CN114252594A - Placenta growth factor detection kit and preparation method and application thereof - Google Patents
Placenta growth factor detection kit and preparation method and application thereof Download PDFInfo
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- CN114252594A CN114252594A CN202111387053.XA CN202111387053A CN114252594A CN 114252594 A CN114252594 A CN 114252594A CN 202111387053 A CN202111387053 A CN 202111387053A CN 114252594 A CN114252594 A CN 114252594A
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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Abstract
The invention relates to a placenta growth factor detection kit and a preparation method and application thereof, wherein an inventor adopts a double-antibody sandwich method combined with a magnetic particle chemiluminescence technology to detect placenta growth factors, and discovers that an immune micro-magnetic particle preparation method optimized by the inventor is adopted, the prepared placenta growth factor capture antibody magnetic particles and an alkaline phosphatase-labeled placenta growth factor detection antibody further prepared by crosslinking agents SPDP, DTT and a sealant MMTS according to a specific proportion are adopted, and the formed placenta growth factor chemiluminescence immunoassay kit can automatically finish detection by taking a full-automatic chemiluminescence immunoassay analyzer as a detection tool, and meanwhile, the detection performance is also obviously improved, the detection sensitivity reaches 0.54pg/mL, the detection linear range is wide, and the detection linear range can reach 3 pg/mL-10000 pg/mL. Indicating that the method has wide detection application prospect.
Description
Technical Field
The invention relates to the field of biological medicines, in particular to a placenta growth factor detection kit and a preparation method and application thereof.
Background
Placental growth factor (PLGF) is synthesized primarily by synbiotic trophoblast cells, binds to tyrosinase receptors located in trophoblast and vascular endothelial cells, and is a protein that autocrine for trophoblast cell function and paracrine for vascular growth. PLGF has unique regulating effect on the functions of trophoblast cell and endothelial cell and can promote angiogenesis.
Placental growth factor is one of the members of the vascular endothelial growth factor family, also known as placental-derived angiogenic factors. In non-pregnant women, there may be a small amount of expression in histiocytes such as thyroid epithelium, keratinocytes and melanoma cells, but less than 10 pg/mL. When a woman is pregnant, i.e. a plurality of PLGFs are synthesized by syncytiotrophoblast, the PLGFs promote the proliferation and differentiation of the trophoblasts through an autocrine mode; on the other hand, the preparation method regulates the formation and physiological recasting of placental blood vessels in a paracrine mode, and simultaneously can play a synergistic role with other vascular endothelial growth factors, so that the biological activity of the preparation method is obviously improved, the development of the placental blood vessels is promoted, an important physiological role is played in the formation process of the placental blood vessels, the normal blood supply and function of a placenta in the pregnancy process are maintained, and the preparation method is suitable for the requirements of maternal and fetal development. The concentration of PLGF increased in maternal blood circulation with increasing gestation weeks, peaked at 26-30 weeks gestation, and declined thereafter until parturition. Meanwhile, the half-life period of the PLGF is only 15 minutes, and one metabolic cycle is about 75 minutes, so that the PLGF can well reflect the instant condition of the placenta function.
Preeclampsia (PE) is a serious complication of pregnancy whose major clinical manifestations are hypertension and proteinuria around 20 weeks after pregnancy. About 3-5% of pregnant women develop preeclampsia and can lead to maternal, fetal, or neonatal death. Preeclampsia, which is associated with thrombocytopenia and elevated liver enzyme activity, can manifest as thrombocytopenia syndrome (hemolysis, elevated liver enzymes, thrombocytopenia). Preeclampsia occurs due to endothelial dysfunction caused by the release of angiogenic factors from the placenta. Alterations in serum PLGF (placental growth factor) and sFlt-1 (soluble tyrosine kinase-1, or vascular endothelial growth factor receptor-1) concentration levels occur in women with pre-eclampsia. Furthermore, the levels of PLGF and sFlt-1 in the blood circulation may precede the appearance of clinical symptoms to identify normal pregnancy and pre-eclampsia. The level of pro-vascular factor PLGF increases in the first 6 months of normal pregnancy and gradually decreases as pregnancy progresses through termination. In contrast, the anti-vascular factor sFlt-1 levels remained stable during early and middle gestation, and did not rise smoothly until termination of pregnancy. Women with pre-eclampsia have higher than normal pregnancy levels of sFlt-1, while levels of PLGF are lower than normal pregnancy levels. Determination of the ratio of sFlt-1 to PLGF is more valuable than detection of sFlt-1 or PLGF alone. Placental endoglin is a member of the TGF- β family, which is upregulated in preeclampsia and released into the maternal circulation as soluble endoglin. Soluble endoglin has been shown to be significantly elevated in severe cases of preeclampsia.
Therefore, the detection of the blood PLGF level of the pregnant women can be clinically used for identifying the existence of oxygen supply pressure of the trophoblast cells of the placental syncytium. Can be used for predicting and assisting diagnosis of gestational hypertension.
The traditional placenta growth factor measuring method includes enzyme linked immunosorbent assay and chemiluminescence assay.
At present, most of domestic methods for detecting PLGF are enzyme-linked immunosorbent assay and fluorescence chromatography. The enzyme-linked immunosorbent assay is long in time consumption and complicated in operation. In addition, the two detection methods have poor detection sensitivity and narrow detection range, and are not fully-automatic tests. The foreign detection method for the PLGF has an electrochemiluminescence immunoassay method, but the detection cost of the method is high, so that the method cannot be applied to pregnancy in areas with limited resources.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a method for preparing immunomagnetic particles.
