CN113447665A - Platelet antibody screening method based on cell membrane fluorescent marker and application thereof - Google Patents
Platelet antibody screening method based on cell membrane fluorescent marker and application thereof Download PDFInfo
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- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
Abstract
The invention belongs to the technical field of antibody detection, and particularly relates to a platelet antibody screening method based on cell membrane fluorescent markers and application thereof, wherein the platelet antibody screening method comprises the following steps: s1: carrying out fluorescence labeling on the blood platelet by using cell membrane fluorescent dye to obtain fluorescence labeled blood platelet; s2: adding a sample to be detected to a solid phase carrier (U-shaped ELISA plate) coated with a second antibody for incubation; s3: and (4) adding the fluorescence labeled platelet obtained in the step (S1) into the incubated sample to be detected, and directly observing the aggregation distribution condition of the platelet through a fluorescence microscope after incubation and centrifugation so as to judge whether the sample to be detected contains the antiplatelet antibody. The screening method has the advantages of simple operation, time and labor saving and low cost, and more importantly, the platelet antibody screening method directly detects the homogeneous antibody in serum or plasma based on the aggregation distribution condition of fluorescence labeled platelets, and has higher detection specificity.
Description
Technical Field
The invention belongs to the technical field of antibody detection, and particularly relates to a platelet antibody screening method based on cell membrane fluorescent labeling and application thereof.
Background
Platelet (PLT) surfaces have a complex antigen system, including antigens common to other tissues or cells, such as ABO antigens and HLA antigens (HLA-a, -B, -C); specific alloantigens present on platelet membrane glycoproteins (Human patelet antigens, HPAs); the GP IV/CD 36, which is present together on platelets, monocytes/macrophages and nucleated red blood cells, and the collagen receptor GP VI on platelets. The methods of pregnancy, blood transfusion, and organ transplantation induce the above platelet-associated antigens to immunize the body to produce antibodies, including HLA antibodies, HPA antibodies, CD36 antibodies, and the like. These antibodies can cause nonhemolytic fever reactions, ineffective platelet infusion, homoimmune thrombocytopenia in both fetal and neonatal species, post-transfusion purpura. In order to ensure the safety and curative effect of clinical blood transfusion, effectively prevent adverse reactions of blood transfusion and ineffective platelet transfusion, save blood resources and assist in diagnosing platelet antibody related immune diseases, platelet antibody detection gradually becomes the routine of immunological detection before clinical blood transfusion.
At present, the methods for detecting platelet antibodies mainly include the following three methods: (1) in the solid phase agglutination (SPRCA), platelet-rich plasma is applied to a microplate to fix platelets to the bottom of the well, and the reaction is indicated by anti-IgG-sensitized erythrocytes. If there is no antibody on the platelet, it indicates that the cells have accumulated at the bottom of the well, forming a cell button, which is a negative result, otherwise it is a positive result. (2) Monoclonal antibody specific platelet antigen capture (MAIPA), platelet specific antigen is fixed, then the serum to be detected is used for reaction, then Horseradish Peroxidase (HRP) labeled anti-human IgG is added for reaction and color development, the absorbance value at 490nm after reaction termination is measured so as to identify platelet specific antibody, a specific diagnosis method is provided for clinical identification of immune and non-immune thrombocytopenia, and the platelet specific antibody can be quantitatively determined, and is shown in figure 1. The method has high sensitivity and strong specificity, can detect HPA-5 antigen with small quantity of antigen on blood platelet, and is the most widely used method at present. (3) Antigen capture enzyme-linked immunosorbent assay (MACE), which comprises the steps of reacting sensitized platelets with monoclonal HLA-I antibodies and GPIIb/IIIa, GPIa/IIa, GPIb/IX and GPIV antibodies, adding anti-human IgG for reaction, developing color, and measuring absorbance value at 405nm after terminating the reaction, wherein the result that the absorbance value is equal to or more than 2 times of the absorbance value of negative control is positive. The method has high specificity and relatively simple and convenient operation, can detect IgG antibodies aiming at platelet specific antigens and HLA antigens, and is suitable for definite diagnosis tests and distinguishing the types of the platelet antibodies.
