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Preparation method for rat-derived polyclonal antibody of thermoduric bacteria

A polyclonal antibody and polyclonal antiserum technology, which is applied in the preparation methods of peptides, chemical instruments and methods, antibacterial immunoglobulins, etc. lock and other problems, to achieve the effect of high purity, good specificity and simple method

Inactive Publication Date: 2012-05-02
SHAANXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, manufacturers mostly use the traditional plate culture counting method to detect heat-resistant bacteria. The biggest disadvantage of the plate culture counting method is that it takes a long time, and the general detection cycle is 4 to 5 days.
The PCR method has the advantages of rapidity and specificity, but it has high requirements for equipment, laboratory conditions and operators, and the steps are complicated, so it is difficult to popularize and apply

Method used

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  • Preparation method for rat-derived polyclonal antibody of thermoduric bacteria
  • Preparation method for rat-derived polyclonal antibody of thermoduric bacteria
  • Preparation method for rat-derived polyclonal antibody of thermoduric bacteria

Examples

Experimental program
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Embodiment 1

[0018] 1. Preparation of thermostable bacteria suspension

[0019] Transfer the standard strain of heat-resistant bacteria (A.acidoterrestris DSM 3922) to 250 mL of 402 medium, place it in an air bath at 45°C for shaking culture for 8 hours, transfer it to Kjeldahl medium, and cultivate it at 41°C for 24 hours, and use Rinse the heat-resistant bacteria cultured on the Kjeldahl medium with sterile saline, transfer the washed heat-resistant bacteria to a sterile conical flask, mix well, centrifuge at 3500 rpm for 30 minutes, discard the supernatant, and use for precipitation. Wash with normal saline for 3 times, add sterile normal saline to the precipitate, and dilute to a concentration of 2×10 9 CFU / mL of thermostable bacterial suspension.

[0020] 2. SD rats were immunized with thermostable bacteria

[0021] Mix 2mL of heat-resistant bacteria suspension and 2mL of immune adjuvant evenly, ultrasonically oscillate until fully emulsified, and immunize male SD rats by multi-poin...

Embodiment 2

[0027] In the step 1 of preparing the thermostable bacteria suspension in Example 1, the precipitate was diluted with sterile physiological saline to a concentration of 1.4×10 8 CFU / mL of heat-resistant bacterial suspension; in step 2 of heat-resistant bacteria immunized SD rats, the concentration of 2mL was 1.4×10 8 Mix CFU / mL heat-resistant bacteria suspension with 2mL immune adjuvant evenly, ultrasonically oscillate until fully emulsified, and immunize male SD rats by multi-point injection through the back muscle. Complete adjuvant, and then 5 booster immunizations with Freund's incomplete adjuvant, the time interval between each immunization was two weeks, the eyeballs were removed for 77 days after immunization, and blood was collected to prepare murine polyclonal antiserum for heat-resistant bacteria. Other steps are identical with embodiment 1.

Embodiment 3

[0029] In the step 1 of preparing the thermostable bacteria suspension in Example 1, the precipitate was diluted with sterile physiological saline to a concentration of 4 × 10 9 CFU / mL of heat-resistant bacteria suspension; in step 2 of heat-resistant bacteria immunized SD rats, the concentration of 2mL was 4×10 9 Mix the CFU / mL heat-resistant bacteria suspension with 2mL immune adjuvant evenly, ultrasonically oscillate until fully emulsified, and immunize male SD rats through multi-point injections in the back muscles. The volume of the immunogen is 0.5mL / rat. Complete adjuvant, and then 6 booster immunizations with Freund's incomplete adjuvant, the time interval between each immunization was two weeks, the eyeballs were removed for 91 days after immunization, and blood was collected to prepare murine polyclonal antiserum for thermostable bacteria. Other steps are identical with embodiment 1.

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Abstract

The invention relates to a preparation method for a rat-derived polyclonal antibody of thermoduric bacteria. The preparation method comprises the steps of: culturing standard strains of the thermoduric bacteria in a culture medium to prepare a 1.4*10<8>-4*10<9> FU / mL thermoduric bacteria suspension, mixing the thermoduric bacteria suspension with an immunological adjuvant at an equal volume, ultrasonically oscillating until sufficient emulsification, immunizing male SD rat through back muscle multipoint injection, excising eyeballs for blood sampling 63-91 days after immunization, preparing rat-derived polyclonal antiserum of the thermoduric bacteria, and respectively carrying out separation and purification on the rat-derived polyclonal antiserum of the thermoduric bacteria by adopting a saturated ammonium sulfate precipitation method and G protein affinity chromatography to prepare the rat-derived polyclonal antibody of the thermoduric bacteria. The preparation method provided by the invention is simple and low in cost; and the prepared rat-derived polyclonal antibody of the thermoduric bacteria has titer up to 12800, excellent specificity, no cross reaction with common food-borne microorganisms and higher purity identified by electrophoresis, and can be used for building an immunochemical detection method of the thermoduric bacteria.

Description

technical field [0001] The invention relates to the field of food microorganism detection, in particular to the preparation, purification and identification of murine polyclonal antibodies of heat-resistant bacteria. Background technique [0002] Heat-resistant bacteria, scientific name Aliyclobacillua acidoterrestris, is a common harmful bacteria in fruit juice production. Due to its heat-resistant and acid-resistant properties, it can survive the pasteurization process in acid fruit juice processing. , when the dormant spores are under suitable temperature conditions, they will rapidly proliferate and cause juice spoilage, seriously affecting the quality and safety of fruit juice. Therefore, thermotolerant bacteria are one of the most important target control microorganisms in the production of concentrated fruit juice. Regarding the detection of heat-resistant bacteria in fruit juice, it is currently divided into the following three categories: traditional plate culture ...

Claims

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Application Information

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IPC IPC(8): C07K16/12C07K16/06C07K1/36C07K1/30C07K1/22
Inventor 李建科夏凯
Owner SHAANXI NORMAL UNIV
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