CN111735942A - Homogeneous phase method chemiluminescence detection method - Google Patents
Homogeneous phase method chemiluminescence detection method Download PDFInfo
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Classifications
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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
- G01N33/54346—Nanoparticles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
Abstract
The invention discloses a homogeneous phase method chemiluminescence detection method, which comprises the steps of detecting a detected object by adopting the reaction principle of a sandwich method, and is characterized in that a kit for detecting the detected object consists of nano-gold photosensitive particles and luminescent particles, wherein the nano-gold photosensitive particles are conjugates formed by coupling a compound of the nano-gold photosensitive particles and a photosensitizer with a compound capable of generating specific binding pairing; the luminescent particles are conjugates of luminescent microspheres and bioactive molecules such as antibodies. The invention adopts nano gold particles as a carrier, electrostatically adsorbs water-soluble photosensitizer and transfers energy to photosensitizer molecules combined with the water-soluble photosensitizer in a non-radiative energy transfer mode to enable the molecules to reach an excited state, so that triplet oxygen is excited to a singlet state, the singlet oxygen content is increased, the difference between the wavelength of the excited light and the emission wavelength is enlarged, and the detection identification degree is improved, thereby enhancing the detection precision, accuracy and sensitivity.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a homogeneous phase method chemiluminescence detection method.
Background
The chemiluminescence method is the most advanced immunoassay methodology at present, and is divided into a direct chemiluminescence method, an enzymatic chemiluminescence method and an electrochemical luminescence method according to different markers; the heterogeneous chemiluminescence method and the homogeneous chemiluminescence method are classified according to the presence or absence of a separation cleaning step. At present, in the aspect of in vitro diagnosis and detection of human medicine, a direct chemiluminescence method and an enzymatic chemiluminescence method are used for detecting a plurality of products at home and abroad, and the methods all belong to heterogeneous chemiluminescence methods. Foreign manufacturers include Yapei, Siemens, Sonin, Beckman, and Xismenkang, while domestic manufacturers include New industries, Anchart, Mike, Mirui, Chengji, Chang Guang Hua Yi, etc. The instrument platforms of all manufacturers are different, especially for domestic manufacturers, the instrument structure is complex, the failure rate is high, the quality is uneven, and the trust of customers on products is influenced. Meanwhile, the manufacturers in China do not innovate the reagent varieties, but only repeat the varieties of others, and the used raw materials (antigen and antibody) have the same source, so that the homogenization is serious and the market competition is strong. At the present stage, only siemens use pure-state oxygen-mediated homogeneous chemiluminescence (light-activated chemiluminescence) products to market, and special LOCI modules are required for detection. The LOCI technology is a one-step chemiluminescence sandwich immunoassay method, and the reagent contains two synthetic bead reagents and a monoclonal antibody of biotics. The first bead reagent (sensor beads) is coated with streptavidin and contains a light sensitive dye; a second bead reagent (chemical bead) coated with another antibody and containing a chemiluminescent dye; incubating the sample with the chemical beads and the biotinylated antibody to form a sandwich complex; then adding sensitive beads, and forming an aggregated immune complex after being combined with biotin; the sensitive beads in the compound can generate singlet oxygen under 680nm light irradiation, the singlet oxygen can initiate a chemiluminescence reaction after being dispersed to the chemical beads, and a chemiluminescence signal generated by the reaction is measured under 612nm wavelength.
The existing light-excited chemiluminescence method needs to adopt a water-insoluble photosensitizer, wherein the yield of singlet oxygen quantum of the unmodified photosensitizer is low, and the detection performance of the reagent is influenced; besides, the photosensitizer used in the existing photoexcitation light system needs to be embedded into polystyrene microspheres in an organic phase, needs to be subjected to complicated purification modes such as centrifugation and ultrafiltration, and needs to be recovered into a water-phase matrix, the photosensitizer is easy to lose in the processing processes such as embedding and purification, the photosensitive effect is reduced, and the existing photoexcitation chemiluminescence technology has the advantages of small difference between the excitation wavelength and the detection wavelength, easy interference and large fluorescent background influence.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provide a homogeneous method chemiluminescence detection method which has the advantages of enhanced detection precision, accuracy and sensitivity and is not easily interfered by exciting light.
In order to achieve the above object, the present invention provides a homogeneous phase method chemiluminescence detection method, which comprises detecting an analyte by using the reaction principle of a sandwich method, and is characterized in that a kit for detecting the analyte comprises a nanogold photosensitive particle and a luminescent microparticle, wherein the nanogold photosensitive particle is a conjugate formed by coupling a compound of the nanogold photosensitive particle and a photosensitizer with a compound capable of generating a specific binding pair; the luminescent particles are conjugates of luminescent microspheres and bioactive molecules such as antibodies.