However, the above prior art still has the following problems: (1) low detection specificity and high false positive rate. Unlike erythrocytes, platelets are a colorless or yellowish small cytoplasm, and individual platelets, whether aggregated or scattered, cannot be readily detected by microscopy or naked eye when unstained; the existing detection technology does not directly detect the binding condition of the platelet and the alloantibody, but indirectly reflects the binding condition of the platelet and the alloantibody through the color development of anti-IgG sensitized indicating red blood cells or HRP-labeled anti-human IgG. Through the gradual amplification of multiple antibodies or enzyme-linked immunosorbent assay, the detection sensitivity is improved, the false positive rate of an experimental result is greatly improved, and the specificity of the experiment is reduced. (2) The operation is complicated and the time consumption is long. The existing technical operation flow does not directly detect the combination condition of the platelet and the homogeneous antibody, but indirectly reacts the combination condition of the platelet and the homogeneous antibody through the color development of anti-IgG sensitized indicating red blood cells or HRP-labeled anti-human IgG, so the operation flow is complicated, a single experiment needs at least 2-3 h, and the time consumption is long. (3) The cost is high. The prior art also relates to amplification reaction of multiple antibodies or enzyme-linked immunosorbent assay, the types of the required antibodies are more (2-4), and in addition, the required antibodies need to be matched with corresponding indicating red blood cells or chromogenic substrates, so that the cost is higher, and further the detection price is high.
Disclosure of Invention
Aiming at the problems of low specificity, tedious operation, long time consumption and high cost existing in the conventional platelet antibody screening, the invention provides a platelet antibody screening method based on cell membrane fluorescent markers and application thereof, and the platelet antibody screening method has the advantages of high detection specificity, simplicity in operation, time and labor saving and low cost.
Based on the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the present invention provides a platelet antibody screening method based on cell membrane fluorescent labeling, comprising the following steps:
s1: carrying out fluorescence labeling on the blood platelet by using cell membrane fluorescent dye to obtain fluorescence labeled blood platelet;
s2: adding a sample to be detected into a solid phase carrier coated with a second antibody for incubation;
s3: and (4) adding the fluorescence labeled platelet obtained in the step (S1) into the incubated sample to be detected, and directly observing the aggregation distribution condition of the platelet through a fluorescence microscope after incubation and centrifugation so as to judge whether the sample to be detected contains the antiplatelet antibody.
The working principle of the platelet antibody screening method of the invention is shown in fig. 2, and specifically comprises the following steps: coating a specific second antibody on a U-shaped bottom ELISA micropore plate, adding serum (or plasma) to be detected and LISS (low ionic strength solution) into corresponding micropores for incubation, washing to remove unbound serum components after reaction is finished, adding cell membrane fluorescent dye to mark platelets, incubating and centrifuging, because the invention utilizes the cell membrane fluorescent labeling dye to label the platelet, based on the principle of the specific combination of the platelet surface antigen and the antibody in the serum to be detected, therefore, the binding, aggregation and distribution conditions of the platelets can be directly observed through a fluorescence microscope, the platelets with different binding degrees have different fluorescence characteristics, so that the fluorescent characteristics observed by the fluorescent microscope can judge whether the sample to be detected contains the platelet antibody, without developing color by anti-IgG sensitized indicator erythrocytes or HRP-labeled anti-human IgG.
If the sample to be detected contains the PLT-resistant alloantibody, namely the sample to be detected contains the antibody capable of being specifically combined with the PLT surface antigen, a second antibody-PLT alloantibody-fluorescence labeled PLT surface antigen complex can be formed, and the complex has the characteristics under a fluorescence microscope: the fluorescent spots are spread on the bottom of the U-shaped bottom reaction hole and show diffuse fluorescence, namely, the spot is judged to be positive (as shown in FIG. 3).
If the sample to be detected does not contain the anti-PLT alloantibody, a second antibody-anti-PLT alloantibody-PLT surface antigen complex cannot be formed, after unbound serum components are removed by washing, the remainder is the platelet marked by the cell membrane fluorescent dye, so the sample to be detected has the characteristics under a fluorescence microscope: the fluorescent sites gather at the bottom of the U-shaped bottom reaction well, and an intense fluorescent spot appears, namely, the determination is negative (as shown in FIG. 3).