The technical scheme is as follows:
(1) pretreatment: pretreating the magnetic particles to obtain a mixed solution 1;
(2) and (3) activation: activating the antibody by using a cross-linking agent;
(3) coupling: adding the activated antibody in the step (2) into the mixed solution 1 obtained in the step (1) to obtain a mixed solution 2, and incubating to obtain an antibody-magnetic bead conjugate;
(4) and (3) sealing: adding a sealing solution into the antibody-magnetic bead conjugate obtained in the step (3) for sealing to obtain an antibody-magnetic bead coupled immunomagnetic particle final product;
wherein the cross-linking agent in step (2) is selected from BS (PEG)5BMPS and BM (PEG)3Any one of the above; in the activation treatment, the mass ratio of the antibody to the crosslinking agent is 103:(0.5～2.0)。
It is also an object of the present invention to provide immunomagnetic particles obtained according to the above-mentioned preparation method.
The invention also aims to provide an application of the immunomagnetic particles in preparation of a kit or a kit detection reagent.
The invention also aims to provide the placenta growth factor capture antibody magnetic particle.
The technical scheme is as follows:
a placenta growth factor capture antibody magnetic particle is prepared by the preparation method of the immune magnetic particle.
The invention also aims to provide a chemiluminescence immunoassay kit for the placenta growth factor.
The technical scheme is as follows:
a placenta growth factor chemiluminescence immunoassay kit comprises the placenta growth factor capture antibody magnetic particles and a placenta growth factor detection antibody marked by a chemiluminescence marker.
The invention also aims to provide a method for quantitatively detecting the placenta growth factor for non-disease diagnosis.
The technical scheme is as follows:
establishing a standard fitting curve according to the placenta growth factor chemiluminescence immunoassay kit;
and obtaining a sample to be detected, detecting by adopting the placenta growth factor chemiluminescence immunoassay kit, recording the luminous value of the sample to be detected, and substituting the luminous value into the standard fitting curve to obtain the concentration of the placenta growth factor in the sample.
The inventor of the invention aims to solve the problems of poor sensitivity, narrow detection range and the like of placenta growth factor (PLGF) detection in the prior art, adopts a double-antibody sandwich method combined with a magnetic particle chemiluminescence technology to detect the placenta growth factor, and in the preparation process of placenta growth factor capture antibody magnetic particles, unexpectedly discovers that the optimized immune micromagnetic particle preparation method is adopted, and particularly, the used cross-linking agent is selected from BS (PEG)5BMPS and BM (PEG)3In any one of the methods, the placenta growth factor capture antibody magnetic particles prepared by a specific proportion reaction and the placenta growth factor detection antibody marked by alkaline phosphatase prepared by the inventor through a specific proportion cross-linking agent SPDP, DTT and a sealing agent MMTS can form the placenta growth factor chemiluminescence immunoassay kit, a full-automatic chemiluminescence immunoassay analyzer can be used as a detection tool to automatically complete detection, the detection performance is also obviously improved, the detection sensitivity reaches 0.54pg/mL, the sensitivity is improved by at least 10 times compared with that of a traditional placenta growth factor detection method, the detection linear range is wide, and the detection linear range can reach 3 pg/mL-10000 pg/mL. Indicating that the method has wide detection application prospect.
Drawings
FIG. 1 is a schematic diagram of the preparation process of the placental growth factor chemiluminescence immunoassay kit of the present invention.
FIG. 2 is a graph of the placental growth factor standards obtained from the test series of PLGF standards in example 2.
FIG. 3 is a statistical chart of the correlation analysis between the results of the chemiluminescent immunoassay kit for placental growth factor and the results of the Roche kit of example 4.
Detailed Description
Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Throughout the specification and claims, the following terms have the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase "in one embodiment" as used in the present disclosure does not necessarily refer to the same embodiment, although it may. Moreover, the phrase "in another embodiment" as used in this disclosure does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined without departing from the scope or spirit of the invention.
Furthermore, as used herein, the term "or" is an inclusive "or" symbol and is equivalent to the term "and/or," unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. Furthermore, throughout the specification the meaning of "a", "an" and "the" include plural referents. The meaning of "in.
In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The present invention relates to abbreviations and terms defined as follows:
BS(PEG)5: pegylated bis (sulfosuccinimidyl) suberic acid
PLGF: placenta growth factor
SPDP: succinimide 3- (2-pyridyldithio) -propionic acid ester
MMTS: methyl sulfur methanesulfonate ester
BMPS: N-beta-Maleimidopropylsuccinimidyl ester
BM(PEG)3:1, 11-bismaleimide-triethylene glycol
DTBP: dithio-alanine dimethyl ester dihydrochloride
DTSSP: 3, 3' -Dithiobis (sulfosuccinimidyl propionate)
AMAS: N-alpha-Maleimidooxysuccinimide ester
The present invention will be described in further detail with reference to specific examples.
