CN114252594B - 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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- CN114252594B CN114252594B CN202111387053.XA CN202111387053A CN114252594B CN 114252594 B CN114252594 B CN 114252594B CN 202111387053 A CN202111387053 A CN 202111387053A CN 114252594 B CN114252594 B CN 114252594B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- 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
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G—PHYSICS
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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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
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Abstract
The invention relates to a placenta growth factor detection kit and a preparation method and application thereof, wherein the placenta growth factor detection kit is prepared by combining a double-antibody sandwich method with a magnetic particle chemiluminescence technology, and the placenta growth factor detection kit is prepared by adopting an immune micro-magnetic particle preparation method optimized by the inventor, and further preparing an alkaline phosphatase marked placenta growth factor detection antibody by the prepared placenta growth factor capture antibody magnetic particles and an inventor through specific proportion cross-linking agents SPDP, DTT and a blocking agent MMTS, and can automatically complete detection by taking a full-automatic chemiluminescence immunoassay analyzer as a detection tool, meanwhile, the detection performance is also obviously improved, the detection sensitivity reaches 0.54pg/mL, and the detection linear range is wide and can reach 3 pg/mL-10000 pg/mL. It is predicted to have wide detection application prospect.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a placenta growth factor detection kit and a preparation method and application thereof.
Background
Placenta growth factor (PLGF) is synthesized mainly by syngeneic trophoblasts, can bind to tyrosinase receptors located in trophoblasts and vascular endothelial cells, and is a protein with autocrine action on trophoblasts and paracrine action on vascular growth. PLGF has unique regulatory effects on trophoblast and endothelial cell functions and can promote neovascularization.
Placental growth factor is one of the vascular endothelial growth factor family members, also known as placenta-derived angiogenic factor. In the female without pregnancy, a small amount of tissue cells such as thyroid epithelium, keratinocytes and melanoma cells can be expressed, but the content is lower than 10pg/mL. When a woman is pregnant, a great amount of PLGF is synthesized by the syngeneic trophoblasts, and the PLGF promotes proliferation and differentiation of the trophoblasts through an autocrine mode on one hand; on the other hand, the placenta angiogenesis and physiological recasting are regulated in a paracrine mode, and meanwhile, the placenta angiogenesis and physiological recasting can also have a synergistic effect with other vascular endothelial growth factors, so that the biological activity of the placenta endothelial growth factors is remarkably improved, the development of the placenta blood vessels is promoted, an important physiological effect is achieved in the placenta vascular forming process, and the normal blood supply and functions of the placenta in the gestation process are maintained, so that the placenta is suitable for the needs of maternal and fetal development. The concentration of PLGF increases in maternal blood circulation with increasing gestational weeks, peaks at gestational weeks 26-30, and thereafter decreases until delivery. Meanwhile, because PLGF half-life is only 15 minutes and one metabolism period is about 75 minutes, PLGF can well reflect the placenta function instant situation.
Preeclampsia (PE) is a serious complication of pregnancy with the main clinical manifestations being hypertension and proteinuria around 20 weeks after pregnancy. About 3-5% of pregnant women develop preeclampsia and can lead to death of the parturient, fetus, or neonate. Preeclampsia is associated with thrombocytopenia and elevated liver enzyme activity and may manifest as thrombocytopenia syndrome (hemolysis, elevated liver enzyme, thrombocytopenia) with varying degrees of clinical symptoms. Preeclampsia occurs due to endothelial dysfunction caused by the release of angiogenic factors from the placenta. Serum PLGF (placental growth factor) and sFlt-1 (soluble tyrosine kinase-1, or vascular endothelial growth factor receptor-1) concentration levels in women suffering from preeclampsia are altered. In addition, levels of PLGF and sFlt-1 in the blood circulation may be used to identify normal pregnancy and preeclampsia prior to the onset of clinical symptoms. The level of the pro-vascular factor PLGF increases in the first 6 months of normal pregnancy and gradually decreases as pregnancy progresses until termination. In contrast, the level of the anti-vascular factor sFlt-1 remained stable in early and mid gestation and did not rise smoothly until gestation was terminated. Women suffering from preeclampsia have sFlt-1 levels above normal pregnancy levels while PLGF levels are below normal pregnancy levels. Determining the ratio of sFlt-1 to PLGF is more valuable than detecting sFlt-1 or PLGF alone. Placental endothelial factor is a member of the TGF- β family that is upregulated in preeclampsia and released into the maternal blood circulation in the form of soluble endothelial factor. Soluble endoglin has been shown to be significantly elevated in severe cases of preeclampsia.
Therefore, detection of maternal blood PLGF levels can be used clinically to identify the presence of oxygenation pressure in placenta-derived trophoblast cells. Can be used for pregnancy-induced hypertension prediction and auxiliary diagnosis.
Traditional placenta growth factor assay methods include enzyme-linked immunosorbent assay and chemiluminescent assay.