If the sample to be detected contains a trace amount of the anti-PLT alloantibody, a trace amount of second antibody-anti-PLT alloantibody-PLT surface antigen complex can be formed, after unbound serum components are removed by washing, the remainder is the platelet marked by cell membrane fluorescent dye and a trace amount of second antibody-anti-PLT alloantibody-PLT surface antigen complex, and the sample to be detected is characterized in that: the fluorescent site does not cover the bottom of the U-shaped reaction hole and shows diffuse fluorescence, namely, the fluorescent site is judged to be weak positive (as shown in figure 3). In practical tests, the positive degree of the detection antibody, such as +, 2+, 3+, 4+ and the like, can be roughly determined according to the size of the diffuse fluorescent ring, and the semi-quantitative detection of the platelet antibody is realized.
The platelet antibody screening method can directly observe the fluorescence condition under a fluorescence microscope, judges the detection result according to the fluorescence characteristic, greatly reduces the false positive rate of the detection of the same antibody and improves the detection specificity compared with the prior step-by-step amplification reaction needing multiple antibodies or enzyme-linked immunosorbent assay. In addition, the operation process is simple, the experiment can be completed within 90min by a single person, and time and labor are saved. In addition, the invention does not need multiple antibodies or amplified reaction of enzyme-linked immunosorbent assay, the types of the antibodies are few, and the antibodies do not need to be matched with corresponding indicating erythrocytes or chromogenic substrates, so the cost is low, the detection price is low, and the invention has higher popularization and application values.
Further, in the above method, the cell membrane fluorescent dye includes PKH26, PKH 67, 1 '-dioctadecyl-3, 3, 3' 3 '-tetramethylenecarbocyanine perchlorate or 3, 3' -dihexadecyloxycarbocyanine perchlorate.
The above-mentioned cell membrane fluorochrome is preferably PKH26, PKH26 is a red fluorochrome specially used for cell membrane marking, it can stably integrate a fluorochrome linked with long tail fat into the lipid region of the platelet membrane, and does not affect the activity of platelet, and the fluorescence intensity can be maintained for several weeks or months when the platelet is marked in vitro and in vivo, the invention uses PKH26 to make cell membrane fluorescent staining on PLT, and the aggregation distribution of PKH26-PLT can be directly observed under the fluorescent microscope after the reaction is finished.
Further, in the above method, the second antibody is a mouse anti-human IgG polyclonal antibody, a rabbit anti-human IgG polyclonal antibody, a goat anti-human IgG polyclonal antibody, a mouse anti-human IgG monoclonal antibody, a rabbit anti-human IgG monoclonal antibody, a goat anti-human IgG monoclonal antibody, or a recombinant anti-human IgG antibody.
Further, in the above method, the solid phase carrier is a U-bottom ELISA plate.
Further, in the above method, the sample to be tested is a serum or plasma sample to be tested.
The invention takes serum or plasma as a detection object, can screen platelet antibodies by collecting blood of a human body to be detected, has convenient and minimally invasive sampling and higher acceptability for people to be detected.
In a second aspect, the present invention provides a platelet antibody screening kit based on cell membrane fluorescent labeling, which comprises platelets labeled with a cell membrane fluorescent dye and a solid support coated with a second antibody.
The working principle of the kit of the invention is the same as the detection principle of the platelet antibody screening method based on the cell membrane fluorescent dye labeling, and whether the sample to be detected contains the antiplatelet antibody or not is judged by analyzing the fluorescence characteristics of the platelet labeled by fluorescence after reaction, and the specific principle is not repeated here.
Further, in the above kit, the cell membrane fluorescent dye includes PKH26, PKH 67, 1 ' -diazadecyl-3, 3,3 ' -tetramethylenepolycarbocyanine perchlorate or 3,3 ' -dihexadecyloxycarbocyanine perchlorate.
