Example 1

[0048] The premolars that need to be extracted due to orthodontic treatment provided by the Oral and Maxillofacial Surgery Clinic of West China Stomatological Hospital of Sichuan University, tooth body and periodontal health. Primary human periodontal ligament fibroblasts (hPDLF) were cultured by tissue block method. The fifth to eighth passage cells were selected as experimental cells.

[0049] Detection of Na by MTT method 2 - Cytotoxicity of EDTA to hPDLF, to screen the appropriate concentration of EDTA for follow-up experiments. Starting from 5mmol / L, dilute in a 1:2 gradient, and set 8 gradient concentrations of Na 2 - EDTA, co-cultivate with hPDLF at 5% CO2, 37°C for 48h. After adding MTT (10 μL, 5 mg / ml), culture was continued for 4 h. Add 100 μL DMSO and place on a shaker for 10 minutes to fully dissolve formazan (blue-purple crystal). Measure OD570nm in e...

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Abstract

The invention provides a monoclonal antibody of disodium EDTA (Na2-EDTA). The monoclonal antibody of the Na2-EDTA can specifically recognize Na2-EDTA and chelates of Ca-EDTA and Mg-EDTA formed by chelating Na2-EDTA with Ca<2+> and Mg<2+>, can be used for further exploring possible biological effects of the Na2-EDTA from a perspective of cellular proteins, and can be specially and stably secreted from a hybridoma cell line of which preservation No. is CCTCC NO: C2014130.

Description

technical field [0001] The invention belongs to the field of biotechnology. This monoclonal antibody can specifically recognize Na 2 -EDTA and its chelated Ca 2+ , Mg 2+ After the ability of the chelated Ca-EDTA and Mg-EDTA, Na used in in vitro cell experiments and in vivo animal experiments 2 -Studies on biological effects of EDTA. To study Na 2 - The biological effect of EDTA provides a new way. Background technique [0002] Ethylenediaminetetraacetic acid (EDTA) is an organic compound, insoluble in cold water, alcohol and general organic solvents, slightly soluble in hot water, soluble in alkaline solution. The solubility of sodium salt is 10.8g / L at 22°C and 23.6g / L at 80°C. The sodium salt of ethylenediaminetetraacetic acid (Na 2 -EDTA) instead of EDTA. Na 2 -EDTA can form relatively stable chelates with multiple five-ring structures with most metal ions, so it is widely used in industry, food and medical fields. But Na 2 -EDTA is almost non-biodegradable an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/44C12N5/20G01N33/577A61K49/00C12R1/91
Inventor 孟玉坤谢翠柳陈雨雪魏大鹏
Owner SICHUAN UNIV
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