The invention provides a homogeneous phase method chemiluminescence detection method, further comprising preparation of nano-gold photosensitive particles and luminescent particles, and is characterized in that the preparation of the nano-gold photosensitive particles comprises the steps of adopting the nano-gold particles, carrying out nano-gold-photosensitizer compounding after obtaining the nano-gold particles, wherein the nano-gold-photosensitizer compounding comprises the steps of adding a water-soluble photosensitizer into the nano-gold particles, stirring overnight in a dark room at room temperature, then centrifuging to finally obtain a precipitated nano-gold-photosensitizer compound, and then re-dissolving the nano-gold-photosensitizer compound by using a buffer solution; the re-dissolved nano gold-photosensitizer compound is coupled with a compound capable of generating specific binding pair, the coupling of the nano gold-photosensitizer and the compound capable of generating specific binding pair is to add the compound capable of generating specific binding pair into the solution of the re-dissolved nano gold-photosensitizer compound, adjust the pH value of the reaction to the isoelectric point of the compound capable of generating specific binding pair, enable the compound capable of generating specific binding pair to generate electrostatic adsorption or covalent binding under the condition of the isoelectric point environment through the charge of the nano gold particles, so that the compound capable of generating specific binding pair is coupled to the surface of the nano gold particles, remove the unbound compound capable of generating specific binding pair through a centrifugal mode, and then add re-dissolving buffer solution for re-dissolving, obtaining the nano-gold photosensitive particles suspended in the re-dissolving buffer.
Herein, the specific binding partner compound refers to a compound that can generate a specific binding partner, such as enzymes and enzymes, small molecules and antibodies, antigens and antibodies, nucleic acids and nucleic acids or nucleic acids and proteins, and the like.
The preparation method of the luminescent particles comprises the steps of purchasing (Alpha accumulator beads) luminescent microspheres of PE company, coating bioactive molecules such as antibodies by adopting a covalent coupling mode, adding 0.5-1mL of Buffer A into 1mL of luminescent microspheres, uniformly mixing, activating by using 0.2-0.5mL of EDC for 10-30min, adding 0.3-1mL of antibodies, carrying out oscillation reaction for 2-5h, sealing for 5-30min, adding Buffer B to a final volume of 50mL, and uniformly stirring to obtain the luminescent particles. Wherein, Buffer A: pH value of 5-6, MES and HEPES, KCl, NaCl and CaCl2、MgCl2The concentration of the plasma salt ions is 1-10%, and a certain ionic strength is provided to promote the activation reaction of the latex microspheres; buffer B: pH value is 6-8, buffer solution containing MES, HEPES, PBS, Tris and the like provides buffer capacity of reaction, and buffer solution containing KCl, NaCl and CaCl2、MgCl2The concentration of plasma salt ions is 1-10% (providing the necessary ionic strength for antibody-microsphere coupling reaction), the concentration of substances containing stable substances such as Bovine Serum Albumin (BSA), sucrose, trehalose, glycerol and the like is 0.1-0.5% (sealing the surfaces of microspheres which are not combined with antibodies, maintaining the stability of immune latex), and the concentration of preservative components Proclin300, sodium azide and the like is 0.01-0.05% (inhibiting the propagation of bacteria and the like).
The nanogold-photosensitizer compound provided by the invention has the advantages of higher photosensitizer utilization rate and higher detection sensitivity.
In order to obtain good composition of the nano gold particles and the photosensitizer and better coupling effect of the nano gold-photosensitizer compound and the specific binding pair compound, the molar ratio of the nano gold particles to the photosensitizer can be 2:1-10, the water-soluble photosensitizer can be a porphyrin derivative, a phthalocyanine derivative or a chlorophyll derivative, such as copper phthalocyanine-3, 4' -tetrasulfonic acid tetrasodium salt or chlorophyll copper sodium salt, and the electrostatic adsorption time can be 1 hour to 2 hours.
The method selects water-soluble photosensitizer, such as chlorophyll derivative (sodium copper chlorophyllin), porphyrin derivative, phthalocyanine derivative (copper phthalocyanine-3, 4' -tetrasulfonic acid tetrasodium salt) and the like, the excitation wavelength range is closer to infrared light 650-780nm, the difference between the excitation wavelength and the emission wavelength is increased, the detection identification degree is improved, and the matrix (mostly 340nm excitation) interference of the blood sample is reduced.
In order to make the re-dissolving effect of the nanogold-photosensitizer compound in the buffer solution more remarkable, the buffer solution can be one of PBS, HEPES, borate, carbonate, Tris-HCl and citric acid; in order to make the re-dissolving effect of the conjugate of the nanogold-photosensitizer complex and the specific binding pair compound more remarkable in a re-dissolving buffer solution, the re-dissolving buffer solution can be one of PBS, HEPES, carbonate and Tris-HCl; in order to improve the stability of the nano-gold photosensitive particle suspension suspended in the re-dissolving buffer, a stabilizer, a surfactant, salt ions and preservative components in a determined proportion can be contained in the re-dissolving buffer, and the stabilizer can be BSA, Blockmaster, Tween and the like; the surfactant can be Triton, Tween, SDS, such as Tween-20, Triton-X100; the salt ion can be sodium chloride, potassium chloride and the like; the antiseptic component may be Proclin300, sodium azide, thimerosal, etc.