At present, most methods for detecting PLGF in China are an enzyme-linked immunosorbent method and a fluorescence chromatography method. The ELISA method is long in time consumption and complex in operation. In addition, the two detection methods have poor detection sensitivity and narrow detection range, and are not fully automatic. The foreign PLGF detection method has an electrochemiluminescence immunoassay method, but the method has high detection cost, so that the method is not suitable for pregnancy in resource-limited areas.
Disclosure of Invention
Based on the above, an object of the present invention is to provide a method for preparing immunomagnetic particles.
The method comprises the following technical scheme:
(1) Pretreatment: pretreating magnetic particles to obtain a mixed solution 1;
(2) Activating: 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) Closing: adding a blocking solution into the antibody-magnetic bead conjugate obtained in the step (3) to block to obtain an immune magnetic particle end product of the antibody-magnetic bead conjugate;
wherein the cross-linking agent in step (2) is selected from BS (PEG) 5 BMPS and BM (PEG) 3 Any one of them; in the activation treatment, the mass ratio of the antibody to the cross-linking agent is 10 3 :(0.5～2.0)。
It is also an object of the present invention to provide immunomagnetic particles obtained according to the above-described preparation method.
The invention also aims at providing an application of the immunomagnetic particles in preparing a kit or a kit detection reagent.
It is also an object of the present invention to provide a magnetic particle of placenta growth factor trap antibody.
The method comprises the following technical scheme:
the placenta growth factor capturing antibody magnetic particle is prepared by the preparation method of the immunomagnetic particle.
The invention also aims at providing a placenta growth factor chemiluminescence immunoassay kit.
The method comprises the following technical scheme:
a placenta growth factor chemiluminescence immunoassay kit comprises the placenta growth factor capturing antibody magnetic particles and placenta growth factor detection antibodies marked by chemiluminescent markers.
The invention also aims to provide a quantitative detection method of placenta growth factor for non-disease diagnosis.
The method comprises the following technical scheme:
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 luminescence value of the sample to be detected, and substituting the luminescence 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 and magnetic particle chemiluminescence technology to detect the placenta growth factor, and unexpectedly discovers that the optimized immune micro-magnetic particle preparation method is adopted by the inventor in the preparation process of placenta growth factor capture antibody magnetic particles, and particularly the cross-linking agent is selected from BS (PEG) 5 BMPS and BM (PEG) 3 In any one of the above, the placenta growth factor capturing antibody magnetic particles prepared by specific proportion reaction and the inventor further pass through specific proportion cross-linking agents SPDP, DTT and blocking agent MThe placenta growth factor detection antibody marked by alkaline phosphatase prepared by MTS can automatically complete detection by taking a full-automatic luminous immunoassay analyzer as a detection tool, meanwhile, the detection performance is obviously improved, the detection sensitivity is up to 0.54pg/mL, compared with the traditional placenta growth factor detection method, the sensitivity is improved by at least 10 times, the detection linear range is wide, and the detection linear range can be up to 3 pg/mL-10000 pg/mL. It is predicted to have wide detection application prospect.
Drawings
FIG. 1 is a schematic diagram of the preparation flow of the placenta growth factor chemiluminescent immunoassay kit of the present invention.
FIG. 2 is a standard plot of placenta growth factor obtained from example 2 over a test series of PLGF calibrators.
FIG. 3 is a statistical chart showing the correlation analysis of the detection results of the placenta growth factor chemiluminescent immunoassay kit and the Roche kit of the present invention in example 4.
Detailed Description
The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. The various chemicals commonly 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. The term "and/or" as used herein 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 invention does not necessarily refer to the same embodiment, although it may. Furthermore, the phrase "in another embodiment" as used in the present invention does not necessarily refer to a different embodiment, although it may. Accordingly, as described below, various embodiments of the present invention may be readily combined without departing from the scope or spirit of the present 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" is included "in" and "on".
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This 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 abbreviations and terms involved in the present invention are 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 thiomethane sulfonate
BMPS: n-beta-maleimidopropyl succinimidyl oxide ester
BM(PEG) 3 :1, 11-bismaleimide-triethylene glycol
DTBP: dithiopropyliminoacid dimethyl ester dihydrochloride
DTSSP:3,3' -dithiobis (sulfosuccinimidyl) propionate
AMAS: n-alpha-maleimidosuccinimide esters
The present invention will be described in further detail with reference to specific examples.
