Preparation method of human IgG immunomagnetic bead for staphylococcus aureus enrichment

An immune magnetic bead and staphylococcus technology, which is applied in the preparation of test samples, measuring devices, instruments, etc., can solve the problems of the optimization of separation and other conditions without detailed research, and the stability and enrichment capacity of magnetic beads are not high. , to achieve the effect of shortening the detection time, improving the lower limit of detection, and simple operation

Inactive Publication Date: 2012-04-25
GUANGDONG OCEAN UNIVERSITY
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AI Technical Summary

Problems solved by technology

[0005] At present, the method of isolating human IgG using the principle of specific binding of SPA molecules to human IgG has been relatively mature, while the study of using human IgG immunomagnetic beads to isolate Staphylococcus aureus is only in the preliminary stage. The optimization of conditions such as the isolation of Staphylococcus has not been studied in detail, and the stability and enrichment capacity of the magnetic beads are not high

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0028] 1. Preparation of human IgG immunomagnetic beads:

[0029] 1. Put the magnetic beads into a centrifuge tube, add activation buffer to wash the magnetic beads three times, each time at an interval of 3 minutes, remove the activation buffer, add 200 μL of activator EDC and NHS each, mix well, and incubate at 24°C for 30 minutes. Carry out magnetic bead activation;

[0030] 2. Wash the magnetic beads with coupling buffer three times, with an interval of 3 minutes each time, add human IgG, mix well, and incubate at 24°C for 24 hours to prepare adult IgG immunomagnetic beads;

[0031] 3. Wash the immunomagnetic beads with washing buffer three times, with an interval of 3 minutes each time, incubate with blocking buffer at 24°C for 1 to 2 hours, and store in storage buffer at 4°C until use;

[0032] 4. Treat the sample according to the sample pretreatment method of the national standard method, add human IgG immunomagnetic beads to the sample, mix well, and incubate at 3...

Embodiment 1

[0035] Use a sterile pipette to draw 25mL of liquid food samples into a sterile Erlenmeyer flask filled with 225mL of phosphate buffer saline or saline, and mix well to make a 1:10 homogeneous sample solution. Add the immunomagnetic beads to the sample at an appropriate concentration, mix well and incubate at 37°C for 1 hour. After the action is completed, place the beads on a magnetic stand to capture the beads, remove the supernatant, and dilute them appropriately with sterile water, and then culture them on a 7.5% sodium chloride nutrient agar plate at 37°C for 18 to 24 hours before counting.

Embodiment 2

[0037]Weigh 25g of semi-solid food to be tested into a sterile homogenizing cup of 225mL phosphate buffer or saline, homogenize at 8 000r / min~10 000r / min for 1~2 minutes, and make a 1:10 sample Uniform solution. Add human IgG immunomagnetic beads to the treated sample, mix thoroughly and incubate at 37°C for 1 hour. Place them on a magnetic stand to capture magnetic beads, remove the supernatant, dilute them appropriately with sterile water, and culture them on 7.5% sodium chloride nutrient agar plate at 37°C for 18-24 hours by pouring method, then count them.

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Abstract

The invention relates to a preparation method of a human IgG immunomagnetic bead (IMB) for staphylococcus aureus enrichment. With a magnetic bead as a carrier and human IgG as a recognition intermediate, the human IgG immunomagnetic bead is prepared through the processes of activation, coupling, washing and blocking. The human IgG immunomagnetic bead incubated in an appropriate buffer solution and under certain conditions can be used for high efficiency capture and enrichment of staphylococcus aureuses in a detection sample. The method of the invention has the advantages of strong specificity, high efficiency and rapidity, simple operation, low cost, and has no need for large equipment as well as specially trained professional operation personnel. Also, the sample needs no special pretreatment. Therefore, the method provided in the invention can be widely used for enrichment separation and detection of staphylococcus aureuses in food, feed, cosmetics, environment and clinics.

Description

technical field [0001] The present invention relates to a preparation method and application of immunomagnetic beads, in particular to a preparation method and application of human IgG immunomagnetic beads for enriching Staphylococcus aureus. The preparation method is used to separate pathogenic Staphylococcus aureus cocci ( Staphylococcus aureus ), including a preparation method and an application method of staphylococcus aureus immunomagnetic beads, belonging to the technical field of pathogenic bacteria isolation. Background technique [0002] Immunomagnetic bead-based separation (IMS) is a new immunological technology developed in recent years. The tiny magnetic beads coated with a certain composite organic material can combine with protein antibodies to form antibody-magnetic bead duplexes under certain conditions. When they are added to the pretreated sample, the specific antibody on the magnetic beads can be combined with the corresponding antigen. Combining to form...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/531G01N33/569G01N1/40
Inventor 孙力军王雅玲刘阳徐德峰聂芳红刘唤明
Owner GUANGDONG OCEAN UNIVERSITY
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