The homogeneous phase method chemiluminescence detection method obtained by the invention adopts nano-gold particles as a carrier, electrostatically adsorbs a water-soluble photosensitizer, when a sandwich method is adopted for detection, a proper light source is selected as an excitation light to irradiate the nano-gold photosensitive particles to excite the nano-gold particles, and energy is transferred to photosensitizer molecules combined with the nano-gold photosensitive particles in a non-radiative energy transfer (non-radiative energy transfer) mode to enable the molecules to reach an excited state, so that triplet oxygen is excited to a singlet state, the singlet oxygen content is increased, the detection accuracy and sensitivity are enhanced, the utilization rate of the photosensitizer is improved, the production process is simplified, the production cost is reduced, the irradiated effective light dose is improved, more photosensitizers are activated and generate stronger photosensitizing effect, the precision of the singlet oxygen quantum yield of the photosensitizer is improved, and the difference between the wavelength of the excitation light and the emission wavelength is increased, the identification degree of detection is improved.
Drawings
FIG. 1 is a schematic diagram of a sandwich assay;
FIG. 2 is a calibration graph of example 1;
FIG. 3 is a linear range diagram of example 1;
FIG. 4 is a calibration graph of example 2;
FIG. 5 is a graph of the linear range of example 2;
FIG. 6 is a linear plot of the accuracy-alignment experiment of example 2;
FIG. 7 is a calibration graph of example 3;
FIG. 8 is a graph of the linear range of example 3;
FIG. 9 is a linear plot of the accuracy-alignment experiment of example 3.
In the figure: exciting light 1, nano gold particles 2, a water-soluble photosensitizer 3, a compound 4 of a specific binding pair, a detection object 5 and luminescent particles 6.
Detailed Description
The present invention will be further described with reference to the following examples and the corresponding test data charts.
Example 1:
in the homogeneous phase chemiluminescence detection method provided in this embodiment, a reaction principle of a sandwich method shown in fig. 1 is used to detect a detection object, and a kit for detecting the detection object is composed of a nanogold photosensitive particle and a luminescent microparticle, where the nanogold photosensitive particle is a conjugate formed by coupling a complex of a nanogold particle and a photosensitizer with a compound capable of generating a specific binding pair; the luminescent particles are conjugates of luminescent microspheres and bioactive molecules such as antibodies.
The compound that can generate a specific binding pair as described in this example is a troponin I monoclonal antibody.
The homogeneous phase method chemiluminescence detection method provided by this embodiment further comprises preparation of nanogold photosensitive particles and luminescent microparticles, wherein the preparation of nanogold photosensitive particles comprises the steps of adopting nanogold particles, obtaining nanogold particles, and then performing nanogold-photosensitizer compounding, wherein the nanogold-photosensitizer compounding is to add a water-soluble photosensitizer into the nanogold particles, stir the mixture at room temperature in a dark room overnight, then obtain a precipitated nanogold-photosensitizer compound through centrifugation, and then re-dissolve the nanogold-photosensitizer compound with a buffer solution; the re-dissolved nano gold-photosensitizer compound and the re-dissolved nano gold-photosensitizer compound are coupled with a troponin I monoclonal antibody, the nano gold-photosensitizer and the troponin I monoclonal antibody are coupled, the troponin I monoclonal antibody is added into the solution of the re-dissolved nano gold-photosensitizer compound, the pH value of the reaction is adjusted to the isoelectric point of the troponin I monoclonal antibody, in the implementation process, the pH value of the isoelectric point is usually controlled to be between plus or minus 0.5 of the isoelectric point, the troponin I monoclonal antibody and the troponin I monoclonal antibody generate electrostatic adsorption under the environment condition of the isoelectric point by the charge of the nano gold particles, so that the troponin I monoclonal antibody is coupled to the surface of the nano gold particles, the unbound troponin I monoclonal antibody is removed by a centrifugal mode, and then a re-dissolving buffer solution is added for re-dissolving, obtaining a coupling body of the nano-gold-photosensitizer compound and the troponin I monoclonal antibody suspended in the redissolution buffer.
In this embodiment, the specific operation steps may be:
(1) preparation of gold nanoparticles
Heating 100ml of 0.01% chloroauric acid aqueous solution to boiling, accurately adding 0.7ml of l% trisodium citrate aqueous solution under stirring, enabling the golden chloroauric acid aqueous solution to become mauve within 2 minutes, continuously boiling for 15 minutes, cooling, and recovering the original volume with distilled water to obtain a 10-50 nanometer gold nanoparticle solution, wherein the gold nanoparticles can also be purchased in the market.
