Method for separating and enriching sperms by adopting IgY antibody immunomagnetic beads

Abstract

The invention relates to the technical field of sperm enrichment, in particular to a method for separating and enriching sperms by adopting IgY antibody immunomagnetic beads, which comprises the steps of sample carrier separation and sperm enrichment, and is characterized in that the sample carrier separation comprises the steps of digestion, low-position centrifugation and high-position centrifugation, and the sperm enrichment comprises the steps of immunomagnetic bead combination, magnetic separation and cleaning. The immunomagnetic bead coupled with the IgY antibody has the characteristics of strong specificity, high temperature resistance and acid and alkali resistance, belongs to a food-grade safety reagent, and is low in production cost, complete DNA can be detected only by injecting the immunomagnetic bead coupled with the IgY antibody for immunization of a hen for multiple times, separating and purifying the antibody after regular egg taking, wrapping the immunomagnetic bead coupled with the IgY antibody and tightening sperms, and 30 sperms. Compared with the prior art, an obvious experiment effect is achieved, laboratory operation is more convenient, the immunomagnetic beads can be combined with an instrument to achieve automatic operation, and sperm enrichment operation of mixed spot detection materials is greatly simplified.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of sperm enrichment, in particular to a method for separating and enriching sperm by using IgY antibody immunomagnetic beads. BACKGROUND

[0002] The role of forensic DNA analysis technology in criminal justice practice is increasingly prominent. DNA testing of mixed stains is often encountered. The carrier type of mixed stain samples is relatively complex. Whether the DNA of sperm cells in the mixed stain can be successfully tested depends on whether the sperm cells can be separated and enriched. The individual source identification of sperm is the key to case investigation and litigation.

[0003] Traditional differential lysis method is used to separate sperm cells, which requires multiple washing and centrifugation, long experimental time, complicated steps, high requirement for pipetting skill during operation, easy loss of sperm, resulting in experimental failure. Especially when a large number of female vaginal epithelial cells exist and the number of sperm cells is small, it is difficult to obtain complete and single sperm cell DNA typing. Sometimes even if the typing result is obtained, it is mostly mixed typing. In practice, when a large number of samples are faced, the enrichment of sperm cells in a short time is often required. However, there is an imbalance between the consumption time and the extraction efficiency. Therefore, the method for separating and enriching sperm by using IgY antibody immunomagnetic beads is proposed to solve the above problems. SUMMARY

[0004] The application aims to provide a method for separating and enriching sperm by using IgY antibody immunomagnetic beads, so as to solve the problem of imbalance between consumption time and extraction efficiency when a large number of samples are faced in practice.

[0005] To achieve the above purpose, the application provides the following technical scheme: A method for separating and enriching sperm by using IgY antibody immunomagnetic beads, comprising sample carrier separation and sperm enrichment, characterized in that: the sample carrier separation comprises digestion, low-speed centrifugation and high-speed centrifugation, and the sperm enrichment comprises immunomagnetic bead combination, magnetic separation and washing. The enrichment method comprises the following steps: Step 1: digestion, moving the inner tube of the new type of stepped sleeve upward, so that the inner tube is arranged at the high position of the outer tube, finally selecting an appropriate amount of sample and placing it in the inner tube, adding separation buffer and closing the cover, moving the inner tube downward, at this time the inner tube is arranged at the low position of the outer tube, at this time the sample is soaked and incubated by the separation solution. Step two: centrifugation, the inner tube is in the low position of the outer tube, then the new stepwise sleeve is put into the centrifuge for centrifugation, realizing low position centrifugation, after low position centrifugation, the inner tube is moved upward, and the inner tube is placed in the high position of the outer tube, the new stepwise sleeve is put into the centrifuge for centrifugation, realizing high position centrifugation, after high position centrifugation, the inner tube is taken out, the inner tube is discarded with the test material, and the mixed liquid in the outer tube is reserved for use; Step three: immune magnetic bead combination, immune magnetic beads are added into the outer tube of the new stepwise sleeve in step three, and then mixed; Step four: magnetic separation, the outer tube of the new stepwise sleeve in step four is placed in a magnetic separator, and after standing, the liquid is sucked; Step five: separation buffer is added into the outer tube of the new stepwise sleeve, mixed by oscillation, then the outer tube is put into the magnetic separator, and after standing, the liquid is sucked, at this time, the magnetic bead-sperm complex adsorbed on the inner side wall of the outer tube can be used for subsequent sperm DNA extraction.

[0006] Preferably, the separation liquid added in step one is 1ml, the incubation temperature is 56℃, and the incubation time is 0.5-1h.

[0007] Preferably, the amount of separation buffer in step one is suitable for completely soaking the test material.

[0008] Preferably, in step two, both high position centrifugation and low position centrifugation are 20000g centrifugation for 3min, and a large-pore U-shaped design is used for the filter column during centrifugation, realizing efficient separation of epithelial cell DNA solution and sperm cells from the test material.

[0009] Preferably, in step two, 100μL of the mixed liquid can be taken out for PSA and sperm cell detection.

