Fluorescent microsphere immune test paper card for detecting quinolone antibiotics and preparation and detection methods

A fluorescent microsphere and quinolone technology, applied in the field of immunological detection, can solve problems such as simultaneous detection of blanks, and achieve the effects of cost saving, convenient detection and high stability

Inactive Publication Date: 2018-05-04
洛阳现代生物技术研究院有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no clear records in the literature on how to use fluorescent microsphere immunochromatography to detect or prepare detection products to detect quinolone antibiotic residues, and it is even blank to use one detection product to detect multiple quinolone antibiotics at the same time

Method used

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  • Fluorescent microsphere immune test paper card for detecting quinolone antibiotics and preparation and detection methods
  • Fluorescent microsphere immune test paper card for detecting quinolone antibiotics and preparation and detection methods
  • Fluorescent microsphere immune test paper card for detecting quinolone antibiotics and preparation and detection methods

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Experimental program
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Effect test

Embodiment 1

[0025] Embodiment 1, test strip preparation

[0026] The preparation process includes: the preparation of the fluorescent microsphere-antibody complex, the preparation of the binding pad, the preparation of the sample pad, the preparation of the detection pad and the assembly of the test strip.

[0027] 1. Preparation of fluorescent microsphere-antibody complexes:

[0028] (1) Cleaning: Take 50 μL of fluorescent microspheres into a 1.5 mL centrifuge tube, add 1 mL of 0.01M MES buffer, shake and mix, centrifuge at 15,000 r / min for 15 min, discard the supernatant, add 1 mL of 0.01M MES buffer, and ultrasonically Loose microspheres, repeat this step three times, the purpose of cleaning the microspheres has been achieved,

[0029] (2) Activation: Add 250 μL of EDC solution to 1 mL of microsphere solution after washing, activate in the dark for 2 h, centrifuge at 15,000 r / min for 15 min, discard the supernatant, add 1 mL of 0.01M MES buffer, and disperse the microspheres by ultras...

Embodiment 2

[0049] Embodiment two, the drawing of standard curve

[0050] 1. Configuration of standard products:

[0051] Enrofloxacin standard was serially diluted to 5ppb, 10ppb, 20ppb, 40ppb, 80ppb with 0.1M PBS pH 7.4,

[0052] 2. Reading:

[0053] Take 50 μL of the above-mentioned standard products, and after reacting for 5 minutes, read in the corresponding card slot of the portable fluorescence immunoassay analyzer. The reading results are shown in Table 1 below:

[0054]

[0055] 3. Verification of the calculation method:

[0056] (1) Four-parameter method, after inputting the corresponding parameters of the enrofloxacin curve into the portable fluorescence immunoassay analyzer, select 5ppb and 20ppb to configure the standard within the standard curve range, click the card, react for 5min, and read the two concentrations as 2.454 ppb and 0.885ppb,

[0057] (2) Segmented linearity, after inputting the corresponding parameters of the enrofloxacin standard curve into the porta...

Embodiment 3

[0060] Embodiment three, taking the detection of quinolone antibiotics in meat products as an example, compare the sensitivity of fluorescent microsphere test strips and colloidal gold test strips

[0061] 1. Homogenize fresh pork samples with a homogenizer;

[0062] 2. Weigh two 5.0±0.05g crushed samples into two 50mL polystyrene centrifuge tubes, add 25ml of 70% ethyl acetate respectively, add 10ng enrofloxacin to tube 2, shake vigorously for 5min with a shaker;

[0063] 3. Place the evenly oscillated sample in a centrifuge and centrifuge at 6000r / min for 5min;

[0064] 4. Take 1mL of centrifuged supernatant and dry it with a nitrogen blower. After drying, accurately add 1mL of sample diluent to fully dissolve the condensate to obtain the sample extract; the sample diluent is pH7.4, 0.05mol / L PBS;

[0065] 5. Dilute with the sample diluent at a ratio of 1:9 (for example: 100 μL sample extract + 900 μL sample diluent);

[0066] 6. Take 50 μL of the diluted liquid in tube 1...

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Abstract

The invention relates to a fluorescent microsphere immune test paper card for detecting quinolone antibiotics and preparation and detection methods and belongs to the field of immunological detection.The test paper card comprises a card shell and a test paper strip. The test paper strip comprises a bottom plate, and a water absorption pad, a detection pad, a combining pad and a sample pad successively overlapped and adhered to the bottom plate; the detection pad is a nitrocellulose membrane provided with a quality control line C and a detection line T, the quality control line C is coated with a goat anti mouse monoclonal antibody and the detection line T is coated with an enrofloxacin-bovine serum albumin conjugate; the combining pad is a glass cellulose membrane of the enrofloxacin monoclonal antibody embedded with a time-resolved fluorescent microsphere label; the sample pad is a dried glass cellulose membrane immersed in a sample treatment liquid; the bottom plate is a PVC plate which does not absorb water, and the water absorption pad is water absorbing filter paper. The test paper card prepared is good in stability, high in sensitivity and high in specificity, can detect 16quinolone antibiotics, and can achieve the purpose of quantitative and batch detection of enrofloxacin quickly through fluorescence signals.

Description

technical field [0001] The invention belongs to the field of immunological detection, and mainly relates to a fluorescent microsphere immunological test paper card for detecting quinolone antibiotics, a preparation method and a detection method. Background technique [0002] Quinolones antibiotics are a class of general-purpose drugs for humans and animals. They target bacterial DNA superhelicase and prevent the enzyme from supercoiling, causing irreversible damage to bacterial DNA and preventing bacterial cells from dividing. Because of its broad-spectrum antibacterial properties, strong antibacterial activity, no cross-resistance with other antibacterial drugs, and low toxicity and side effects, it is widely used in the prevention and treatment of animal diseases in animal husbandry, aquaculture and other aquaculture industries. With the continuous expansion of the scale of animal breeding, the blind and excessive use of antibiotics in the breeding process will lead to the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/58G01N33/558G01N33/533
CPCG01N33/9446G01N33/533G01N33/558G01N33/582G01N33/585
Inventor 李秀梅杨志耿玉静张小飞杨二霞姬应宾
Owner 洛阳现代生物技术研究院有限公司
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