Calcitonin luminescence detection kit and detection method and application thereof

Abstract

The invention relates to a calcitonin luminescence detection kit and a detection method and application thereof. Specifically, the invention provides the high-sensitivity calcitonin luminescence detection kit, the high-sensitivity calcitonin luminescence detection kit comprises a first reagent and a second reagent, and the first reagent comprises magnetic beads connected with first protein; the second reagent comprises alkaline phosphatase connected with the second protein. The kit improves the detection accuracy and the sample coincidence rate.

Description

Technical Field

[0001] This disclosure relates to the field of calcitonin detection technology, specifically to a calcitonin magnetic microparticle chemiluminescence detection kit, its detection method, and its application. Background Technology

[0002] Caltonin (CT) is a polypeptide hormone secreted by the parafollicular cells of the thyroid gland (also known as calcitonin cells or C cells).

[0003] Human calcitonin is a single-chain peptide composed of 32 amino acid residues. This hormone has a molecular weight of 3418 Da and is linked by a disulfide bond between cysteine ​​residues at positions 1 and 7, forming a 7-amino acid ring at the amino terminus. Normal serum calcitonin concentrations are 0–18.5 pg / ml in men and 0–11.5 pg / ml in women, with a half-life of less than 1 hour. It is primarily degraded and excreted by the kidneys. Its main function is to lower serum calcium, being secreted when serum calcium levels are elevated, inhibiting calcium release from bone, increasing urinary phosphorus, and lowering both serum calcium and phosphorus. The target organs of calcitonin are mainly bone and the kidneys. It primarily promotes osteoblast activity, leading to bone salt deposition in osteoid, and inhibits calcium ion absorption in the gastrointestinal tract and renal tubules, thus lowering serum calcium concentration.

[0004] Calcitonin is an important indicator for detecting thyroid dysfunction. The reference range for calcitonin in normal individuals is ≤10 pg / ml. For the detection of calcitonin (CT), which requires high sensitivity, traditional platform CT detection has poor sensitivity in the reference range, and therefore urgently needs improvement. Summary of the Invention

[0005] In view of this, this disclosure provides a calcitonin luminescence detection kit based on alkaline phosphatase chemiluminescence, which is automated, highly sensitive, and suitable for dynamic observation during treatment.

[0006] According to some implementation schemes, a calcitonin luminescent detection kit is provided, which includes a first reagent and a second reagent.

[0007] In some embodiments, the first reagent comprises:

[0008] Magnetic beads coated with first-line calcitonin antibodies at concentrations ranging from 0.2 mg / mL to 0.8 mg / mL (mg / mL based on bead mass).

[0009] 10mM to 500mM HEPES buffer with pH 7 to 8

[0010] 0.1g / L to 5g / L trehalose

[0011] 0.1g / L to 5g / L Tween20

[0012] Biological preservative PC, 0.1 g / L to 5 g / L.

[0013] In some embodiments, the second reagent comprises:

[0014] 0.2 mg / L to 4 mg / L alkaline phosphatase linked to a second calcitonin antibody,

[0015] 10mM to 500mM MOPS buffer with pH 7 to 8

[0016] 0.1 g / L to 5 g / L bovine serum albumin

[0017] 0.1g / L to 5g / L Tween20

[0018] Biological preservative PC, 0.1 g / L to 5 g / L.

[0019] The primary and secondary antibodies target different epitopes of calcitonin. As an example, the primary antibody targets (or specifically binds to) residues 1-15 of calcitonin, while the secondary antibody targets (or specifically binds to) residues 17-32 of calcitonin; and vice versa.

[0020] In some specific implementations, the first reagent comprises:

[0021] 0.4 mg / mL magnetic beads coated with calcitonin antibody,

[0022] 200mM HEPES buffer, pH 7.2

[0023] 2g / L trehalose,

[0024] 1g / L Tween20;

[0025] 1 g / L of biological preservative PC.

[0026] In some embodiments, the second reagent comprises:

[0027] 2 mg / L alkaline phosphatase linked to a second calcitonin antibody,

[0028] 200mM MOPS buffer, pH 8.0

[0029] 1g / L bovine serum albumin,

[0030] 1g / L Tween20

[0031] 1 g / L of biological preservative PC.

