CN115428783A - Erythrocyte membrane fragment freeze-drying protective solution, use method and application - Google Patents
Erythrocyte membrane fragment freeze-drying protective solution, use method and application Download PDFInfo
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Abstract
The invention discloses a erythrocyte membrane fragment freeze-drying protective solution, a preparation method of erythrocyte membrane freeze-drying fragments and application, wherein the freeze-drying protective solution comprises the following components in parts by weight: i) 20mM glucose, 12-16 mM lactose, 141mM trehalose, 39mM NaCl, 5.3mM KCl, 0.125mM KH 2 PO 4 And 1.2mM Na 2 HPO 4 (ii) a And ii) 0.3 to 0.39mM adenine, 8.19 to 8.73mM mannitol, 2.1% by weight bovine serum albumin, 4.2% by weight sodium citrate, 100mM inosine and 0.03 to 0.05mmol/L activated carbon. The erythrocyte membrane freeze-drying fragment prepared by the freeze-drying protective solution can be stored for one year at normal temperature without obviously reducing the antigenicity, greatly prolongs the storage time of the erythrocyte membrane fragment, and is beneficial to implementation and application of blood type reverse typing detection standardization.
Description
Technical Field
The invention belongs to the field of blood type antibody detection, and particularly relates to a erythrocyte membrane fragment freeze-drying protective solution, a using method and application.
Background
ABO blood group is determined by both erythrocyte surface antigens and antibodies in the blood. According to the different antigens on the surface of erythrocyte membrane in human blood, different blood types can be distinguished. The red blood cells of the type A blood are provided with blood group antigen A (hereinafter referred to as "A antigen"), and blood group antibody B (hereinafter referred to as "B antibody") is contained in the blood serum; the red blood cells of the type B blood are provided with blood group antigen B (hereinafter referred to as "B antigen"), and blood group antibody A (hereinafter referred to as "A antibody") is contained in the blood serum; the red blood cells of AB type blood have both A antigen and B antigen, and the serum has no A antibody and B antibody; the red blood cells of the blood group O have no A antigen and B antigen, and the serum has both A antibody and B antibody. The positive typing detection refers to that standard serum which can react with A antigen and B antigen on erythrocyte membrane is mixed with human blood sample to be detected to observe whether agglutination reaction is generated. The reverse typing assay refers to the measurement of antibodies in the serum of a sample using known red blood cell reagents of type A or B. When the ABO blood type is to be typed, it is preferable that both positive and negative typing are performed, so that the blood type can be more accurately determined.
At present, the blood type detection is clinically carried out, and a microcolumn gel blood type card is mainly adopted. In the existing anti-typing detection, the used erythrocyte reagent is mostly prepared by a transfusion unit (such as a hospital) and the erythrocyte preservation solution is adopted to preserve the complete erythrocyte. Because the components and the physicochemical properties of the erythrocyte preservation solution used in different units are different, the quality of the erythrocyte reagent for retrotyping can be unstable, so that the preservation of blood group antigens on erythrocytes is influenced, and finally, the detection result of serum antibodies is inaccurate. This not only makes the countertyping test difficult to standardize, but also even affects clinical transfusion safety. Other anti-typing methods utilize red cell membrane fragments and adopt enzyme-linked immunosorbent assay for detection, although the sensitivity is generally considered to be higher than that of agglutination technology, the problem that blood group antigens are difficult to store exists.
