Hybridoma cell capable of secreting anti retinol binding protein monoclonal antibody and application

A monoclonal antibody, hybridoma cell technology, applied in the field of retinol binding protein immunodetection, can solve the problems of easy degradation, short half-life, unstable β2 microglobulin, etc.

Active Publication Date: 2014-01-22
FAPON BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

β2 microglobulin is very unstable and easy to degrade when the pH is less than 5.5, and the half-life of β2 microglobulin in urine is short, and 70% of it is degraded after 4 hours in the environment of pH 4.0, while RBP is in the same Only 5% degradation under the conditions

Method used

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  • Hybridoma cell capable of secreting anti retinol binding protein monoclonal antibody and application
  • Hybridoma cell capable of secreting anti retinol binding protein monoclonal antibody and application
  • Hybridoma cell capable of secreting anti retinol binding protein monoclonal antibody and application

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Experimental program
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Effect test

Embodiment 1

[0027] The establishment of embodiment 1 hybridoma cell line and the preparation of anti-RBP monoclonal antibody

[0028] 1. Antigen Immunization

[0029] The same amount of recombinant RBP antigen (produced by Shenzhen Faipeng Biological Co., Ltd., product number BA-PAB-MU0032) was mixed with complete Freund's adjuvant (appearance: amber cell suspension; components: Paraffin Oil85%, Mannide Monooleate15%, Mycobacterium smegmatis 1mg / mL) were mixed to obtain an oily emulsion. The emulsion was subcutaneously injected into the back site of BALB / c mice (Guangzhou Provincial Experimental Animal Center, 6-week-old female, 5) at a dose of 0.2 mL, and the intraperitoneal booster immunization was performed 14 days after the first immunization (equal antigen and Freund's Incomplete adjuvant mixture), after boosting immunization to four injections, tail blood was collected for titer detection, and the titer met the fusion requirements.

[0030] Three days before the fusion, the same d...

Embodiment 2

[0073] Embodiment 2RBP latex immunoturbidimetric assay kit

[0074] 1. The main reagents of the detection kit of the present embodiment are prepared as follows:

[0075] (1) Reagent R1 includes: 45mmol / L Tris buffer, 85mmol / L polyethylene glycol, 5mmol / L sodium azide, 8.5mmol / L sodium edetate and 5mmol / L bovine serum albumin.

[0076] (2) Reagent R2 includes: 45mmol / L Tris buffer, 5% (v / v) latex particles bound with RBP monoclonal antibody, 5mmol / L Tween-20, 3mmol / L sodium azide, 8.5mmol / L Sodium edetate, 5mmol / L bovine serum albumin.

[0077] (3) Calibrators: The calibrators used to detect serum are serum matrix, with concentrations of 130mg / L, 100mg / L, 50mg / L, 25mg / L, 12.5mg / L, 0mg / L, or similar proportions ; The calibrator used to detect urine is urine matrix, the concentration is respectively 4mg / L, 2mg / L, 1mg / L, 0.5mg / L, 0.25mg / L, 0mg / L, or the content of similar ratio.

[0078] 2 detection process

[0079] (1) Reagent preparation, reagent R1 includes 45mmol / L Tris bu...

Embodiment 3

[0082] Application of embodiment 3RBP latex immunoturbidimetric assay kit

[0083] Use the reagent of this method invention, adopt Hitachi 7170 full-automatic biochemical instrument, be 110mg / L to adding concentration, 60mg / L, 15mg / L RBP serum sample and 3.5mg / L, 2mg / L, 0.2mg / L RBP urine The samples were tested, and each sample was tested 5 times, and the average value, standard deviation, and coefficient of variation were calculated to investigate the stability of the kit. The results are shown in Table 2 and Table 3. Simultaneously, use the reagent of the invention of this method to contrast foreign certain commodity reagent, adopt Hitachi 7170 automatic biochemical analyzer, measure 50 serum samples simultaneously, carry out correlation analysis to measured value, linear equation is Y=0.98733X-2.89949, correlation The coefficient R=0.98602, see the result figure 2 .

[0084] Table 2 Serum sample addition recovery test

[0085]

[0086] Table 3 Urine sample addition r...

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Abstract

The invention relates to a hybridoma cell capable of secreting an anti retinol binding protein monoclonal antibody, wherein the hybridoma cell has the preservation number of CCTCC No:C2013132. The hybridoma cell has high secreting yield, and the anti retinol binding protein monoclonal antibody obtained by secretion has the advantages of high affinity and high specificity, can be widely applied in the field of retinol binding protein detection, such as the detection reagent or detection equipment preparation fields and the like, and has significant advantages in specificity, sensitivity, detection rate and other aspects compared with a conventional detection method. In addition, the invention also relates to the monoclonal antibody secreted by the hybridoma cell, the hybridoma cell and an application of the monoclonal antibody.

Description

technical field [0001] The invention relates to the field of immune detection of retinol binding protein, in particular to a hybridoma cell capable of secreting anti-retinol binding protein monoclonal antibody and its application. Background technique [0002] At present, the urinary small molecular proteins used clinically for the early diagnosis of renal impairment include β2 microglobulin and RBP (retinol binding protein). β2 microglobulin is very unstable and easy to degrade when the pH is less than 5.5, and the half-life of β2 microglobulin in urine is short, and 70% of it is degraded after 4 hours in the environment of pH 4.0, while RBP is in the same Only 5% degradation under the conditions. Therefore, RBP is more stable and reliable in the diagnosis of renal function injury than β2 microglobulin. [0003] RBP is a low-molecular protein with a molecular weight of about 21KD. It is mainly synthesized by liver cells. Its main function is to transport retinol from the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/28G01N33/68G01N33/577C12R1/91
Inventor 何志强王刘花彭亮刘莉莉
Owner FAPON BIOTECH INC
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