SERUM THYMIDINE KINASE 1 DETECTION KIT BASED ON AUTOMATIC CHEMILUMINESCENCE ANALYZER

Abstract

Disclosed are a kit and use thereof. The kit includes a first polyclonal antibody that has been immobilized or suitable for immobilization on a solid carrier, a second polyclonal antibody labeled with a marker. Both the first polyclonal antibody and the second polyclonal antibody are both chicken anti human-thymidine kinase IgY-polyclonal antibodies. Both the first polyclonal antibody and the second polyclonal antibody are suitable for specifically binding to thymidine kinase 1. The kit is suitable for the risk assessment of micro malignant tumors/precancerous diseases and tumors that are not detectable by human population screening images.

Claims

1. A kit, comprising: a first polyclonal antibody that has been immobilized or is suitable for immobilization on a solid carrier; and a second polyclonal antibody labeled with a marker, wherein both the first polyclonal antibody and the second polyclonal antibody are chicken anti human thymidine kinase 1 IgY-polyclonal antibodies, and both the first polyclonal antibody and the second polyclonal antibody are suitable for specifically binding to thymidine kinase 1.

2. The kit of claim 1, wherein epitopes recognized by the first polyclonal antibody and the second polyclonal antibody include: a third peptide fragment at a carbon terminal, the third peptide fragment including a sequence shown in SEQ ID NO: 3; and at least two peptide fragments selected from the following peptide fragments: a first peptide fragment at the carbon terminal, the first peptide fragment including a sequence shown in SEQ ID NO: 1; a second peptide fragment at the carbon terminal, the second peptide fragment including a sequence shown in SEQ ID NO: 2; a fourth peptide fragment at the carbon terminal, the fourth peptide fragment including a sequence shown in SEQ ID NO: 4; a fifth peptide fragment at the carbon terminal, the fifth peptide fragment including a sequence shown in SEQ ID NO: 5.

3. The kit of claim 1, wherein the first polyclonal antibody and the second polyclonal antibody are obtained by immunizing different chickens with an antigen, and the antigen is a polypeptide at a carbon terminal of human thymidine kinase 1.

4. The kit of claim 3, wherein the antigen includes a sequence shown in SEQ ID NO: 6.

5. The kit of claim 1, further comprising: a substrate luminescence catalyst coupled with a marker identifier that specifically recognizes the marker; and a luminescence substrate that emits a light signal under action of the substrate luminescence catalyst.

6. The kit of claim 5, wherein the marker is biotin, and the marker identifier is streptavidin.

7. The kit of claim 1, wherein the solid carrier is magnetic particles.

8. The kit of claim 1, further comprising a calibrator, a quality control product, an anti-reagent, a diluent and a washing solution.

9. A use of the kit of claim 1 for detecting thymidine kinase 1.

10. A use of the kit of claim 1 for assessing risk of small malignant tumors and tumors/precancerous diseases that cannot be detected by images.

11. A method for determining abnormal cell proliferation in a subject, comprising: using the kit of claim 1 to determine a content of thymidine kinase 1 in a serum of the subject; and evaluating whether the cell proliferation in the subject is abnormal based on the content of the thymidine kinase 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The above and/or additional aspects and advantages of the present disclosure will become obvious and easy to understand from the description of the embodiments in conjunction with the following drawings.

[0060] FIG. 1 shows a standard curve diagram according to an embodiment of the present disclosure.

[0061] FIG. 2 shows results of the detection of serum TK1 concentration distribution and percentage of population according to an embodiment of the present disclosure, A is kit 1, B is kit 2, and C is kit 3.

[0062] FIG. 3 shows a result diagram of correlation between dilution concentration of the lysate of tumor cell TK1 positive cell line and expression of TK1 in the kit according to the embodiment of the present disclosure.

[0063] FIG. 4 shows a schematic diagram of correlation between human STK1p concentration and tumor growth according to an embodiment of the present disclosure.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0064] The embodiments of the present disclosure are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The same or similar reference numerals indicate the same or similar elements or elements with the same or similar functions. The embodiments described below with reference to the drawings are exemplary, and are only used to explain the present disclosure, but should not be understood as limiting the present disclosure.