The blood group antigen detection is carried out by using freshly prepared red cell membrane fragments, wherein most of the red cell membrane fragments are currently prepared (a few are stored at 4 ℃ and 20 ℃ below zero for a short time), the process is complex, and the preparation process is time-consuming and labor-consuming. The existing short-term preservation method of the erythrocyte membrane fragments also has a plurality of defects, such as less than one week of preservation days, and the antigenicity of the erythrocyte membrane fragments is reduced along with the increase of the preservation days. Therefore, it is not suitable for mass production using the red cell membrane fragments as raw materials, and there may occur a problem that the lot-to-lot variation is serious. Some existing technical schemes try to store erythrocyte membrane fragments by a freeze-drying method, for example, patent CN202110568806.0 discloses a freeze-drying protective solution for erythrocyte membrane fragments, a freeze-drying method and application thereof, and freeze-dried erythrocyte membrane fragments prepared by using the freeze-drying protective solution and the freeze-drying method provided by the patent can be stored for 28 days at normal temperature, and meanwhile, the antigenicity of blood group antigens on the freeze-dried erythrocyte membrane fragments is still effectively kept to be similar to that of blood group antigens on freshly prepared erythrocyte membrane fragments. However, the 28-day preservation time is still not sufficient for standardization of anti-typing detection, so it is necessary to further optimize the technical solution of erythrocyte membrane freeze-drying protection to prolong the preservation time of erythrocyte membrane fragments.
Disclosure of Invention
In view of this, an object of the present invention is to provide a technical solution for freeze-drying protection solution for red cell membrane fragments. The erythrocyte membrane fragment freeze-drying protection solution comprises:
i) 20mmol/L glucose, 12-16 mmol/L lactose, 141mmol/L trehalose, 39mmol/L NaCl, 5.3mmol/L KCl, 0.125mmol/L KH 2 PO 4 And 1.2mmol/L Na 2 HPO 4 (ii) a And
ii) 0.3 to 0.39mmol/L of adenine, 8.19 to 8.73mmol/L of mannitol, 2.1% by weight of bovine serum albumin, 4.2% by weight of sodium citrate, 100mmol/L of inosine and 0.03 to 0.05mmol/L of activated carbon.
In some embodiments, the erythrocyte membrane fragment lyophilization protection solution comprises:
i) 20mmol/L glucose, 12mmol/L lactose, 141mmol/L trehalose, 39mmol/L NaCl, 5.3mmol/L KCl, 0.125mmol/L KH 2 PO 4 And 1.2mmol/L Na 2 HPO 4 (ii) a And
ii) 0.39mmol/L adenine, 8.56mmol/L mannitol, 2.1% wt bovine serum albumin, 4.2% wt sodium citrate, 100mmol/L inosine and 0.03mmol/L activated carbon. In some other embodiments, the erythrocyte membrane fragment lyophilization protection solution comprises:
i) 20mmol/L glucose, 16mmol/L lactose, 141mmol/L trehalose, 39mmol/L NaCl, 5.3mmol/L KCl, 0.125mmol/L KH 2 PO 4 And 1.2mmol/L Na 2 HPO 4 (ii) a And
ii) 0.3mmol/L adenine, 8.19mmol/L mannitol, 2.1% wt bovine serum albumin, 4.2% wt sodium citrate, 100mmol/L inosine and 0.05mmol/L activated carbon.
In some other embodiments, the erythrocyte membrane fragment lyophilization protection solution comprises:
i) 20mmol/L glucose, 14mmol/L lactose, 141mmol/L trehalose, 39mmol/L NaCl, 5.3mmol/L KCl, 0.125mmol/L KH 2 PO 4 And 1.2mmol/L Na 2 HPO 4 (ii) a And
ii) 0.37mmol/L adenine, 8.73mmol/L mannitol, 2.1% wt bovine serum albumin, 4.2% wt sodium citrate, 100mmol/L inosine and 0.04mmol/L activated carbon.
In another aspect, the present invention also provides a method for preparing a freeze-dried fragment of an erythrocyte membrane, comprising:
1) Obtaining fragments of the fresh red cell membranes;
2) Mixing the fresh red blood cell membrane fragments with the red blood cell membrane fragment freeze-drying protection solution of any one of claims 1 to 4 to obtain a mixed product;
3) Firstly, placing the mixed product at the temperature condition of 4 ℃ and keeping the temperature condition for 30min, then changing the temperature condition into a condition of reducing the temperature to-70 ℃ at the cooling rate of 10 ℃/min and keeping the temperature for 2h to obtain a precooled product;
4) And (3) carrying out freeze-drying treatment on the precooled product to obtain a red cell membrane freeze-dried fragment.