(2) Synthesis of nanogold-photosensitizer complexes
Adding copper phthalocyanine-3, 4 '-tetrasulfonic acid tetrasodium salt into the nano gold particle solution, wherein the molar ratio of nano gold to the copper phthalocyanine-3, 4' -tetrasulfonic acid tetrasodium salt is 2: 1. The mixture was stirred overnight in a dark room at room temperature, centrifuged at 12000rpm to obtain the final complex precipitate, which was reconstituted with 50ml PB buffer.
(3) Coupling of nanogold-photosensitizer complex and troponin I monoclonal antibody
Adding 100 mu L of troponin I monoclonal antibody into the re-dissolved nano-gold-photosensitizer compound solution, adjusting the reaction pH to 8.0, coupling the troponin I monoclonal antibody to the surface of the nano-gold particles through electrostatic adsorption, removing unbound troponin I monoclonal antibody through centrifugation at 15000rpm for 15min after 2 hours, and adding 50ml of HEPES re-dissolving buffer solution for re-dissolving, wherein the re-dissolving buffer solution contains 0.5% of BSA, 0.1% of Tween-20, 0.9% of sodium chloride and 0.02% of proclin 300.
(4) Preparation of luminescent particles
The luminescent microspheres are purchased from Alpha Acceptor beads produced and sold by PE company, and the troponin I monoclonal antibody is coated in a covalent coupling mode. Adding 0.5mL of Buffer A0.5mL into 1mL of luminescent microspheres, uniformly mixing, activating with 0.2mL of EDC for 10min, adding 0.3mL of antibody, shaking for reaction for 2h, sealing for 5min, adding Buffer B to a final volume of 50mL, and uniformly stirring to obtain the luminescent particles.
Wherein: buffer A: pH value of 5-6, MES and HEPES, KCl, NaCl and CaCl2、MgCl2The concentration of plasma salt ion is 1-10%, and a certain ionic strength is provided to promote the activation reaction of the latex microsphere.
Buffer B: pH value is 6-8, one buffer solution containing MES, HEPES, PBS and Tris provides buffer capacity of reaction, and the buffer solution contains KCl, NaCl and CaCl2、MgCl2Wherein one of the salt ions has a concentration of 1-10% (providing the ionic strength required for antibody-microsphere coupling reaction), and contains one of the stable substances such as Bovine Serum Albumin (BSA), sucrose, trehalose, and glycerol at a concentration of 0.1% -0.5% (blocking the surface of the microsphere not bound with antibody)Maintaining the stability of immune latex), and the preservative component can be 0.01-0.05% (inhibiting the propagation of bacteria, etc.) of Proclin300, sodium azide, etc.
The kit composed of the nanogold photosensitive particles and the luminescent particles provided by the embodiment is used for chemiluminescence detection of troponin, when the kit is used, a detection object is detected according to a sandwich method detection principle shown in figure 1, and through detection, the kit has the following performances:
calibration curve:
the reagent is balanced to the ambient temperature before use, 20 mul of gradient calibrator is respectively added into calibration holes by adopting an instrument self-carried calibration program, and 30 mul of nanogold photosensitive particles and 30 mul of luminescent particles are sequentially added for calibration test.
Tables 1 to 1: detection result of calibration product
From the calibration curves obtained in Table 1-1 and FIG. 2, it can be seen that: the reaction degree is higher, and the detection range is wider. Detection example 1 reagent linear range:
and (3) diluting the high-value serum sample with negative serum or physiological saline in a gradient manner, repeatedly measuring twice, and calculating the correlation between the theoretical concentration and the actually measured average value by using a least square method column equation.
Tables 1 to 2: table of the linear range of detection for the kit prepared by the method described in this example:
from tables 1-2, the linear range: the high-value serum sample is diluted by negative serum or normal saline in a gradient way, the measurement is repeated twice, the correlation between the theoretical concentration and the measured mean value is calculated by a least square method column equation, and the linear high value can be judged to reach 85.27. As shown in fig. 3, the linearity of the detection result obtained in this example is good, and the detection reagent has a good correlation (R2 ═ 0.9995).
The kit prepared by the method of the embodiment has the following detection precision:
precision is an important index for measuring the variation of the reagent between batches and is an important basis for evaluating the effectiveness of the products to be marketed, and usually comprises the precision between batches and the precision between batches.
The evaluation method of the precision in the batch comprises the following steps: using low (L) and high (H) value samples, independent analysis was performed on 2 batches of product, the assay was repeated 10 times for each batch, and the average of the 10 measurements was calculated
And Standard Deviation (SD), according to the formula
Calculating Coefficient of Variation (CV)
The method for evaluating the batch precision comprises the following steps: using low (L) and high (H) value samples, 3 batches of the product were analyzed independently, the assay was repeated 10 times for each batch, and the average of the 20 measurements was calculated
And Standard Deviation (SD), according to the formula
The Coefficient of Variation (CV) was calculated.