Some embodiments of the present invention provide a method for preparing immunomagnetic particles, comprising the steps of:
(1) pretreatment: pretreating the magnetic particles to obtain a mixed solution 1;
(2) and (3) activation: activating the antibody by using a cross-linking agent;
(3) coupling: adding the activated antibody in the step (2) into the mixed solution 1 obtained in the step (1) to obtain a mixed solution 2, and incubating to obtain an antibody-magnetic bead conjugate;
(4) and (3) sealing: adding a sealing solution into the antibody-magnetic bead conjugate obtained in the step (3) for sealing to obtain an antibody-magnetic bead coupled immunomagnetic particle final product;
wherein the cross-linking agent in step (2) is selected from BS (PEG)5BMPS and BM (PEG)3Any one of the above; in the activation treatment, the mass ratio of the antibody to the crosslinking agent is 103(0.5 to 1.5), and more preferably, the mass ratio of the antibody to the crosslinking agent is 103:1.0。
In some of the examples, the crosslinking agent in step (2) of the above preparation process is preferably BS (PEG)5Antibodies with crosslinking agent BS (PEG)5The mass ratio used is1031.0 due to BS (PEG)5The PEG-modified immunoassay method is a polymer with 5 PEGs, and a coupling long arm is arranged, so that the distance between an antibody and a magnetic bead can be increased as a bridge, the steric hindrance of the antibody and the magnetic bead is reduced, more antibodies can be connected to the magnetic bead in unit area, and more antibodies can be combined with an antigen subsequently, so that the stability and the sensitivity of the immunoassay method are improved.
In some embodiments, in step (1) of the preparation method, the mixed solution 1 is obtained by washing and resuspending the magnetic particles with a buffer solution, and then uniformly dispersing the magnetic particles in an ammonium sulfate solution; further, the concentration of the ammonium sulfate solution is 2-5M, and the pH value is 8-10. More preferably, the ammonium sulfate solution used has a concentration of 3M and a pH of 9.5, and at this time, ammonium sulfate acts as a reaction accelerator to accelerate the binding of the subsequent magnetic beads to the protein.
In some embodiments, in step (1) of the preparation method, the buffer is preferably a PBS buffer and a BBS buffer; more preferably, the magnetic microparticles are washed with 0.01M PBS buffer at pH 7.4, the supernatant is magnetically separated, and then resuspended in 0.1M BBS buffer at pH 9.5.
In some embodiments, in step (1) of the preparation method, the magnetic particle is modified by tosyl, that is, when the active group of the magnetic particle is tosyl, the magnetic particle can be coupled with protein, so that the coupled protein can be combined with an object to be detected, and then the high-efficiency separation and detection of the object to be detected can be realized by the action of an external magnetic field.
In some embodiments, in step (1) of the above preparation method, the tosylated magnetic particles have a particle size of 0.9 μm to 1.8. mu.m.
In some embodiments, in step (4) of the above preparation method, the blocking solution is 0.05M Tris buffer containing 0.5% BSA and having a pH of 7.4.
In some embodiments, in step (4) of the preparation method, in the final product of the antibody-magnetic bead coupled immunomagnetic particles, the mass ratio of the antibody to the magnetic bead is 1: (10-50). More preferably, the mass ratio of the antibody to the magnetic beads is 1: 20.
In some embodiments, in step (2) of the above preparation method, the protein comprises an antigen and an antibody, and further, the protein is an antibody, and more preferably, a placental growth factor monoclonal antibody.
Some embodiments of the present invention also provide an immunomagnetic particle obtained according to the above preparation method.
Some embodiments of the invention also provide application of the immunomagnetic particles in preparation of kits or kit detection reagents.
Some embodiments of the present invention also provide a placenta growth factor capture antibody magnetic microparticle prepared by the above preparation method.
Some embodiments of the present invention further provide a placental growth factor chemiluminescence immunoassay kit, which comprises the placental growth factor capture antibody magnetic microparticles and a placental growth factor detection antibody labeled with a chemiluminescence marker.
In some embodiments, the chemiluminescent marker labeled placental growth factor detection antibody is obtained by mixing, crosslinking and blocking agent treatment of a chemiluminescent substance activated by DTT and a placental growth factor detection antibody activated by a crosslinking agent.
In some embodiments, the preparation method of the chemiluminescent marker-labeled placental growth factor detection antibody specifically comprises the following steps:
(1) antibody activation: activating the antibody by using a cross-linking agent;
(2) activation of chemiluminescence substance: activating the chemiluminescent marker by using dithiothreitol;
(3) coupling: mixing and incubating the chemiluminescent marker activated in the step (2) and the antibody activated in the step (1) to obtain a chemiluminescent marker-antibody conjugate;
(4) and (3) sealing: adding a blocking agent into the chemiluminescent marker-antibody conjugate obtained in the step (3) for blocking to obtain a chemiluminescent marker-antibody final product;
the cross-linking agent is selected from any one of SPDP, DTBP, DTSSP and AMAS, and the blocking agent is MMTS. Further, the cross-linking agent is preferably SPDP, when the cross-linking agent is a 5mg/mL SPDP solution, the SPDP is used as an antibody activator, the antibody can be activated with the maximum efficiency, and the coupling efficiency of the subsequent antibody and the chemiluminescent marker can be greatly improved. Thereby improving the detection sensitivity.
In some embodiments, the antibody and the cross-linking agent are used at the same concentration in the activation treatment in step (1) by incubating at room temperature for 1 hour.
In some of these embodiments, the activation treatment of the chemiluminescent label in step (2) is DTT at a concentration of 5mg/mL and incubation at room temperature for 1 h.
In some embodiments, the blocking agent used in step (4) is MMTS at 5mg/mL, and further, desalting and purifying treatment is performed, preferably, the desalted and purified conjugate is recovered to 0.1mg/mL with PBS buffer solution at 0.01M and pH 7.4, and then an equal volume of glycerol is added and mixed to obtain the chemiluminescent-labeled placental growth factor detection antibody.
In some embodiments, the above-described chemiluminescent label for the placental growth factor detection antibody is selected from one of acridinium ester, ruthenium terpyridyl, adamantane, luminol, a derivative of luminol, isoluminol, a derivative of isoluminol, horseradish peroxidase, and alkaline phosphatase. Of course, in other embodiments, the label in the antibody labeled with the label is not limited to the above, and may be other substances that can be used in the chemiluminescent immunoassay platform.