Further, the volume ratio of the fresh red blood cell membrane fragments to the red blood cell membrane fragment freeze-drying protection solution is 1:10.
further, the freeze-drying treatment comprises primary drying and secondary drying, wherein in the primary drying process, the temperature and the pressure of a partition plate of a freeze dryer are respectively kept at minus 50 ℃ and 2-4 Pa, and the treatment time is 18 hours; in the secondary drying process, the temperature and the pressure of a clapboard of the freeze dryer are respectively kept at 20 ℃ and 2-4 Pa, and the processing time is 10 hours.
On the other hand, the invention also provides application of the red cell membrane freeze-drying fragments prepared by the use method of the red cell membrane fragment freeze-drying protection solution in blood type reverse typing detection.
Furthermore, the freeze-dried fragment of the erythrocyte membrane is used as an antigen raw material to be coated on a nitrocellulose membrane and is applied to an Elisa plate or a dot hybridization detection.
Advantageous technical effects
The freeze-dried erythrocyte membrane fragments prepared by the freeze-drying protective solution provided by the invention can be stably stored for a long time (for example, for up to one year) at normal temperature without obviously reducing the antigenicity of blood group antigens on the membrane fragments. The freeze-drying protective solution provided by the invention can greatly prolong the preservation time of erythrocyte membrane fragments, and is beneficial to implementation and application of blood type reverse typing detection standardization: for example, the lyophilized red cell membrane fragments prepared by the lyophilized protection solution of the present invention can be prepared as a standard for application to areas where transport and storage conditions are relatively more limited.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising a … …" does not exclude the presence of another identical element in a process, method, article, or apparatus that comprises the element.
As used in this specification, the term "about" typically means +/-5% of the stated value, more typically +/-4% of the stated value, more typically +/-3% of the stated value, more typically +/-2% of the stated value, even more typically +/-1% of the stated value, and even more typically +/-0.5% of the stated value.
In this specification, certain embodiments may be disclosed in a range of formats. It should be understood that this description of "within a certain range" is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, the range
The description of (a) should be read as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, e.g., 1,2,3,4,5 and 6. The above rules apply regardless of the breadth of the range.
The "fragment of erythrocyte membrane" as used herein refers to the membrane of erythrocyte in an incomplete state. Red blood cells are typically treated by hypotonic or repeated freezing and thawing, etc. to disrupt the red blood cell membranes, followed by centrifugation to separate the membrane debris from other components (e.g., hemoglobin) to obtain red blood cell membrane debris in the form of a precipitate. The components of the red cell membrane fragments include lipids and membrane proteins, including various blood group antigens.
The "fresh red cell membrane fragments" as used herein refers to newly prepared red cell membrane fragments, which are generally preserved for no more than 1 day after preparation.
The "lyoprotectant" refers to an agent that is mixed with red blood cell membrane fragments during the lyophilization process and that can maintain the antigenicity of the red blood cell membrane fragments during the lyophilization (and subsequent storage and reconstitution) process.
The "antigenicity" of the invention refers to the ability of blood group antigens on erythrocyte membranes or erythrocyte membrane fragments to specifically bind with corresponding blood group antibodies: the stronger the binding capacity, the higher or stronger the antigenicity; conversely, if the binding capacity is reduced, the lower or less antigenic is.
The erythrocyte membrane fragment freeze-drying protective solution provided by the invention comprises glucose, lactose, trehalose, naCl, KCl and KH 2 PO 4 、Na 2 HPO 4 Adenine, mannitol, bovine serum albumin, sodium citrate, inosine and activated carbon. Mixing the freshly prepared red blood cell membrane fragments with the freeze-drying protection solution provided by the invention, and freeze-drying to obtain the freeze-dried red blood cell membrane fragments.
The freeze-dried red cell membrane fragments can be reconstituted with water or buffer solution before use, and can be used in antigen-antibody affinity assay (e.g., for detecting blood group antibodies in serum).
