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Hybridoma cell DBP-4E10 and dibutyl phthalate monoclonal antibody produced by hybridoma cell DBP-4E10

A DBP-4E10, dibutyl phthalate technology, applied in microorganisms, biological testing, biochemical equipment and methods, etc., can solve the problems of complex processing process, time-consuming operation, expensive analysis cost, etc., and achieve good specificity. , the effect of high sensitivity

Active Publication Date: 2015-06-17
HOPE ANALYTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the pretreatment process of this type of analysis method system to separate and extract the pollutants to be measured from the sample and remove the interference of impurities is very complicated, time-consuming, and expensive to analyze.

Method used

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  • Hybridoma cell DBP-4E10 and dibutyl phthalate monoclonal antibody produced by hybridoma cell DBP-4E10

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Screening of hybridoma cell line DBP-4E10

[0022] 1. Antigen synthesis and animal immunization

[0023] 4-Nitrophthalic anhydride undergoes esterification reaction with n-butanol under raw conditions to obtain 4-nitrophthalate dibutyl, and then undergoes reduction reaction with zinc powder under acidic conditions to obtain hapten derivatives 4-aminophthalate dibutyl ester, phthalate hapten derivatives with aniline react with NaNO2 and concentrated HCl at low temperature to generate corresponding diazonium salts; The salt is coupled with the carrier protein to obtain the corresponding artificial antigen.

[0024] Synthesis of 4-nitrophthalate dibutyl

[0025] Dissolve 2.5 grams of 4-nitrophthalic acid in 50 mL of n-butanol, slowly drop 1 mL of concentrated H2SO4 into it as a catalyst, and react at 120 ° C with constant stirring. After the reaction, wash three times with 10% NaCO3, 10 mL each time. Butanol and water were removed by rotary evaporation at 80...

Embodiment 2

[0041] Example 2: Preparation, purification and identification of antibodies

[0042] The obtained hybridoma cell line DBP-4E10 obtained in Example 1 was injected into BALB / c mice pre-treated with Freund's incomplete adjuvant, the ascites of the mice was collected, purified and treated to obtain anti-phthalic acid Monoclonal antibody to dibutyl ester.

[0043] The specific operation is as follows: centrifuge the ascites at 3000r / min at 4°C for more than 5min, absorb the supernatant, mix the supernatant with 2 times the volume of acetate buffer, and slowly add n-octanoic acid while stirring. The volume was 33 μL, mixed at room temperature for 30 minutes, and allowed to stand at 4°C for 2 hours to fully precipitate. Then centrifuge at 12000r / min at 4°C for 30min, discard the precipitate, filter the obtained supernatant with a sand core funnel, add 1 / 10 of the filtrate volume of 0.1mol / L pH7.4 phosphate buffer, add 2 mol / L Adjust the pH to 7.4 with sodium hydroxide solution, pr...

Embodiment 3

[0048] Embodiment 3: the application of antibody

[0049] The anti-dibutyl phthalate monoclonal antibody secreted by the hybridoma cell line DBP-4E10 was used in the dibutyl phthalate monoclonal ELISA addition recovery test, and the specific steps were as follows:

[0050] (1) Coating: use a 96-well polystyrene microplate plate, dilute DBP-BSA to 1 μg / mL with 0.05mol / L pH 9.6 carbonic acid buffer, and use a multi-channel adjustable pipette gun (referred to as the discharge gun) to Add 100 μL to each well, cover the microtiter plate with plastic wrap, and place it at 4°C overnight.

[0051] (2) Plate washing: The next day, take the microplate out from 4°C, wash with PBST, 250 μl per well, wash 4 times, and pat dry on a towel after washing.

[0052] (3) Sealing: add 200 μL of 1% BSA PBS to each well with a row gun, cover the microplate plate with a lid after adding, and place it at 37°C for 2 hours. Wash with PBST, 250 μl per well, wash 4 times, and pat dry on a towel after wa...

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Abstract

The invention provides a hybridoma cell DBP-4E10, an anti-dibutyl phthalate DBP monoclonal antibody secreted by hybridoma cell DBP-4E10 and an application thereof. The hybridoma cell strain DBP-4E10 is used for preparing the dibutyl phthalate DBP antibody with high potency, titer measured by an enzyme linked immunosorbent assay (ELISA) of mouse ascites antibody can reach 3.12*10<6>; the anti-dibutyl phthalate DBP monoclonal antibody has high sensitivity, has 50% inhibition concentration IC50 of 0.22 mg / mL on the dibutyl phthalate DBP, and used for determining the content of dibutyl phthalate DBP, and reality application value is large.

Description

technical field [0001] The invention relates to a hybridoma cell line DBP-4E10 and the dibutyl phthalate monoclonal antibody produced therefrom. Background technique [0002] Phthalate esters (PAEs) are a class of environmental hormones that have estrogenic activity, interfere with the normal function of the endocrine system, cause mutations, distortions, and cancer cell proliferation. In 2005, the European Union assessed dioctyl phthalate (DEHP), dibutyl phthalate (DBP) and butyl benzyl phthalate (BBP) as "secondary reproductive toxicity". Among them, DBP is considered to be the most toxic phthalate compound. [0003] PAEs compounds are the main plasticizers and softeners in the plastics industry, and are widely used in the manufacture of food packaging materials, containers, medical supplies and plastics. Due to their wide application and release from various products, phthalates have penetrated into almost every corner of our lives. Studies have shown that the daily in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/44G01N33/74G01N33/577
Inventor 时国庆孙清邓乾民栗时锋高永杰曹雨
Owner HOPE ANALYTECH INC
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