CN111596060A - Chemiluminescence immunoassay kit for homologous isomer of prostate specific antigen and preparation method thereof - Google Patents
Chemiluminescence immunoassay kit for homologous isomer of prostate specific antigen and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a chemiluminescence immunoassay kit for homologous isomers of prostate specific antigen, belonging to the technical field of in-vitro detection. The kit comprises: a calibrator, a magnetic particle reaction solution R1, a tracer conjugate reaction solution R2 and a capture antibody reaction solution R3; the magnetic particle reaction solution R1 contains streptavidin magnetic beads, buffer solution and preservative; the tracer conjugate reaction liquid R2 contains an acridinium ester labeled p2PSA antibody or a tPSA antibody, NaCl, a buffer solution, a preservative, a blocking agent, a protein stabilizer and a surfactant; the capture antibody reaction solution R3 contains a biotin-labeled tPSA antibody or p2PSA antibody, NaCl, a buffer solution, a preservative, a protein stabilizer and a surfactant. The invention also provides a preparation method of the kit. The chemiluminescence immunoassay kit for the prostate specific antigen homologous isomer has the advantages of high detection sensitivity, good reliability and low cost, and is suitable for popularization and use.
Description
Technical Field
The invention belongs to the technical field of in-vitro detection, and particularly relates to a chemiluminescence immunoassay kit for homologous isomers of prostate specific antigen and a preparation method thereof.
Background
Prostate cancer is a common malignant tumor which endangers the health of middle-aged and old men, serum prostate-specific antigen (PSA) detection is combined with digital rectal examination to be a preliminary screening method for early detection of prostate cancer, and a prostate puncture biopsy can be performed for screening suspicious cases to help diagnosis. However, when the total PSA (tPSA) of a patient is 4.0 to 10.0. mu.g/L, which is the gray zone in the conventional view, it is difficult to perform clear differential diagnosis using PSA. Although free PSA (fPSA) detection and calculation of fPSA to tPSA ratio (f/tPSA) can be performed simultaneously for identification, the effect is not obvious.
Serum prostate specific antigen isoform 2 (isoform-2) pro state-specific, p2PSA) is a isoform of PSA precursor, with non-hydrolytic stability, tumor-specificity, tissue region-specificity, and more studies have shown: compared with the traditional PSA and the like, the p2PSA (%, p2PSA) and the Prostate Health Index (PHI) obtained by detection and calculation can obviously improve the diagnosis specificity of the Prostate cancer (PCa), reduce unnecessary Prostate puncture biopsy, and show good application value in tumor malignancy prediction and active monitoring of low-risk and limited PCa patients.
Quantitative detection of the prostate specific antigen isoform (p2PSA) in serum aids in the diagnosis of prostate cancer. In China, the clinical application of serum prostate specific antigen homologous isomer (p2PSA) is mainly based on the reagent application of foreign Beckmann companies, and the reagent imported from foreign countries is very expensive, so that the reagent brings great economic burden to patients and is not beneficial to popularization at the basic level. Therefore, a serum prostate specific antigen isoform detection kit with high detection sensitivity and good reliability needs to be developed so as to reduce the use cost of patients and improve the popularization and use.
Disclosure of Invention
The invention aims to solve the technical problems in the prior art, and provides a chemiluminescence immunoassay kit for homologous isomers of prostate specific antigen with high detection sensitivity and good reliability and a preparation method thereof, so as to reduce the use cost of patients and improve the popularization and use.
In order to solve the technical problems, the technical scheme of the invention is as follows:
the invention provides a chemiluminescence immunoassay kit for homologous isomers of prostate specific antigen, which comprises: a calibrator, a magnetic particle reaction solution R1, a tracer conjugate reaction solution R2 and a capture antibody reaction solution R3;
the magnetic particle reaction solution R1 contains streptavidin magnetic beads, buffer solution and preservative;
the tracer conjugate reaction liquid R2 contains an acridinium ester labeled p2PSA antibody or an acridinium ester labeled tPSA antibody, NaCl, a buffer solution, a preservative, a blocking agent, a protein stabilizing agent and a surfactant;
the capture antibody reaction solution R3 contains a biotin-labeled tPSA antibody or a biotin-labeled p2PSA antibody, NaCl, a buffer solution, a preservative, a protein stabilizer and a surfactant.
In the technical scheme, the mass concentration of the magnetic beads of the streptavidin in the magnetic particle reaction solution R1 is 0.03-0.1%; the concentration of the acridinium ester labeled p2PSA antibody or the acridinium ester labeled tPSA antibody in the tracer conjugate reaction liquid R2 is 0.1-3 mu g/mL; the concentration of the biotin-labeled tPSA antibody or the biotin-labeled p2PSA antibody in the capture antibody reaction solution R3 is 0.5-8. mu.g/mL.