The results of the intra-and inter-batch fine density tests were as follows:
tables 1 to 3: the kit prepared by the method of the embodiment has the following internal precision and batch-to-batch precision:
as can be seen from tables 1-3, the kits prepared by the methods described in this example all had batch precision<5％Inter-batch precision is equal<5％The kit prepared by the method of the embodiment has good repeatability and small random error when being used for detection.
The detection accuracy of the kit prepared by the method of the embodiment is as follows:
the accuracy is the coincidence degree of the measured value and the actual value, and the detection error of the reaction reagent.
Performing three-time repeated detection with NIST SRM2921 as sample, and calculating average value
The relative deviation B from the reference value T is calculated by the formula:
tables 1 to 4: the detection accuracy of the kit prepared by the method of the embodiment
| Reference target value (ng/mL) | 31.2 |
| Test value 1 | 32.695 |
| |
32.765 |
| |
32.775 |
| Mean value of test | 32.745 |
| Relative deviation% | 4.95 |
The test result shows that the B is (32.745-31.2)/31.2 multiplied by 100 percent to 4.95 percent, and the relative deviation is less than 10 percent.
As can be seen from tables 1-4, the kit prepared by the method of this example has a small deviation of detection accuracy, the relative deviation is within 5%, which indicates that the measured value is close to the theoretical value, and the kit prepared by the method of this example has a small detection error.
Table 1-5 sensitivities:
the measurement was repeated 20 times with the calibrator at 0ng/mL, and the average value of the 20 times was calculated
And Standard Deviation (SD) D, analytical sensitivity calculation formula:
the sensitivity can reach 0.009 ng/mL.
Example 2:
this example provides a homogeneous method chemiluminescence detection method, which is used for detecting myoglobin chemiluminescence, and the specific binding pair compound is myoglobin monoclonal antibody.
In this embodiment, the specific operation steps may be:
(1) the nano gold particle solution of 10 nm to 50 nm can be purchased from the market.
(2) Synthesis of nanogold-photosensitizer complexes
Adding copper phthalocyanine-3, 4 '-tetrasulfonic acid tetrasodium salt into the nano gold particle solution, wherein the molar ratio of nano gold to copper phthalocyanine-3, 4' -tetrasulfonic acid tetrasodium salt is 2: 5. The mixture was stirred overnight in a dark room at room temperature, centrifuged at 12000rpm to obtain the final complex precipitate, which was reconstituted with 50ml PB buffer.
(3) Coupling of nano-gold-photosensitizer compound and myoglobin monoclonal antibody
Adding 150 mu g of myoglobin monoclonal antibody into the re-dissolved nano-gold-photosensitizer compound solution, adjusting the reaction pH to 7.4, coupling the myoglobin monoclonal antibody to the surface of the nano-gold particles through electrostatic adsorption, removing the unbound myoglobin monoclonal antibody through centrifugation at 15000rpm for 15min after 1 hour, and adding 50ml of PB re-dissolving buffer solution for re-dissolving, wherein the re-dissolving buffer solution contains 0.5% of triton-X100, 1.5% of sodium chloride and 0.02% of proclin 300.
(4) Preparation of luminescent particles
The luminescent microspheres are purchased from Alpha Acceptor beads produced and sold by PE company, and the myoglobin monoclonal antibody is coated by adopting a covalent coupling mode. Adding 0.75mL of Buffer A0.75mL into 1mL of luminescent microsphere, mixing uniformly, activating with 0.3mL of EDC for 20min, adding 0.7mL of antibody, shaking for reaction for 3.5 h, sealing for 15min, adding Buffer B to a final volume of 50mL, and stirring uniformly to obtain the luminescent microparticle.
Wherein: buffer A: pH value of 5-6, MES and HEPES, KCl, NaCl and CaCl2、MgCl2The concentration of plasma salt ion is 1-10%, and a certain ionic strength is provided to promote the activation reaction of the latex microsphere.
Buffer B: pH value is 6-8, one buffer solution containing MES, HEPES, PBS, Tris and the like provides buffer capacity of reaction, and the buffer solution contains KCl, NaCl, CaCl2、MgCl2And the concentration of one of the salt ions is 1-10% (providing the ionic strength required by the antibody-microsphere coupling reaction), the concentration of one of the substances containing a stable substance such as Bovine Serum Albumin (BSA), sucrose, trehalose, glycerol and the like is 0.1% -0.5% (sealing the surface of the microsphere which is not combined with the antibody and maintaining the stability of immune latex), and the preservative component can be 0.01% -0.05% (inhibiting the propagation of bacteria and the like) of Proclin300, sodium azide and the like.
