CN114994304B - cTnI dry-type homogeneous phase chemiluminescence detection kit - Google Patents
cTnI dry-type homogeneous phase chemiluminescence detection kit Download PDFInfo
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- 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
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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
Abstract
The invention belongs to the technical field of biology, and particularly relates to a cTnI dry-type homogeneous chemiluminescence detection kit. The kit provided by the invention comprises biotin-labeled anti-cTnI 24-40aa chimeric human antibody freeze-dried beads, anti-cTnI 41-49aa antibody and anti-TnC antibody-coated receptor microsphere freeze-dried beads, streptavidin-coated donor microsphere freeze-dried beads, sample injection diluent and a detection card. The kit provided by the invention adopts a cTnI + cTnI-TnC form to detect cTnI antigen, and selects an antibody pair which can preferentially identify a relatively stable amino acid sequence, thereby solving the problem of missed detection; the method can also solve the uncertain influence caused by mixing the targets, improve the detection sensitivity, ensure the stability and reliability of the marking process and the detection result, and have higher luminous value and better sensitivity.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a cTnI dry-type homogeneous chemiluminescence detection kit.
Background
Cardiac troponin I (cTnI) is expressed in cardiac muscle tissue, is not expressed in fetal, healthy human, or adult skeletal muscle in a disease state, and thus has high specificity to cardiac muscle. Approved by the U.S. national Food and Drug Administration (FDA) in 1994-1995 for use in the diagnosis of clinical Acute Myocardial Infarction (AMI). Troponin (Tn) belongs to a regulatory protein and consists of three subunits, namely troponin C (TnC), troponin I (TnI) and troponin T (TnT). TnI is a basic protein, consisting of 209 amino acids, is an extended molecule, and forms a binary complex with TnC by taking antiparallel orientation. TnC is a calcium ion receptor that participates in regulating the activation process of the filament. TnT is an asymmetric protein with a globular C-terminal domain that has a site for binding to tropomyosin. cTnI exists in cardiomyocytes in different forms, e.g. in free form in the cytosol cTnI represents only a small fraction, the majority being in complex form in association with troponin T and C subunits. When the myocardium is damaged, the cTnI in the peripheral blood is mostly (more than 90%) in the form of a cTnI-TnC complex. The cTnI-cTnT complex is only found in the blood of less than 50% of patients, and therefore, the importance of detection is far less important than the cTnI-cTnC complex; other forms + less cTnI (e.g. oxidized, reduced, phosphorylated, dephosphorylated, proteolytically degraded forms). When the integrity of the myocardial cell membrane is disrupted, cTnI is first released from the cytoplasm and, therefore, serum levels rise rapidly, while bound cTnI is slowly released from the structural proteins of the myocardial cells on account of its large relative molecular mass. After myocardial injury, the increase of the myocardial troponin I in peripheral blood generally occurs within 5-8 h, reaches the maximum value within 12-24 h, and the high level is maintained for 7-10 d. In addition, the investigators found that the fragment located between amino acid residues 33-110 was highly stable, able to generate a longer sustained signal, and analyzed probably due to its complex formation with cTnC. Among proteolytic fragments of cTnI, a fragment having 96 to 116 amino acid residues is highly resistant to proteolysis, and a TnI fragment which forms a dimer with TnC in serum is stable and may have the entire inhibitory region. The inhibitory region of cTnI plays an important role in the immunological activity and stability of the cTnI-cTnC complex.
The determination of cTnI began in the middle of the 80's of the 20 th century, with two-antibody competitive radioimmunoassay and competitive ELISA assays being used in addition. At present, the cTnI is mostly detected by a chemiluminescence method. Labeling anti-cTnI antibodies with chemiluminescent materials greatly improves the accuracy and sensitivity of cTnI determination, but in the heterogeneous labeling immunoassay technology, chemiluminescence removes free labels which do not participate in binding through 'separation washing', so that the repeatability of detection is reduced; the homogeneous phase labeling immunoassay technology has the characteristics that the whole process has no separation washing process, no washing error exists, but the conventional light-excited homogeneous phase luminescent reagent mainly adopts a liquid phase product, and the transportation and storage stability of the reagent in a liquid state is greatly limited. And the current common cTnI measuring method is not standardized, different antibody recognition sites are adopted in different methods, most of literature data adopts an antibody for recognizing cTnI epitope (comprising easily hydrolyzed phosphorylation fragments) instead of an antibody for resisting a cTnI-TnC complex to measure the cTnI, and problems of missing detection or insufficient sensitivity, specificity and the like can be caused.
The Chinese patent application CN104090105A discloses a method for detecting HEV antibody, a kit and a preparation method of the kit, the provided kit can also reduce the occurrence of missed detection, but the detection is realized by adding antigen detection sites; further, chinese patent application CN107918022A discloses a cTnI detection kit and a method for using the same, and the provided kit contains components such as a calibrator, a washing solution, a substrate solution, a pretreatment solution, a specific antibody, an enzyme conjugate working solution, and a magnetic bead conjugate working solution, but the provided kit has low detection sensitivity and is prone to leak detection.
In conclusion, the problems of easy detection omission, low sensitivity, insufficient specificity and the like generally exist in the prior art.
Disclosure of Invention
Aiming at the defects generally existing in the prior art, the invention provides a cTnI dry-type homogeneous chemiluminescence detection kit. The kit provided by the invention adopts a cTnI + cTnI-TnC form to detect cTnI antigen, and selects an antibody pair which can preferentially identify a relatively stable amino acid sequence, thereby solving the problem of missed detection; the method can also solve the uncertain influence caused by mixing the labels, improve the detection sensitivity, ensure the stable and reliable marking process and detection result and have higher luminous value and better sensitivity.