In the above technical solution, the preservative in the magnetic particle reaction solution R1, the tracer conjugate reaction solution R2, and the capture antibody reaction solution R3 is NaN3And Proclin300 or Proclin950, wherein the mass concentration of the preservative is 0.01-1%.
In the above embodiment, the concentrations of NaCl in the tracer conjugate reaction solution R2 and the capture antibody reaction solution R3 are 0.15 mol/L.
In the technical scheme, the protein stabilizing agent in the tracer conjugate reaction solution R2 and the capture antibody reaction solution R3 is one or more of BSA, casein and BGG, and the mass concentration of the protein stabilizing agent is 0.1-10%.
In the technical scheme, the surfactants in the tracer conjugate reaction solution R2 and the capture antibody reaction solution R3 are Tween, polyethylene glycol or Triton, and the mass concentration of the surfactant is 0.01-1%.
In the technical scheme, the buffer solution in the magnetic particle reaction solution R1, the tracer conjugate reaction solution R2 and the capture antibody reaction solution R3 is 2- (N-morpholine) ethanesulfonic acid (MES), piperazine-NN-bis (2-ethanesulfonic acid) (PIPES), 3- (N-morphiline) -2-hydroxypropanesulfonic acid (MOPSO), 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) or Tris-hydrochloric acid, the pH value of the buffer solution is 6.0-8.0, and the concentration of the buffer solution is 10-600 mmoL/L.
In the technical scheme, the blocker in the tracer conjugate reaction solution R2 is IgG, Roche or Scantibody, and the concentration range of the blocker is 10-500 μ g/mL.
The invention also provides a preparation method of the chemiluminescence immunoassay kit for the homologous isomer of the prostate specific antigen, which comprises the following steps:
the method comprises the following steps: preparation of magnetic particle reaction solution R1
Mixing the buffer solution and the preservative to prepare an R1 basic buffer solution;
taking streptavidin magnetic beads, washing the streptavidin magnetic beads with PBS buffer solution for three times, and then resuspending the magnetic beads into R1 basic buffer solution to prepare magnetic particle reaction solution R1;
step two: preparation of tracer conjugate reaction solution R2
Mixing NaCl, a buffer solution, a preservative, a blocking agent, a protein stabilizer and a surfactant to prepare an R2 basic buffer solution;
marking the p2PSA antibody or the tPSA antibody with an acridine ester solution dissolved in a solvent for 1-12 hours, keeping out of the sun during the reaction, purifying after the marking reaction is finished, collecting the purified stock solution for antibody quantification to obtain the p2PSA antibody or the tPSA antibody marked by acridine ester;
placing the p2PSA antibody marked by acridinium ester or the tPSA antibody marked by acridinium ester in an R2 basic buffer solution to prepare a tracer conjugate reaction solution R2;
step three: preparation of capture antibody reaction solution R3
Mixing NaCl, a buffer solution, a preservative, a protein stabilizer and a surfactant to prepare an R3 basic buffer solution;
marking the tPSA antibody or the p2PSA antibody with a biotin solution dissolved in a solvent for 2-24h, keeping the reaction away from light, after the marking reaction is finished, purifying, collecting the purified stock solution, and quantifying the antibody to obtain the biotin-labeled tPSA antibody or the biotin-labeled p2PSA antibody;
placing a biotin-labeled tPSA antibody or a biotin-labeled p2PSA antibody in an R3 basic buffer solution to prepare a capture antibody reaction solution R3;
step four: and (5) preparing a calibrator.
In the technical scheme, in the second step, the p2PSA antibody or the tPSA antibody and the acridinium ester solution are mixed according to the molar concentration of 1: 1-20 for labeling; tPSA antibody or p2PSA antibody and biotin solution in step three were mixed at molar concentration 1: the scale of 1-50 was labeled.
The invention has the beneficial effects that:
the invention relates to a prostate specific antigen homologous isomer (p2PSA) chemiluminescence immunoassay kit, which takes streptavidin magnetic particles as a solid phase carrier, detects by a double-antibody sandwich principle, labels a p2PSA antibody (or tPSA antibody) by combining a chemiluminescence substance with higher luminous intensity and sensitivity, adopts a nitric acid/sodium hydroxide chemiluminescence system, realizes quantitative detection of the p2PSA by a double-antibody sandwich method, selects acridinium as a labeling material of a chemiluminescence immunoassay system, and has the energy transition generated when an excited state returns to a ground state as direct chemiluminescence without participation of enzyme, thereby saving time and cost; the streptavidin magnetic beads and the analog marked by the biotin can be firmly combined together, so that nonspecific adsorption is reduced, the accuracy of a test sample is improved, and the anti-interference capability is strong. The chemiluminescence immunoassay kit for the prostate specific antigen homologous isomer (p2PSA) is a full-automatic measurement sample, directly gives numerical values, reduces artificial operation errors, realizes unattended operation, shortens the time required by clinical detection, has high detection precision, and has small system error because a reagent and an instrument form a closed system.
