Colloidal gold immunological test paper for detecting paeniBacillus larvae of bee, preparation and applications thereof

A technology of bacillus and bee larvae, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of unfavorable rapid detection, time-consuming detection methods, and high technical requirements for operation, and achieve easy promotion and use, low cost, and detection repeatability Good results

Inactive Publication Date: 2018-04-13
COMPREHENSIVE TECH SERVICE CENT YILI ENTRY EXIT INSPECTION & QUARANTINE BUREAU +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problems in the prior art that the detection method of honeybee larval bacillus takes a long time, requires high operating technology, and is not conducive to rapid detection on the spot, the present inv

Method used

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  • Colloidal gold immunological test paper for detecting paeniBacillus larvae of bee, preparation and applications thereof
  • Colloidal gold immunological test paper for detecting paeniBacillus larvae of bee, preparation and applications thereof
  • Colloidal gold immunological test paper for detecting paeniBacillus larvae of bee, preparation and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0036] Example 1: Purification of PLMP polyclonal antibody

[0037] 1. Serum pretreatment

[0038] Use a clean syringe to draw 6 mL of rabbit serum and filter it with a 0.45 μm filter membrane.

[0039] 2. Caprylic acid precipitates non-IgG protein

[0040] Take 50 mL of autoclaved centrifuge tube, add 24 mL of acetic acid buffer and 6 mL of rabbit serum filtered by 0.45μm membrane, shake and mix, and adjust the pH to 4.5 with 1mol / L NaOH. Add 2.25 mL of n-octanoic acid, stir at room temperature for 30 min, centrifuge at 10000 rpm for 20 min, and discard the precipitate. Syringe to suck the remaining liquid part, after filtering with 0.45μm filter membrane, add 1 / 10 volume of 0.01mol / L PBS solution (PH=7.4), then use 1mol / L NaOH to adjust the pH to 7.4, and place in an ice bath for follow-up experiment.

[0041] 3. Ammonium sulfate precipitation of IgG

[0042] In an ice bath environment, add an equal volume of saturated ammonium sulfate to the above solution, stir and mix for 30 minu...

Example Embodiment

[0043] Example 2: Preparation of gold label pad

[0044] 1. Determination of the optimum pH value of colloidal gold

[0045] This test is to add 0.2mol / L K 2 CO 3 The amount is the standard. Take 10 1.5mL centrifuge tubes and mark them with numbers, and add 1mL of colloidal gold solution to each centrifuge tube. Add a certain amount of 0.2mol / L K to each centrifuge tube 2 CO 3 Mix thoroughly (as shown in Table 3-2), then add 30 μg of purified polyclonal antibody to each tube, mix again, and let stand at room temperature for 30 min. Slowly add 20 μL of 10% NaCl solution to each centrifuge tube, mix well and let stand at room temperature for 2 hours. Observe the color change of 10 centrifuge tubes and the appearance of black particles that have precipitated and aggregated. Choose the K of the tube that starts without gathering 2 CO 3 The amount of solution added is the optimum K of 0.2mol / L for colloidal gold in this test 2 CO 3 The amount of addition is the optimum pH value. Use...

Example Embodiment

[0059] Example 3: Preparation of detection line and quality control line

[0060] 1. Determination of the concentration of the coating antibody of the detection line

[0061] On the NC membrane of the test line, the concentrations diluted with PBS were respectively 0.4mg / mL, 0.6mg / mL, 0.8mg / mL, 1.0mg / mL, 1.2mg / mL, 1.4mg / mL, 1.6mg / mL, 1.8mg / mL of 1μLPLMP polyclonal antibody, on the NC membrane of the quality control line, drop 1.0μL of goat anti-rabbit IgG antibody diluted with PBS to a concentration of 1.0mg / mL. Use 10μg / mL purified PLMP protein for detection, observe the color development of the detection line, and determine the optimal concentration of coating antibody.

[0062] When the C line was coated with 1.5μL goat anti-rabbit IgG antibody with a concentration of 1mg / mL, 8 concentration gradients were set up for the PLMP protein polyclonal antibody, and the PLMP protein was used for detection. The color becomes lighter at 0.4mg / mL, and the color gradually becomes darker wh...

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Abstract

The invention discloses colloidal gold immunological test paper for detecting the paeniBacillus larvae of bee, preparation and applications thereof. The colloidal gold immunological test paper specifically comprises a sample pad, a gold standard pad, a nitrocellulose membrane, a water absorption pad and a bottom plate, wherein the gold standard pad contains colloidal gold-labeled PLMP protein polyclonal antibody, and the nitrocellulose membrane has a PLMP protein polyclonal antibody pre-coated detection line and a goat anti-rabbit IgG solution pre-coated quality control line. According to thepresent invention, the prepared colloidal gold immunological test paper has advantages of strong specificity, good sensitivity, good detection repeatability and stable performance, and provides the negative results for negative bacteria liquids, wherein the specificity can be 100%, the minimum detection concentrations of the bacterial liquid and the positive sample of the paeniBacillus larvae respectively are 10<5> CFU/mL and 10<6> CFU/mL, and the preservation period of the colloidal gold immunological test paper is 3 months.

Description

technical field [0001] The present invention mainly relates to the technical field of molecular biology, specifically, the present invention relates to the technical field of polyclonal antibody preparation. Background technique [0002] American foulbrood (AFB) is an acute and severe infectious disease of bee larvae and bee pupae caused by Paenibacilus larvae (P. larvae). The main pathogenic factors of Bacillus larvae are some extracellular secreted proteases. Studies have shown that these proteases are zinc-containing metalloproteases (Paenibacilus larvae metaloproteases, PLMP) and are involved in larval degeneration. The disease is serious, destructive and only infects bee larvae. In the early stage of infection, the body color of the larvae changes from normal pearly white to brownish-yellow, brown or even dark brown. The final corpse can pull out 2cm-3cm filaments, giving off a fishy smell, and finally clings to the nest wall with dark brown scales. P.larvae spores ar...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/558G01N33/532
CPCG01N33/532G01N33/558G01N33/56911G01N2333/32
Inventor 王振宝季新成员丽娟何晓杰雷程红葛婷叶尔保勒叶尔兰·阿不都买金阿斯喀·夏热甫汉哈森
Owner COMPREHENSIVE TECH SERVICE CENT YILI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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