Some embodiments of the present invention provide a method of preparing immunomagnetic microparticles comprising the steps of:
(1) Pretreatment: pretreating magnetic particles to obtain a mixed solution 1;
(2) Activating: 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) Closing: adding a blocking solution into the antibody-magnetic bead conjugate obtained in the step (3) to block to obtain an immune magnetic particle end product of the antibody-magnetic bead conjugate;
wherein the cross-linking agent in step (2) is selected from BS (PEG) 5 BMPS and BM (PEG) 3 Any one of them; in the activation treatment, the mass ratio of the antibody to the cross-linking agent is 10 3 (0.5-1.5), more preferably, the mass ratio of the antibody to the crosslinking agent is 10 3 :1.0。
In some of these embodiments, the crosslinking agent in step (2) of the above preparation method is preferably BS (PEG) 5 Antibody and crosslinking agent BS (PEG) 5 The mass ratio of the use is 10 3 1.0 due to BS (PEG) 5 The method is a polymer with 5 PEG, and is provided with a coupling 'long arm', so that the method can be used as a 'bridge' to increase the distance between the antibody and the magnetic beads and reduce the steric hindrance of the antibody and the magnetic beads, so that more antibodies can be connected to the magnetic beads in unit area, and then more antibodies can be combined with the antigen, thereby improving the stability and the sensitivity of an immunoassay method.
In some embodiments, in the step (1) of the preparation method, the mixed solution 1 is obtained by uniformly dispersing magnetic particles in an ammonium sulfate solution after washing and resuspension with a buffer solution; further, the concentration of the ammonium sulfate solution is 2-5M, and the pH value is 8-10. More preferably, the concentration of the ammonium sulfate solution used is 3M and the pH is 9.5, and at this time, ammonium sulfate is used as a reaction accelerator, so that the subsequent binding speed of the magnetic beads and the protein can be increased.
In some embodiments, in the above preparation method step (1), the buffer is preferably a PBS buffer or a BBS buffer; more preferably, the magnetic particles are washed with 0.01M PBS buffer at pH 7.4, and the supernatant is magnetically separated and resuspended in 0.1M BBS buffer at pH 9.5.
In some embodiments, in the step (1) of the preparation method, the magnetic particles are modified by tosyl, that is, when the active group of the magnetic particles is tosyl, the magnetic particles can be coupled with protein, so that the coupled protein can be combined with the to-be-detected object to realize efficient separation and detection of the to-be-detected object by using the action of an external magnetic field.
In some embodiments, in the above preparation method step (1), the tosylated magnetic particles have a particle diameter of 0.9 μm to 1.8 μm.
In some of these embodiments, in step (4) of the above preparation method, the blocking solution is 0.05M Tris buffer with 0.5% BSA, pH 7.4.
In some embodiments, in the preparation method step (4), the mass ratio of the antibody to the magnetic beads in the antibody-magnetic bead coupled immunomagnetic particle end product 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, further, the protein is an antibody, more preferably, a placenta growth factor monoclonal antibody.
Some embodiments of the present invention also provide an immunomagnetic particle obtained according to the above-described preparation method.
Some embodiments of the invention also provide the use of the immunomagnetic particles described above in the preparation of a kit or kit detection reagent.
Some embodiments of the invention also provide a placenta growth factor capturing antibody magnetic particle, which is prepared by the preparation method.
Some embodiments of the invention also provide a placenta growth factor chemiluminescent immunoassay kit comprising the placenta growth factor capture antibody magnetic particle and a placenta growth factor detection antibody labeled with a chemiluminescent label.
In some embodiments, the placenta growth factor detecting antibody marked by the chemiluminescent label is obtained by mixing and crosslinking the placenta growth factor detecting antibody activated by the DTT activated chemiluminescent substance and the placenta growth factor detecting antibody activated by the crosslinking agent, and treating the mixture with the blocking agent.
In some embodiments, the method for preparing the placenta growth factor detection antibody marked by the chemiluminescent label specifically comprises the following steps:
(1) Antibody activation: activating the antibody by using a cross-linking agent;
(2) Chemiluminescent activation: activating the chemiluminescent label with dithiothreitol;
(3) Coupling: mixing the chemiluminescent label activated in the step (2) with the antibody activated in the step (1), and incubating to obtain a chemiluminescent label-antibody conjugate;
(4) Closing: adding a blocking agent into the chemiluminescent label-antibody conjugate obtained in the step (3) for blocking to obtain a chemiluminescent label-antibody final product;
the cross-linking agent is selected from any one of SPDP, DTBP, DTSSP, AMAS, and the blocking agent is MMTS. Further, the cross-linking agent is preferably SPDP, and when the cross-linking agent is 5mg/mL SPDP solution, the SPDP is used as an antibody activator, so that the antibody can be activated with maximum efficiency, and the coupling efficiency of the subsequent antibody and the chemiluminescent label can be greatly improved. Thereby enabling to improve the detection sensitivity.
In some of these embodiments, the antibody and the cross-linking agent are used in equal concentrations during the activation treatment in step (1) by incubation at room temperature for 1 hour.
In some of these embodiments, the activation of the chemiluminescent label in step (2) is performed using DTT at a concentration of 5mg/mL and incubated for 1h at room temperature.