The preparation method of the chemiluminescence immunoassay kit for the prostate specific antigen homologous isomer has simple process, and the prepared kit has the advantages of high detection sensitivity, good reliability, low cost and suitability for popularization and use.
Drawings
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
FIG. 1 is a calibration curve of the kit of example 1.
Detailed Description
The invention idea of the invention is as follows: chemiluminescence immunoassay (CLIA) is an emerging immunoassay technology developed after enzyme immunoassay, radioimmunoassay, fluorescence immunoassay and time-resolved fluorescence immunoassay. Because it has high specificity of immunoreaction and high sensitivity of luminous reaction, it has been widely used in monitoring and analysis of various hormones, special proteins and medicines in recent years by clinical laboratories and scientific research units at home and abroad. The method has the advantages of high sensitivity, strong specificity, wide linear range, simple operation, good reagent stability, simple operation and easy realization of automation, and is an ideal clinical trace biochemical test analysis means. There are several common chemiluminescent systems, the most important of which are: HRP-luminol system, acridine ester luminescent system, electrochemical luminescent system and the like. Acridinium ester luminescent systems have their particular advantages over other systems: firstly, the acridinium ester labeling process is simple, the labeling substance is stable in luminescence, the kit is long in validity period, and the cost is relatively low; the light is flash, the light is fast, concentrated and strong, the fast detection is convenient to realize, the sensitivity and the precision of the detection are high, the requirement on the instrument is simple, and the full-automatic operation is convenient to realize; and secondly, the acridinium ester luminescent system is simple, the alkaline-hydrogen peroxide can directly emit light without a reinforcing agent or a catalyst, interference factors are few, the background is extremely low, and the signal-to-noise ratio is high.
Based on the situation, the chemiluminescence immunoassay kit for the acridinium ester prostate specific antigen homologous isomer (p2PSA) with high specificity and high sensitivity is developed and developed, the prostate specific antigen homologous isomer (p2PSA) in serum is quantitatively detected, and the kit plays an auxiliary role in diagnosis of prostate cancer, is feasible in theoretical and practical meanings and has wide market prospect.
The invention provides a chemiluminescence immunoassay kit for a prostate specific antigen homologous isomer (p2PSA), which comprises: a calibrator, a magnetic particle reaction solution R1, a tracer conjugate reaction solution R2 and a capture antibody reaction solution R3;
the concentration of the calibrator is 0pg/mL, 10pg/mL, 20pg/mL, 50pg/mL, 100pg/mL and 500 pg/mL. The calibrator matrix formula comprises phosphate buffer solution with pH7.4, 0.15mol/L NaCl and BSA with mass concentration of 5%.
The magnetic particle reaction solution R1 contains streptavidin magnetic beads, buffer solution and preservative; in the magnetic particle reaction solution R1, the mass concentration of streptavidin magnetic beads is preferably 0.03-0.1%; the preferred particle size of the streptavidin magnetic bead is 1-3 μm;
the tracer conjugate reaction liquid R2 contains an acridinium ester labeled p2PSA antibody or a tPSA antibody, NaCl, a buffer solution, a preservative, a blocking agent, a protein stabilizer and a surfactant; in the tracer conjugate reaction liquid R2, the concentration of the acridinium ester labeled p2PSA antibody or tPSA antibody is preferably 0.1-3 μ g/mL, and the concentration of NaCl is preferably 0.15 mol/L;
the capture antibody reaction solution R3 contains a biotin-labeled tPSA antibody or p2PSA antibody, NaCl, a buffer solution, a preservative, a protein stabilizer and a surfactant, wherein in the capture antibody reaction solution R3, the concentration of the biotin-labeled tPSA antibody or p2PSA antibody is preferably 0.5-8 μ g/mL, and the concentration of NaCl is preferably 0.15 mol/L;
according to the present invention, the buffer of the magnetic particle reaction solution R1, the tracer conjugate reaction solution R2, and the capture antibody reaction solution R3 is preferably at pH6.0 to 8.0, more preferably at pH6.0 to 7.4, and 2- (N-morpholino) ethanesulfonic acid (MES), piperazine-NN-bis (2-ethanesulfonic acid) (PIPES), sodium 3- (N-coderphinyl) -2-hydroxypropanesulfonate (MOPSO), 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), or Tris-hydrochloric acid is used as a buffer salt, and the concentration of the buffer salt is preferably 10 to 600mmoL/L, more preferably 25 to 400 mmoL/L.
According to the present invention, the preservative in the magnetic particle reaction solution R1, the labeled conjugate reaction solution R2, and the capture antibody reaction solution R3 is preferably sodium azide (NaN)3) Proclin300 or Proclin950, and the mass concentration of the preservative is preferably 0.01-1%.
According to the invention, the blocking agent in the tracer conjugate reaction solution R2 can be IgG, Roche or Scantibody commercial blocking agent, and the concentration range of the blocking agent is preferably 10-500. mu.g/mL, more preferably 50-200. mu.g/mL.