In order to achieve the technical effects, the invention adopts the technical scheme that:
a cTnI dry-type homogeneous phase chemiluminescence detection kit comprises the following components: the kit comprises biotin-labeled anti-cTnI 24-40aa chimeric human antibody freeze-dried beads, anti-cTnI 41-49aa antibody and anti-TnC antibody-coated receptor microsphere freeze-dried beads, streptavidin-coated donor microsphere freeze-dried beads, sample injection diluent and a detection card.
Preferably, the amino acid sequence of cTnI 24-40 is shown in SEQ ID NO. 1; the amino acid sequence of the cTnI 41-49 is shown in SEQ ID NO. 2.
NYRAYATEPHAKKKSKI（SEQ ID NO.1）；
SASRKLQLK（SEQ ID NO.2）；
Preferably, the preparation process of the anti-cTnI 41-49aa antibody labeled microsphere is as follows:
(1) Putting 100 mu L of receptor microsphere suspension with the concentration of 10mg/mL into a test tube, centrifuging for 15min at the rotating speed of 14000rpm, removing supernatant, adding pH 5-6 MES solution with twice volume, cleaning twice, centrifuging for 15min at the rotating speed of 14000rpm, removing supernatant, adding 200 mu L of pH 5-6 MES solution, and performing ultrasonic dispersion to prepare primary microsphere suspension I;
(2) Respectively adding 50-70 mu g of Anti-cTnI 41-49aa antibody (Anti-cTnI Hytest 4TC21cc-19C7 cc), 2-4 mu L of 10mg/mL EDC-MES solution and 1-3 mu L of 10% Tween 20-deionized water into the primary microsphere suspension I prepared in the step (1), uniformly mixing, shaking a shaking table at 30-37 ℃ for 2h, adding 1mg of bovine serum albumin and 1.2 mu L of ethanolamine after the reaction is finished, uniformly mixing, placing the mixture on the shaking table at 30-37 ℃ for sealing for 30min, centrifuging at 14000rpm for 15min, removing the supernatant, adding a buffer solution (1) with twice volume, washing twice, adding the buffer solution (1) and storing.
Preferably, the preparation process of the anti-TnC antibody labeled microsphere is as follows:
(1) Putting 100 mu L of receptor microsphere suspension with the concentration of 10mg/mL into a test tube, centrifuging for 15min at the rotating speed of 14000rpm, removing supernatant, adding MES solution with the volume twice as large as pH 5-6, cleaning twice, centrifuging for 15min at the rotating speed of 14000rpm, removing supernatant, adding 200 mu L of MES solution with the pH of 5-6, and performing ultrasonic dispersion to prepare primary microsphere suspension I;
(2) Respectively adding 2-4 mu L of 10mg/mL EDC-MES solution and 4-8 mu L of 10mg/mL NHS-MES into the primary microsphere suspension I prepared in the step (1), uniformly mixing, oscillating for 0.5h in a shaking table at 30-37 ℃, centrifuging to remove supernatant after the reaction is finished, adding 200 mu L of HEPES with pH of 7.4-8.0.05M, cleaning for 2 times, centrifuging for 15min at the same rotating speed of 14000rpm, removing supernatant, adding 200 mu L of HEPES solution with pH of 7.4-8, and performing ultrasonic dispersion to prepare a secondary microsphere suspension II;
(3) Adding 50-70 mu g of Anti-TnC antibody (Anti-TnC hybrid 4T27cc-7B9 cc) and 1-3 mu L of 10% Tween 20-deionized water solution into the secondary microsphere suspension II prepared in the step (2), uniformly mixing, shaking for 4 hours in a shaking table at 30-37 ℃, adding 1mg of bovine serum albumin and 1.2 mu L of ethanolamine after the reaction is finished, uniformly mixing, placing on the shaking table at 30-37 ℃, sealing for 30 minutes, centrifuging for 15 minutes at the rotating speed of 14000rpm, removing supernatant, adding twice the volume of buffer (1), cleaning twice, adding 50 mu L of buffer (1), and storing.
Preferably, the preparation process of the biotin-labeled anti-cTnI 24-40aa antibody-labeled microsphere is as follows: adding 50 mu g of Anti-cTnI 24-40aa antibody (Anti-cTnI hystest RC4T21-RecR 33) and 0.95-1.89 mu g of sigma long-chain biotin into 50 mu L of CB buffer solution with pH9.5 and 0.05M, shaking for 2h at 25 ℃, adding 0.5-1mg of glycine after reaction, and shaking and sealing for 30min at 25 ℃. And (3) dialyzing the biotin-labeled anti-cTnI 24-40aa antibody solution in PBS buffer solution with the pH value of 7.4 and the concentration of 0.01M for 24 hours to obtain the recombinant human factor.