In some embodiments, the blocking agent used in step (4) is MMTS at 5mg/mL, and further, a desalting purification treatment is performed, preferably, the above desalted and purified conjugate is recovered to 0.1mg/mL with 0.01M PBS buffer at pH 7.4, and then an equal volume of glycerol is added and mixed to obtain the placenta growth factor detection antibody labeled with the chemiluminescent label.
In some embodiments, the chemiluminescent label is selected from the group consisting of acridinium esters, ruthenium terpyridyl, adamantane, luminol, derivatives of luminol, isoluminol, derivatives of isoluminol, horseradish peroxidase, and alkaline phosphatase. Of course, in other embodiments, the labels in the label-labeled antibodies are not limited to the above, but may be other substances useful in chemiluminescent immunoassay platforms.
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 alkaline phosphatase-labeled placenta growth factor detection antibody is 100 (0.5-2.0). More preferably, the mass ratio of the placenta growth factor detection antibody to the crosslinking agent is 100:1.0.
In some embodiments, the placenta growth factor chemiluminescent immunoassay kit further comprises a placenta growth factor standard, further wherein the placenta growth factor standard is prepared by using a calibration buffer solution, and the calibration buffer solution comprises 45 mM-55 mM Tris,0.05% -0.15% BSA and 0.9% NaCl. Preferably, the calibration buffer comprises 50mM Tris,0.1%BSA,0.9%NaCl,pH7.5.
In some of these embodiments, the disc growth factor chemiluminescent immunoassay kit further comprises a chemiluminescent substrate solution, further preferably an APS substrate solution.
Some embodiments of the invention also provide a quantitative detection method for placenta growth factor of non-disease diagnosis purpose.
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 luminescence value of the sample to be detected, and substituting the luminescence 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 includes: taking 20-100 mu L of serum sample, adding 20-100 mu L of placenta growth factor capturing antibody magnetic particles and 20-100 mu L of alkaline phosphatase labeled placenta growth factor detecting antibody, reacting for 5-15 min, performing magnetic separation, adding 100-300 mu L of substrate solution, mixing uniformly, sending the reaction mixture into a darkroom 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 capturing antibody magnetic particles and 50 mu L of placenta growth factor detecting antibody marked by alkaline phosphatase are added for reaction for 10min, then magnetic separation is carried out, then 200 mu L of substrate solution is added, after uniform mixing, the reaction mixture is sent into a darkroom by an instrument, and finally the luminescence value is recorded.
EXAMPLE 1 preparation of PLGF chemiluminescent immunoassay kit
(1) Preparation of placenta growth factor monoclonal antibody coated tosylated magnetic microparticles:
taking a suspension containing 80mg of tosylated magnetic particles (Magnospere TM, cat# MS 160) with a particle size of 0.9 μm to 1.8 μm, magnetically separating the supernatant, washing the magnetic particles with 0.01M PBS buffer with a pH of 7.4, magnetically separating the supernatant, re-suspending the supernatant with 0.1M BBS buffer with a pH of 9.5, adding 3mL of 3M ammonium sulfate solution with a pH of 9.5, dispersing uniformly to obtain a mixed solution 1, and then adding 4. Mu.g of BS (PEG) to 4mg of placenta growth factor monoclonal antibody (PLGF-34#) 5 (PEGylated bis (sulfosuccinimidyl) suberic acid, thermoFisher SCIENTIFIC) activated placental growth factor monoclonal antibody, and adding the activated antibody to the mixture 1 to obtain a mixture 2. Then the mixture 2 is placed on a rotary shaking table for the first incubation treatment, and after magnetic separation of the supernatant, the mixture is treated with 0.05M pH7Washing Tris buffer solution of 4, removing supernatant, adding a solution containing 0.5% of BSA, performing a second incubation treatment, washing with 0.05M Tris buffer solution with pH of 7.4 after magnetic separation of supernatant, and finally re-suspending to 10mg/mL Tris buffer solution with pH of 7.4 and 0.05M Tris buffer solution containing 0.5% of BSA to obtain toluene yellow acylated magnetic particles coated with placenta growth factor monoclonal antibody, wherein the corresponding magnetic bead mother liquor is named as follows: PLGF-R1- (1) is stored at 4℃for further use.