The erythrocyte membrane fragments can be stored for one year at normal temperature after being frozen in the presence of the freeze-drying protective solution provided by the invention, and the sensitivity is close to the antigenicity of freshly prepared cell membrane fragments in an antigen-antibody affinity detection experiment.
The freeze-drying protective solution provided by the invention is used for freeze-drying and storing erythrocyte membrane fragments, and is favorable for standardization of blood type reverse typing detection.
The following examples used some of the instruments and consumables as shown in the following table:
the first embodiment is as follows: preparation of erythrocyte membrane fragments
The red blood cell debris was prepared as follows:
1) Taking 3mL of anticoagulated mixed whole blood (taken from 6-10 blood samples of A type (or other blood types)), adding the anticoagulated mixed whole blood into a 15mL centrifuge tube filled with 10mL0.01mol/L PBS (pH 7.2), centrifuging for 5min at 3000r/min, and removing the supernatant, white blood cells and platelet layers under the supernatant to obtain about 1.5mL of packed red blood cells;
2) Washing with 4 deg.C pre-cooled 0.01mol/L PBS (pH7.2) with volume 3 times of packed red blood cells, and centrifuging at 4 deg.C for 15min at 5000 r/min;
3) V =40, 1, adding 4 ℃ precooled 0.01mol/L PBS (pH7.2) and mixing the precipitate, standing at 4 ℃ for 2h, centrifuging at 9000r/min for 20min, and discarding the supernatant;
4) Repeating the step 3) for 4 times until no red blood cells are visible to the naked eye, and obtaining about 800 microliter of precipitate which is the red blood cell membrane fragment sample carrying the membrane antigen.
Multiple preparations were carried out in this way, and the red cell membrane fragments obtained were either used immediately (for membrane antigen detection or lyophilization) or were dispensed directly and stored at 4 ℃ or-20 ℃ respectively.
Example two: preparation of freeze-dried protective solution
The lyophilized protection solution was prepared using water for injection, according to the components and concentrations shown in the following table.
TABLE 1 different Freeze-drying protection solution formulations
(mM in mmol/L abbreviation)
Lactose and trehalose are used in the present invention to sufficiently stretch the fragments of the cell membrane, protect the antigens on the cell membrane from falling off and maintain homeostasis.
The invention creatively adds the active carbon as a protective component of the cell membrane antigen into the formula of the protective solution. Due to the porous structural characteristics of the activated carbon, the activated carbon can be used as a skeleton component to adsorb other nutrient components (such as lactose and trehalose) in the formula liquid, and the activated carbon can be suspended in the formula in a sol form and uniformly distributed through the precise proportioning of the invention, and can also play a role in uniformly distributing the nutrient components in the formula liquid, so that the nutrient components in the formula of the protective liquid can more effectively play a role.
Example three: freeze-drying treatment of erythrocyte membrane fragments
Mixing the fresh erythrocyte membrane fragments prepared in example 1 and the freeze-drying protective solutions prepared in example 2 according to the volume ratio of 1:10, and adding the mixture into a penicillin bottle with the volume of 5mL to ensure that the liquid content of each penicillin bottle is 1mL. Placing the penicillin bottle into a refrigerator at 4 ℃ for precooling for 30 minutes, then reducing the temperature to-70 ℃ according to the cooling rate of 10 ℃ per minute, and keeping the temperature for more than 2 hours to freeze the substance to be freeze-dried. Then, vacuumizing and primary drying are carried out, so that the temperature and the pressure of a clapboard of the freeze dryer are respectively kept at minus 50 ℃ and about 2-4 Pa for 18 hours. And finally, performing secondary drying, wherein the temperature of the partition plate is set to be 20 ℃, the pressure is set to be about 2-4 Pa, and the duration is 10 hours, so as to prepare the freeze-dried erythrocyte membrane fragments.
The freeze-dried erythrocyte membrane fragments are stored at normal temperature. Reconstituted with an equal volume of water for injection (same volume as the fragments of fresh red cell membrane before lyophilization) before use.