Preferably, the streptavidin-coated donor microspheres are prepared as follows:
(1) Putting 100 mu L of donor microsphere suspension with the concentration of 10mg/mL into a test tube, centrifuging for 15min at the rotating speed of 14000rpm, removing supernatant, adding pH 5-6 MES solution with twice volume, cleaning twice, centrifuging for 15min at the rotating speed of 14000rpm, removing supernatant, adding 200 mu L of pH 5-6 MES solution, and performing ultrasonic dispersion to prepare primary microsphere suspension I;
(2) Respectively adding 50-100 mu g of avidin and 2-4 mu L of 10mg/mL EDC-MES solution into the primary microsphere suspension I prepared in the step (1), uniformly mixing, shaking a shaking table at 30-37 ℃ for 2h, after the reaction is finished, 1mg of bovine serum albumin and 1.2 mu L of ethanolamine, uniformly mixing, sealing the mixture on the shaking table at 30-37 ℃ for 30min, centrifuging the mixture at the rotating speed of 14000rpm for 15min, removing supernatant, adding a buffer (1) with twice volume, washing the mixture twice, and adding the buffer (1) with 50 mu L for storage.
Preferably, the freeze-dried pellet is prepared as follows: adjusting the volume of the effluent of the bead dropping machine to 10 mu L, wherein the anti-cTnI 41-49aa antibody and anti-TnC antibody coated receptor microsphere working solution, biotin-labeled anti-cTnI 24-40aa antibody working solution or streptavidin coated donor microsphere working solution are dropped into small droplets by the bead dropping machine, and the dropped droplets are in a globular shape after being frozen by liquid nitrogen; transferring the liquid nitrogen frozen pellets to a freeze dryer which is pre-frozen to-40 ℃, and then carrying out freeze drying; after the drying is finished, three-component freeze-dried pellets can be respectively obtained.
Preferably, the anti-cTnI 41-49aa antibody and the anti-TnC antibody coated receptor microsphere working solution are respectively diluted by buffer solution (2) to working concentrations of 0.5mg/mL and 0.25mg/mL; the biotin-labeled anti-cTnI 24-40aa antibody working solution is diluted by a buffer solution (3) until the working concentration is 2.5 mu g/mL; the streptavidin-coated donor microsphere working solution is diluted by buffer solution (1) to a working concentration of 0.25mg/mL.
Preferably, the buffer (1) is prepared from 0.25M TRIS, 0 to 0.9% of sodium chloride, 5 to 7.5% of bovine serum albumin, 0.5 to 1.0% of Tween20, 0.5% of Proclin-300 and deionized water, and has a pH value of 8.5; the buffer solution (2) is prepared by 0.25M TRIS, 0 to 0.9 percent of sodium chloride, 5 to 7.5 percent of bovine serum albumin, 0.5 to 1.0 percent of Twenn 20, 0.5 percent of Proclin-300, 0.01 to 0.05 percent of phenol red sodium salt and deionized water, and the pH value is 8.5; the buffer (3) is prepared from 0.25M TRIS, 0 to 0.9% of sodium chloride, 5 to 7.5% of bovine serum albumin, 0.5 to 1.0% of Tween20, 0.5% of Proclin-300, 0.01 to 0.05% of sunset yellow and deionized water, and has a pH value of 8.5.
In order to solve the problems of high sensitivity and specificity of cTnI antigen detection, unstable placement of a liquid reagent at normal temperature, need of cold chain transportation and the like, the design and preparation method of a cTnI dry-type homogeneous phase chemiluminescence detection kit is developed, and on the homogeneous phase luminescence methodology, an inventor respectively screens and integrates antibodies with different recognition epitopes and optimizes the antibody protein ratio through a large number of experiments: (1) The receptor microsphere coated antibody contains an IC (integrated circuit) compound recognition antibody and a cTnI epitope recognition antibody; (2) the biotin labeled antibody is an antibody for recognizing cTnI epitope; (3) Selection of partial recognition epitope antibodies to prevent HAMA response murine anti-constant regions were substituted for the human fragment. Finally, antibody pairs capable of preferentially recognizing the more stable amino acid sequences were selected: 1+2 combination of biotin-labeled anti-cTnI 24-40aa chimeric human antibody, receptor microsphere-labeled anti-cTnI 41-49aa antibody and anti-TnC antibody is used for high sensitivity and specificity detection of cTnI, wherein (a) we surprisingly find that different antibodies are labeled on biotin or receptor microspheres, and influence on sensitivity and light value is large. Therefore, in the methodology, the antibody marking position has great influence on the detection result, and the optimal effect can be achieved without placing any marking position; (b) We found that the anti-TnC antibody contained at one end of the marker can compensate for the omission of detection, but the low-value sensitivity is poorer than that of the antibodies recognizing the cTnI epitope both in the biotin marker and the receptor microsphere marker. (c) The research of the marking mode aiming at two antibodies marked by receptor microspheres shows that the CV is tested to be poor under the condition that the antibody mixed marking is compared with the single marking and then the mixed solution preparation is carried out, and the optimal marking pH values of 41-49aa antibodies and TnC antibodies are different, wherein the optimal marking pH value of 41-49aa antibodies is 5-6, the optimal marking pH value of TnC antibodies is 7.4-8.0, and different marking modes (one-step method or two-step method marking) are adopted aiming at the two antibodies so as to be beneficial to the performance of the kit. (d) The inventor prepares the reagent into the freeze-dried pellet so as to complete the subsequent detection in one step, the method effectively avoids the complex steps of elution, separation and the like in the test process of the kit, and the reagent has good storage stability at normal temperature.