When the homogeneous phase method chemiluminescence detection method provided in this embodiment is used, a kit is composed of a conjugate of a nanogold-photosensitizer complex suspended in a redissolving buffer solution and a myoglobin monoclonal antibody and luminescent microparticles, and detection of a detection object can be performed according to the sandwich method detection principle shown in fig. 1, and the kit has the following properties after detection:
calibration curve:
the reagent is balanced to the ambient temperature before use, 20 mul of gradient calibrator is respectively added into calibration holes by adopting an instrument self-carried calibration program, and 30 mul of nanogold photosensitive particles and 30 mul of luminescent particles are sequentially added for calibration test.
Tables 1 to 1: detection result of calibration product
From the calibration curves obtained in Table 1-1 and FIG. 4, it can be seen that: the reaction degree is higher, and the detection range is wider. Detection example 1 reagent linear range:
and (3) diluting the high-value serum sample with negative serum or physiological saline in a gradient manner, repeatedly measuring twice, and calculating the correlation between the theoretical concentration and the actually measured average value by using a least square method column equation.
Tables 1 to 2: table of the linear range of detection for the kit prepared by the method described in this example:
from tables 1-2, the linear range: and (3) diluting the high-value serum sample with negative serum or normal saline in a gradient manner, repeatedly measuring twice, calculating the correlation between the theoretical concentration and the actually measured mean value by using a least square method column equation, and judging that the linear high value can reach 800 ng/mL. As shown in fig. 5, the linearity of the detection result obtained in this example is good, and the detection reagent has a good correlation (R2 ═ 0.9988).
The kit prepared by the method of the embodiment has the following detection precision:
precision is an important index for measuring the variation of the reagent between batches and is an important basis for evaluating the effectiveness of the products to be marketed, and usually comprises the precision between batches and the precision between batches.
The evaluation method of the precision in the batch comprises the following steps: by low (L), high(H) Value samples, independent analysis of 2 batches of product, 10 replicates per batch, calculation of the average of 10 measurements
And Standard Deviation (SD), according to the formula
Calculating Coefficient of Variation (CV)
The method for evaluating the batch precision comprises the following steps: using low (L) and high (H) value samples, 3 batches of the product were analyzed independently, the assay was repeated 10 times for each batch, and the average of the 20 measurements was calculated
And Standard Deviation (SD), according to the formula
The Coefficient of Variation (CV) was calculated.
The results of the intra-and inter-batch fine density tests were as follows:
tables 1 to 2: the kit prepared by the method of this example has both batch-to-batch precision and batch-to-batch precision
As can be seen from tables 1-2, the kits prepared by the methods described in this example all had batch precision<5％Inter-batch precision is equal<5％The kit prepared by the method of the embodiment has good repeatability and small random error when being used for detection.
The detection accuracy of the kit prepared by the method of the embodiment is as follows:
the accuracy is the coincidence degree of the measured value and the actual value, and the detection error of the reaction reagent. The project has no international agreed calibrator or international agreed reference measuring program, and can not trace to the SI condition in the measurement. Accuracy was assessed by comparison to a commercially available registered product.
The test method comprises simultaneously testing and analyzing 20 different clinical patient samples with self-made reagent and comparison reagent, calculating correlation coefficient r with the measured values of all samples, and calculating if r is not less than 0.975 (or r is not less than 02Not less than 0.95), the measured values of the two test methods are comparable, and the test result can meet the clinical requirement.
Tables 1 to 3: the detection accuracy of the kit prepared by the method of the embodiment
The accuracy-alignment experimental linear graph is shown in fig. 6.
From tables 1-3, the correlation coefficient r between the self-made reagent and the comparison reagent2Not less than 0.95, the measured values of the two test methods are comparable, and the test result can meet the clinical requirement.
Table 1-5 sensitivities:
the measurement was repeated 20 times with the calibrator at 0ng/mL, and the average value of the 20 times was calculated
And Standard Deviation (SD) D, analytical sensitivity calculation formula:
the sensitivity can reach 0.57 ng/mL.
Example 3:
the homogeneous phase chemiluminescence detection method provided by this embodiment is used for chemiluminescence detection of cardiac fatty acid binding protein, and the specific binding partner compound is a cardiac fatty acid binding protein monoclonal antibody.
In this embodiment, the specific operation steps may be:
(1) the preparation of the nano gold particles can be purchased from the same example 1 or the market;
(2) synthesis of nanogold-photosensitizer complexes
Adding 100 mu L of copper phthalocyanine-3, 4 '-tetrasulfonic acid tetrasodium salt into the nano gold particle solution, wherein the molar ratio of the nano gold to the copper phthalocyanine-3, 4' -tetrasulfonic acid tetrasodium salt is 2: 2. The mixture was stirred overnight in a dark room at room temperature, centrifuged at 12000rpm to give a final complex precipitate, which was reconstituted with 50ml PB.