According to the invention, the protein stabilizer in the tracer conjugate reaction liquid R2 and the capture antibody reaction liquid R3 is preferably one or more of BSA, casein and BGG, and the mass concentration of the protein stabilizer is 0.1-10%.
According to the invention, the surfactant in the tracer conjugate reaction solution R2 and the capture antibody reaction solution R3 is preferably Tween, polyethylene glycol or Triton, and the mass concentration of the surfactant is preferably 0.01-1%.
The invention also provides a preparation method of the chemiluminescence immunoassay kit for the homologous isomer (p2PSA) of the prostate specific antigen, which comprises the following steps:
the method comprises the following steps: preparation of magnetic particle reaction solution R1
Mixing the buffer solution and the preservative to prepare an R1 basic buffer solution;
taking commercial Streptavidin magnetic beads (selected from JSR company, model number MS300/Streptavidin), the particle size of which is 3 μm, washing with PBS (20mmol/L PB and 150mmol/L NaCl) buffer solution with pH7.2 for three times, then re-suspending the magnetic beads into R1 basic buffer solution, and preparing into magnetic particle reaction solution R1 with mass concentration of 0.03% -0.1%;
step two: preparation of tracer conjugate reaction solution R2
Mixing NaCl, a buffer solution, a preservative, a blocking agent, a protein stabilizer and a surfactant to prepare an R2 basic buffer solution;
marking a p2PSA antibody (or a tPSA antibody) and an acridine ester solution dissolved in a solvent according to the molar concentration of 1 (1-20), wherein the marking buffer solution is pH7.4PB, the marking time is 1-12 hours, the reaction process needs to be protected from light, after the marking reaction is finished, an AKTA purifier (G25 gel column) is used for purifying, the flow rate is 3mL/min, and the purified stock solution in a collecting pipe is subjected to antibody quantification by using an ultraviolet spectrophotometer to obtain the p2PSA antibody (or the tPSA antibody) marked by the acridine ester; the solvent is preferably DMF, the concentration of the acridine ester solution is preferably 3mg/mL, and the concentration of the labeling buffer solution is preferably 20 mmol/L;
placing acridinium ester labeled p2PSA antibody (or tPSA antibody) in R2 basic buffer solution to prepare tracer conjugate reaction solution R2 with concentration of 0.1-3 μ g/mL;
step three: preparation of capture antibody reaction solution R3
Mixing NaCl, a buffer solution, a preservative, a protein stabilizer and a surfactant to prepare an R3 basic buffer solution;
labeling a tPSA antibody (or a p2PSA antibody) with a biotin solution dissolved in a solvent according to the molar concentration of 1 (1-50), wherein the labeling buffer solution is pH7.4PB, the labeling time is 2-24h, the reaction process needs to be protected from light, after the labeling reaction is finished, an AKTA purifier (G25 gel column) is used for purifying, the flow rate is 3mL/min, and the purified stock solution in a collection tube is subjected to antibody quantification by using an ultraviolet spectrophotometer to obtain the biotin-labeled tPSA antibody (or the p2PSA antibody); the solvent is preferably DMF, the concentration of the biotin solution is preferably 3mg/mL, and the concentration of the labeling buffer solution is preferably 20 mmol/L;
the biotin-labeled tPSA antibody (or p2PSA antibody) is placed in an R3 basic buffer solution to prepare a capture antibody reaction solution R3 with the concentration of 0.5-8 mug/mL.
Step four: calibration article
Quantitatively adding the high-concentration antigen into PBS buffer solution containing 5% BSA to prepare a calibrator with theoretical concentrations of 0pg/mL, 10pg/mL, 20pg/mL, 50pg/mL, 100pg/mL and 500pg/mL, and then assigning a value by using a primary calibrator, wherein the assigned concentration is the using concentration of the calibrator.
According to the invention, the determination method of the kit comprises the following steps:
adding a sample to be detected, R2 and R3 reaction liquid into a reaction cup in sequence, and incubating for 10 minutes, wherein in the process, the antibody and the antigen form a sandwich complex; then, adding the R1 reaction solution into the reaction cup, and incubating for 10 minutes; adding a cleaning solution and cleaning; and finally, adding the pre-excitation liquid and the excitation liquid into the reaction mixture, exciting a luminescence reaction, measuring the number of generated light quanta, wherein the content of the p2PSA in the sample is in direct proportion to the number of the light quanta.
The present invention will be described in further detail with reference to specific examples.
The structure of the acridinium esters used in the following examples is:
the biotin used has the structure
Example 1
1.1 kit construction
1.2 preparation method of kit
(1) Preparation of magnetic particle reaction solution R1
Streptavidin magnetic beads with the particle size of 3 μm were taken, washed three times with PBS buffer solution with pH7.2, and then the magnetic beads were resuspended in an appropriate amount of R1 basic buffer solution (other components except the magnetic beads) to prepare a magnetic particle reaction solution R1 with the mass concentration of 0.1%.
