Colloidal gold test strip for simultaneous detection of five staphylococcus aureus enterotoxins and preparation method thereof

A technology of staphylococcal intestinal and colloidal gold test paper, which is applied in the field of immunoassay, can solve the problems of poor detection sensitivity, long time-consuming experiment, high cost, etc., and achieve the effect of broad application prospects

Active Publication Date: 2016-02-24
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Traditional detection methods are time-consuming, costly, and poor in dete

Method used

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  • Colloidal gold test strip for simultaneous detection of five staphylococcus aureus enterotoxins and preparation method thereof
  • Colloidal gold test strip for simultaneous detection of five staphylococcus aureus enterotoxins and preparation method thereof
  • Colloidal gold test strip for simultaneous detection of five staphylococcus aureus enterotoxins and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1. Preparation of monoclonal antibody against Staphylococcus aureus enterotoxin

[0047] 1) Experimental animals: 5 BALB / c mice aged 7 weeks were selected for immunization. 2) Antigen configuration: Dilute the immunogen with physiological saline to prepare a 1 mg / mL solution. 3) Immunization: the first immunization in the first week, 15 μg per mouse, multi-point subcutaneous injection after emulsification of complete Freund’s adjuvant; second immunization, 15 μg per mouse, multi-point subcutaneous injection after emulsification of Freund’s incomplete adjuvant ; In the sixth week, three immunizations were carried out, 7.5 μg per mouse, and multi-point subcutaneous injection after emulsification of Freund's incomplete adjuvant. 4) After the third immunization, the titer was determined by indirect competition method. After the titer reached the requirement, a sprint immunization was carried out. Three days after the immunization, blood was collected from the o...

Embodiment 2

[0048] Example 2. Determination steps of Staphylococcus aureus enterotoxin double-antibody sandwich method:

[0049] a. Coating: Coat the microtiter plate with 6 μg / mL JN2011-01, 100 μL / well, overnight at 4°C;

[0050] b. Washing: Wash the reaction plate three times with PBST, 3 min each time, 200 μL / well, and then dry the reaction plate;

[0051] c. Blocking: CBS containing 0.2% gelatin, 200 μL / well, blocking at 37°C for 2 hours;

[0052] d. Washing: Same as b;

[0053] e. Sample: Dilute the SEA mother solution with PBS to a series concentration of 0.125, 0.25, 0.5, 1, 2, 4, 8ng / mL, and set up a PBS blank control; add 100 μL of sample to each well, and incubate at 37°C for 1 hour;

[0054] f. Washing: Same as b;

[0055] g. Add enzyme-labeled antibody (JN2011-02-HRP, 4 μg / mL), 100 μL / well, incubate at 37°C for 1 hour;

[0056] h. Washing: Same as b;

[0057] i. Color development: add substrate TMB 100 μL / well, and develop color for 15 minutes;

[0058] j. Termination: a...

Embodiment 3

[0060] Embodiment 3. simultaneously detects the preparation method of the colloidal gold test strip of 5 kinds of Staphylococcus aureus enterotoxin A, B, C, D, E:

[0061] a, the synthesis of colloidal gold:

[0062] 30nm gold nanoparticles were synthesized by citric acid reduction method. Add 300mL of 0.01% chloroauric acid solution into the cleaned flask, and heat to complete boiling under magnetic stirring. Quickly add 4.8 mL of 1% trisodium citrate solution to the solution. The solution was boiled for 10 min until the color changed to bright wine red. Cool the flask at room temperature and store at 4°C;

[0063] b. Preparation of gold standard detection antibody:

[0064] Colloidal gold particles were labeled with detection antibodies JN2011-02, 1F6, 1H8, 10F1, 2E3 corresponding to Staphylococcus aureus enterotoxins A, B, C, D, and E, respectively. The specific process is: adjust the pH of 1mL colloidal gold solution to 8 with 0.1M potassium carbonate solution; add 10...

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Abstract

The invention provides a colloidal gold test strip for simultaneous detection of five staphylococcus aureus enterotoxins A, B, C, D and E, and a preparation method thereof, belonging to the technical field of immunoassay. The invention discloses preparation and screening of sandwiched ELISA antibody pairs against the five enterotoxins, and the colloidal gold test strip for simultaneous detection of the five enterotoxins and the preparation method thereof. The test strip is composed of a sample pad, a cellulose nitrate film on which five test lines and one quality control line are sprayed, a water-absorbing pad and a PVC base plate. The five test lines respectively coat capture antibodies corresponding to enterotoxin E, D, C, B and A from front to back, and the quality control line coats a goat anti-mouse IgG secondary antibody. Under the condition of existence of a target object, colloidal gold detection antibodies corresponding to the five gold nanoparticle-labeled enterotoxins are respectively bonded with the target object to form a sandwich structure and develop a color at corresponding test lines. The test strip can realize simultaneous detection of the five enterotoxins in food; a test method is simple, fast and sensitive and has good specificity; and minimum naked-eye detection limits of the five enterotoxins A, B, C, D and E are 5 ng/mL, 5 ng/mL, 5 ng/mL, 2.5 ng/mL and 10 ng/mL.

Description

technical field [0001] The invention relates to a colloidal gold test strip for simultaneously detecting five kinds of Staphylococcus aureus enterotoxins A, B, C, D and E and a preparation method thereof, belonging to the technical field of immune analysis. Background technique [0002] Staphylococcus aureus is a common foodborne pathogen. The bacteria widely exist in nature, and are distributed on human skin, air and water. Food is easily contaminated by Staphylococcus aureus, which can multiply rapidly at a suitable temperature and produce Staphylococcal enterotoxin (Staphylococcal enterotoxin, SE). A small amount of enterotoxin can cause food poisoning in humans. There are many types of Staphylococcus aureus enterotoxins, but according to literature reports, 95% of Staphylococcus aureus food poisoning is caused by SEA, SEB, SEC, SED, and SEE. [0003] Staphylococcus aureus enterotoxin is a worldwide health problem. Food poisoning caused by Staphylococcus aureus enteroto...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N21/78
CPCG01N21/78G01N33/577
Inventor 匡华王文彬胥传来徐丽广马伟刘丽强吴晓玲宋珊珊胡拥明
Owner JIANGNAN UNIV
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