Further, in the kit, the solid phase carrier is a U-shaped bottom ELISA microplate; the second antibody is mouse anti-human IgG polyclonal antibody, rabbit anti-human IgG polyclonal antibody, goat anti-human IgG polyclonal antibody, mouse anti-human IgG monoclonal antibody, rabbit anti-human IgG monoclonal antibody, goat anti-human IgG monoclonal antibody or recombinant anti-human IgG antibody.
Further, the kit also comprises positive quality control serum and negative quality control serum; the positive quality control serum is serum containing anti-HPA antibody and/or anti-HLA antibody; the negative quality control serum is serum without antiplatelet alloantibodies.
In a third aspect, the invention provides the use of the kit in platelet antibody screening.
Compared with the prior art, the invention has the following beneficial effects:
the platelet antibody screening method is characterized in that platelet is labeled in advance by cell membrane fluorescent dye, a second antibody is added, and different fluorescence characteristics are presented under a fluorescence microscope by utilizing the fact that a sample to be detected contains an antibody specifically combined with a platelet surface antigen or does not contain the antibody, so that whether the sample to be detected contains the platelet antibody or not is judged.
Drawings
FIG. 1 is a schematic diagram of the detection of a monoclonal antibody specific platelet antigen by a capture method;
FIG. 2 is a schematic diagram of the platelet antibody screening method based on the cell membrane fluorescent labeling;
FIG. 3 is a graph showing fluorescence determined as negative, weakly positive, and positive according to the present invention.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples. It will be understood by those skilled in the art that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.
Example 1
This example provides a platelet antibody screening kit based on cell membrane fluorescent labeling, and takes cell membrane fluorescent dye PKH26 as an example to illustrate the platelet antibody screening method of the present invention.
1. Composition of the kit
(1) U-bottom ELISA plate: a U-shaped microporous plate contains 8 holes multiplied by 12 and 96 detection holes, and all the micropores are coated with mouse anti-human IgG monoclonal antibodies.
(2) Low ionic strength salt solution (LISS solution): the LISS solution was purple and non-fluorescent.
(3) Platelet suspensions labeled with PKH26 (hereinafter referred to as PKH 26-PLT).
(4) Positive quality control serum: sera containing anti-HPA antibodies and/or anti-HLA antibodies.
(5) Negative quality control serum: serum free of anti-platelet alloantibodies.
(6) PBS/Tween washes.
In addition to the above six materials already in the kit, the following tools are also required for completing the platelet antibody screening method of the present invention:
a multichannel pipette with the measuring range of 100 mu L, a disposable pipette head, a microporous plate centrifuge, a 37 ℃ incubator, a microporous plate oscillator and a fluorescence microscope.
2. Preparation method of kit
(1) Preparation of U-shaped bottom ELISA (enzyme-Linked immuno sorbent assay) microporous plate coated with mouse anti-human IgG (immunoglobulin G) monoclonal antibody
A. Coating: sterilizing a polystyrene plate by ultraviolet irradiation (30W ultraviolet lamp, 75cm irradiation for 12h), diluting a mouse anti-human IgG monoclonal antibody with a coating buffer solution into an antibody diluent with the concentration of 0.5-100 mu g/mL (10 mu g/mL is the optimal antibody diluent concentration), adding 100 mu L and 10 mu g/mL of the antibody diluent into a hole of the polystyrene plate, placing the enzyme label plate at 4 ℃ for overnight binding for 24h, and washing to remove the unbound antibody diluent.
B. And (3) sealing: after washing, the plate was blocked by adding 0.01mol/L PBS buffer (containing 10g/L bovine serum albumin), and then the plate was left at 4 ℃ overnight.
C. Washing the plate: taking out the coated microporous plate, and balancing at room temperature; then, the confining liquid is thrown off, PBS washing liquid is uniformly added on the plate, each hole is filled with the washing liquid, the plate is placed for about 0.5min, and then the washing liquid is thrown off; repeating the steps for four times, and sucking the residual washing liquid in the plate on absorbent paper for later use.
(2) Preparation of PKH 26-labeled platelet (PKH 26PLT) suspension
A. Take 2X 107～2×109Single platelet (at 2X 10)8Optimally) the suspension was placed in a conical bottom polypropylene tube, washed once with PBS/Tween washes, and then placed in a centrifuge at 400g for 5min to allow the platelets to aggregate into a loose pellet.