[0010] Preferably, in step three, the surface of the immune magnetic beads is coupled with specific sperm antibodies, namely IgY antibodies, which only react immunologically with sperm in the mixed liquid to form a complex.

[0011] Preferably, in step three, 20μL of immune magnetic beads is added, and the mixing temperature is 37℃, and the mixing time is 30-40min.

[0012] Preferably, in step four, the outer tube needs to be placed in the magnetic separator for 1min, and the immune magnetic beads need to be prevented from being sucked away during the sucking process.

[0013] Preferably, the washing of the magnetic beads in step five needs to be washed three times, each time 1ml of separation buffer needs to be added, the mixing time is 1min, the outer tube is placed in the magnetic separator for 1min, and the immunomagnetic beads need to be prevented from being sucked away during the suction process.

[0014] Preferably, the preparation method of the immunomagnetic beads is to immunize hens with human sperm cells to produce IgY antibodies, precipitate the antibodies by the polyvinyl alcohol method, and then couple the purified antibodies with magnetic beads to obtain the immunomagnetic beads.

[0015] Compared with the prior art, the application has the following beneficial effects: In the application, the IgY antibody-coupled immunomagnetic beads have the characteristics of strong specificity, high temperature resistance, and acid and alkali resistance, and are food-grade safe reagents with low production cost. Only a few times of injection of the hens, periodic collection of the eggs, separation and purification of the antibodies, and coupling of the IgY antibody-coupled immunomagnetic beads are needed to coat the sperm firmly, and 30 sperm can be used to test the complete DNA, thereby obtaining a relatively obvious experimental effect, which is more convenient for laboratory operation. The immunomagnetic beads can also be combined with instruments to realize automatic operation, greatly simplifying the sperm enrichment operation of mixed stain samples. In addition, the method using the immunomagnetic beads can also be combined with instruments to realize automatic operation, saving manpower, reducing the error of personnel operation, and improving the laboratory work efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The operation flowchart of the application. DETAILED DESCRIPTION

[0017] Please refer to Figure 1 The application provides a technical solution: A method for separating and enriching sperm by using IgY antibody immunomagnetic beads, which comprises sample carrier separation and sperm enrichment. The sample carrier separation comprises digestion, low-speed centrifugation and high-speed centrifugation. The sperm enrichment comprises immunomagnetic bead combination, magnetic separation and washing. The enrichment method comprises the following steps: Step one: digestion, move the inner tube of the new type of stepped sleeve upward, so that the inner tube is placed at the high position of the outer tube, then select an appropriate amount of sample and place it in the inner tube, add separation buffer and close the cover, then move the inner tube downward, so that the inner tube is arranged at the low position of the outer tube, and then soak and warm the sample in the separation buffer; Step two: centrifugation, place the inner tube at the low position of the outer tube, then place the new type of stepped sleeve in the centrifuge for centrifugation to realize low-speed centrifugation. After the low-speed centrifugation is completed, move the inner tube upward so that the inner tube is placed at the high position of the outer tube, then place the new type of stepped sleeve in the centrifuge for centrifugation to realize high-speed centrifugation. After the high-speed centrifugation is completed, take out the inner tube, and discard the inner tube with the sample, and reserve the mixed liquid in the outer tube for use. Step three: immune magnetic beads binding, add immune magnetic beads in the outer tube of the new step three ladder type sleeve, and then mix well; Step four: magnetic separation, place the outer tube of the new step four ladder type sleeve in the magnetic separator, and after standing, the liquid is sucked; Step five: add separation buffer in the outer tube of the new step five ladder type sleeve, mix well by shaking, then place the outer tube in the magnetic separator, and after standing, the liquid is sucked. At this time, the magnetic bead-sperm complex adsorbed on the inner side wall of the outer tube can be used for subsequent sperm DNA extraction.

[0018] The new step ladder type sleeve used in the present application is a new two-step ladder type centrifugal filtration sleeve with application number "202321377062.5". The separation liquid added in step one is 1ml, the incubation temperature is 56℃, and the incubation time is 0.5-1h. The amount of separation buffer used in step one is preferably completely soaked in the sample. In step two, both high-speed centrifugation and low-speed centrifugation are 20000g centrifugation for 3min. During the centrifugation process, the filter column adopts a large-pore U-shaped design to realize efficient separation of epithelial cell DNA solution and sperm cells from the sample. In step two, 100μL of the mixed solution can be taken out for PSA and sperm cell detection. In step three, the surface of the immune magnetic beads is coupled with specific sperm antibodies, namely IgY antibodies. The antibodies only react immunologically with sperm in the mixed solution to form a complex. In step three, 20μL of immune magnetic beads are added, and the mixing temperature is 37℃, and the mixing time is 30-40min. In step four, the magnetic separation requires that the outer tube be placed in the magnetic separator for 1min. At the same time, during the suction process, the immune magnetic beads need to be prevented from being sucked away. In step five, the magnetic beads need to be washed three times. Each time, 1ml of separation buffer is added, mixed well by shaking for 1min, and the outer tube is placed in the magnetic separator for 1min. At the same time, during the suction process, the immune magnetic beads need to be prevented from being sucked away. The preparation method of the immune magnetic beads is as follows: immunize hens with human sperm cells to produce IgY antibodies, precipitate the antibodies by polyvinyl alcohol method, purify the antibodies, and then couple the antibodies with magnetic beads to obtain immune magnetic beads.