(2) Preparation of alkaline phosphatase-labeled placental growth factor monoclonal antibodies:
taking a solution containing 1mg of placenta growth factor monoclonal antibody (Wanfu Biotechnology Co., ltd., product No. PLGF-30 #), centrifuging, removing supernatant, replacing the liquid in the original antibody with 0.01M PBS buffer with pH of 7.4 to 5mg/mL, adding 2. Mu.L of SPDP (succinimidyl 3- (2-pyridyldithio) -propionate with concentration of 5mg/mL, thermoFisher SCIENTIFIC), incubating for 1h at room temperature, and activating the antibody; then 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 then mixed together and incubated at room temperature for 1h to crosslink. Finally, 4 mu L of MMTS (methyl thiomesylate, thermoFisher SCIENTIFIC) with the concentration of 5mg/mL is added into the crosslinked mixture for sealing, then desalting and purifying are carried out, the desalted and purified conjugate is recovered to 0.1mg/mL by using a PBS buffer with the pH of 7.4 and 0.01M, then glycerol with the same volume is added, and uniform mixing is carried out, thus obtaining the alkaline phosphatase marked placenta growth factor monoclonal antibody conjugate, and the corresponding enzyme-labeled mother liquor is named as follows: PLGF-R2- (1) is stored at-20 ℃ for standby.
(3) Preparation of placenta growth factor calibration:
placenta growth factor (R & D Systems, inc.) was formulated with a standard 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 dispensed at 0.5mL per tube for storage at-20 ℃.
Example 2 detection method of PLGF chemiluminescent immunoassay kit
The detection principle of the kit of the invention is as follows: the magnetic particles coated with placenta growth factor (PLGF) capture antibody are combined with alkaline phosphatase-labeled placenta growth factor (PLGF) detection antibody and placenta growth factor in a sample, calibrator or quality control to form a "sandwich" complex. Under the action of an externally applied magnetic field, separating the compound formed by the immune reaction from other unbound substances, cleaning the compound, and adding an enzymatic chemiluminescent substrate. The substrate is catalytically cracked under the action of enzyme to form an unstable excited state intermediate, and photons are emitted when the excited state intermediate returns to the ground state to form a luminescence reaction, so that the luminescence intensity of the reaction can be detected by using a chemiluminescent instrument. Meanwhile, the luminous marker alkaline phosphatase is not basically consumed, and the luminous agent in the reaction system is sufficiently 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, kwanfu Biotechnology Co., ltd.) is used as a detection tool, the methodology mode is double antibody sandwich, namely 50 mu L of serum sample, 50 mu L of placenta growth factor monoclonal antibody coated tosylated magnetic particles and 50 mu L of alkaline phosphatase labeled placenta growth factor monoclonal antibody are sequentially added into the instrument, after reaction for 10min, magnetic separation is carried out, then 200 mu L of substrate solution is added into the reaction mixture, after uniform mixing, the reaction mixture is sent into a darkroom by the instrument, and finally the luminescence value is recorded.
The PLGF calibration prepared in example 1 was tested by the method described above, and a standard curve was drawn as shown in FIG. 2.
And then testing the actual sample, and calculating the sample concentration according to the sample luminescence value.
Example 3 optimization of PLGF chemiluminescent immunoassay kit
(1) Screening of antibody activators in preparation of placenta growth factor monoclonal antibody-coated tosylated magnetic microparticles:
mixed solution 1 was prepared as described in reference example 1, followed by the addition of 4. Mu.g of BS (PEG) to 4mg of placenta growth factor monoclonal antibody (PLGF-34 # available from Guangzhou Wanfu Biotechnology Co., ltd.) 5 4 μg BMPS (N-. Beta. -maleimidopropyl butylene oxide imide ester, thermoFisher SCIENTIFIC), 4 μg BM (PEG) 3 (1, 11-bismaleimide-triethylene glycol, thermoFisher SCIENTIFIC) activating placenta growth factor monoclonal antibodies, and then adding the activated antibodies into the mixed solution 1 to obtain 3 mixed solutions 2. Then placing the 3 kinds of mixed solution 2 on a rotary shaking table for the first incubation treatment, magnetically separating supernatant, washing with 0.05M Tris buffer with pH of 7.4, removing supernatant, adding 0.5% BSA solution for the second incubation treatment, magnetically separating supernatant, washing with 0.05M Tris buffer with pH of 7.4, and finally re-suspending with 0.05M Tris buffer with pH of 7.4 to 10mg/mL to obtain 3 pieces of placenta growth factor monoclonal antibody-coated toluene yellow acylated magnetic particles (BS (PEG) 5 The magnetic mother-of-pearl liquid corresponding to the coupling agent is named as: PLGF-R1- (1); the magnetic mother-of-pearl solution corresponding to the BMPS coupling agent is named as follows: PLGF-R1- (2); BM (PEG) 3 The magnetic mother-of-pearl liquid corresponding to the coupling agent is named as: PLGF-R1- (3)) is stored at 4 ℃ for standby, and then the three magnetic bead mother liquors are respectively matched with corresponding alkaline phosphatase labeled placenta growth factor monoclonal antibodies to test and verify the sensitivity, repeatability and linearity indexes of the reagent, and the specific results are shown in the following table 3-1.
TABLE 3-1 results of Performance verification of magnetic microparticles coated PLGF antibodies with different coupling agents
As can be seen from Table 3-1, the signal to noise ratio (S/N) of the PLGF-R1- (1) bead coating is maximum, the repeatability is within 5% and the linearity R is optimal when the PLGF-R1- (1) is compared with the PLGF-R1- (2) and the PLGF-R1- (3) bead coatings.