Example four: erythrocyte membrane fragment antigenicity detection (Elisa method)
1. The detection principle is as follows:
coating 100 mu L of red cell membrane fragments to be detected on an enzyme label plate, and adding a standard substance antibody, wherein the antibody can be specifically combined with membrane antigens on the red cell membrane fragments. After washing off the free components, horseradish peroxidase-labeled secondary antibody was added. The membrane antigen is combined with a standard substance antibody and a horseradish peroxidase-labeled secondary antibody to form an immune complex, free components are washed away, a color development liquid substrate TMB is added, the TMB presents blue under the catalysis of horseradish peroxidase, and the color becomes yellow after the stop solution is added. Measuring OD value with a microplate reader at 450nm wavelength, wherein the antigenicity of the specific antigen on the erythrocyte membrane fragments is in direct proportion to the OD450 value, and comparing the antigenicity with the OD450 value detected by freshly prepared erythrocyte membrane fragments to determine the antigenicity change of the erythrocyte membrane fragments stored in other modes.
The red cell membrane fragments to be detected comprise: (1) fresh red cell membrane fragments; (2) Storing the red cell membrane fragments for different days at the temperature of 4 ℃; (3) Storing red cell membrane fragments at-20 ℃ for different days; (4) Red cell membrane fragments were stored for different days and reconstituted after lyophilization.
2. The specific detection steps are as follows:
coating: diluting 100 mu L of erythrocyte membrane fragments to be detected by 10000 times by using coating buffer solution, adding the diluted fragments into an ELISA plate according to 100 mu L/hole, and coating overnight at 4 ℃;
washing the plate: discarding the liquid in the hole the next day, lightly tapping on a paper towel to remove the residual liquid, adding washing solution (280-300 μ L/hole), washing for 2-3 times, each time for 3-5min;
and (3) sealing: adding the sealing liquid into the pores at a rate of 200-250 μ L/pore, standing at room temperature for 2 hr, and removing the sealing liquid from the pores;
standard antibody treatment: diluting the standard substance antibody by a washing solution by a multiple ratio, wherein the dilution ratio is 2 1 ，2 2 ，2 3 ，2 4 ，2 5 ，2 6 And 2 7 ；
Sample adding: sequentially adding diluted standard substance antibodies into 50 mu L/hole in sequence, and incubating for 30min at room temperature;
washing the plate: discarding the liquid in the hole, lightly tapping on a paper towel to remove the residual liquid, adding a washing solution (280-300 μ L/hole), and washing for 2-3 times, each time for 3-5min;
adding an enzyme-labeled antibody: adding newly prepared enzyme-labeled antibody according to 50 mu L/hole, and incubating for 30min at room temperature;
washing the plate: discarding the liquid in the hole, lightly tapping on a paper towel to remove the residual liquid, adding a washing solution (280-300 μ L/hole), and washing for 2-3 times, each time for 3-5min;
adding a substrate solution for color development: adding TMB substrate solution according to 100 mu L/hole, and reacting for 30min at room temperature in a dark place;
and (3) terminating the reaction: 50. Mu.L of stop solution was added to each reaction well.
And (4) judging a result: OD was measured with an enzyme-labeled instrument (to add only coating buffer blank Kong Diaoling).
3. The experimental results are as follows:
1. detection result of protection effect of freeze-drying protection solution (1 # -3 #) on erythrocyte membrane fragment antigenicity
TABLE 2 antigenic test results stored for 30 days
TABLE 3 antigenic test results stored for 60 days
TABLE 4 preservation of 90 days antigenicity test results
TABLE 5 antigenic assay results stored for 120 days
TABLE 6 preservation of 360 days antigenicity test results
Discussion of experimental results:
the freeze-drying protective solution 1# to 3# can effectively protect antigens on freeze-dried erythrocyte membrane fragments, so that the antigens can be stored for 360 days at normal temperature without remarkable reduction of the antigenicity. Particularly 1# and 2#, after being preserved for 360 days at normal temperature, the antigenicity of the freeze-dried erythrocyte membrane fragments is almost consistent with that of freshly prepared erythrocyte membrane fragments; although slightly lower than freshly prepared erythrocyte membranes, the 3# still achieves effective protection against the antigenicity.