In some embodiments, the chemiluminescent label is alkaline phosphatase, and the mass ratio of the placenta growth factor detection antibody to the cross-linking agent in the placenta growth factor detection antibody labeled by alkaline phosphatase is 100 (0.5-2.0). More preferably, the mass ratio of the placenta growth factor detecting antibody to the cross-linking agent is 100: 1.0.
In some embodiments, the kit further comprises a placenta growth factor calibrator, and the placenta growth factor calibrator is prepared by using a calibration buffer solution, wherein the calibration buffer solution comprises 45mM to 55mM Tris, 0.05% to 0.15% BSA, and 0.9% NaCl. Preferably, the calibration buffer comprises 50mM Tris, 0.1% BSA, 0.9% NaCl, pH 7.5.
In some embodiments, the above-mentioned dish growth factor chemiluminescence immunoassay kit further comprises a chemiluminescence substrate solution, and further preferably an APS substrate solution.
Some embodiments of the invention also provide a method for quantitatively detecting placental growth factor for non-disease diagnostic purposes.
The method comprises the following steps:
establishing a standard fitting curve according to the placenta growth factor chemiluminescence immunoassay kit;
and obtaining a sample to be detected, detecting by adopting the placenta growth factor chemiluminescence immunoassay kit, recording the luminous value of the sample to be detected, and substituting the luminous value into the standard fitting curve to obtain the concentration of the placenta growth factor in the sample.
In some embodiments, the sample to be tested is serum.
In some embodiments, the quantitative detection method specifically comprises: and (2) taking 20-100 mu L of serum sample, adding 20-100 mu L of placenta growth factor capture antibody magnetic particles and 20-100 mu L of placenta growth factor detection antibody marked by alkaline phosphatase, reacting for 5-15 min, performing magnetic separation, then adding 100-300 mu L of substrate solution, mixing uniformly, sending the reaction mixture into a dark room by an instrument, and finally recording the luminescence value. Preferably, 50 mu L of serum sample is taken, 50 mu L of placenta growth factor capture antibody magnetic particles and 50 mu L of placenta growth factor detection antibody marked by alkaline phosphatase are added, after reaction for 10min, magnetic separation is carried out, then 200 mu L of substrate solution is added, after uniform mixing, the reaction mixture is sent into a dark room by an instrument, and finally, the luminescence value is recorded.
EXAMPLE 1 preparation of a PLGF chemiluminescent immunoassay kit
(1) Preparation of tosylated magnetic microparticles coated with placental growth factor monoclonal antibody:
taking a suspension containing 80mg of tosylated magnetic particles (Magnosphere TM, cat # MS160) having a particle size of 0.9 to 1.8 μ M, magnetically separating the supernatant, washing the magnetic particles with 0.01M of PBS buffer solution having pH of 7.4, magnetically separating the supernatant, resuspending the supernatant with 0.1M of BBS buffer solution having pH of 9.5, adding 3mL of 3M ammonium sulfate solution having pH of 9.5, dispersing the solution uniformly to obtain a mixed solution 1, and adding 4. mu.g of (PEG) to 4mg of placental growth factor monoclonal antibody (PLGF-34 #) (Guangzhou Wanfu Biotechnology GmbH, cat #)5(Pegylated bis (sulfosuccinimidyl) suberic acid, ThermoFisher SCIENTIFIC) activating the placental growth factor monoclonal antibody, and adding the activated antibody to the mixed solution 1 to obtain a mixed solution 2. Then placing the mixed solution 2 on a rotary shaking bed for carrying out primary incubation treatment, washing with 0.05M Tris buffer solution with pH 7.4 after magnetically separating supernatant, removing supernatant, then adding 0.5% BSA solution for carrying out secondary incubation treatment, washing with 0.05M Tris buffer solution with pH 7.4 after magnetically separating supernatant, finally re-suspending to 10mg/mL with 0.05M Tris buffer solution with 0.5% BSA and pH 7.4 to obtain the toluoylated magnetic particles coated by the placenta growth factor monoclonal antibody, wherein the corresponding magnetic bead mother solution is named as: PLGF-R1-R, stored at 4 ℃ for use.
(2) Preparing a placenta growth factor monoclonal antibody marked by alkaline phosphatase:
taking a solution containing 1mg of placenta growth factor monoclonal antibody (product number: PLGF-30#), centrifuging to remove supernatant, replacing the liquid in the original antibody with 0.01M PBS buffer solution with pH 7.4 to 5mg/mL, adding 2 μ L of SPDP (succinimide 3- (2-pyridyl dithio) -propionate, ThermoFisher SCIENTIFIC) with concentration of 5mg/mL, incubating at room temperature for 1h, and activating the antibody; then adding 2 μ L of DTT with the concentration of 5mg/mL into 50 μ L of alkaline phosphatase with the concentration of 20mg/mL, and incubating for 1h at room temperature to activate the alkaline phosphatase; the activated antibody and alkaline phosphatase were mixed together and incubated at room temperature for 1 hour for crosslinking. Finally, adding 4 μ L of MMTS (methyl sulfur methanesulfonate) with the concentration of 5mg/mL into the crosslinked mixture for blocking, desalting and purifying, recovering the desalted and purified conjugate to 0.1mg/mL by using 0.01M PBS buffer solution with the pH of 7.4, adding equal volume of glycerol, and uniformly mixing to obtain the placenta growth factor monoclonal antibody conjugate labeled by alkaline phosphatase, wherein the corresponding enzyme labeling mother liquor is named as: PLGF-R2-R, stored at-20 ℃ for use.