(3) Coupling of nano gold-photosensitizer compound and heart-type fatty acid binding protein monoclonal antibody
Adding 100 mu L of heart-type fatty acid binding protein monoclonal antibody into a nano-gold particle-photosensitizer, adjusting the reaction pH to 7.4, coupling the heart-type fatty acid binding protein monoclonal antibody to the surface of the nano-gold particle through electrostatic adsorption, removing the unbound heart-type fatty acid binding protein monoclonal antibody through centrifugation at 15000rpm for 15min after 2h, and adding 50ml of PB buffer solution to redissolve, wherein the buffer solution contains 0.5% of BSA, 0.5% of triton-X100, 1.5% of sodium chloride and 0.02% of proclin 300.
(4) Preparation of luminescent particles:
the luminescent microspheres are purchased from Alpha Acceptor beads produced and sold by PE company, and the heart-type fatty acid binding protein monoclonal antibody is coated by adopting a covalent coupling mode. Adding 1mL of Buffer A into 1mL of luminescent microspheres, uniformly mixing, activating with 0.5mL of LEDC for 30min, adding 1mL of antibody, carrying out shake reaction for 5h, sealing for 30min, adding Buffer B to a final volume of 50mL, and uniformly stirring to obtain the luminescent particles.
Wherein: buffer A: pH value of 5-6, MES and HEPES, KCl, NaCl and CaCl2、MgCl2The concentration of plasma salt ion is 1-10%, and a certain ionic strength is provided to promote the activation reaction of the latex microsphere.
Buffer B: pH value is 6-8, one buffer solution containing MES, HEPES, PBS, Tris and the like provides buffer capacity of reaction, and the buffer solution contains KCl, NaCl, CaCl2、MgCl2Etc. of one kind of themThe concentration of 1-10% (providing the ionic strength required by antibody-microsphere coupling reaction), the concentration of one substance containing stable substances such as Bovine Serum Albumin (BSA), sucrose, trehalose, glycerol and the like is 0.1% -0.5% (sealing the microsphere surface not combined with the antibody and maintaining the stability of immune latex), and the preservative component can be 0.01% -0.05% (inhibiting the propagation of bacteria and the like) of Proclin300, sodium azide and the like.
The homogeneous phase chemiluminescence detection method provided in this embodiment is used for chemiluminescence detection of cardiac fatty acid binding protein, and when the kit is used, the kit is composed of a conjugate of a nanogold-photosensitizer complex suspended in a redissolving buffer solution and a cardiac fatty acid binding protein monoclonal antibody, and luminescent microparticles, and can detect a detected object according to the sandwich method detection principle shown in fig. 1, and the kit has the following properties after detection:
calibration curve:
the kit is balanced to the ambient temperature before use, 20 mul of gradient calibrator is respectively added into a calibration hole by adopting a self-contained calibration program of the instrument, and 30 mul of nanogold photosensitive particles and 30 mul of luminescent particles are sequentially added for calibration test.
Tables 1 to 1: detection result of calibration product
From the calibration curves obtained in Table 1-1 and FIG. 7, it can be seen that: the reaction degree is higher, and the detection range is wider. Detection example 1 reagent linear range:
and (3) diluting the high-value serum sample with negative serum or physiological saline in a gradient manner, repeatedly measuring twice, and calculating the correlation between the theoretical concentration and the actually measured average value by using a least square method column equation.
Tables 1 to 2: table of the linear range of detection for the kit prepared by the method described in this example:
from tables 1-2, the linear range: and (3) diluting the high-value serum sample with negative serum or normal saline in a gradient manner, repeatedly measuring twice, calculating the correlation between the theoretical concentration and the actually measured mean value by using a least square method column equation, and judging that the linear high value can reach 160 ng/mL. As shown in fig. 8, the linearity of the detection result obtained in this example is good, and the detection reagent has a good correlation (R2 ═ 0.9998).
The kit prepared by the method of the embodiment has the following detection precision:
precision is an important index for measuring the variation of the reagent between batches and is an important basis for evaluating the effectiveness of the products to be marketed, and usually comprises the precision between batches and the precision between batches.
The evaluation method of the precision in the batch comprises the following steps: using low (L) and high (H) value samples, independent analysis was performed on 2 batches of product, the assay was repeated 10 times for each batch, and the average of the 10 measurements was calculated
And Standard Deviation (SD), according to the formula
Calculating Coefficient of Variation (CV)
The method for evaluating the batch precision comprises the following steps: using low (L) and high (H) value samples, 3 batches of the product were analyzed independently, the assay was repeated 10 times for each batch, and the average of the 20 measurements was calculated
And Standard Deviation (SD), according to the formula
The Coefficient of Variation (CV) was calculated.
The results of the intra-and inter-batch fine density tests were as follows:
tables 1 to 3: the kit prepared by the method of this example has both batch-to-batch precision and batch-to-batch precision
As can be seen from tables 1-3, the kits prepared by the methods described in this example all had batch precision<5％Inter-batch precision is equal<5％The kit prepared by the method of the embodiment has good repeatability and small random error when being used for detection.