(2) Preparation of tracer conjugate reaction solution R2
a. Preparation of acridinium ester-labeled p2PSA antibody:
taking an appropriate amount of p2PSA antibody, and mixing with acridinium ester (3mg/mL) dissolved in DMF according to a molar concentration of 1: labeling at the ratio of 7.5, wherein the labeling buffer is pH7.4PB (20mmol/L), the labeling time is 4h, and the reaction process needs to be protected from light. After the labeling reaction was completed, the reaction mixture was purified by an AKTA purification apparatus (G25 gel column) at a flow rate of 3mL/min, and the purified stock solution in the collection tube was subjected to antibody quantitation by an ultraviolet spectrophotometer at a quantitative concentration of 268.5. mu.g/mL.
b. Preparation of tracer conjugate reaction solution R2:
a proper amount of acridinium ester labeled p2PSA antibody stock solution is put into an R2 basic buffer solution to prepare a tracer conjugate reaction solution R2 with the concentration of the p2PSA antibody of 0.2 mu g/mL.
(3) Preparation of capture antibody reaction solution R3
a. Preparation of biotin-labeled tPSA antibody:
taking a proper amount of tPSA antibody, and labeling the tPSA antibody and biotin (3mg/mL) dissolved in DMF according to the molar concentration ratio of 1:20, wherein the labeling buffer solution is pH7.4PB (20mmol/L), the labeling time is 4h, and the reaction process needs to be protected from light. After the labeling reaction was completed, the reaction mixture was purified by an AKTA purification apparatus (G25 gel column) at a flow rate of 3mL/min, and the purified stock solution in the collection tube was subjected to antibody quantification by an ultraviolet spectrophotometer at a quantitative concentration of 267.1. mu.g/mL.
b. Preparation of capture antibody reaction solution R3:
a suitable amount of stock solution of the biotin-labeled tPSA antibody was added to a basic buffer solution R1 (containing components other than the antibody) to prepare a capture antibody reaction solution R3 at a concentration of 0.7. mu.g/mL.
1.3 test method of kit
Firstly, adding 30 mu L of sample, 50 mu L R2 and 50 mu L R3 reaction solution into a reaction cup in sequence, and incubating for 5 minutes, wherein the antibody and the antigen form a sandwich complex in the process; secondly, adding 40 mu L R1 reaction solution into the reaction cup, and incubating for 5 minutes; thirdly, adding cleaning fluid and cleaning twice; and fourthly, adding the pre-excitation liquid and the excitation liquid into the reaction mixture to excite the luminescence reaction, and measuring the generated light quantum number.
Example 2
1.1 kit construction
1.2 preparation method of kit
(1) Preparation of magnetic particle reaction solution R1
Streptavidin magnetic beads with the particle size of 3 μm were taken, washed three times with PBS buffer solution with pH7.2, and then the magnetic beads were resuspended in an appropriate amount of R1 basic buffer solution (other components except the magnetic beads) to prepare a magnetic particle reaction solution R1 with the mass concentration of 0.1%.
(2) Preparation of tracer conjugate reaction solution R2
a. Preparation of acridinium ester-labeled tPSA antibody:
taking a proper amount of tPSA antibody, and mixing with acridinium ester (3mg/mL) dissolved in DMF according to the molar concentration of 1: 3, labeling, wherein the labeling buffer solution is pH7.4PB (20mmol/L), the labeling time is 12h, and the reaction process needs to be protected from light. After the labeling reaction was completed, the reaction mixture was purified by an AKTA purification apparatus (G25 gel column) at a flow rate of 3mL/min, and the stock solution purified in the collection tube was subjected to antibody quantitation by an ultraviolet spectrophotometer at a quantitative concentration of 198.4. mu.g/mL.
b. Preparation of tracer conjugate reaction solution R2:
taking a proper amount of acridinium ester labeled tPSA antibody stock solution to prepare a tracer conjugate reaction solution R2 with the concentration of the tPSA antibody of 1 mu g/mL in an R2 basic buffer solution.
(3) Preparation of capture antibody reaction solution R3
a. Preparation of biotin-labeled p2PSA antibody:
an appropriate amount of p2PSA antibody and biotin (3mg/mL) dissolved in DMF are labeled according to the molar concentration ratio of 1:40, the labeling buffer solution is pH7.4PB (20mmol/L), the labeling time is 8h, and the reaction process needs to be protected from light. After the labeling reaction was completed, the reaction mixture was purified by an AKTA purification apparatus (G25 gel column) at a flow rate of 3mL/min, and the purified stock solution in the collection tube was subjected to antibody quantitation by an ultraviolet spectrophotometer at a quantitative concentration of 218.9. mu.g/mL.
b. Preparation of capture antibody reaction solution R3:
a suitable amount of biotin-labeled p2PSA antibody stock solution was put in an R1 base buffer (other components except for the antibody) to prepare a capture antibody reaction solution R3 at a concentration of 2. mu.g/mL.