(3) Preparing a placenta growth factor calibration sample:
placenta growth factor (R & D Systems, Inc.) was prepared with a calibrator buffer (50mM Tris, 0.1% BSA, 0.9% NaCl, pH7.5) at a concentration of 0pg/mL, 10pg/mL, 50pg/mL, 100pg/mL, 500pg/mL, 1000pg/mL, 2500pg/mL, 5000pg/mL, 8000pg/mL, 11000pg/mL, and was dispensed at 0.5mL per tube and stored at-20 ℃ for later use.
Example 2 detection method of PLGF chemiluminescence immunoassay kit
The detection principle of the kit is as follows: the magnetic particles coated with the placenta growth factor (PLGF) capture antibody are combined with the placenta growth factor (PLGF) detection antibody marked by alkaline phosphatase and the placenta growth factor in a sample, a calibrator or a quality control product to form a 'sandwich' compound. Separating the complex formed by immunoreaction from other unbound substances under the action of an external magnetic field, washing the complex, and adding an enzymatic chemiluminescent substrate. The substrate is catalytically cracked under the action of enzyme to form an unstable excited intermediate, and when the excited intermediate returns to the ground state, photons are emitted to form a luminescence reaction, namely, a chemiluminescence apparatus is used for detecting the luminescence intensity of the reaction. Meanwhile, the luminous marker alkaline phosphatase is not consumed basically, the luminous agent in the reaction system is excessive, so that the luminous signal is strong and stable, the luminous time is long, the luminous intensity is in direct proportion to the content of the placenta growth factor in the sample in the detection range, and the concentration of the placenta growth factor in the sample can be calculated by referring to a standard curve.
A full-automatic chemiluminescence immunoassay analyzer (model FC-302, Guangzhou Wanfu biotechnology Co., Ltd.) is used as a detection tool, the methodology mode is double-antibody sandwich, namely, 50 mu L of serum sample is sequentially added into the analyzer, 50 mu L of tosylated magnetic particles coated by placenta growth factor monoclonal antibody and 50 mu L of placenta growth factor monoclonal antibody marked by alkaline phosphatase are added, after reaction is carried out for 10min, magnetic separation is carried out, 200 mu L of substrate liquid is added into the magnetic particles, after uniform mixing, the analyzer sends the reaction mixture into a dark room, and finally, the luminescence value is recorded.
The PLGF standards prepared in example 1 were examined by the above method, and the plotted standard curve was shown in fig. 2.
Then, the actual sample is tested, and the concentration of the sample is calculated according to the luminous value of the sample.
EXAMPLE 3 optimization of the PLGF chemiluminescent immunoassay kit
(1) Screening of antibody activator in preparation of tosylated magnetic microparticles coated with placental growth factor monoclonal antibody:
mixed solution 1 prepared as described in reference example 1 was added with 4. mu.g of BS (PEG) to 4mg of a placental growth factor monoclonal antibody (product No. PLGF-34, Van., guangzhou, Mobil Biotechnology Co., Ltd.)54. mu.g of BMPS (N-. beta. -maleimidopropylsuccinimidyl ester, ThermoFisher SCIENTIFIC), 4. mu.g of BM (PEG)3(1, 11-bismaleimide-triethylene glycol, ThermoFisher SCIENTIFIC) activating the placenta growth factor monoclonal antibody, and respectively adding the activated antibody into the mixed solution 1 to obtain 3 mixed solutions 2. Then, the 3 mixed solutions 2 were placed on a rotary shaking table to perform a first incubation treatment, after magnetic separation of the supernatant, they were washed with 0.05M Tris buffer (pH 7.4), after removal of the supernatant, a solution containing 0.5% BSA was added to perform a second incubation treatment, after magnetic separation of the supernatant, they were washed with 0.05M Tris buffer (pH 7.4), and finally resuspended in 10mg/mL Tris buffer (pH 7.4) containing 0.5% BSA to obtain 3 magnetic toluoylated magnetic particles coated with placental growth factor monoclonal antibodies (BS (PEG))5The mother liquor of magnetic beads corresponding to the coupling agent is named as: PLGF-R1-R; the mother liquor of magnetic beads corresponding to the BMPS coupling agent is named as: PLGF-R1-2; BM (PEG)3The mother liquor of magnetic beads corresponding to the coupling agent is named as: PLGF-R1- ③), stored at 4 ℃ for later use, and then used separatelyThe three magnetic bead mother solutions are matched with corresponding placenta growth factor monoclonal antibodies marked by alkaline phosphatase, and the test verification is carried out on the sensitivity, the repeatability and the linear indexes of the reagent, and the specific results are shown in the following table 3-1.
TABLE 3-1 Performance test results of PLGF antibodies coated with magnetic particles of different coupling agents
As can be seen from Table 3-1, when compared with the magnetic bead coating materials of PLGF-R1-phi and PLGF-R1-phi and PLGF-R1-phi, the signal to noise ratio (S/N) of the magnetic bead coating materials of PLGF-R1-phi is the largest, the repeatability is within 5%, and the linearity R is the best.
