CN103344755B - Method for preparing magnetized and hydroformyled sheep red blood cell - Google Patents

Abstract

The invention discloses a preparation method of magnetized aldhylated sheep erythrocytes, which comprises the following steps: preparation of nanometer magnetic beads; preparation of aldehylated sheep erythrocytes; preparation of magnetized aldolized sheep erythrocytes; magnetization of aldolized sheep erythrocytes coating protein; sample detection; result observe. The prepared magnetized formaldehyde sheep erythrocytes are used as indicator cells for agglutination tests, carriers for antigen-antibody agglutination tests, and detection carriers for enzyme, chemiluminescence or fluorescent substance labeling tests. The magnetized formaldehyde sheep erythrocytes prepared by the present invention have a long shelf life and have paramagnetism at the same time, which can realize rapid aggregation and redispersion, avoid the cumbersome centrifugation process in the traditional agglutination test, and can be used to prepare artificial particles with uniform sizes Sexual antigen antibody; after replacing the solid phase carrier, the coating and detection reactions are carried out in a homogeneous phase, the reaction is sufficient and sensitive, and the cost is lower than that of the microtiter plate. It is mainly used for special red blood cell blood group antibodies and other soluble antigen antibody screening. or quantitative testing.

Description

A kind of preparation method of magnetized formaldehyde sheep erythrocyte

technical field

The invention relates to the field of biotechnology, in particular to a method for preparing magnetically-formylated sheep erythrocytes.

Background technique

Agglutination reaction is a serological reaction, the process is the combination of particulate antigens (intact pathogenic microorganisms or red blood cells, etc.) It can be divided into direct agglutination reaction and indirect agglutination reaction.

Direct agglutination is the agglutination phenomenon that occurs when granular antigens (such as bacteria, red blood cells, etc.) are directly combined with corresponding antibodies, and it is mainly divided into slide method and test tube method. The slide method is a qualitative test method in which known antibodies can be used to detect unknown antigens. The test tube method is a classic method of quantitative testing. Known antigens can be used to detect the presence or absence of a certain antibody and the content of the antibody in the tested serum, which is used to assist clinical diagnosis or for epidemiological investigation and research.

The indirect agglutination reaction is to first adsorb the soluble antigen (or antibody) on the surface of a granular carrier of a certain size irrelevant to immunity to make artificial granular antigen, and then react with the corresponding antibody (or antigen). Aggregation can occur under suitable conditions in the presence of a dielectric. The microspheres used as carriers can be natural particulate matter, such as human (O-type) and animal (sheep, rabbit, etc.) into, such as polystyrene latex microspheres, etc. Because the carrier particles increase the reaction area of the soluble antigen, when the antigen on the particle is combined with a small amount of antibody, it is enough to have a reaction visible to the naked eye, and the sensitivity is much higher than that of direct agglutination reaction.

The granular antigen in the agglutination reaction uses fresh cells or bacteria, which are not suitable for long-term storage and cumbersome operation. Although the results of the agglutination test are relatively intuitive, there are problems of low sensitivity and poor accuracy, and the experimental results are not easy. Standardization, which has caused certain obstacles to the application and popularization of agglutination reaction in the immunological detection of routine antigen-antibody reaction. Therefore, on the basis of agglutination tests, detection technologies such as enzymes, chemiluminescence or fluorescent labels have been developed, and artificial granular substances are used to increase the reaction area of soluble antigens and sensitive detection methods to make the detection of soluble antigens and antibodies more accurate and faster. At present, artificially synthesized or natural polymer materials are used to make some microspheres for the detection of soluble antigens and antibodies, but the synthesis process of this type of microspheres is cumbersome, the experimental conditions are harsh, the price of purchased finished products is high, and there are particles of different sizes. Uniformity and low protein labeling efficiency, so it has not been widely used clinically so far.