[0019] The principles and implementations of the present application are described herein with specific examples. The above examples are only used to help understand the method and its core idea of the present application. The above description is only the preferred embodiments of the present application. It should be pointed out that due to the limited nature of the language expression, there are objectively infinite specific structures, and for ordinary skilled persons in the art, without departing from the principles of the present application, a number of improvements, refinements or changes can be made, or the above technical features can be combined in an appropriate manner; these improvements, refinements, changes or combinations, or without improvement, directly apply the concept and technical solution of the present application to other occasions, should be regarded as the protection scope of the present application.

Claims

1. A method for separating and enriching spermatozoa by using IgY antibody immunomagnetic beads, comprising sample carrier separation and spermatozoa enrichment, characterized in that: The sample carrier separation includes digestion, low-speed centrifugation and high-speed centrifugation, and the sperm enrichment includes immunomagnetic bead binding, magnetic separation and cleaning. ​ The enrichment method comprises the following steps: Step one: digestion, moving the inner tube of the new stepped sleeve upward to place the inner tube at the high position of the outer tube, finally selecting an appropriate amount of sample and placing it in the inner tube, adding separation buffer and closing the cover, moving the inner tube downward, at this time the inner tube is arranged at the low position of the outer tube, at this time the sample is soaked and warmed by the separation solution; Step two: centrifugation, the inner tube is at the low position of the outer tube, then the new stepped sleeve is placed in the centrifuge for centrifugation operation to realize low-speed centrifugation, after low-speed centrifugation, the inner tube is moved upward to place the inner tube at the high position of the outer tube, the new stepped sleeve is placed in the centrifuge for centrifugation operation to realize high-speed centrifugation, after high-speed centrifugation, the inner tube is taken out, the inner tube and the sample are disposed of, and the mixed solution in the outer tube is reserved for use; Step three: immunomagnetic bead binding, adding immunomagnetic beads in the outer tube of the new stepped sleeve in step three, and then mixing; Step four: magnetic separation, placing the outer tube of the new stepped sleeve in step four in the magnetic separator, and after standing, the liquid is sucked; Step five: cleaning, adding separation buffer in the outer tube of the new stepped sleeve, oscillating and mixing, then placing the outer tube into the magnetic separator, standing after, and sucking the liquid, at this time the magnetic bead-sperm complex adsorbed on the inner side wall of the outer tube can be used for subsequent sperm DNA extraction.

2. The method for separating and enriching sperm by IgY antibody immunomagnetic beads according to claim 1, characterized in that: The separation solution added in step one is 1ml, the warming temperature is 56℃, and the warming time is 0.5-1h.

3. The method for separating and enriching sperm by IgY antibody immunomagnetic beads according to claim 1, characterized in that: The amount of separation buffer in step one is preferably completely soaked in the sample.

4. The method for separating and enriching sperm by IgY antibody immunomagnetic beads according to claim 1, characterized in that: In step two, both high-speed centrifugation and low-speed centrifugation are 20000g centrifugation for 3min, and a large-pore U-shaped filter column is used in the centrifugation process to realize efficient separation of epithelial cell DNA solution and sperm cells from the sample.

5. The method for separating and enriching sperm by IgY antibody immunomagnetic beads according to claim 1, characterized in that: In step two, 100μL of the mixed solution can be taken out for PSA and sperm cell detection.

6. The method for separating and enriching sperm by IgY antibody immunomagnetic beads according to claim 1, characterized in that: In step three, the surface of the immunomagnetic beads is coupled with specific sperm antibodies, i.e. IgY antibodies, which only react immunologically with sperm in the mixed solution to form a complex.

7. The method for separating and enriching sperm by IgY antibody immunomagnetic beads according to claim 1, characterized in that: In step three, 20μL of immunomagnetic beads are added, the mixing temperature is 37℃, and the mixing time is 30-40min.

8. The method for separating and enriching sperm by IgY antibody immunomagnetic beads according to claim 1, characterized in that: In step four, the magnetic separation requires that the outer tube be placed in the magnetic separator for 1min, and the immunomagnetic beads need to be prevented from being sucked away during the sucking process.

9. The method for separating and enriching sperm by IgY antibody immunomagnetic beads according to claim 1, characterized in that: In step five, the cleaning of the magnetic beads requires cleaning three times, each time 1ml of separation buffer is added, the oscillation and mixing time is 1min, the outer tube is placed in the magnetic separator for 1min, and the immunomagnetic beads need to be prevented from being sucked away during the sucking process.

10. The method for separating and enriching sperm by using IgY antibody immunomagnetic beads according to claim 6, characterized in that: The preparation method of the immunomagnetic beads is as follows: immunizing hens with human sperm cells to produce IgY antibodies, precipitating the antibodies by the polyvinyl alcohol method, purifying the antibodies, and coupling the antibodies with magnetic beads to obtain the immunomagnetic beads.

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