The diluent in the kit adopts a form of adding a preservative (Proclin 300) into physiological saline, and the detection mode of the detection card (see patent CN 202111293823.4) and the detection mode of the detection card are as follows: and (3) respectively taking 1 freeze-dried small ball, subpackaging the 1 freeze-dried small ball into a reaction detection hole of the detection card, sealing the reagent card after the subpackaging is finished, and putting the reagent card into a sealing bag filled with a drying agent for sealing. When a sample is tested, a medical easily-torn film above a reagent card is removed, a proper amount of sample injection diluent is added, the sample is added into a filter hole of a detection card, the reagent detection card is placed into an instrument, the instrument pumps negative pressure to a reaction detection hole, the sample and the sample injection diluent enter the reaction detection hole from a channel through a blood filtering material along with the pressure, the liquid entering the detection hole redissolves 3 freeze-dried pellets, the instrument vibrates the reagent card for several seconds, one-step incubation is carried out, after 15min incubation is finished, a photon signal is read, the concentration of the sample to be tested is calculated according to a standard curve, and the quantitative detection result of the sample is directly obtained.
Compared with the prior art, the dry-type homogeneous chemiluminescence detection kit for cTnI provided by the invention has the following advantages:
(1) According to the kit provided by the invention, a cTnI + cTnI-TnC form is adopted to detect a cTnI antigen, antibody selection avoids cTnI phosphorylation sites and easily hydrolyzed fragments as much as possible, cTnI is released into blood in a binding state form when being combined with myocardial injury, and an antibody pair capable of preferentially recognizing a relatively stable amino acid sequence is selected, so that the problem of missed detection is solved;
(2) The anti-cTnI 41-49aa antibody and the anti-TnC antibody coated receptor microsphere do not adopt a mixed labeling mode, and respectively select respective optimal labeling modes, and the two antibodies are respectively labeled and then research the final concentrations of the two antibodies in the working solution to determine the optimal addition amount, thereby solving the uncertain influence caused by mixed labeling, improving the detection sensitivity, ensuring the labeling process and the detection result to be stable and reliable, and being beneficial to the research of different variables of the experiment;
(3) The antibody is used for biotin labeling or coated on a receptor microsphere, and has great influence on cTnI sensitivity. The invention discovers that the anti-cTnI 24-40aa antibody is marked on the biotin, so that a higher luminous value or better sensitivity can be obtained.
Drawings
Fig. 1 is a representation of the cTnI morphology that may be present in blood;
figure 2 is a format for detection of cTnI antigen.
Detailed Description
The present invention is further explained with reference to the following specific examples, but it should be noted that the following examples are only illustrative of the present invention and should not be construed as limiting the present invention, and all technical solutions similar or equivalent to the present invention are within the scope of the present invention. The method and the device are operated according to the conventional technical method and the content of the instrument instruction, wherein the specific technology or condition is not indicated in the embodiment; the reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1 preparation of cTnI Dry homogeneous chemiluminescent assay kit
The preparation process of the cTnI dry-type homogeneous phase chemiluminescence detection kit is as follows:
preparing an S1, an anti-cTnI 41-49aa antibody and an anti-TnC antibody coated receptor microsphere and preparing a working solution:
preparation of anti-cTnI 41-49aa antibody labeled microspheres: (1) Putting 100 mu L of acceptor microsphere suspension with the concentration of 10mg/mL into a test tube, centrifuging for 15min at the rotating speed of 14000rpm, removing supernatant, adding pH5 MES solution with twice volume for cleaning twice, centrifuging for 15min at the rotating speed of 14000rpm, removing supernatant, adding 200 mu L of pH5 MES solution, and performing ultrasonic dispersion to prepare primary microsphere suspension I;
(2) Respectively adding 50 mu g of anti-cTnI 41-49aa chimeric human antibody, 2 mu L of 10mg/mL EDC-MES solution and 2 mu L of 10% Tween 20-deionized water into the primary microsphere suspension I prepared in the step (1), uniformly mixing, shaking for 2h in a shaking table at 30 ℃, adding 1mg of bovine serum albumin and 1.2 mu L of ethanolamine after the reaction is finished, uniformly mixing, placing on the shaking table at 30 ℃, sealing for 30min, centrifuging for 15min at the rotating speed of 14000rpm, removing supernatant, adding buffer (1) with twice volume, washing twice, adding 50 mu L of buffer (1), and storing. The buffer (1) was prepared from 0.05M TRIS, 0.9% sodium chloride, 1.5% bovine serum albumin, 0.2% Tween20, 0.1% Proclin-300 and deionized water, and had a pH of 8.5.
Preparing anti-TnC antibody labeled microspheres: (1) Putting 100 mu L of acceptor microsphere suspension with the concentration of 10mg/mL into a test tube, centrifuging for 15min at the rotating speed of 14000rpm, removing supernatant, adding pH5 MES solution with twice volume for cleaning twice, centrifuging for 15min at the rotating speed of 14000rpm, removing supernatant, adding 200 mu L of pH5 MES solution, and performing ultrasonic dispersion to prepare primary microsphere suspension I; (2) Respectively adding 2 mu L of 10mg/mL EDC-MES solution and 4 mu L of 10mg/mL NHS-MES into the primary microsphere suspension I prepared in the step (1), uniformly mixing, shaking for 0.5h in a shaking table at 30 ℃, centrifuging to remove a supernatant after the reaction is finished, adding 200 mu L of HEPES with pH 8.0.05M to clean for 2 times, centrifuging for 15min at the same rotating speed of 14000rpm, removing the supernatant, adding 200 mu L of HEPES buffer solution with pH8.0, and performing ultrasonic dispersion to prepare a secondary microsphere suspension II; (3) Adding 50 mu g of anti-TnC antibody and 2 mu L of 10% Tween 20-deionized water solution into the secondary microsphere suspension II prepared in the step (2), uniformly mixing, shaking for 4 hours in a shaking table at 30 ℃, adding 1mg of bovine serum albumin and 1.2 mu L of ethanolamine after the reaction is finished, uniformly mixing, placing on the shaking table at 30 ℃, sealing for 30min, centrifuging for 15min at the rotating speed of 14000rpm, removing the supernatant, adding a buffer solution (1) with twice volume, washing twice, and adding 50 mu L of the buffer solution (1) for storage.