Thus, tosylated superparamagnetic micro coated with placenta growth factor monoclonal antibodyIn the pellet, 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 1mg of each of placenta growth factor monoclonal antibodies (PLGF-34 # were supplied from Kyowa Biotechnology Co., ltd.) were added with 0.5. Mu.g of BS (PEG), respectively 5 1.0 μg of BS (PEG) 5 And 2.0 μg of BS (PEG) 5 Activating placenta growth factor monoclonal antibody, and adding the activated antibody into the mixed solution 1 to obtain 3 mixed solutions 2. Then the 3 kinds of mixed solution 2 are placed on a rotary shaking table for the first incubation treatment, after the supernatant is separated magnetically, the supernatant is washed with 0.05M Tris buffer with pH of 7.4, after the supernatant is removed, a solution containing 0.5% BSA is added for the second incubation treatment, after the supernatant is separated magnetically, the supernatant is washed with 0.05M Tris buffer with pH of 7.4, finally the supernatant is resuspended to 10mg/mL Tris buffer containing 0.5% BSA with pH of 7.4, and 3 pieces of toluene yellow acylated magnetic microparticles (0.5 mug BS (PEG) coated with placenta growth factor monoclonal antibodies are obtained respectively 5 The magnetic mother-of-pearl liquid corresponding to the coupling agent is named as: PLGF-R1- (4); 1.0 μg of BS (PEG) 5 The magnetic mother-of-pearl liquid corresponding to the coupling agent is named as: PLGF-R1- (5); 2.0 μg BS (PEG) 5 The magnetic mother-of-pearl liquid corresponding to the coupling agent is named as: PLGF-R1- (6)) is stored at 4 ℃ for later use, and then the three magnetic bead mother liquors are respectively matched with corresponding alkaline phosphatase labeled placenta growth factor monoclonal antibodies to test and verify the sensitivity, repeatability and linearity indexes of the reagent, and the specific results are shown in the following tables 3-2.
TABLE 3-2 different concentrations of BS (PEG) 5 Coupling agent magnetic particle coated PLGF antibody performance verification result
As can be seen from Table 3-2, the signal to noise ratio (S/N), repeatability and linearity of the bead coatings of PLGF-R1- (5) and PLGF-R1- (6) are comparable to those of the bead coatings of PLGF-R1- (5) and PLGF-R1- (6), but are better than that of PLGF-R1- (4), and finally PLGF-R1- (5) is preferred.
Thus, in the placenta growth factor monoclonal antibody coated tosylated superparamagnetic particles, the antibody and antibody activator BS (PEG) used 5 Is preferably 10 3 :1.0。
(3) Screening of crosslinking agent in preparation of alkaline phosphatase-labeled placenta growth factor monoclonal antibody:
reference example 1, 1mg of each placental growth factor monoclonal antibody (product number: PLGF-30# by Wanfu Biotechnology Co., ltd., guangzhou), 5mg/mL of the liquid in the original antibody was replaced with 0.01M PBS buffer at pH 7.4, 2. Mu.L of SPDP at 5mg/mL, 2. Mu.L of DTBP at 5mg/mL (dimethyl dithiopropyliminodihydrochloride, thermoFisher SCIENTIFIC), 2. Mu.L of DTSSP at 5mg/mL (3, 3' -dithiobis (sulfosuccinimidyl propionate), thermoFisher SCIENTIFIC) and 2. Mu.L of AMAS (N-. Alpha. -maleimidosuccinimidyl ester, thermoFisher SCIENTIFIC) at 5mg/mL were added, and incubated at room temperature for 1h, respectively, to activate the antibody; then 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 respectively, and incubating for 1h at room temperature to activate the alkaline phosphatase; the activated antibody and alkaline phosphatase were then mixed together and incubated at room temperature for 1h to crosslink. Finally, 4 mu L of MMTS with the concentration of 5mg/mL is added into the mixture after the crosslinking for sealing, then desalination and purification are carried out, PBS buffer solution with the pH of 7.4 and 0.01M is used for recovering the combination after the desalination and purification to 0.1mg/mL, then equal volume of glycerol is added and uniformly mixed, thus obtaining the placenta growth factor monoclonal antibody combination marked by alkaline phosphatase (the enzyme-labeled mother liquor corresponding to the SPDP crosslinking agent is named PLGF-R2- (1), the magnetic bead mother liquor corresponding to the DTBP crosslinking agent is named PLGF-R2- (2), the magnetic bead mother liquor corresponding to the DTSSP crosslinking agent is named PLGF-R2- (3), the magnetic bead mother liquor corresponding to the AMAS crosslinking agent is named PLGF-R2- (4)), the magnetic bead is preserved at the temperature of-20 ℃ for standby, and then the three enzyme-labeled mother liquors are respectively matched with toluene sulfonylated magnetic particles coated by the corresponding placenta growth factor monoclonal antibody, and the sensitivity, the repeatability and the linearity indexes of the reagent are tested, and the results are shown in the following table 3-3.