[0032] In some embodiments, the calcitonin luminescent detection kit further includes calibrators and / or quality control materials.

[0033] In some implementations, the calibrator comprises:

[0034] 0.05M to 0.5M MES buffer, pH 6.5 to 8.5.

[0035] 0.5g / L to 5g / L Tween20

[0036] 5g / L to 50g / L bovine serum albumin, and

[0037] 0, 30, 100, 500, 1000, 2000 pg / mL calcitonin.

[0038] In some implementations, the calibrator comprises:

[0039] 0.2M MES buffer, pH 6.8

[0040] 2g / L Tween20

[0041] 30g / L bovine serum albumin, and

[0042] 0, 30, 100, 500, 1000, 2000 pg / mL calcitonin.

[0043] In some implementations, the quality control material comprises:

[0044] 0.05 to 0.5 M MES buffer, pH 6.5 to 8.5

[0045] 0.5g / L to 5g / L Tween20

[0046] 5g / L to 50g / L bovine serum albumin, and

[0047] Calcitonin at 10 pg / mL and 100 pg / mL;

[0048] In some implementations, the quality control material comprises:

[0049] 0.2M MES buffer, pH 6.8

[0050] 2g / L Tween20

[0051] 30g / L bovine serum albumin, and

[0052] Calcitonin at 10 pg / mL and 100 pg / mL.

[0053] This disclosure also provides instructions for using the calcitonin chemiluminescence detection kit, including the following steps:

[0054] 1) Provide samples;

[0055] 2) Expose the sample to the first and second reagents;

[0056] 3) Add a chemiluminescent substrate and measure the luminescence intensity.

[0057] In the specific implementation plan, the method of using the calcitonin chemiluminescence detection kit includes the following steps:

[0058] 1) Provide 10 to 100 μL of sample (or calibrator or quality control sample);

[0059] 2) Incubate the sample (or calibrator or quality control) with the first and second reagents at 37°C for 5 to 20 minutes;

[0060] 3) Under the action of a magnetic field, the reactants obtained in step 2) are washed 3 to 5 times to obtain the final reaction product;

[0061] 4) The final reaction product is contacted with the chemiluminescent substrate, and the luminescence intensity is detected after 1 to 2 minutes;

[0062] 5) Calculate the concentration of calcitonin in the sample based on the standard curve.

[0063] In the specific implementation plan, the volume ratio of sample (or calibrator or quality control): first reagent: second reagent is 1-3:1-3:1-3.

[0064] In the specific implementation plan, the volume ratio of sample (or calibrator or quality control sample): first reagent: second reagent is 1:1:1. Attached Figure Description

[0065] Figure 1 Correlation plot of 100 real samples compared with Roche reagent kit. Detailed Implementation

[0066] The present disclosure will be further described in detail below with reference to the embodiments, but the implementation of the present disclosure is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and test examples are conventional means well known to those skilled in the art, and the materials and reagents used are all commercially available.

[0067] Example 1. Preparation method of calcitonin magnetic microparticle chemiluminescence detection kit

[0068] The calcitonin magnetic microparticle chemiluminescence detection kit includes a first reagent and a second reagent. Optionally, calibrators and quality control samples may also be included.

[0069] 1. First reagent:

[0070] 0.4 mg / mL calcitonin antibody linked to carboxyl magnetic beads (3 μm),

[0071] 200mM HEPES buffer, pH 7.2

[0072] 2g / L trehalose,

[0073] 1g / L Tween20;

[0074] 1 g / L of biological preservative PC.

[0075] The first calcitonin antibody was purchased from HaiTai Biotechnology and is a standard component of the calcitonin quantitative detection kit. It can be obtained through methods well-known to those skilled in the art and is not limited to a specific antibody clone. The first calcitonin antibody was linked to the carboxyl magnetic beads using conventional labeling methods.

[0076] 2. Second reagent:

[0077] Second calcitonin antibody-linked alkaline phosphatase,

[0078] 200mM MOPS buffer, pH 8.0

[0079] 1g / L bovine serum albumin,

[0080] 1g / L Tween20

[0081] 1 g / L of biological preservative PC.

