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Fluorescent microsphere detection card for quantitative determination of beta-lactamase residues in milk

A lactamase and quantitative detection technology, applied in the biological field, can solve the problems of long detection time and high detection cost, and achieve the effects of detection sensitivity, high specificity and high detection sensitivity

Active Publication Date: 2018-08-24
SHANDONG LVDU BIO SICIENCE & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The above quantitative detection methods all have the disadvantages of long detection time, some need large-scale instrument auxiliary test, and high detection cost.

Method used

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  • Fluorescent microsphere detection card for quantitative determination of beta-lactamase residues in milk
  • Fluorescent microsphere detection card for quantitative determination of beta-lactamase residues in milk
  • Fluorescent microsphere detection card for quantitative determination of beta-lactamase residues in milk

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Preparation of paired monoclonal antibodies against β-lactamase

[0047] 1.1 Immunization program:

[0048] Mix β-lactamase (sigma, product number P0389) with 501 adjuvant (Shandong Lvdu Biotechnology Co., Ltd., product number LD003) at a volume ratio of 1:1, and treat 8-10-week-old Balb / c mice ( (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) for immunization, leg intramuscular injection, 50 μg / animal, and then immunized once every two weeks, with the same dose and site as above; after 5 immunizations, booster immunization, intraperitoneal injection, without adjuvant dose, double the dose;

[0049] 1.2 Cell Fusion:

[0050] The splenocytes in Balb / c mice were mixed with the myeloma cells in the peritoneal cavity at a ratio of 20:1, centrifuged at 800 rpm for 7 minutes, the supernatant was discarded, 1 mL of 50% PEG (W / W) was added within 30 seconds, and statically Set aside for 1 min, slowly add 1 mL of serum-free DMEM medi...

Embodiment 2

[0056] Example 2: Preparation of rapid detection card for quantitative detection of β-lactamase residues

[0057] 2.1 Preparation of fluorescent microspheres with anti-β-lactamase monoclonal antibody for detection

[0058] Add 10 μl of 20% Tween-20 solution, 2 mg of 13E4 anti-β-lactamase monoclonal antibody purified on SPA column and 20 μl NaCNBH 3 (25 mg / mL, prepared in 0.1 M MES buffer solution pH6.0, ready to use), 37 ° C for 2 h in the dark; add 300 μl N,O-bistrimethylsilylacetamide (100 0.1 M pH 6.0 MES buffer) solution, 37°C for 6 h in the dark; centrifuge at 12,000 rpm for 30 minutes at 4°C; discard the supernatant and wash twice with 10 mL of pH 6.0 MES buffer; The detection microsphere suspension preservation solution is suspended, and the formula is composed of 0.01M Tris buffer at pH 8.5, containing 3% sucrose, 10% bovine serum albumin, 0.5% polyethylene glycol 20000, and 0.1% sodium azide.

[0059] Microsphere Pad Treatment:

[0060] Polyester 6613 was chosen a...

Embodiment 3

[0067] Example 3: Performance determination of the detection card for quantitative detection of β-lactamase residues

[0068] 3.1 Determination of the minimum detection limit

[0069] Take 3 batches of test cards, and each batch of test cards detects blank milk, 0.3×10 -6 U / mL(U / g), 1×10 -6 U / mL(U / g), 2×10 -6U / mL (U / g) standard addition sample, 3×10 -6 U / mL (U / g) standard add sample to determine the minimum detection limit of the test card, when the enzyme content is greater than or equal to 1×10 -6 When U / mL (U / g), the card reader displays the value, so the minimum detection limit of the test card is 1×10 -6 U / mL (U / g).

[0070] Table 1: Test strip sensitivity test results

[0071]

[0072] 3.2 False positive rate, false negative rate

[0073] False positives are the results of experiments showing positive results due to the interference of certain conditions on samples that were originally negative. Otherwise it is a false negative. According to relevant regulati...

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Abstract

The invention relates to a fluorescent microsphere detection card for quantitative determination of beta-lactamase residues in milk and a preparation method thereof. The method comprises the followingconcrete steps: 1) preparation of an anti-beta-lactamase pairing monoclonal antibody, comprising immune procedure, cell fusion, hybridomas screening and cloning, and antibody preparation and purification; and 2) the fluorescent microsphere quantitative detection card for beta-lactamase residues and its preparation method, comprising preparation of the fluorescent microsphere anti-beta-lactamase monoclonal antibody, microspheres pad treatment, membrane spraying, C and T line determination, and performance determination. The lowest detection limit of the beta-lactamase residues in milk is 1*10<-6> U / mL (U / g), a milk sample is directly detected, any sample processing is not required, and the beta-lactamase residues in the milk realize quantitative determination in a rapid, sensitive and accurate mode.

Description

technical field [0001] The invention belongs to the field of biological technology and the technical field of quantitative detection of food safety, in particular to a fluorescent microsphere quantitative detection card for semi-quantitative rapid quantitative detection of β-lactamase residue in milk and a preparation method thereof. Background technique [0002] β-lactamase (β-lactamase) is an enzyme produced by bacteria that can hydrolyze β-lactam ring antibiotics. The production of β-lactamase is the most common mechanism of bacterial resistance to (β-lactam) antimicrobials, and it is widely involved in many important pathogens of community-acquired infections and nosocomial infections. Among various drug-resistant mechanisms Accounted for 80%. Because β-lactamase can decompose the residual β-lactam antibiotics in milk and cover up traces of antibiotics, it is included in the list of "non-edible substances that may be illegally added to food", and China does not allow it...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/573G01N33/558
CPCG01N33/558G01N33/573G01N33/577G01N2333/986
Inventor 武玉香唐世云沈志强
Owner SHANDONG LVDU BIO SICIENCE & TECH
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