TABLE 3-3 results of Performance validation of alkaline phosphatase-labeled PLGF antibodies with different crosslinking agents
As can be seen from the above tables 3 to 3, the signal to noise ratio (S/N) of the PLGF-R2- (1) enzyme labels is maximum, the repeatability is within 5% and the linearity R is optimal when the PLGF-R2- (1) enzyme labels are compared with the PLGF-R2- (2), PLGF-R2- (3) and PLGF-R2- (4) enzyme labels.
Thus, in the preparation of alkaline phosphatase-labeled placental growth factor monoclonal antibodies, the cross-linking agents used are preferably SPDP and DTT, and the blocking agent is MMTS.
(4) Screening of crosslinker concentration in preparation of alkaline phosphatase-labeled placental growth factor monoclonal antibodies:
referring to example 1, 1mg of each placental growth factor monoclonal antibody (product number: PLGF-30# of Kwanfu Biotechnology Co., ltd.) was used to replace the liquid in the original antibody to 5mg/mL with 0.01M PBS buffer at pH 7.4, and then 1. Mu.L, 2. Mu.L and 4. Mu.L of SPDP at 5mg/mL were added, respectively, and incubated for 1h at room temperature to activate the antibody; then 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 respectively, and incubating for 1h at room temperature to activate the alkaline phosphatase; the activated antibody and alkaline phosphatase were then mixed together and incubated at room temperature for 1h to crosslink. Finally, 4 mu L of MMTS with the concentration of 5mg/mL is added into the mixture after the crosslinking for sealing, then desalting and purifying are carried out, the joint compound after the desalting and purifying is recovered to 0.1mg/mL by using a PBS buffer solution with the concentration of 0.01M and the pH of 7.4, then equal volume of glycerol is added, the joint compound is uniformly mixed, and then an alkaline phosphatase marked placenta growth factor monoclonal antibody joint compound (1 mu L of enzyme marked mother liquor corresponding to SPDP crosslinking agent with the concentration of 5mg/mL is named as PLGF-R2- (5), 2 mu L of enzyme marked mother liquor corresponding to SPDP crosslinking agent with the concentration of 5mg/mL is named as PLGF-R2- (6), 4 mu L of enzyme marked mother liquor corresponding to SPDP crosslinking agent with the concentration of 5mg/mL is named as PLGF-R2- (7)) is stored at the temperature of-20 ℃ for standby, and then toluene sulfonylated magnetic particles coated by the corresponding placenta growth factor monoclonal antibody are respectively matched with the three enzyme marked mother liquor, and the sensitivity, the repeatability and the specific results of the reagent are tested to be shown in a linear test table of 3-4.
Tables 3-4 results of Performance verification of SPDP crosslinking agent alkaline phosphatase-labeled PLGF antibodies at various concentrations
As can be seen from tables 3 to 4, the signal to noise ratio (S/N), reproducibility and linearity of the magnetic bead coatings of PLGF-R2- (5), PLGF-R2- (6) and PLGF-R2- (7) are comparable to those of the enzyme markers of PLGF-R2- (5), but better than that of PLGF-R2- (5), and finally PLGF-R2- (6) is preferred.
Therefore, in the preparation of the alkaline phosphatase-labeled placenta growth factor monoclonal antibody, the mass ratio of the antibody to the cross-linking agent SPDP is preferably 100:1.0.
EXAMPLE 4 evaluation of Performance of PLGF chemiluminescent immunoassay kit
PLGF calibration was tested by the method of example 2, and a standard curve was drawn as shown in FIG. 2.
And then testing the actual sample, and calculating the sample concentration according to the sample luminescence value.
(1) Detection of sensitivity:
referring to the CLSI EP17-A document recommended experimental protocol, the sensitivity of the placenta growth factor chemiluminescent immunoassay kit was calculated to be 0.54pg/mL.
(2) Detection of linearity:
linear analysis was performed on the standard samples with concentrations of 0pg/mL, 10pg/mL, 50pg/mL, 100pg/mL, 500pg/mL, 1000pg/mL, 2500pg/mL, 5000pg/mL, 8000pg/mL, 11000pg/mL, and the linear correlation coefficient was calculated, r= 0.9996, and the linear range of placenta growth factor sample detection by the kit was 3pg/mL to 10000pg/mL.
(3) And (3) detecting precision:
two placenta growth factor samples with concentrations of 100pg/mL and 5000pg/mL were taken, each sample was tested 10 times, three kits were used for detection, and the intra-batch and inter-batch differences of the kits were calculated, and the results showed that the intra-batch differences of the kits were less than 5% and the inter-batch differences were less than 10%, and the specific results are shown in Table 4 below.