1.3 test method of kit
Firstly, adding 30 mu L of sample, 60 mu LR3 reaction solution and 50 mu LR1 reaction solution into a reaction cup in sequence, and incubating for 18 minutes, wherein antigen-antibody-streptomycin-magnetic bead complexes are formed in the process; a second part, adding cleaning liquid into the reaction cup and cleaning once; thirdly, continuously adding 60 mu LR2 reaction liquid into the reaction cup to form an antigen-antibody complex, and incubating for 5 minutes; fourthly, adding cleaning fluid and cleaning once; and fifthly, adding the pre-excitation liquid and the excitation liquid into the reaction mixture to excite the luminescence reaction, and measuring the generated light quantum number.
Example 3
1.1 kit construction
1.2 preparation method of kit
(1) Preparation of magnetic particle reaction solution R1
Streptavidin magnetic beads with the particle size of 1 μm were taken, washed three times with PBS buffer solution with pH7.2, and then the magnetic beads were resuspended in an appropriate amount of R1 basic buffer solution (other components except the magnetic beads) to prepare a magnetic particle reaction solution R1 with the mass concentration of 0.03%.
(2) Preparation of tracer conjugate reaction solution R2
a. Preparation of acridinium ester-labeled p2PSA antibody:
taking an appropriate amount of p2PSA antibody, and mixing with acridinium ester (3mg/mL) dissolved in DMF according to a molar concentration of 1:20, labeling with pH7.4PB (20mmol/L) as a labeling buffer for 1h, and avoiding light during the reaction. After the labeling reaction was completed, the reaction mixture was purified by an AKTA purification apparatus (G25 gel column) at a flow rate of 3mL/min, and the purified stock solution in the collection tube was subjected to antibody quantification by an ultraviolet spectrophotometer at a quantitative concentration of 247.2. mu.g/mL.
b. Preparation of tracer conjugate reaction solution R2:
and (3) taking a proper amount of acridinium ester labeled p2PSA antibody stock solution to prepare a tracer conjugate reaction solution R2 with the p2PSA antibody concentration of 1 mu g/mL in an R2 basic buffer solution.
(3) Preparation of capture antibody reaction solution R3
a. Preparation of biotin-labeled tPSA antibody:
taking a proper amount of tPSA antibody, and labeling the tPSA antibody and biotin (3mg/mL) dissolved in DMF according to the molar concentration ratio of 1:50, wherein the labeling buffer solution is pH7.4PB (20mmol/L), the labeling time is 2h, and the reaction process needs to be protected from light. After the labeling reaction was completed, the reaction mixture was purified by an AKTA purification apparatus (G25 gel column) at a flow rate of 3mL/min, and the purified stock solution in the collection tube was subjected to antibody quantitation by an ultraviolet spectrophotometer at a quantitative concentration of 227.6. mu.g/mL.
b. Preparation of capture antibody reaction solution R3:
a suitable amount of stock solution of the biotin-labeled tPSA antibody was added to a basic buffer solution R1 (containing components other than the antibody) to prepare a capture antibody reaction solution R3 at a concentration of 2. mu.g/mL.
1.3 test method of kit
Step one, uniformly mixing 30 mu L of sample and 270 mu L of cleaning solution, adding 50 mu L of diluted sample, 60 mu LR2 and 60 mu LR3 reaction solution into a reaction cup in sequence, and incubating for 10 minutes, wherein in the process, the antibody and the antigen form a sandwich compound; secondly, adding 50 mu L R1 reaction solution into the reaction cup, and incubating for 10 minutes; thirdly, adding cleaning fluid and cleaning once; and fourthly, adding the pre-excitation liquid and the excitation liquid into the reaction mixture to excite the luminescence reaction, and measuring the generated light quantum number.
According to examples 1 to 3, the chemiluminescent immunoassay kit for homologous isomers of prostate specific antigens can be prepared by replacing the reagents used therein with any substance within the aforementioned limits and limiting the reagent concentration to any value within the aforementioned limits, which is not given by way of example.
The following is evaluation data of the chemiluminescence immunoassay kit for the prostate specific antigen isoform (p2PSA) of example 1, and examples 2 to 3 show that the detection effect is similar without changing the sample, and are not repeated.
Fig. 1 and table 1 show the calibration curve and calibration data of the kit of example 1, and the calibration curve of the kit is obtained by plotting the concentration of the calibrator against the number of photons by using a 5P fitting method. And (4) bringing the light quantum number of the clinical sample into a calibration curve to obtain the concentration value of the clinical sample.