Therefore, in the placental growth factor monoclonal antibody coated tosylated superparamagnetic microparticles, the antibody activator used is preferably BS (PEG)5。
(2) Screening of antibody activator concentration in preparation of placenta growth factor monoclonal antibody coated tosylated magnetic microparticles:
mixed solution 1 was prepared as described in reference example 1, and then each 1mg of placenta growth factor monoclonal antibody (product No. PLGF-34, Vanfu Biotechnology Co., Ltd., Guangzhou) was added to 0.5. mu.g of BS (PEG)51.0. mu.g of BS (PEG)5And 2.0. mu.g of BS (PEG)5Activating the placenta growth factor monoclonal antibody, and adding the activated antibody into the mixed solution 1 to obtain 3 mixed solutions 2. Then, the 3 mixed solutions 2 were placed on a rotary shaking table to perform a first incubation treatment, after magnetic separation of the supernatant, they were washed with 0.05M Tris buffer at pH 7.4, after removal of the supernatant, a solution containing 0.5% BSA was added to perform a second incubation treatment, after magnetic separation of the supernatant, they were washed with 0.05M Tris buffer at pH 7.4, and finally resuspended in 10mg/mL Tris buffer at pH 7.4 containing 0.5% BSA to obtain 3 magnetic particles (0.5. mu.g of BS (PEG))5The mother liquor of magnetic beads corresponding to the coupling agent is named as: PLGF-R1- ((R)); 1.0 μ g of BS (PEG)5The mother liquor of magnetic beads corresponding to the coupling agent is named as: PLGF-R1-fifthly; 2.0 μ g of BS (PEG)5The mother liquor of magnetic beads corresponding to the coupling agent is named as: PLGF-R1-sixth), stored at 4 deg.C for use, and then the three mother liquid of magnetic beads are matched with corresponding placenta growth factor monoclonal antibody marked by alkaline phosphatase to test and verify the sensitivity, repeatability and linearity indexes of the reagent, the specific results are shown in the following table 3-2.
TABLE 3-2 different concentrations of BS (PEG)5Performance verification result of coupling agent magnetic particle coated PLGF antibody
As can be seen from tables 3-2, the signal-to-noise ratio (S/N), the repeatability and the linearity of the PLGF-R1-p and the PLGF-R1-c.o.m.p.m. of the PLGF-R1-p.m. of the PLGF-R1-p.m. of the PLGF-R1-c.p.m. of the PLGF-R1-p.m. of the PLGF-R1-p.m. of the magnetic bead coating are almost the same, but the performances are better than those of the PLGF-R1-p.m.
Thus, in the placental growth factor monoclonal antibody-coated tosylated superparamagnetic microparticles, the antibody and antibody activator BS (PEG) used5Is preferably 103:1.0。
(3) Screening a cross-linking agent in the preparation of the placenta growth factor monoclonal antibody marked by alkaline phosphatase:
referring to example 1, 1mg each of the placental growth factor monoclonal antibodies (Fukupffer Biotechnology Co., Ltd., Guangzhou, cat # PLGF-30) was substituted with PBS buffer of 0.01M and pH 7.4 to 5mg/mL, followed by addition of 2. mu.L of SPDP at a concentration of 5mg/mL, 2. mu.L of DTBP (dimethyl dithioalaninate dihydrochloride, ThermoFisher SCIENTIFIC) at a concentration of 5mg/mL, 2. mu.L of DTSSP (3, 3' -dithiobis (sulfosuccinimidyl propionate), ThermoFisher SCIENTIFIC) at a concentration of 5mg/mL, and 2. mu.L of AMAS (N-. alpha. -maleimidoxysuccinimide ester, ThermoFisher SCIENTIFIC) at a concentration of 5mg/mL, and incubation was carried out for 1 hour at room temperature to activate the antibodies; then respectively adding 2 mu L of DTT with the concentration of 5mg/mL into 50 mu L of alkaline phosphatase with the concentration of 20mg/mL, and incubating for 1h at room temperature to activate the alkaline phosphatase; the activated antibody and alkaline phosphatase were mixed together and incubated at room temperature for 1 hour for crosslinking. Finally, respectively adding 4 muL of MMTS with the concentration of 5mg/mL into the crosslinked mixture for sealing, desalting and purifying, recovering the desalted and purified combination to 0.1mg/mL by using PBS buffer solution with the pH value of 7.4 and 0.01M, then adding glycerol with the same volume, uniformly mixing to obtain the placenta growth factor monoclonal antibody combination marked by alkaline phosphatase (the enzyme-labeled mother solution corresponding to the SPDP crosslinking agent is named as PLGF-R2-phi, the magnetic bead mother solution corresponding to the DTBP crosslinking agent is named as PLGF-R2-phi, the magnetic bead mother solution corresponding to the DTSSP crosslinking agent is named as PLGF-R2-phi, the magnetic bead mother solution corresponding to the AMAS crosslinking agent is named as PLGF-R2-phi), storing at the temperature of-20 ℃ for later use, respectively matching the three enzyme-labeled mother solutions, and the tosylated magnetic particles coated by the corresponding placenta growth factor monoclonal antibody, the sensitivity, repeatability and linearity indexes of the reagent are tested and verified, and the specific results are shown in tables 3-3 below.
TABLE 3-3 Performance test results of alkaline phosphatase-labeled PLGF antibodies with different crosslinking agents
As can be seen from the above table 3-3, compared with the enzyme markers PLGF-R2-R2-R, PLGF-R2-R and PLGF-R2-R, the enzyme marker PLGF-R2-R has the largest signal-to-noise ratio (S/N), the repeatability within 5% and the optimal linearity R.
Therefore, in the preparation of the placenta growth factor monoclonal antibody marked by alkaline phosphatase, the used cross-linking agent is preferably SPDP and DTT, and the blocking agent is MMTS.