[0082] The alkaline phosphatase conjugated with the second calcitonin antibody was diluted with buffer at dilution ratios ranging from 1:1000 to 1:20000 (specifically, 1:2000 corresponds to 2 mg / L). Alkaline phosphatase labeling is a commonly used marker in chemiluminescent immunoassays. Utilizing AMPPD as a luminescent substrate, the alkaline phosphatase generates a light signal upon oxidation. By detecting these light signals, the concentration of the target molecule in the sample can be quantitatively determined.

[0083] 3. Calibrators

[0084] Calcitonin calibrators with concentrations of 0, 30, 100, 500, 1000, and 2000 pg / mL were prepared using calibration buffer as the solvent. The calibration buffer was a solution containing 0.05-0.5 M MES, 0.5 g / L to 5 g / L Tween 20, 5 g / L to 50 g / L bovine serum albumin, and a pH of 6.5 to 8.5. Preferably, the concentration of MES was 0.2 M, the concentration of Tween 20 was 2 g / L, the concentration of bovine serum albumin was 30 g / L, and the pH was 6.8.

[0085] The lyophilized calibrators were prepared using conventional lyophilization methods.

[0086] 4. Quality control products

[0087] Using a quality control buffer as a solvent, prepare calcium-lowering quality control samples at concentrations of 10 pg / mL and 100 pg / mL. The quality control buffer is a solution containing 0.05-0.5 M MES, 0.5 g / L to 5 g / L Tween 20, 5 g / L to 50 g / L bovine serum albumin, and a pH of 6.5 to 8.5. Preferably, the concentration of MES is 0.2 M, the concentration of Tween 20 is 2 g / L, the concentration of bovine serum albumin is 30 g / L, and the pH is 6.8.

[0088] The freeze-dried products of the quality control materials are prepared by freeze-drying in accordance with conventional freeze-drying methods.

[0089] Example 2. Detection Method

[0090] 1. The volume ratio of the sample (or calibrator, quality control) to the first reagent and the second reagent is 1-3:1-3:1-3; preferably, the volume ratio of the sample (or calibrator, quality control) to the first reagent and the second reagent is 1:1:1.

[0091] 2. The detection method based on the aforementioned calcitonin magnetic microparticle chemiluminescence detection kit includes the following steps:

[0092] (1) Add 10 to 100 μL (preferably 50 μL) of sample, calibrator or quality control material to the reaction vessel;

[0093] (2) Add the first and second reagents to the reaction vessel and mix well. Incubate at 37°C for 5 to 20 minutes (preferably 10 minutes).

[0094] (3) Under the action of an external magnetic field, the reactants in the reaction vessel are washed 3 to 5 times to obtain the final reaction product;

[0095] (4) Add the chemiluminescent exciter to the reaction vessel and measure the luminescence intensity after 1 to 2 minutes;

[0096] (5) The working curve after the kit program is calibrated (the luminescence value is on the vertical axis and the calibrator concentration is on the horizontal axis, which is the working curve) can automatically calculate the calcitonin concentration value based on the sample light intensity.

[0097] Optimization Example 1. The Influence of Surface Groups on Sensitivity and Batch-to-Batch Variation of Magnetic Beads

[0098] The reagent kit composition in Example 1 was optimized. It is as follows:

[0099] 1. Surface groups and particle size

[0100] Table 1A

[0101] Surface groups Average particle size (μm) Biotin 3 μm Carboxyl 3 μm Carboxyl 1.6 μm p-Toluenesulfonyl 3 μm p-Toluenesulfonyl 1.6 μm

[0102] 2. Experimental data:

[0103] Table 1B

[0104]

[0105] Based on the results, magnetic beads with carboxyl-modified surfaces and an average particle size of 3 μm were preferred.

[0106] Optimization Example 2. Magnetic beads coated with calcitonin I antibody

[0107] The reagent kit composition in Example 1 was optimized. It is as follows:

[0108] Carboxyl magnetic beads with a particle size of 3.0 μm were selected, and the following tests were performed.