Preparation: the acceptor microsphere stock solution coated with anti-cTnI 41-49aa antibody and anti-TnC antibody was diluted with the buffer (2) to working concentrations of 0.5mg/mL and 0.25mg/mL, respectively. The buffer solution (2) is prepared from 0.25M TRIS, 0 to 0.9 percent of sodium chloride, 5 to 7.5 percent of bovine serum albumin, 0.5 to 1.0 percent of Tween20, 0.5 percent of Proclin-300, 0.01 to 0.05 percent of phenol red sodium salt and deionized water, and the pH value is 8.5;
s2, preparing a biotin-labeled anti-cTnI 24-40aa chimeric human antibody and preparing a working solution:
preparation: adding 50 mu g of anti-cTnI 24-40aa chimeric human antibody and 1.89 mu g of sigma long-chain biotin into 50 mu L of CB buffer solution (sodium bicarbonate buffer solution) with the pH value of 9.5 and the M of 0.05, shaking for 2 hours at 25 ℃, adding 1mg of glycine after reaction, and shaking and sealing for 30 minutes at 25 ℃; the biotin-labeled anti-cTnI 24-40aa chimeric human antibody solution was dialyzed for 24h in 0.01M PBS buffer at pH 7.4.
Preparation: the biotin-labeled anti-cTnI 24-40aa chimeric human antibody labeling solution is further diluted with a buffer solution (3) to a working concentration of 2.5. Mu.g/mL. The buffer (3) was prepared from 0.25M TRIS, 0.9% sodium chloride, 7.5% bovine serum albumin, 1.0% Tween20, 0.5% Proclin-300, 0.01% sunset yellow and deionized water, and had a pH of 8.5.
S3, preparation of avidin-coated donor microsphere and preparation of working solution
(1) Putting 100 mu L of donor microsphere suspension with the concentration of 10mg/mL into a test tube, centrifuging for 15min at the rotating speed of 14000rpm, removing supernatant, adding pH5 MES solution with twice volume for cleaning twice, centrifuging for 15min at the rotating speed of 14000rpm, removing supernatant, adding 200 mu L of pH5 MES solution, and performing ultrasonic dispersion to prepare primary microsphere suspension I; (2) Respectively adding 50 mu g of avidin and 2 mu L of 10mg/mL EDC-MES solution into the primary microsphere suspension I prepared in the step (1), uniformly mixing, shaking for 2h in a shaking table at 30 ℃, uniformly mixing 1mg of bovine serum albumin and 1.2 mu L of ethanolamine after the reaction is finished, placing on the shaking table at 30 ℃, sealing for 30min, centrifuging for 15min at the rotating speed of 14000rpm, removing the supernatant, adding buffer solution (1) with the volume twice for cleaning twice, and adding 50 mu L of buffer solution (1) for storage.
Preparation: the stock solution of the donor microspheres coated with avidin was diluted with the buffer (1) to a working concentration of 0.25mg/mL.
S4, preparing liquid nitrogen freezing pellets:
adjusting the volume of the effluent of the bead dropping machine to 10 mu L, and dropping the acceptor microsphere working solution coated by the anti-cTnI 41-49aa antibody and the anti-TnC antibody, the biotin-labeled anti-cTnI 24-40aa antibody working solution or the streptavidin-coated donor microsphere working solution in small droplets by the bead dropping machine, wherein the dropped droplets are in a globular shape after being frozen by liquid nitrogen. The liquid nitrogen frozen pellets were transferred to a freeze dryer that had been prefrozen to-40 ℃ and then freeze-dried. After drying, three components of freeze-dried pellets can be respectively obtained.
S5, drying:
the liquid nitrogen frozen pellets were transferred to a freeze dryer that had been prefrozen to-40 ℃ and then freeze-dried. And after drying, respectively taking 1 of the 3 freeze-dried pellets, subpackaging the 1 freeze-dried pellets into a reaction detection hole of a reagent detection card, sealing the reagent card after subpackaging is finished, and putting the reagent card into a sealing bag filled with a drying agent (the main component is calcium chloride) for sealing.
S6, detection result:
(1) Linear range determination: the sample type is procoagulant tube serum, an Yapei hypersensitive troponin I determination kit (chemiluminescence microparticle immunoassay) is selected to determine a blank sample L (less than the lower limit of detection) and a high-concentration sample H, and the S1-S5 concentration samples are mixed according to the following modes: s1= L, S2=0.75L +0.25H, S3=0.5L +0.5H, S4=0.25L +0.75H, S5= H, respectively marked with numbers 1, 2, 3, 4, 5. The kit is adopted for detection, and each sample is repeated for 3 times. Table 1 shows the comparison result between the cTnI concentration measured in the present invention and the cTnI concentration measured in Yapei test, R 2 =0.9994。
TABLE 1 Linear Range test results
LOB determination: and (3) repeatedly testing blank for 20 times to obtain an RLU value of a test result, calculating the average value and standard deviation SD of the RLU value to obtain +2SD, and calculating to obtain a concentration value of 7.355pg/mL according to a calibration curve equation of the kit. A blank is selected as negative base (manufacturer: nanjing Songyataceae product number: S-4SP 1).