Table 2 shows the blank limit test data of example 1 of the present invention;
table 3 shows the linear test data of example 1 of the present invention;
table 4 shows the repeatability of the test data of example 1 of the present invention;
table 5 shows the test data of the lot-to-lot differences of example 1 of the present invention;
table 1 p2PSA calibration test data
| Theoretical concentration (pg/mL) | Quantum number of light |
| 0.00 | 129 |
| 10.00 | 2677 |
| 20.00 | 4972 |
| 50.00 | 11905 |
| 100.00 | 23526 |
| 500.00 | 116801 |
| 5000.00 | 1090286 |
(1) Margin limit
Parallelly measuring the primary calibrator A with zero value or the sample diluent for 20 times, recording the signal value, calculating the average M and standard deviation SD, calculating the value of M +2SD, measuring the primary calibrator B with adjacent concentration for 3 times, recording the signal value, and averaging. And (3) performing two-point linear regression fitting according to the concentration-signal value result between the zero-value first-level calibrator (A) and the adjacent concentration first-level calibrator (B) to obtain a linear equation, substituting the signal value corresponding to M +2SD into the equation, wherein the obtained concentration is a blank limit, and the result is less than 0.50 pg/mL.
Table 2p 2PSA margin test data
(2) Linearity
Diluting the high value sample close to the upper limit of the linear range to at least 5 concentrations according to a certain proportion, wherein the low value concentration sample is close to the lower limit of the linear range. And (3) repeatedly detecting samples with each concentration for 3 times, calculating an average value, performing linear fitting on the result average value and the dilution ratio by using a least square method, and calculating a linear correlation coefficient r, wherein the result meets the requirement (the linear range is 0.5 pg/mL-5000 pg/mL, and the linear correlation coefficient r is more than or equal to 0.9900).
Table 3 p2PSA linearity test data
(3) Repeatability of
The method comprises the steps of repeatedly detecting samples with different concentrations for 10 times by using samples with different concentrations, calculating the average value (M) and the Standard Deviation (SD) of 10 measurement results, and calculating the Coefficient of Variation (CV) according to the formula (2), wherein the Coefficient of Variation (CV) is less than or equal to 8.0%.
CV＝SD/M×100％…………………………………(2)
In the formula: CV — coefficient of variation;
SD — standard deviation of measurement;
m-mean of measurements.
Table 4p 2PSA repeatability test data
(4) Difference between batches
And 3 batch number kits are used for respectively detecting the same low and high samples with different concentrations, so that the inter-batch variation Coefficient (CV) among the 3 batch number kits is less than or equal to 15.0 percent.
TABLE 5p 2PSA run-to-run test data
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. A chemiluminescence immunoassay kit for homologous isomers of prostate specific antigen is characterized by comprising: a calibrator, a magnetic particle reaction solution R1, a tracer conjugate reaction solution R2 and a capture antibody reaction solution R3;
the magnetic particle reaction solution R1 contains streptavidin magnetic beads, buffer solution and preservative;
the tracer conjugate reaction liquid R2 contains an acridinium ester labeled p2PSA antibody or an acridinium ester labeled tPSA antibody, NaCl, a buffer solution, a preservative, a blocking agent, a protein stabilizing agent and a surfactant;
the capture antibody reaction solution R3 contains a biotin-labeled tPSA antibody or a biotin-labeled p2PSA antibody, NaCl, a buffer solution, a preservative, a protein stabilizer and a surfactant.
2. The chemiluminescent immunoassay kit for homologous isomers of prostate specific antigen as claimed in claim 1, wherein the mass concentration of the streptavidin magnetic beads in the magnetic particle reaction solution R1 is 0.03% -0.1%; the concentration of the acridinium ester labeled p2PSA antibody or the acridinium ester labeled tPSA antibody in the tracer conjugate reaction liquid R2 is 0.1-3 mu g/mL; the concentration of the biotin-labeled tPSA antibody or the biotin-labeled p2PSA antibody in the capture antibody reaction solution R3 is 0.5-8. mu.g/mL.
3. The kit for chemiluminescent immunoassay of homologous isomers of prostate specific antigen as claimed in claim 1, wherein the preservative in the magnetic particle reaction solution R1, the tracer conjugate reaction solution R2 and the capture antibody reaction solution R3 is NaN3Proclin300 or Proclin950, wherein the preservative has a mass concentration of 0.01％-1％。
4. The kit for chemiluminescent immunoassay of a prostate specific antigen homolog according to claim 1, wherein the concentrations of NaCl in the tracer conjugate reaction solution R2 and the capture antibody reaction solution R3 are 0.15 mol/L.
5. The chemiluminescence immunoassay kit of homologous isomers of prostate specific antigen according to claim 1, wherein the protein stabilizer in the tracer conjugate reaction solution R2 and the capture antibody reaction solution R3 is one or more of BSA, casein and BGG, and the mass concentration of the protein stabilizer is 0.1% -10%.
6. The chemiluminescent immunoassay kit for homologous isomers of prostate specific antigen as claimed in claim 1, wherein the surfactants in the tracer conjugate reaction solution R2 and the capture antibody reaction solution R3 are tween, polyethylene glycol or triton, and the mass concentration of the surfactant is 0.01% -1%.