Contents of the invention

The main technical problem to be solved by the present invention is to provide a preparation method of magnetized aldylated sheep erythrocytes. The composite particles prepared by the present invention not only have the characteristics of indicator particles, but also have the characteristics of solid phase carrier and can be made into artificial granules. Antigen and antibody have a long storage period and have paramagnetic properties, which can achieve rapid aggregation and redispersion, avoiding the cumbersome centrifugation process in traditional agglutination tests, and are very convenient. They can be used as indicator cells and antigen-antibody screening for traditional agglutination tests The carrier of agglutination test and the carrier of qualitative or quantitative detection in the enzyme, chemiluminescence or fluorescent substance labeling test of antigen antibody, this kind of carrier can replace the solid phase carrier and prepare artificial granular antigen antibody with uniform size, including Both the detection and detection reactions are carried out in a homogeneous phase, which is not only cheaper than ordinary microtiter plates, but also makes the reaction more complete and more sensitive.

In order to solve the above-mentioned technical problems, a technical solution adopted by the present invention is to provide a method for preparing magnetically-formylated sheep erythrocytes, comprising the following steps:

(1) Preparation of nano magnetic beads;

(2) Preparation of aldehydated sheep red blood cells;

(3) Preparation of sheep erythrocytes by magnetization and formaldehyde;

(4) Magnetized formaldehyde sheep erythrocyte coating protein;

(5) Sample testing;

(6) Result observation.

In a preferred embodiment of the present invention, the specific steps of step (1) preparation of nano-magnetic beads are: preparing iron salt solution and ammonia solution, adding surfactant to the prepared iron salt solution and stirring, then adding Prepared ammonia solution to obtain nanometer magnetic beads.

In a preferred embodiment of the present invention, the specific steps for the preparation of the step (2) formaldehydeized sheep erythrocytes are: dilute the sheep erythrocytes with physiological saline, add glutaraldehyde, mix well and let stand, wash to remove excess glutaraldehyde Aldehydes, made into a suspension for later use.

In a preferred embodiment of the present invention, the specific steps of the step (3) preparation of magnetically-formulated sheep red blood cells are as follows: determine the magnetization conditions through pre-experiments, mix and oscillate the formaldehyde-formulated sheep red blood cells and nano-magnetic beads, and then wash with magnetic force adsorption , to wash away the unmagnetized red blood cells.

In a preferred embodiment of the present invention, the specific steps of the step (4) of magnetizing the protein coated sheep red blood cells are as follows: mix the magnetized formaldehyde sheep red blood cells and the protein, let them stand still, and then mix them with a roller , and then let it sit for washing to make a suspension for later use.

In a preferred embodiment of the present invention, the particle size of the magnetic nano beads is 10-500 nm.

In a preferred embodiment of the present invention, the volume percentage concentration of sheep red blood cells is 2-5%, the mass percentage concentration of glutaraldehyde is 1-2%, and the two are mixed at a reaction volume ratio of 1:1.

In a preferred embodiment of the present invention, the volume ratio of the aldehylated sheep erythrocytes and the magnetic nano beads is 15:1.

In a preferred embodiment of the present invention, the mass concentration of the protein is 1-5 mg/mL, the volume percentage concentration of the magnetized formaldehyde sheep red blood cells is 20-50%, and the volume ratio of the two is 3:1 .

In a preferred embodiment of the present invention, the magnetically-formylated sheep red blood cells can be used as indicator cells for agglutination tests, carriers for antigen-antibody screening agglutination tests, and qualitative or Carrier for quantitative detection.

The beneficial effects of the present invention are:

1. The sheep red blood cells involved in the present invention have a wide range of sources, low cost, uniform size, and the sheep red blood cells after formaldehyde are not easy to hemolyze and can be stored for more than 3 months, so they are a good indicator particle. There are a wide range of applications in testing.

2. The present invention innovatively introduces nano-magnetic beads onto the surface of aldylated sheep erythrocytes to form magnetized aldolized sheep erythrocytes. Such a composite particle can be prepared not only with the characteristics of indicator particles but also with the characteristics of a solid-phase carrier Adult artificial granular antigen has a long shelf life and is paramagnetic, which can realize rapid aggregation and redispersion, avoiding the cumbersome centrifugation process in traditional agglutination tests, which is very convenient.