TABLE 2 blank test
The linear range of the kit of the invention is: 7.355 to 46892.6 pg/mL.
(2) Precision: mu.L of cTnI serum samples with known concentration are taken and added into the filtration wells of the reagent detection card, 20 wells of each sample are added with 150. Mu.L of diluent, and after incubation for 15min at 37 ℃, liCA Reader is used for reading.
TABLE 3 kit precision results
The result shows that the precision of the kit in batches is less than or equal to 10 percent, and the kit can be used for detecting cTnI samples.
Example 2 screening of pairing patterns
Considering that the cTnI exists in a cTnI-TnC compound and a free state after myocardial damage, different degrees of hydrolytic degradation can occur in blood, the ratio of the two can not be accurately judged, and the condition of missed detection can be caused by simply using the two epitopes to measure the cTnI. We divided two technical routes to study:
the most stable region of cTnI is 30-110 aa, because the structure of the C terminal of TnC can be tightly combined with the helical structure formed by 33-86 aa on the cTnI molecule, and the effect of stabilizing cTnI is achieved. Binding of TnC and cTnI alters the protein conformation and also occludes the cTnI epitope. Furthermore, since the effects of HAMA response are most prevalent for immunodiagnostics, most immunodiagnostic antibodies are murine mAbs, and powerful tools for addressing the effects of HAMA effects are chimeric antibodies (chimerism: replacement of the constant region of a murine antibody by a human fragment) or fully humanized antibodies, or the use of other antibodies of animal origin.
The immunological activity of the cTnI monomer and the complex is not the same. Aiming at complex detection, anti-cTnI and Anti-cTn-complex/Anti-TnC are used as basic antibodies to respectively mark biotin and receptor microspheres, and cross-pairing is carried out to test a sample. Aiming at the detection of cTnI, different epitopes of Anti-cTnI and Anti-cTnI are selected to respectively mark biotin and receptor microspheres and are subjected to cross pairing. Among them, studies have shown that: phosphorylation of proteins occurs primarily at several amino acids, serine and threonine, followed by tyrosine. And the phosphorylation sites on the cTnI antigen molecule comprise 22/23 th serine, 43/45 th serine, 149 th tyrosine and 144 th tyrosine. They together affect the molecular structure and function of cTnI. We selected the chimeric/non-chimeric human antibody in the market and carried out the experiment synchronously. To sum up, anti-cTnI we selected several epitopes: 24-40 (27-40 (including chimeric)), 40-50 (41-49), 83-106 (83-93, 85-92), 189-198, avoiding phosphorylation sites as much as possible. As cross-pairing data is more, a pair with better correlation of a detection sample and higher low-value sensitivity is selected for specific analysis, wherein the optimal biotin marker is selected to be an anti-24-40 aa epitope antibody, and an anti-41-49 aa epitope antibody and an anti-TnC antibody are selected from an antibody marked by a receptor microsphere. The comparison results of the different conditions of the better group are screened out as follows.
TABLE 4 antibody formats and Effect of immune origin
From table 4 we can conclude that different antibody formats of the same epitope have a greater impact on low value testing. Wherein, the constant region of the mouse monoclonal antibody replaces the chimeric human antibody or adopts goat polyclonal antibody, the low-value test sensitivity is improved compared with the recognition of the 20-40aa mouse monoclonal antibody, and the light value of individual samples tested by the mouse monoclonal antibody is higher, probably due to HAMA effect. In order to reduce the effect of HAMA effect, the antibody can be selected by replacing the mouse anti-constant region with human or other animal immune antibodies.
TABLE 5 missed-check test results
Table 5 shows that 1 sample is missed when the anti-24-40 aa antibody biotin marker and the anti-41-49 aa antibody receptor microsphere are coated to detect the sample. In the screening pairing process, the anti-24-40 aa antibody biotin marker and the coated anti-TnC antibody receptor microsphere are found to have no false negative when the sample is tested, but the low-value detection gradient of the combination mode is poorer than that of the anti-24-40 aa antibody biotin marker and the coated anti-41-49 aa antibody receptor microsphere. In order to prevent the condition of missing detection or low concentration value in the test process, the components of the receptor microsphere working solution are introduced into an anti-TnC antibody on the basis of an anti-41-49 aa antibody. By testing the same sample we found that the addition of appropriate amount of TnC can compensate for the omission and maintain good gradient and sensitivity of the test results.
TABLE 6 antibody marker positions
When cTnI pairing screening is carried out on the platform, the fact that light values obtained by labeling the antibody on biotin and receptor microspheres are possibly inconsistent is found, and even the situation that the sensitivity of a detection sample is deteriorated in some pairing conditions is found. Therefore, the present invention has studied the position of the antibody label. The anti-24-40 aa chimeric human antibody is marked on biotin, so that the detection result is better.