The detection accuracy of the kit prepared by the method of the embodiment is as follows:
the accuracy is the coincidence degree of the measured value and the actual value, and the detection error of the reaction reagent.
The accuracy is the coincidence degree of the measured value and the actual value, and the detection error of the reaction reagent. The project has no international agreed calibrator or international agreed reference measuring program, and can not trace to the SI condition in the measurement. Accuracy was assessed by comparison to a commercially available registered product.
The test method is characterized in that a self-made reagent and a comparison reagent are adopted to simultaneously test and analyze 20 different clinical patient samples, correlation coefficient r is calculated by using the measured values of all samples in duplicate, if r is more than or equal to 0.975 (or r2 is more than or equal to 0.95), the measured values of the two test methods are comparable, and the test result can meet the clinical requirement.
Tables 1 to 4: the detection accuracy of the kit prepared by the method of the embodiment is as follows:
the accuracy alignment experiment is shown in figure 9.
As can be seen from tables 1-4 and FIG. 9, the correlation coefficient r between the home-made reagent and the comparison reagent2Not less than 0.95, the measured values of the two test methods are comparable, and the test result can meet the clinical requirement.
Table 1-5 sensitivities:
the measurement was repeated 20 times with the calibrator at 0ng/mL, and the average value of the 20 times was calculated
And Standard Deviation (SD) D, analytical sensitivity calculation formula:
the sensitivity can reach 0.01 ng/mL.
Claims (8)
1. A homogeneous phase method chemiluminescence detection method, it includes adopting the reaction principle of the sandwich method to detect the detected object, its characteristic is that the kit to detect the detected object is made up of photosensitive particle and luminescent particle of nanometer gold, the photosensitive particle of said nanometer gold is the couplet after the compound of the nanometer gold particle and photosensitizer couples with compound of the specific binding pair; the luminescent particles are conjugates of luminescent microspheres and bioactive molecules such as antibodies.
2. The homogeneous-phase chemiluminescence detection method of claim 1, wherein the method comprises the preparation of nanogold photosensitive particles and luminescent microparticles, wherein the preparation of nanogold photosensitive particles comprises the steps of using nanogold particles, obtaining nanogold particles, and then carrying out nanogold-photosensitizer compounding, wherein the nanogold-photosensitizer compounding comprises the steps of adding a water-soluble photosensitizer into the nanogold particles, stirring overnight at room temperature in a dark room, then centrifuging to finally obtain a precipitated nanogold-photosensitizer compound, and then re-dissolving the nanogold-photosensitizer compound by using a buffer solution; the re-dissolved nano gold-photosensitizer compound is coupled with a compound capable of generating specific binding pair, the coupling of the nano gold-photosensitizer and the compound capable of generating specific binding pair is to add the compound capable of generating specific binding pair into the solution of the re-dissolved nano gold-photosensitizer compound, adjust the pH value of the reaction to the isoelectric point of the compound capable of generating specific binding pair, enable the compound capable of generating specific binding pair to generate electrostatic adsorption or covalent binding under the condition of the isoelectric point environment through the charge of the nano gold particles, so that the compound capable of generating specific binding pair is coupled to the surface of the nano gold particles, remove the unbound compound capable of generating specific binding pair through a centrifugal mode, and then add re-dissolving buffer solution for re-dissolving, obtaining the nano-gold photosensitive particles suspended in the re-dissolving buffer.
3. The homogeneous chemiluminescence detection method of claim 2, wherein the luminescent particles are prepared by purchasing Alpha receptor beads from PE, coating biologically active molecules such as antibodies by covalent coupling, adding 0.5-1mL Buffer A into 1mL luminescent microspheres, mixing, activating with 0.2-0.5mL EDC for 10-30min, adding 0.3-1mL antibody, shaking for 2-5h, sealing for 5-30min, adding Buffer B to a final volume of 50mL, and stirring to obtain the luminescent particles.
4. The homogeneous-phase chemiluminescence detection method according to claim 2, wherein the molar ratio of the gold nanoparticles to the photosensitizer is 2: 1-10.
5. The homogeneous-phase chemiluminescence detection method of claim 2, wherein the water-soluble photosensitizer is a porphyrin derivative, a phthalocyanine derivative, or a chlorophyll derivative.
6. A homogeneous method chemiluminescent detection method of claim 2 or claim 4 or claim 5 wherein the electrostatic adsorption is for a period of 1 hour to 2 hours.
7. A homogeneous method chemiluminescent detection method of claim 2 or 4 or 5 wherein the buffer is one of PBS, HEPES, borate, carbonate, Tris-HCl, citric acid; the re-dissolving buffer solution is one of PBS, HEPES, carbonate and Tris-HCl.
8. The homogeneous chemiluminescent detection method of claim 6 wherein the buffer is one of PBS, HEPES, borate, carbonate, Tris-HCl, citric acid; the re-dissolving buffer solution is one of PBS, HEPES, carbonate and Tris-HCl.
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