[0109] Table 2. Concentration Selection

[0110]

[0111] Based on the results, the preferred concentration of magnetic beads coated with the first calcitonin antibody is 0.4 mg / ml.

[0112] Optimization Example 3. Second Antibody Concentration

[0113] The reagent kit composition in Example 1 was optimized. It is as follows:

[0114] Table 3

[0115]

[0116] Conclusion: Based on the results, 2 mg / L is the optimal concentration.

[0117] Test Case 1. Correlation

[0118] The reagent prepared in Example 1 was used to simultaneously test 100 samples with a calcitonin assay kit (electrochemiluminescence method) from existing technologies (such as Roche Diagnostics), and the measured values ​​were compared to calculate the deviation between the two kits.

[0119] The results showed that the reagent kit disclosed herein had a correlation greater than 0.9998 with the prior art reagent kit, and the measurement deviation was less than 15%. The reagent kit disclosed herein fully meets the clinical testing requirements for calcitonin.

[0120] Table 4

[0121]

[0122] Test Example 2A. Accelerated Stability

[0123] The calibrators in Example 1 were tested, and their values ​​were measured after 3, 5, 7, and 10 days of acceleration. The results showed that the measurement deviation was less than 5%, reflecting the good stability of the test kit in Example 1 and its long-term storage capability.

[0124] Table 5A. Calibration stability

[0125]

[0126] Test Example 2B. Accelerated Stability

[0127] The calibrators from Example 1 were tested, and the measured values ​​were examined after transportation. The results showed that the measured value deviation was less than 5% after transportation, reflecting the good stability of the test kit in Example 1.

[0128] Table 5B. Transportation Stability

[0129]

[0130] Test Example 3. Precision

[0131] Two quality control samples and four serum samples were tested separately, with the tests repeated 10 times. The mean of the 10 results was calculated, along with the standard deviation and coefficient of variation. The coefficient of variation represents the precision of the assay. The results showed that the precision was less than 3% for all samples, indicating that the kit has good precision.

[0132] Table 6. Precision Results

[0133]

[0134] Test Example 4. Sensitivity

[0135] The detection limit was determined using the reagent prepared in Example 1. Tests were conducted over 5 days, 8 times per day, with 40 repeated tests, resulting in 80 measured values. The mean, standard deviation, and coefficient of variation were calculated. A coefficient of variation less than 20% was considered sufficient for sensitivity. The results showed that even at a detection value as low as 0.5 pg / mL, the coefficient of variation was less than 20%, indicating that the kit prepared in Example 1 has high sensitivity.

[0136] Table 7. Sensitivity Results

[0137]

[0138]

[0139] Test Example 5. Total Imprecision

[0140] Using the reagents prepared in Example 1, two quality control samples (quality control 1 and quality control 2) at approximately two different concentrations were tested continuously for 20 days. The average value and the coefficient of variation between days were calculated. The results showed that, after 20 days of continuous testing, the day-to-day precision of this kit was less than 4%, indicating that the kit has good day-to-day precision.

[0141] Table 8. Overall Imprecision Results

[0142]

[0143]

[0144] Test Example 6. Accelerated Stability of Reagents

[0145] Twenty-five samples were simultaneously tested using the 4°C reagent from Example 1 and the reagent accelerated for 7 days. The results showed that the measured values ​​of the accelerated samples deviated by less than 10% from those of the 4°C samples. The 7-day acceleration simulated the state of the reagent after one year, indicating that the kit disclosed herein can be stored for a long time without statistically significant changes in the measured values.