(4) Screening the concentration of a cross-linking agent in the preparation of the placenta growth factor monoclonal antibody marked by alkaline phosphatase:
referring to example 1, 1mg of each placental growth factor monoclonal antibody (manufactured by Funikou Biotechnology Co., Ltd., Guangzhou, cat # PLGF-30) was used to replace the solution in the original antibody with 0.01M PBS buffer at pH 7.4 to 5mg/mL, followed by addition of 1. mu.L, 2. mu.L and 4. mu.L of SPDP at a concentration of 5mg/mL, and incubation was carried out at room temperature for 1 hour to activate the antibody; then respectively adding 2 mu L of DTT with the concentration of 5mg/mL into 50 mu L of alkaline phosphatase with the concentration of 20mg/mL, and incubating for 1h at room temperature to activate the alkaline phosphatase; the activated antibody and alkaline phosphatase were mixed together and incubated at room temperature for 1 hour for crosslinking. Finally, respectively adding 4 muL of MMTS with the concentration of 5mg/mL into the crosslinked mixture for sealing, desalting and purifying, recovering the desalted and purified combination to 0.1mg/mL by using PBS buffer solution with the concentration of 0.01M and the pH value of 7.4, then adding glycerol with the same volume, uniformly mixing to obtain alkaline phosphatase-labeled placenta growth factor monoclonal antibody combination (enzyme-labeled mother solution corresponding to 1 muL of SPDP crosslinking agent with the concentration of 5mg/mL is named as PLGF-R2-; enzyme-labeled mother solution corresponding to 2 muL of SPDP crosslinking agent with the concentration of 5mg/mL is named as PLGF-R2-; enzyme-labeled mother solution corresponding to 4 muL of SPDP crosslinking agent with the concentration of 5mg/mL is named as PLGF-R2-seventy), storing at the temperature of 20 ℃ for standby, subsequently respectively using the three enzyme-labeled mother solutions, matching with toluene sulfonylated magnetic particles coated by corresponding placenta growth factor monoclonal antibodies, the sensitivity, repeatability and linearity indexes of the reagent are tested and verified, and the specific results are shown in tables 3-4 below.
TABLE 3-4 SPDP crosslinker-alkaline phosphatase-labeled PLGF antibody Performance validation results at various concentrations
As can be seen from tables 3-4, compared with PLGF-R2-fifthly, PLGF-R2-sixth and PLGF-R2-enzyme markers, PLGF-R2-sixth and PLGF-R2-seventh magnetic bead coatings have the same signal to noise ratio (S/N), repeatability and linearity as the PLGF-R2-sixth magnetic bead coatings, but the performances are better than those of PLGF-R2-fifthly, and the PLGF-R2-sixth magnetic bead coatings are preferred finally.
Therefore, in the preparation of the placental growth factor monoclonal antibody labeled with alkaline phosphatase, the mass ratio of the antibody used to the crosslinking agent SPDP is preferably 100: 1.0.
Example 4 evaluation of the Performance of the PLGF chemiluminescent immunoassay kit
The PLGF standards were examined using the method of example 2, and the plotted standard curve was obtained as shown in fig. 2.
Then, the actual sample is tested, and the concentration of the sample is calculated according to the luminous value of the sample.
(1) Detection of sensitivity:
the sensitivity of the placental growth factor chemiluminescence immunoassay kit was calculated according to the recommended protocol of the CLSI EP17-A document, and the sensitivity was found to be 0.54 pg/mL.
(2) And (3) linear detection:
the linear analysis is carried out on calibration samples with the concentrations of 0pg/mL, 10pg/mL, 50pg/mL, 100pg/mL, 500pg/mL, 1000pg/mL, 2500pg/mL, 5000pg/mL, 8000pg/mL and 11000pg/mL, the linear correlation coefficient is calculated, r is 0.9996, and in addition, the linear range of the detection of the kit on the placenta growth factor sample is 3 pg/mL-10000 pg/mL.
(3) And (3) detection of precision:
two placenta growth factor samples with the concentration of 100pg/mL and 5000pg/mL are taken, each sample is tested for 10 times, three batches of kits are used for detection, the difference between the kits in each batch and between the kits is calculated, the result shows that the difference between the kits in each batch is less than 5%, the difference between the kits in each batch is less than 10%, and the specific result is shown in the following table 4.
(4) Interference experiments:
taking mixed serum and adding interferents respectively comprises the following steps: bilirubin (10mg/dL), hemoglobin (500mg/dL), and triglyceride (1000mg/dL) were added at a mass ratio of 1:20, and the measurement values of the mixed serum and the mixed serum to which various interfering substances were added were measured. The deviation between the two was calculated to be within ± 10% of the acceptable range, and the specific results are shown in table 4 below. The result shows that the interference performance reaches the file standard of NCCLS, and the method can be used for accurately evaluating the condition of the placenta growth factor in a clinical laboratory.
TABLE 4 detection results of PLGF chemiluminescence immunoassay kit
(5) And (3) detection of clinical performance:
197 clinical specimens were tested with the kit of the present invention in example 1, and the luminescence value was measured, and the concentration was calculated from the standard curve (shown in FIG. 2).
Meanwhile, the concentration of the clinical sample is measured by using a placenta growth factor detection kit (electrochemical luminescence method) of Roche.
The detection concentration of the kit prepared in the example of the present invention is analyzed and compared with the concentration measurement result of the placenta growth factor detection kit (electrochemiluminescence method) of roche, wherein the clinical correlation of the placenta growth factor is R2 ═ 0.9733, which indicates that the kit of the present invention has good correlation with the roche kit. The clinical relevance results are shown in figure 3.