3. It can not only be used as the indicator cell of the traditional agglutination test, but also can be used as the carrier of antigen antibody screening agglutination test and the carrier of qualitative or quantitative detection in the enzyme, chemiluminescence or fluorescent substance labeling test of antigen antibody, instead of solid After the phase carrier, the coating and detection reactions are carried out in a homogeneous phase, which is not only cheaper than ordinary microtiter plates, but also makes the reaction more complete and more sensitive. It is mainly used for special red blood cell blood group antibodies and other soluble antigen antibodies. Screening or quantitative detection tests.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings that need to be used in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative work, wherein:

Fig. 1 is a scanning electron microscope image of the magnetically-formylated sheep erythrocytes obtained in a preferred embodiment of the method for preparing magnetically-formylated sheep erythrocytes of the present invention.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Embodiment one:

Provides an agglutination test model using aldehyde-magnetized sheep erythrocytes to detect soluble antibodies, detects other specific antibodies, and coats the corresponding specific antigens on aldehyde-magnetized sheep erythrocytes, which can be applied to antibody detection. Including the following steps:

1. Preparation of nanomagnetic beads

0.85g FeCl ₃ ·6H ₂ O and 0.30g FeCl ₂ ·4H ₂ O were dissolved in 200 mL of distilled water under nitrogen protection. Add an appropriate amount of surfactant, and slowly add 1.5 mol/L ammonia solution to the above solution under strong stirring. When the pH of the solution rises to 6~7, a large number of black Fe ₃ O ₄ particles will be produced in the solution; continue Add ammonia to pH = 8 to complete the hydrolysis. Aged at 80°C for 0.5 h. The resulting solid was separated, washed three times with distilled water, and then dispersed in 100 mL of distilled water under the action of ultrasound to obtain a colloidal solution of Fe ₃ O ₄ which was collected for later use.

2. Preparation of sheep red blood cells

10 mL of fresh goat blood was centrifuged at 1500 rpm for 10 min to collect red blood cells, which were washed three times with normal saline to prepare 2 mL of red blood cell suspension for later use.

3. Glutaraldehyde configuration

Prepare 10 mL of 2% glutaraldehyde with normal saline and set aside.

4. Formulation of sheep erythrocytes

(1) Dilute sheep red blood cells to a concentration of 5% with normal saline, take 5 mL, add an equal volume of 2% glutaraldehyde, mix gently, and let stand at 4°C for 30 minutes;

(2) Wash 3 times with normal saline to remove excess glutaraldehyde, and make a suspension with normal saline for later use.

5. Magnetization of Aldehydated Sheep Red Blood Cells

(1) Determine the magnetization conditions through the pre-experiment, the volume ratio of aldehylated red blood cells and magnetic beads is 15:1, take 1 mL of each, mix and shake for 2 minutes;

(2) Magnetic adsorption washing to wash away unmagnetized red blood cells.

6. Aldehyde-magnetized sheep red blood cells coated with blood group polypeptide antigen

(1) The mass concentration of blood group peptide antigen is 2 mg/mL, diluted with 0.15M PBS with a pH value of 7.2;

(2) Magnetized formaldehyde sheep red blood cells were replaced with 0.15M PBS with a pH value of 7.2 and diluted to a mass percentage concentration of 50%;

(3) The volume ratio of magnetized formaldehyde sheep red blood cells and blood group polypeptide antigen is 3:1, mix them, and let stand at room temperature for 30 minutes;

(4) Mix with a roller for 5 hours at a speed of 100 r/min;

(5) After standing at room temperature for 30 minutes, wash with 0.15 M PH7.2 PBS 3 times to make a suspension for later use.

7. Sample testing

(1) Put 100 μl of the aldylated magnetized sheep red blood cell suspension coated with blood group polypeptide antigen into the well of a 96-well microtiter plate, add 50 μl each of normal sample/negative sample/blank sample, and shake for 1 min;

(2) After incubating in a water bath at 37°C for 30 minutes, shake and mix evenly during the period, and use a programmed magnetic oscillator to absorb and wash 3 times;

(3) Add goat anti-human IgM, incubate in a 37°C water bath for 30 minutes, shake and mix to observe the results;

(4) There is agglutination in positive results, but no agglutination in negative and blank controls.