B. After centrifugation, the supernatant was carefully aspirated, taking care not to remove any cell particles, leaving only a trace amount of supernatant, forming a cell suspension.
C. A 2 x cell suspension is prepared, then 1mL of diluent (an aqueous solution intended to maintain the viability of the cells while facilitating the lysis and staining of the dye, supplied by the PKH26 kit) is added to the cell suspension and resuspended gently with a pipette and ensuring complete dispersion. Vortex oscillation is contraindicated and the cell particles do not stay in the diluent for too long.
D. Before dyeing, a 2X dye solution (4X 10) is first prepared6M), the specific method comprises the following steps: adding 4 mu L of PKH26 ethanol dye solution into 1mL of diluent, and uniformly mixing to obtain the dye.
E. 1mL of 2 × cell suspension (step C) was quickly added to 1mL of 2 × dye solution (step D) and mixed immediately, the final concentration after mixing would be 108Individual cells/mL and 2X 106 M PKH 26. Incubating the mixed solution for 1-5 minShaking up. Since the dyeing process is very rapid, there is no benefit for a long time.
F. The staining was stopped by adding an equal amount of serum or other suitable protein solution (1% BSA) to the incubated mixture and incubating for 1min to allow binding of excess dye.
G. The supernatant was then removed, ensuring that the cells were not removed. The cell particles were then resuspended in 10mL PBS and transferred to a fresh sterile conical tube, 400g at room temperature, centrifuged for 5min, and the cells were washed again with 10mL PBS to ensure removal of unbound dye.
H. After the last wash, the cells were resuspended in 10ml PBS to assess cell recovery, cell survival and fluorescence intensity. After centrifugation to remove the supernatant, 5mL of LISS solution was added and resuspended (cell concentration at 4X 10)6～4×108In the range of 4X 107Optimally), a suspension of PKH26-PLT is prepared for use.
3. Method for detecting platelet antibody by using kit
The method for screening the platelet antibody based on the cell membrane fluorescent marker is described by taking the using method of the kit as an example, and the specific detection method is as follows:
(1) and taking out the U-shaped bottom ELISA microporous plate coated by the mouse anti-human IgG monoclonal antibody as required, wherein the microporous plate is provided with a positive quality control hole and a negative quality control hole, the positive quality control hole is used for adding positive quality control serum, and the negative quality control hole is used for adding negative quality control serum.
(2) 100 μ L of purple LISS solution was added to each well of the U-bottom ELISA plate using a pipette gun.
(3) And respectively adding 50 mu L of positive quality control serum, negative quality control serum and to-be-detected plasma or serum into the positive quality control hole, the negative quality control hole and the detection hole of the U-shaped bottom ELISA micropore plate by using a pipette.
(4) Shaking the U-shaped bottom ELISA microplate on a micropore oscillator at 800rpm/min for 10s, and incubating the U-shaped bottom ELISA microplate at 37 ℃ for 15min (sealing the microplate during incubation) when the color of LISS liquid in a blank hole changes from purple to blue.
(5) After incubation, plates were washed manually three times with 150 μ L PBS/Tween wash to remove unbound serum, and after each wash, the PBS/Tween wash was gently decanted and tapped off.
(6) Immediately after washing, 100. mu.L of PKH26-PLT suspension was added to each well, and the plate was incubated at 37 ℃ for 30min (the plate was sealed and protected from light).
(7) And after the incubation is finished, placing the U-shaped bottom ELISA microplate in a centrifuge for 5min at 200rpm/min without interruption.
(8) And observing and interpreting results through a fluorescence microscope after the centrifugation is finished.
4. Determination of results
The fluorescence characteristic of the positive or weak positive reaction hole is that PKH26-PLT is flatly laid at the bottom of the reaction hole and shows diffuse red fluorescence; the PKH26-PLT in the positive reaction well completely spreads out at the bottom of the reaction well, while the PKH26-PLT in the weak positive reaction well does not completely spread out at the bottom of the reaction well, but does not exhibit aggregation.