(4) Interference experiments:
taking mixed serum and adding interferents respectively comprises the following steps: bilirubin (10 mg/dL), hemoglobin (500 mg/dL) and triglyceride (1000 mg/dL) were added in a mass ratio of 1:20, and the measurement values of the mixed serum and the mixed serum after adding various interferents were measured, respectively. The deviation between the two was calculated to be within an acceptable range of + -10%, and the specific results are shown in Table 4 below. The result shows that the interference reaches the file standard of NCCLS, and can be used for accurately evaluating the placenta growth factor condition in clinical laboratories.
TABLE 4PLGF chemiluminescent immunoassay kit detection results
(5) Detection of clinical performance:
197 clinical samples were taken and tested with the kit of example 1 of the present invention, luminescence values were determined, and concentrations were calculated based on a standard curve (as shown in fig. 2).
The concentration of the clinical samples was also measured using the placenta growth factor assay kit (electrochemiluminescence method) from roche company.
The detection concentration of the kit prepared in the embodiment of the invention is analyzed and compared with the concentration measurement result of the placenta growth factor detection kit (electrochemiluminescence method) of the Roche company, wherein the clinical relevance of the placenta growth factor is R2= 0.9733, which indicates that the kit has good relevance with the Roche kit. The results of the clinical correlation are shown in FIG. 3.
Example 5 comparative experiments with PLGF chemiluminescent immunoassay kit
Placenta growth factor samples at concentrations of 0pg/mL and 10pg/mL were tested by chemiluminescence detection and enzyme-linked immunosorbent assay (R & D Systems, inc., cat# DPG 00), respectively, and the data are shown in Table 5-1 below for the comparison of the detection sensitivities of the two methods:
TABLE 5-1
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As can be seen from the 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 a conventional detection ELISA method.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (9)
1. The placenta growth factor chemiluminescent immunoassay kit is characterized by comprising placenta growth factor capture antibody magnetic particles and placenta growth factor detection antibodies marked by chemiluminescent markers;
the preparation method of the placenta growth factor capturing antibody magnetic particle comprises the following steps:
(1) Pretreatment: pretreating magnetic particles to obtain a mixed solution 1, wherein the magnetic particles are modified by adopting toluene sulfonyl;
(2) Activating: activation of antibodies with a crosslinking agent, which is BS (PEG) 5 The antibody is placenta growth factor monoclonal antibody, and the mass ratio of the antibody to the cross-linking agent is 10 3 ：（0.5~2.0）；
(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) Closing: adding a blocking solution into the antibody-magnetic bead conjugate obtained in the step (3) to block to obtain an immune magnetic particle end product of the antibody-magnetic bead conjugate;
the placenta growth factor detection antibody marked by the chemiluminescent marker is obtained by mixing and crosslinking a chemiluminescent substance activated by DTT and a placenta growth factor detection antibody activated by a crosslinking agent and treating the mixture with a blocking agent;
the cross-linking agent is selected from any one of SPDP, DTBP, DTSSP, and the mass ratio of the placenta growth factor detection antibody to the cross-linking agent is 100 (0.5-2.0); the blocking agent is MMTS;
the chemiluminescent label is alkaline phosphatase.
2. The kit for chemiluminescent immunoassay of placenta growth factor of claim 1 wherein in step (1), the mixed solution 1 is obtained by dispersing magnetic particles uniformly in an ammonium sulfate solution after washing and resuspension with a buffer solution.
3. The placenta growth factor chemiluminescent immunoassay kit of claim 2 wherein the ammonium sulfate solution has a concentration of 2-5 m and a ph of 8-10.
4. The placenta growth factor chemiluminescent immunoassay kit of claim 1, wherein in step (1), the tosylated magnetic particles have a particle size of 0.9-1.8 μm.
5. The kit for chemiluminescent immunoassay of placenta growth factor according to any one of claims 1-4, wherein in step (4), the mass ratio of antibody to magnetic beads in the final product of antibody-magnetic bead coupled immunomagnetic particles is 1 (10-50).
6. The placenta growth factor chemiluminescent immunoassay kit of claim 1, wherein the mass ratio of placenta growth factor detection antibody to cross-linking agent in the alkaline phosphatase-labeled placenta growth factor detection antibody is 100:1.0.
7. The placenta growth factor chemiluminescent immunoassay kit of any one of claims 1-4, wherein the cross-linking agent is SPDP.
8. The placenta growth factor chemiluminescent immunoassay kit of any one of claims 1-4, further comprising a placenta growth factor standard.
9. The placenta growth factor chemiluminescent immunoassay kit of claim 8 wherein the placenta factor standard is formulated with a calibration buffer comprising 45mM~55mM Tris,0.05% -0.15% BSA and 0.9% NaCl.
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