8. Detection and interpretation of results.

Embodiment two:

The invention provides a test model of using aldehyde-magnetized sheep erythrocytes for antibody fluorescence detection, in which specific antigens are coated on aldehyde-magnetized sheep erythrocytes for antibody detection. Including the following steps:

1. Preparation of nano magnetic beads

0.85g FeCl ₃ ·6H ₂ O and 0.30g FeCl ₂ ·4H ₂ O were dissolved in 200 mL of distilled water under nitrogen protection. Add an appropriate amount of surfactant, and slowly add 1.5 mol/L ammonia solution to the above solution under strong stirring. When the pH of the solution rises to 6~7, a large number of black Fe ₃ O ₄ particles will be produced in the solution; continue Add ammonia to pH = 8 to complete the hydrolysis. Aged at 80°C for 0.5 h. The resulting solid was separated, washed three times with distilled water, and then dispersed in 100 mL of distilled water under the action of ultrasound to obtain a colloidal solution of Fe ₃ O ₄ which was collected for later use.

2. Preparation of sheep red blood cells

10 mL of fresh goat blood was centrifuged at 1500 rpm for 10 min to collect red blood cells, which were then washed three times with normal saline to prepare 2 mL of red blood cell suspension for later use.

3. Glutaraldehyde configuration

Prepare 10 mL of 2% glutaraldehyde with normal saline and set aside.

4. Formulation of sheep erythrocytes

(1) Dilute sheep red blood cells to a concentration of 5% with normal saline, take 5 mL, add an equal volume of 2% glutaraldehyde, mix gently, and let stand at 4°C for 30 minutes;

(2) Wash 3 times with normal saline to remove excess glutaraldehyde, and make a suspension with normal saline for later use.

5. Magnetization of Aldehydated Sheep Red Blood Cells

(1) Determine the magnetization conditions through the pre-experiment, the volume ratio of aldehydated erythrocytes and nano-magnetic beads is 15:1, take 1 mL each, mix and shake for 2 minutes,

(2) Magnetic adsorption washing to wash away unmagnetized red blood cells.

6. Aldehyde-magnetized sheep erythrocytes coated with human IgG

(1) The mass concentration of IgG is 2mg/mL, diluted with 0.15M PBS with a pH value of 7.2;

(2) Aldehyde-magnetized sheep red blood cells were replaced with 0.15M PBS with a pH value of 7.2 and diluted to a concentration of 50% by mass;

(3) Aldehyde-magnetized sheep red blood cells and IgG volume ratio 3:1, mix the two, and let stand at room temperature for 30 minutes;

(4) Mix with a roller for 5 hours at a speed of 100rpm;

(5) After standing at room temperature for 30 minutes, wash 3 times with 0.15M PBS with a pH value of 7.2, and make a suspension for use.

7. Sample testing

(1) Put the aldehyde-magnetized sheep erythrocyte suspension coated with human IgG in the wells of a 96-well microtiter plate;

(2) Add FITC-labeled goat anti-human IgG, incubate in a water bath at 37°C for 30 min, and wash twice;

(3) Read the fluorescence signal value with flow cytometer or fluorescence detector.