2. Antigenicity detection results of freeze-drying protective solution (1 # -5 #) and comparative experimental examples (1-1 # -1-6 #) of the invention
TABLE 7 antigenicity test results of the experimental examples and comparative examples of the present invention stored for 30 days
TABLE 8 antigenic detection results of the experimental examples and comparative examples of the present invention stored for 60 days
TABLE 9 antigenic detection results of the experimental examples and comparative examples of the present invention stored for 90 days
TABLE 10 antigenic assay results of the experimental examples and comparative examples of the present invention stored for 120 days
TABLE 11 antigenic detection results of the experimental examples and comparative examples of the present invention stored for 360 days
Discussion of experimental results:
1. ) The 4# and 5# lyophilized protective solutions were obtained by adjusting the component values based on the 1# to 3# lyophilized protective solutions of the present invention. It was found that the 4# and 5# lyophilized protective solutions maintained antigenicity comparable to the 1# to 3# protective solutions when stored for 30-60 days, but the results were close to each other as a whole, although the antigenicity was reduced. After 60 days of storage, it can be seen that the OD values of the 4# and 5# groups have exhibited significant differences compared to 1# to 3#, especially from the dilution factor of 2 2 And starting. The antigenicity of the lyophilized red cell membrane fragments preserved with # 4 and # 5 protective solutions showed a growing difference from that of freshly prepared red cell membrane fragments starting from 90 days of preservation.
2) Comparative examples 1-1# and 1-2# lyophilized protective solutions were prepared by removing some important components based on the 1# protective solution of the present invention (1-1 # removed adenine, mannitol, bovine serum albumin, sodium citrate, inosine, and activated charcoal; removing glucose, lactose, trehalose, naCl, KCl, KH from No. 1-2 2 PO 4 、Na 2 HPO 4 And activated carbon). It can be seen that the protective effect on the antigenicity of the lyophilized erythrocyte membrane is far less than that of the protective solution # 1 of the present invention and is far different from that of the freshly prepared erythrocyte debris when the lyophilized erythrocyte membrane is preserved for 30 days. It is considered that the two protective solutions of the comparative examples do not already protect the antigenicity of the membranes of the freeze-dried erythrocytes when stored for 30 days. In all comparative example lyophilized protective solutions, 1-2# protected antigenicity on lyophilized erythrocyte membranesThe effect was the worst, therefore, the antigen OD was not measured when stored for 360 days.
3) Comparative examples 1-3# to 1-5# lyophilized protective solutions were obtained by removing lactose, trehalose and activated carbon, respectively, on the basis of the protective solution 1# of the present invention. It can be seen that the lack of any of these three components negatively affects the protection of antigenicity on the membranes of the lyophilized erythrocytes (as demonstrated by not being as antigenic as # 1). The No. 1-6 is a protective solution with the 3 components removed, and the protective effect of the protective solution is more different from that of the No. 1-3-1-5.
Example five erythrocyte Membrane fragment antigen sensitive detection (column agglutination method)
1. Principle of detection
In the embodiment, the red cell membrane fragments preserved by the 1# freeze-drying protective solution are redissolved by water or buffer solution; the resolubilization product is coupled with the microsphere (red microsphere indication method), and the sensitivity of the antigen of the resolubilization product is shown through the enhancement effect of the marker on the red microsphere.
The present example uses a column agglutination method, which utilizes the agglutination of erythrocytes visible to the naked eye caused by the reaction of blood group antigens and antibodies to determine the result.
And (4) judging a result:
agglutination is characterized in that the red blood cells stay on the upper surface of the gel separation medium or are distributed in the gel separation medium; non-agglutination is indicated by the red blood cells remaining at the bottom of the microcolumn through the gel separation medium. Agglutination of the red blood cells is a positive result, indicating the presence of the corresponding antigen/antibody in the sample; non-agglutination is a negative result, indicating that the corresponding antigen/antibody is not present in the sample.