Example 5 comparative experiment with PLGF chemiluminescent immunoassay kit
Placental growth factor samples at concentrations of 0pg/mL and 10pg/mL were tested using chemiluminescence detection and enzyme-linked immunosorbent assay (R & D Systems, inc., cat # DPG00), respectively, and the sensitivity of detection was compared for the two methods, as shown in table 5-1 below:
TABLE 5-1
As can be seen from the above table, the detection sensitivity of the placenta growth factor chemiluminescence detection kit provided by the invention is improved by about 10 times compared with that of the conventional enzyme-linked immunosorbent assay.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (15)
1. A preparation method of immunomagnetic particles is characterized by comprising the following steps:
(1) pretreatment: pretreating the magnetic particles to obtain a mixed solution 1;
(2) and (3) activation: activating the antibody by using a cross-linking agent;
(3) coupling: adding the activated antibody in the step (2) into the mixed solution 1 obtained in the step (1) to obtain a mixed solution 2, and incubating to obtain an antibody-magnetic bead conjugate;
(4) and (3) sealing: adding a sealing solution into the antibody-magnetic bead conjugate obtained in the step (3) for sealing to obtain an antibody-magnetic bead coupled immunomagnetic particle final product;
wherein the cross-linking agent in step (2) is selected from BS (PEG)5BMPS and BM (PEG)3Any one of the above; in the activation treatment, the mass ratio of the antibody to the crosslinking agent is 103：(0.5～2.0)。
2. The preparation method according to claim 1, wherein in step (1), the mixed solution 1 is obtained by washing and resuspending magnetic particles with a buffer solution, and then uniformly dispersing the magnetic particles in an ammonium sulfate solution; further, the concentration of the ammonium sulfate solution is 2-5M, and the pH value is 8-10.
3. The method according to any one of claims 1 to 2, wherein in the step (1), the magnetic fine particles are modified with tosyl group.
4. The production method according to claim 3, wherein in the step (1), the tosylated magnetic particles have a particle diameter of 0.9 to 1.8 μm.
5. The preparation method according to any one of claims 1 to 4, wherein in the step (4), the mass ratio of the antibody to the magnetic bead in the final product of the antibody-magnetic bead coupled immunomagnetic particles is 1 (10-50).
6. The method according to any one of claims 1 to 5, wherein in the step (2), the antibody is a placental growth factor monoclonal antibody.
7. An immunomagnetic microparticle obtained by the production method according to any one of claims 1 to 6.
8. Use of immunomagnetic particles according to claim 7 for the preparation of a kit or a kit detection reagent.
9. A placental growth factor capture antibody magnetic microparticle prepared by the preparation method according to claim 6.
10. A placental growth factor chemiluminescent immunoassay kit comprising the placental growth factor capture antibody magnetic microparticles of claim 9 and a chemiluminescent label labeled placental growth factor detection antibody.
11. The placental growth factor chemiluminescent immunoassay kit of claim 10, wherein the chemiluminescent label-labeled placental growth factor detection antibody is obtained by mixing, crosslinking, and blocking agent treatment of a DTT-activated chemiluminescent substance and a crosslinker-activated placental growth factor detection antibody; the cross-linking agent is selected from any one of SPDP, DTBP, DTSSP and AMAS, and the blocking agent is MMTS.
12. The placental growth factor chemiluminescent immunoassay kit of any one of claims 10 to 11, wherein the chemiluminescent label for the placental growth factor detection antibody is selected from the group consisting of luminol, isoluminol, alkaline phosphatase, ruthenium terpyridyl, acridinium ester.
13. The placental growth factor chemiluminescence immunoassay kit according to claim 12, wherein the chemiluminescent label is alkaline phosphatase, and the mass ratio of the placental growth factor detection antibody to the cross-linking agent in the alkaline phosphatase-labeled placental growth factor detection antibody is 100 (0.5-2.0).
14. The placental growth factor chemiluminescent immunoassay kit of any one of claims 10 to 13, further comprising a placental growth factor calibrator, further wherein said placental growth factor calibrator is formulated with a calibration buffer comprising 45mM to 55mM Tris, 0.05% to 0.15% BSA and 0.9% NaCl.
15. A non-disease diagnosis purpose placenta growth factor quantitative determination method comprises the following steps:
the placental growth factor chemiluminescent immunoassay kit of any one of claims 10-14, establishing a standard fit curve;
obtaining a sample to be tested, detecting by using the placenta growth factor chemiluminescence immunoassay kit of any one of claims 10-14, recording the luminescence value of the sample to be tested, and substituting the luminescence value into the standard fitting curve to obtain the concentration of the placenta growth factor in the sample.
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| CN115561230A (en) * | 2022-11-02 | 2023-01-03 | 江苏三联生物工程股份有限公司 | Application of dithiothreitol in preparation of product based on electrochemiluminescence immunoassay |
| CN116718777A (en) * | 2023-05-30 | 2023-09-08 | 北京健平金星医疗器械有限公司 | Preparation and application of PLGF detection kit |
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| US20210172938A1 (en) * | 2019-12-04 | 2021-06-10 | Progenity, Inc. | Assessment of preeclampsia using assays for free and dissociated placental growth factor |
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| CN115561230A (en) * | 2022-11-02 | 2023-01-03 | 江苏三联生物工程股份有限公司 | Application of dithiothreitol in preparation of product based on electrochemiluminescence immunoassay |
| CN115561230B (en) * | 2022-11-02 | 2023-12-05 | 江苏三联生物工程股份有限公司 | Application of dithiothreitol in preparation of electrochemiluminescence immunoassay-based product |
| CN116718777A (en) * | 2023-05-30 | 2023-09-08 | 北京健平金星医疗器械有限公司 | Preparation and application of PLGF detection kit |
| CN116718777B (en) * | 2023-05-30 | 2024-02-13 | 北京健平金星生物医药有限公司 | Preparation and application of PLGF detection kit |
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