The beneficial effects of the present invention are: the sheep red blood cells involved in the present invention have a wide range of sources, low cost, uniform size, and the sheep red blood cells after formaldehyde are not easy to hemolyze and can be stored for more than 3 months, so they are a good indicator particle , is widely used in traditional agglutination tests. The present invention innovatively introduces nano-magnetic beads into the surface of aldylated sheep erythrocytes to form magnetized aldolized sheep erythrocytes. Such a composite particle not only has the characteristics of indicator particles but also has the characteristics of a solid phase carrier and can be prepared as an artificial Type granular antigen can significantly extend the shelf life, and at the same time, it is magnetic under the action of a magnetic field, which can realize rapid aggregation and redispersion, avoiding the cumbersome centrifugation process in traditional agglutination tests, which is very convenient. Not only can it be used as indicator cells in traditional agglutination tests, it can also be used as a carrier for antigen-antibody screening agglutination tests and a carrier for qualitative or quantitative detection in antigen-antibody enzymes, chemiluminescence or fluorescent substance labeling tests, instead of solid phase carriers Finally, the coating and detection reactions are carried out in a homogeneous phase, which is not only cheaper than ordinary microtiter plates, but also makes the reaction more complete and more sensitive. It can be mainly used for special red blood cell blood group antibodies and other soluble antigen antibody screening. or quantitative testing.

The above descriptions are only examples of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the content of the description of the present invention, or directly or indirectly used in other related technical fields, shall be The same reasoning is included in the patent protection scope of the present invention.

Claims (9)

1. magnetize a preparation method for hydroformylation sheep red blood cell, it is characterized in that, comprise the following steps:

(1) nanometer magnetic bead preparation;

(2) hydroformylation sheep red blood cell preparation;

(3) preparation of hydroformylation sheep red blood cell is magnetized;

(4) hydroformylation sheep red blood cell coating protein is magnetized;

(5) sample detection;

(6) result is observed, and wherein said magnetization hydroformylation sheep red blood cell can as carrier that is qualitative in the enzyme of the carrier of the indicator cells of agglutination test, antigen-antibody screening agglutination test and antigen-antibody, chemiluminescent substance or fluorescent substance mark test or detection by quantitative.

2. the preparation method of magnetization hydroformylation sheep red blood cell according to claim 1, it is characterized in that, concrete steps prepared by described step (1) nanometer magnetic bead are: preparation iron salt solutions and ammonia soln, in the iron salt solutions prepared, add tensio-active agent and stir, then adding the ammonia soln prepared and obtain nanometer magnetic bead.

3. the preparation method of magnetization hydroformylation sheep red blood cell according to claim 1, it is characterized in that, concrete steps prepared by described step (2) hydroformylation sheep red blood cell are: with physiological saline by dilution after sheep red blood cell washing, add glutaraldehyde, leave standstill after mixing, the unnecessary glutaraldehyde of washing removing, makes suspension for subsequent use.

4. the preparation method of magnetization hydroformylation sheep red blood cell according to claim 1, it is characterized in that, concrete steps prepared by described step (3) magnetization hydroformylation sheep red blood cell are: by preliminary experiment determination magnetization condition, hydroformylation sheep red blood cell and nanometer magnetic bead mixing vibration, then magnetic-adsorption washing, washes away the red corpuscle be not magnetized.

5. the preparation method of magnetization hydroformylation sheep red blood cell according to claim 1, it is characterized in that, the concrete steps of described step (4) magnetization hydroformylation sheep red blood cell coating protein for: will magnetize hydroformylation sheep red blood cell and albumen mixing is standing afterwards, again with the mixing of roller bearing instrument, and then leave standstill washing, make suspension for subsequent use.

6. according to the preparation method of the magnetization hydroformylation sheep red blood cell described in claim 2, it is characterized in that, described nanometer magnetic bead particle diameter is 10-500 nm.

7. according to the preparation method of the magnetization hydroformylation sheep red blood cell described in claim 3, it is characterized in that, the hematocrit percentage concentration of described sheep red blood cell is 2-5%, and the mass percent concentration of glutaraldehyde is 1-2%, and both press the reaction volume of 1:1 than mixing.

8. according to the preparation method of the magnetization hydroformylation sheep red blood cell described in claim 4, it is characterized in that, the hematocrit of described hydroformylation sheep red blood cell and nanometer magnetic bead is than being 15:1.

9. according to the preparation method of the magnetization hydroformylation sheep red blood cell described in claim 5, it is characterized in that, the mass concentration of described albumen is 1-5 mg/mL, and the hematocrit percentage concentration of magnetization hydroformylation sheep red blood cell is 20-50%, and the long-pending ratio of both bodies is 3:1.