The cohesive strength was as follows:
4+ erythrocytes agglutinate on the upper surface of the gel separation medium and form an annular band;
the majority of erythrocytes with 3+ agglutination are retained in the upper half of the gel separation medium;
2+ agglutinated erythrocytes are distributed in the whole gel separation medium, and a small amount of erythrocytes are arranged at the bottom of the microcolumn;
1+ most agglutinated red blood cells stay at the lower half part of the gel separation medium, and more red blood cells are arranged at the bottom of the microcolumn;
negative reaction, all red blood cells pass through the gel gap, forming a flat red blood cell agglutination band at the bottom of the microcolumn. In some cases, the cells were judged to be negative because they did not agglutinate and formed a cloudy red color on the flat agglutination belt.
2. The method comprises the following specific operation steps:
red microsphere mark (5 μm)
1. Adding 500. Mu.L MES (0.05M, pH 6.1) into 25. Mu.L microsphere;
2. adding 10 μ L of 30mg/mL N-hydroxy thiosuccinimide (NHS) and 10 μ L of 50mg/mL EDC, and performing rotary activation for 30min;
3. centrifuging at 13000rpm for 15min; discarding the supernatant, adding 1mL MES, and performing ultrasonic treatment for 1min;
4. adding 0.1mg of redissolved erythrocyte membrane fragments, reacting for 1-2min, performing ultrasonic treatment for 30s, and performing rotary reaction for 2h;
5. add 25. Mu.L 20% BSA (final concentration 0.5%), block for 2h;
6. finally, centrifugation was carried out and the supernatant was discarded, and 400. Mu.L of erythrocyte preservation solution was added.
The control adopts erythrocyte indicator, and the preparation method comprises: washing the anticoagulated or anticoagulated blood sample to remove part of impurities and obtain packed red blood cells; adding a suitable proportion of a commercially available erythrocyte preservation solution into the packed erythrocytes to prepare an erythrocyte suspension, namely the erythrocyte indicator (needing to be stored in a refrigerator at 4 ℃).
3. Results of the experiment
TABLE 12 comparison of column agglutination test results
In this example, the column agglutination method is used to prove that the antigen sensitivity of the microsphere-coupled erythrocyte membrane fragment is significantly higher than that of the erythrocyte indicator. Samples that were stored lyophilized with the lyoprotectant # 1 of the present invention and then immediately reconstituted (day 0) were dilutedRelease to 2 7 At the beginning, the sensitivity was shown to be higher than that of the red blood cell indicator. When the red blood cell indicator is stored at 4 ℃ for 5 months, agglutination reaction hardly occurs; the erythrocyte membrane fragments preserved by the freeze-drying protective solution 1# can still keep high antigen sensitivity when preserved for 12 months.
It should be clarified that the dilution in detecting the antibody (standard) by the column agglutination method in example five is 2 16 ～2 18 In example four, the dilution by the Elisa method is only 2 7 The reason why the dilution factor is different is mainly that the two experimental systems are different. The concentration of antigen that can be coated onto an Elisa plate is low (about 0.1 mg/ml) when performing the Elisa assay, and the concentration of antigen coated onto a strip is high (about 3 mg/ml) when performing the column agglutination assay, so that the dilution factor for the antibody is low when performing the Elisa assay, and the higher dilution factor for the antibody is required when performing the column agglutination assay.
The present invention is not limited to the above-described embodiments, which are only illustrative and not restrictive, and those skilled in the art can make many modifications without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A freeze-drying protective solution for erythrocyte membrane fragments is characterized in that: the method comprises the following steps:
i) 20mmol/L glucose, 12-16 mmol/L lactose, 141mmol/L trehalose, 39mmol/L NaCl, 5.3mmol/L KCl, 0.125mmol/L KH 2 PO 4 And 1.2mmol/L Na 2 HPO 4 (ii) a And
ii) 0.3 to 0.39mmol/L of adenine, 8.19 to 8.73mmol/L of mannitol, 2.1% by weight of bovine serum albumin, 4.2% by weight of sodium citrate, 100mmol/L of inosine and 0.03 to 0.05mmol/L of activated carbon.