7. The chemiluminescent immunoassay kit for homologous isomers of prostate specific antigen as claimed in claim 1, wherein the buffer solution in the magnetic particle reaction solution R1, the tracer conjugate reaction solution R2 and the capture antibody reaction solution R3 is 2- (N-morpholino) ethanesulfonic acid (MES), piperazine-NN-bis (2-ethanesulfonic acid) (PIPES), 3- (N-codeine) -2-hydroxypropanesulfonic acid sodium (MOPSO), 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) or Tris-hydrochloric acid, and has a ph of 6.0-8.0 and a concentration of 10-600 mmoL/L.
8. The kit for chemiluminescent immunoassay of homologous isomers of prostate specific antigen according to claim 1, wherein the blocking agent in the tracer conjugate reaction solution R2 is IgG, roche or Scantibody, and the concentration of the blocking agent is in the range of 10-500 μ g/mL.
9. A preparation method of a chemiluminescence immunoassay kit for homologous isomers of prostate specific antigen is characterized by comprising the following steps:
the method comprises the following steps: preparation of magnetic particle reaction solution R1
Mixing the buffer solution and the preservative to prepare an R1 basic buffer solution;
taking streptavidin magnetic beads, washing the streptavidin magnetic beads with PBS buffer solution for three times, and then resuspending the magnetic beads into R1 basic buffer solution to prepare magnetic particle reaction solution R1;
step two: preparation of tracer conjugate reaction solution R2
Mixing NaCl, a buffer solution, a preservative, a blocking agent, a protein stabilizer and a surfactant to prepare an R2 basic buffer solution;
marking the p2PSA antibody or the tPSA antibody with an acridine ester solution dissolved in a solvent for 1-12 hours, keeping out of the sun during the reaction, purifying after the marking reaction is finished, collecting the purified stock solution for antibody quantification to obtain the p2PSA antibody or the tPSA antibody marked by acridine ester;
placing the p2PSA antibody marked by acridinium ester or the tPSA antibody marked by acridinium ester in an R2 basic buffer solution to prepare a tracer conjugate reaction solution R2;
step three: preparation of capture antibody reaction solution R3
Mixing NaCl, a buffer solution, a preservative, a protein stabilizer and a surfactant to prepare an R3 basic buffer solution;
marking the tPSA antibody or the p2PSA antibody with a biotin solution dissolved in a solvent for 2-24h, keeping the reaction away from light, after the marking reaction is finished, purifying, collecting the purified stock solution, and quantifying the antibody to obtain the biotin-labeled tPSA antibody or the biotin-labeled p2PSA antibody;
placing a biotin-labeled tPSA antibody or a biotin-labeled p2PSA antibody in an R3 basic buffer solution to prepare a capture antibody reaction solution R3;
step four: and (5) preparing a calibrator.
10. The method for preparing the chemiluminescence immunoassay kit for the homologous isomers of the prostate specific antigen according to claim 9, wherein in the second step, the ratio of the p2PSA antibody or the tPSA antibody to the acridinium ester solution is determined according to the molar concentration of 1: 1-20 for labeling; tPSA antibody or p2PSA antibody and biotin solution in step three were mixed at molar concentration 1: the scale of 1-50 was labeled.
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| CN113552353A (en) * | 2021-07-12 | 2021-10-26 | 江南大学 | Magnetic particle chemiluminescence kit for diagnosis of PCa and CRPC diseases |
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| CN1656379A (en) * | 2002-05-24 | 2005-08-17 | 海布里特克有限公司 | Method of analyzing non-complexed forms of prostate specific antigen in a sample to improve prostate cancer detection |
| CN105929151A (en) * | 2016-06-30 | 2016-09-07 | 深圳市亚辉龙生物科技股份有限公司 | Chemiluminiscence immune detection kit for homologous isomer of prostate-specific antigen and preparation method of chemiluminiscence immune detection kit |
| CN109187973A (en) * | 2018-08-17 | 2019-01-11 | 迪瑞医疗科技股份有限公司 | For detecting the chemiluminescence immunoassay kit of neuronspecific enolase |
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| CN1656379A (en) * | 2002-05-24 | 2005-08-17 | 海布里特克有限公司 | Method of analyzing non-complexed forms of prostate specific antigen in a sample to improve prostate cancer detection |
| CN105929151A (en) * | 2016-06-30 | 2016-09-07 | 深圳市亚辉龙生物科技股份有限公司 | Chemiluminiscence immune detection kit for homologous isomer of prostate-specific antigen and preparation method of chemiluminiscence immune detection kit |
| CN109187973A (en) * | 2018-08-17 | 2019-01-11 | 迪瑞医疗科技股份有限公司 | For detecting the chemiluminescence immunoassay kit of neuronspecific enolase |
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| CN113552353A (en) * | 2021-07-12 | 2021-10-26 | 江南大学 | Magnetic particle chemiluminescence kit for diagnosis of PCa and CRPC diseases |
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