Finally, antibody pairs capable of preferentially recognizing the more stable amino acid sequences were selected: the combination of 1+2 of biotin-labeled-anti-cTnI 24-40aa chimeric human antibody, receptor microsphere-labeled-anti-cTnI 41-49aa antibody and anti-TnC antibody is used for the high sensitivity and specificity detection of cTnI,
example 3 selection of the means for coating the antibody with the receptor microspheres
Because the inventor explores the form of mixing the respectively marked antibody-receptor microspheres into the same working solution in different concentrations when in leak-repairing detection and sensitivity experiments, the method is favorable for quick experiments and obtaining the required concentration of the antibody-receptor microspheres added into the working solution. From the previous experiments, we obtained the receptor microspheres labeled with anti-41-49 aa antibody and anti-TnC antibody. For the selected microspheres we performed the following experiments: either a mixed label or a single label. Wherein: labeling alone we can select the appropriate labeling modality for the antibody. The antibody is coated on the acceptor microsphere by a one-step method label and a two-step method label. Wherein the one-step method marks EDC, antibody and microsphere for synchronous reaction; the two-step method comprises the steps of firstly reacting EDC and NHS with microspheres and then reacting the microspheres with antibodies at higher pH.
TABLE 7 comparison of mixed and individual marking modes
Firstly, the control labeling modes of the invention are all one-step methods, two antibodies adopt mixed labeling and single labeling, three tubes are labeled simultaneously, and labeling is carried out in two days. From the results in table 7, it was found that the CV of the mixed standard is higher than that of the single marker, and the individual tube markers affect the test sensitivity, and it is possible that the better inter-batch difference cannot be satisfied by the one-step mixed standard antibody method of the present invention.
TABLE 8 comparison of one-step and two-step labeling modes for individual labels
The one-step method of the invention is used for coupling the antibody with the pH value of 5-6, the two-step method is used for coupling with the pH value of 7.4-8, and other pH values are not studied too much. In view of the above conditions, the inventors labeled the anti-41-49 aa antibody and anti-TnC antibody under different conditions, respectively. Table 8 detection sensitivity is the result of comparing only the same antibody in two ways. It was found that the optimal pH for labeling of the anti-41-49 aa antibody and anti-TnC antibody was different under the labeling method of the present invention. Some performance may be lost with the use of label blending. The inventors propose to label different kinds of antibodies in order to optimize the label.
In conclusion, the two antibodies coated by the receptor microsphere of the kit are marked by adopting a single method, the anti-41-49 aa antibody is marked by adopting a one-step method, and the anti-TnC antibody is marked by adopting a two-step method.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
SEQUENCE LISTING
<110> Xiamen Baotai Biotechnology Ltd, xiamen Baotai Biotechnology Ltd
<120> cTnI dry-type homogeneous phase chemiluminescence detection kit
<130> 2022.5.17
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 17
<212> PRT
<213> Amino acid sequence of cTnI 24-40 (Amino acid sequence of cTnI 24-40)
<400> 1
Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys Lys Lys Ser Lys
1 5 10 15
Ile
<210> 2
<211> 9
<212> PRT
<213> cTnI 41-49(Amino acid sequence of cTnI 41-49)
<400> 2
Ser Ala Ser Arg Lys Leu Gln Leu Lys
1 5
Claims (4)
1. A cTnI dry-type homogeneous phase chemiluminescence detection kit is characterized by comprising the following components: the kit comprises biotin-labeled anti-cTnI 24-40aa chimeric human antibody lyophilized beads, lyophilized beads prepared from anti-cTnI 41-49aa antibody-coated receptor microspheres and anti-TnC antibody-coated receptor microspheres, streptavidin-coated donor microsphere lyophilized beads, a sample injection diluent and a detection card; the marking pH of the anti-TnC antibody is 7.4 to 8.0, and the anti-TnC antibody is marked by adopting a two-step method, wherein the preparation process comprises the following steps:
(1) Putting 100 mu L of receptor microsphere suspension with the concentration of 10mg/mL into a test tube, centrifuging for 15min at the rotating speed of 14000rpm, removing supernatant, adding MES solution with the volume twice as large as pH 5-6, cleaning twice, centrifuging for 15min at the rotating speed of 14000rpm, removing supernatant, adding 200 mu L of MES solution with the pH of 5-6, and performing ultrasonic dispersion to prepare primary microsphere suspension I;
(2) Sequentially adding 2-4 mu L of 10mg/mL EDC-MES solution and 4-8 mu L of 10mg/mL NHS-MES into the primary microsphere suspension I prepared in the step (1), uniformly mixing, oscillating for 0.5h in a shaking table at 30-37 ℃, centrifuging to remove supernatant after the reaction is finished, adding 200 mu L of HEPES with pH of 7.4-8.0.05M, cleaning for 2 times, centrifuging for 15min at the same rotating speed of 14000rpm, removing supernatant, adding 200 mu L of HEPES solution with pH of 7.4-8, and performing ultrasonic dispersion to prepare a secondary microsphere suspension II;