[0146] Table 9. Accelerated stability results

[0147]

[0148]

[0149] The first reagent in this technical solution comprises carboxyl magnetic beads coated with a first calcitonin antibody. The first calcitonin antibody on these magnetic beads specifically binds to calcitonin in the sample, forming a calcitonin-first calcitonin antibody-magnetic bead complex. The second reagent contains alkaline phosphatase linked to a second calcitonin antibody. This alkaline phosphatase-labeled second calcitonin antibody recognizes and binds to the calcitonin immobilized on the magnetic beads, forming a complex. Due to the use of magnetic beads, direct precipitation in an external magnetic field separates the complex formed by the immune reaction from other unbound substances. Next, a washing solution is used to remove unbound components and other interfering substances, ensuring that only the specifically bound complex remains on the magnetic beads (the complex formed by alkaline phosphatase-second calcitonin antibody-calcitonin-first calcitonin antibody-magnetic bead). Upon addition of a chemiluminescent exciter, the alkaline phosphatase is oxidized and emits a light signal. The light intensity is directly proportional to the amount of calcitonin in the sample (this scheme uses a sandwich design, where an alkaline phosphatase-labeled second calcitonin antibody, carboxyl magnetic beads coated with a first calcitonin antibody, and calcitonin in the sample specifically bind). By measuring the light signal intensity, the actual concentration of calcitonin in the sample can be calculated.

[0150] In summary, the reagent kit disclosed herein exhibits good sample correlation, good stability, and satisfactory results in terms of precision and sensitivity.

Claims

1. A calcitonin luminescent detection kit, comprising a first reagent and a second reagent; wherein: The first reagent comprises: Magnetic beads coated with 0.2 mg / mL to 0.8 mg / mL calcitonin antibody, 10 mM to 500 mM HEPES buffer at pH 7 to 8. 0.1g / L to 5g / L trehalose 0.1g / L to 5g / L Tween20 0.1 g / L to 5 g / L of biological preservative PC; The second reagent comprises: 0.2 mg / L to 4 mg / L alkaline phosphatase conjugated with a second calcitonin antibody, 10 mM to 500 mM MOPS buffer at pH 7 to 8. 0.1 g / L to 5 g / L bovine serum albumin 0.1g / L to 5g / L Tween20 0.1 g / L to 5 g / L of biological preservative PC; The average particle size of the magnetic beads is 1.6 μm to 3 μm.

2. The calcitonin luminescent detection kit according to claim 1, wherein: The first reagent comprises: 0.4 mg / mL magnetic beads coated with calcitonin antibody, 200mM HEPES buffer, pH 7.2 2g / L trehalose, 1g / L Tween20; 1 g / L of biological preservative PC; The second reagent comprises: 2 mg / L alkaline phosphatase linked to a second calcitonin antibody, 200mM MOPS buffer, pH 8.0 1g / L bovine serum albumin, 1g / L Tween20 1 g / L of biological preservative PC.

3. The calcitonin luminescent detection kit according to claim 1 or 2, wherein the magnetic beads are selected from: The average particle size is 1.6 μm and the surface is modified with p-toluenesulfonyl groups. The average particle size is 1.6 μm and the surface is modified with carboxyl groups. The average particle size is 3 μm and the surface is modified with p-toluenesulfonyl groups. The average particle size is 3 μm and the surface is modified with carboxyl groups; preferably, the magnetic beads have an average particle size of 3 μm and the surface is modified with carboxyl groups.

4. The calcitonin luminescence detection kit according to claim 1, further comprising calibrators and / or quality control products.

5. The calcitonin luminescent detection kit according to claim 4, wherein: The calibrator comprises: 0.05M to 0.5M MES buffer, pH 6.5 to 8.

5. 0.5g / L to 5g / L Tween20 5g / L to 50g / L bovine serum albumin, and 0, 30, 100, 500, 1000, 2000 pg / mL calcitonin; Preferably, the calibrator comprises: 0.2M MES buffer, pH 6.8 2g / L Tween20 30g / L bovine serum albumin, and 0, 30, 100, 500, 1000, 2000 pg / mL calcitonin.

6. The calcitonin luminescent detection kit according to claim 4, wherein: The quality control materials include: 0.05 to 0.5 M MES buffer, pH 6.5 to 8.5 0.5g / L to 5g / L Tween20 5g / L to 50g / L bovine serum albumin, and Calcitonin at 10 pg / mL and 100 pg / mL; Preferably, the quality control material comprises: 0.2M MES buffer, pH 6.8, 2 g / L Tween 20, 30 g / L bovine serum albumin, and 10 pg / mL and 100 pg / mL calcitonin.

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