2. The erythrocyte membrane fragment freeze-drying protection solution as claimed in claim 1, wherein the freeze-drying protection solution comprises: the method comprises the following steps:
i) 20mmol/L glucose, 12mmol/L lactose, 141mmol/L trehalose, 39mmol/L NaCl, 5.3mmol/L KCl, 0.125mmol/L KH 2 PO 4 And 1.2mmol/L Na 2 HPO 4 (ii) a And
ii) 0.39mmol/L adenine, 8.56mmol/L mannitol, 2.1% wt bovine serum albumin, 4.2% wt sodium citrate, 100mmol/L inosine and 0.03mmol/L activated carbon.
3. The erythrocyte membrane fragment freeze-drying protection solution of claim 1, which is characterized in that: the method comprises the following steps:
i) 20mmol/L glucose, 16mmol/L lactose, 141mmol/L trehalose, 39mmol/L NaCl, 5.3mmol/L KCl, 0.125mmol/L KH 2 PO 4 And 1.2mmol/L Na 2 HPO 4 (ii) a And
ii) 0.3mmol/L adenine, 8.19mmol/L mannitol, 2.1% wt bovine serum albumin, 4.2% wt sodium citrate, 100mmol/L inosine and 0.05mmol/L activated carbon.
4. The erythrocyte membrane fragment freeze-drying protection solution as claimed in claim 1, wherein the freeze-drying protection solution comprises: the method comprises the following steps:
i) 20mmol/L glucose, 14mmol/L lactose, 141mmol/L trehalose, 39mmol/L NaCl, 5.3mmol/L KCl, 0.125mmol/L KH 2 PO 4 And 1.2mmol/L Na 2 HPO 4 (ii) a And
ii) 0.37mmol/L adenine, 8.73mmol/L mannitol, 2.1% wt bovine serum albumin, 4.2% wt sodium citrate, 100mmol/L inosine and 0.04mmol/L activated carbon.
5. A method for preparing a freeze-dried fragment of an erythrocyte membrane is characterized by comprising the following steps:
1) Obtaining fragments of the fresh red cell membranes;
2) Mixing the fresh red blood cell membrane fragments with the red blood cell membrane fragment freeze-drying protection solution of any one of claims 1 to 4 to obtain a mixed product;
3) Firstly, placing the mixed product at the temperature condition of 4 ℃ and keeping the temperature condition for 30min, then changing the temperature condition into a temperature reduction rate of 10 ℃/min, cooling to-70 ℃ and keeping the temperature for 2h to obtain a precooled product;
4) And carrying out freeze-drying treatment on the precooled product to obtain a erythrocyte membrane freeze-dried fragment.
6. The method of claim 5, wherein: the volume ratio of the fresh erythrocyte membrane fragments to the erythrocyte membrane fragment freeze-drying protection solution is 1:10.
7. the method of claim 5, wherein: the freeze-drying treatment comprises primary freeze-drying and secondary freeze-drying, wherein the freeze-drying temperature and pressure are respectively kept at minus 50 ℃ and 2-4 Pa in the primary freeze-drying process, and the treatment time is 18 hours; in the secondary freeze-drying process, the freeze-drying temperature and the pressure are respectively kept at 20 ℃ and 2-4 Pa, and the treatment time is 10 hours.
8. The lyophilized fragment of erythrocyte membrane prepared by the method of any one of claims 5 to 7.
9. Use of the lyophilized fragment of erythrocyte membrane of claim 8 in blood group reverse typing test.
10. The use according to claim 9, wherein the lyophilized fragment of red cell membrane is coated on nitrocellulose membrane as an antigen material for use in an Elisa plate or dot blot hybridization assay.
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