The fluorescence of the negative reaction well was characterized by the concentration of PKH26-PLT in the center of the bottom of the reaction well, which showed an intense fluorescent spot, as shown in FIG. 3.
5. The kit of the invention verifies the detection effect
In order to determine the detection accuracy of the novel cell membrane fluorescence-labeled platelet antibody screening method, 6 samples with screening results of-4 + by a solid phase agglutination method are taken as samples, the platelet antibody screening method is used for verification, the results are shown in table 1, when the reaction intensity of the anti-platelet alloantibody is in the range of-to +/-2 (sample 1 and sample 2), the results detected by the method are negative, and the sample 2 is verified to be negative by a monoclonal antibody specific platelet antigen capture method, which shows that the detection accuracy of the platelet antibody screening method is superior to that of the conventional solid phase agglutination method. When the reaction strength of the antiplatelet homogeneous antibody is in the range of + to 4+, the screening method can effectively detect the existence of the antibody, the antibody reaction strengths are consistent, the specific reaction results are shown in table 1, and the results prove that: the method has higher accuracy in detecting the platelet antibody.
TABLE 1 comparison of results of solid phase agglutination methods with cell membrane fluorescence labeled platelet antibody screening methods
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. A platelet antibody screening method based on cell membrane fluorescent labeling is characterized by comprising the following steps:
s1: carrying out fluorescence labeling on the blood platelet by using cell membrane fluorescent dye to obtain fluorescence labeled blood platelet;
s2: adding a sample to be detected into a solid phase carrier coated with a second antibody for incubation;
s3: and (4) adding the fluorescence labeled platelet obtained in the step (S1) into the incubated sample to be detected, and directly observing the aggregation distribution condition of the platelet through a fluorescence microscope after incubation and centrifugation so as to judge whether the sample to be detected contains the antiplatelet antibody.
2. The platelet antibody screening method of claim 1, wherein the cell membrane fluorochrome comprises PKH26, PKH 67, 1 ' -diazadecyl-3, 3,3 ' -tetramethylenepolycarbocyanine perchlorate or 3,3 ' -dihexadecyloxycarbocyanine perchlorate.
3. The platelet antibody screening method of claim 1, wherein the second antibody is a mouse anti-human IgG polyclonal antibody, a rabbit anti-human IgG polyclonal antibody, a goat anti-human IgG polyclonal antibody, a mouse anti-human IgG monoclonal antibody, a rabbit anti-human IgG monoclonal antibody, a goat anti-human IgG monoclonal antibody, or a recombinant anti-human IgG antibody.
4. The platelet antibody screening method of claim 1, wherein the solid support is a U-bottom ELISA plate.
5. The method for screening platelet antibodies according to claim 1, wherein said sample to be tested is a serum or plasma sample to be tested.
6. A platelet antibody screening kit based on cell membrane fluorescent labeling is characterized by comprising platelets labeled by cell membrane fluorescent dye and a solid phase carrier coated with a second antibody.
7. The platelet antibody screening kit of claim 6, wherein the cell membrane fluorochrome comprises PKH26, PKH 67, 1 ' -diazadecyl-3, 3,3 ' -tetramethylenepolycarbocyanine perchlorate or 3,3 ' -dihexadecyloxycarbocyanine perchlorate.
8. The platelet antibody screening kit of claim 6, wherein the solid support is a U-bottom ELISA microplate; the second antibody is a mouse anti-human IgG polyclonal antibody, a rabbit anti-human IgG polyclonal antibody, a goat anti-human IgG polyclonal antibody, a mouse anti-human IgG monoclonal antibody, a rabbit anti-human IgG monoclonal antibody, a goat anti-human IgG monoclonal antibody or a recombinant anti-human IgG antibody.
9. The platelet antibody screening kit of claim 6, further comprising a positive quality control serum and a negative quality control serum; the positive quality control serum is serum containing anti-HPA antibody and/or anti-HLA antibody; the negative quality control serum is serum without antiplatelet alloantibodies.
10. Use of the platelet antibody screening kit of claim 6 for platelet antibody screening.
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