(3) Respectively and sequentially adding 50-70 mu g of anti-TnC antibody and 1-3 mu L of 10% Tween 20-deionized water solution into the secondary microsphere suspension II prepared in the step (2), uniformly mixing, shaking for 4 hours in a shaking table at 30-37 ℃, adding 1mg of bovine serum albumin and 1.2 mu L of ethanolamine after the reaction is finished, uniformly mixing, sealing for 30min in the shaking table at 30-37 ℃, centrifuging for 15min at the rotating speed of 14000rpm, removing the supernatant, adding a buffer solution (1) with twice volume, cleaning twice, and adding 50 mu L of the buffer solution (1) for storage; the preparation process of the streptavidin-coated donor microsphere is as follows:
A. putting 100 mu L of donor microsphere suspension with the concentration of 10mg/mL into a test tube, centrifuging for 15min at the rotating speed of 14000rpm, removing supernatant, adding pH 5-6 MES solution with twice volume, cleaning twice, centrifuging for 15min at the rotating speed of 14000rpm, removing supernatant, adding 200 mu L of pH 5-6 MES solution, and performing ultrasonic dispersion to prepare primary microsphere suspension I;
B. respectively and sequentially adding 50-100 mu g of streptavidin and 2-4 mu L of 10mg/mL EDC-MES solution into the primary microsphere suspension I prepared in the step A, uniformly mixing, shaking a shaking table at 30-37 ℃ for 2h, after the reaction is finished, 1mg of bovine serum albumin and 1.2 mu L of ethanolamine, uniformly mixing, sealing the mixture on the shaking table at 30-37 ℃ for 30min, centrifuging the mixture for 15min at the rotating speed of 14000rpm, removing supernatant, adding a buffer solution (1) with the volume twice as large as the volume of the mixture, washing the mixture twice, and adding 50 mu L of the buffer solution (1) for storage;
the receptor microsphere coated by the anti-cTnI 41-49aa antibody and the receptor microsphere coated by the anti-TnC antibody are respectively diluted by buffer solution (2) to working concentrations of 0.5mg/mL and 0.25mg/mL; the biotin-labeled anti-cTnI 24-40aa chimeric human antibody working solution is diluted by a buffer solution (3) until the working concentration is 2.5 mu g/mL; the donor microsphere working solution coated by the streptavidin is diluted by a buffer solution (1) until the working concentration is 0.25mg/mL; the buffer solution (1) is prepared from 0.25M TRIS, 0 to 0.9 percent of sodium chloride, 5 to 7.5 percent of bovine serum albumin, 0.5 to 1.0 percent of Tween20, 0.5 percent of Proclin-300 and deionized water, and the pH value is 8.5; the buffer solution (2) is prepared from 0.25M TRIS, 0 to 0.9 percent of sodium chloride, 5 to 7.5 percent of bovine serum albumin, 0.5 to 1.0 percent of Tween20, 0.5 percent of Proclin-300, 0.01 to 0.05 percent of phenol red sodium salt and deionized water; the buffer solution (3) is prepared by 0.25M TRIS, 0 to 0.9 percent of sodium chloride, 5 to 7.5 percent of bovine serum albumin, 0.5 to 1.0 percent of Tweeen 20, 0.5 percent of Proclin-300, 0.01 to 0.05 percent of sunset yellow and deionized water, and the pH value is 8.5; the labeled pH value of the 41-49aa antibody is 5-6, and the antibody is labeled by a one-step method, and the specific preparation process is as follows:
a. putting 100 mu L of receptor microsphere suspension with the concentration of 10mg/mL into a test tube, centrifuging for 15min at the rotating speed of 14000rpm, removing supernatant, adding pH 5-6 MES solution with the volume twice as large as that of the suspension, cleaning twice, centrifuging for 15min at the rotating speed of 14000rpm, removing supernatant, adding 200 mu L of pH 5-6 MES solution, and performing ultrasonic dispersion to prepare primary microsphere suspension I;
b. and (b) respectively and sequentially adding 50-70 mu g of anti-cTnI 41-49aa antibody, 2-4 mu L of 10mg/mL EDC-MES solution and 1-3 mu L of 10% Tween 20-deionized water into the primary microsphere suspension I prepared in the step (a), uniformly mixing, shaking for 2h in a shaking table at 30-37 ℃, adding 1mg of bovine serum albumin and 1.2 mu L of ethanolamine after the reaction is finished, uniformly mixing, placing on the shaking table at 30-37 ℃, sealing for 30min, centrifuging for 15min at the rotating speed of 14000rpm, removing supernatant, adding twice volume of buffer (1), washing twice, adding 50 mu L of buffer (1), and storing.
2. The dry homogeneous chemiluminescent assay kit of claim 1 wherein the amino acid sequence of cTnI 24-40aa is shown in SEQ ID No. 1; the amino acid sequence of the cTnI 41-49aa is shown in SEQ ID NO. 2.
3. The dry homogeneous chemiluminescent assay kit of claim 1 wherein the biotin labeled anti-cTnI 24-40aa chimeric human antibody is prepared as follows: adding 50 mu g of anti-cTnI 24-40aa chimeric human antibody and 0.95-1.89 mu g of sigma long-chain biotin into 50 mu L of CB buffer solution with pH9.5 and 0.05M, oscillating for 2h at 25 ℃, adding 0.5-1mg of glycine after reaction, oscillating and sealing for 30min at 25 ℃, and dialyzing the biotin-labeled anti-cTnI 24-40aa chimeric human antibody solution for 24h in PBS buffer solution with pH7.4 and 0.01M to obtain the anti-cTnI 24-40aa chimeric human antibody.
4. The dry homogeneous chemiluminescent assay kit of claim 1 wherein the lyophilized pellet is prepared as follows: adjusting the volume of the effluent of the bead dropping machine to 10 mu L, wherein the anti-cTnI 41-49aa antibody coated receptor microsphere working solution, the anti-TnC antibody coated receptor microsphere working solution, the biotin labeled anti-cTnI 24-40aa chimeric human antibody working solution or the streptavidin coated donor microsphere working solution are dropped into small drops by the bead dropping machine, and the dropped drops are in a globular shape after being frozen by liquid nitrogen; transferring the liquid nitrogen frozen pellets to a freeze dryer which is pre-frozen to-40 ℃, and then carrying out freeze drying; and after drying, respectively obtaining three-component freeze-dried pellets.
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