[0065] It should be noted that the terms “first” and “second” are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Thus, the features defined with “first” and “second” may explicitly or implicitly include one or more of these features. Further, in the description of the present disclosure, unless otherwise specified, “a plurality of” means two or more.

[0066] The present disclosure will be described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and should not be understood as limiting the present disclosure.

[0067] The solution of the present disclosure will be explained below in conjunction with examples. Those skilled in the art will understand that the following embodiments are only used to illustrate the present disclosure and should not be regarded as limiting the scope of the present disclosure. Where specific techniques or conditions are not indicated in the embodiments, the procedures shall be carried out in accordance with the techniques or conditions described in the literature or in accordance with the product specification. The reagents or instruments used without the manufacturer's indication are all conventional products that can be purchased commercially, for example, purchased from Sigma.

Embodiment 1

[0068] The kit of the embodiment of the present disclosure includes a calibrator, a quality control product, a blocking agent, a magnetic particle reagent coupled with a first antibody, and a second antibody labeled with a biotin, a streptavidin-labeled alkaline phosphatase, a luminescence substrate, an anti-reagent, a diluent, a washing solution. The preparation method is as follows.

[0069] 1. Coupling Magnetic Particle Reagent with the First Antibody

[0070] 1) Adding fully mixed tosyl magnetic particle concentrate into a reaction flask, placing the reaction flask in a magnetic field for 15 minutes, removing a supernatant after all the tosyl magnetic particles are absorbed by the magnetic field, adding 10 times the volume of magnetic particle activation buffer to the reaction flask, shaking and washing for 10 minutes, then placing the reaction flask in the magnetic field for 15 minutes, and removing the supernatant; repeating cleaning tosyl magnetic particles 2 times; finally, diluting the tosyl magnetic particle solution to 10 mg/ml, mixing well and setting aside;

[0071] 2) Adding the first antibody to the tosyl magnetic particle solution prepared in step 1) according to a mass ratio of tosyl magnetic particle solution: the first antibody=100:1 to perform a ligation reaction, adding 1/10 of the total volume of the magnetic particle catalytic buffer, and reacting at 37° C. for 18 hours in a mixed state;

[0072] 3) Adding 10% BSA of 1/20 of the total volume of the solution to the magnetic particle solution prepared in step 2), and reacting at 37° C. for 6 hours in a mixed state;

[0073] 4) Placing the reaction flask in a magnetic field for 15 minutes, cleaning the tosyl magnetic particles 3 times with magnetic particle cleaning solution after the tosyl magnetic particles are absorbed into the magnetic field, then adjusting to 1 mg/ml and storing at 4° C. to prepare the first antibody coupled with magnetic particles.

[0074] The method for preparing the magnetic particle activation buffer is dissolving 5.18˜7.36 g of boric acid in 900 ml of deionized water, adjusting pH to 8-10 with NaOH, diluting to 1 L and filtering with 0.45 μm filter membrane.

[0075] The method for preparing the magnetic particle catalytic buffer is dissolving 100-150 g of ammonium sulfate in 1 L of magnetic particle activation buffer, and filtering with 0.45 μm filter membrane after complete dissolution.

[0076] The magnetic particle cleaning solution is TBST buffer with pH 7.4.

[0077] 2. Second Antibody Labeled with the Biotin

[0078] using PBS to prepare a 2-5 mg second antibody solution, and use DMSO to prepare a 5-50 mM biotin solution, adding the biotin solution to the antibody solution and mixing well, reacting in an ice bath for 2 hours or at room temperature for 30 minutes to prepare the second antibody labeled with the biotin, and diluting the second antibody solution labeled with the biotin to 0.2 ug/ml. The second antibody is a polyclonal antibody obtained by immunizing the chicken with the sequence shown in SEQ ID NO: 6 as an antigen.

[0079] 3. Luminescence Substrate

[0080] APS-5 (purchased from Wason Biotech)

[0081] 4. Blocking Agent

[0082] Skimmed milk powder

[0083] 5. Diluent

[0084] dissolving 0.1 g-10 g blocking agent in 1 L Tris buffer, adding 0.1 ml -5 ml preservative, completely dissolving and filtering with 0.22 μm filter membrane.

[0085] 6. Anti-Reagent

[0086] diluting the biotinylated anti-thymidine kinase 1-IgY polyclonal antibody to a final concentration of 0.2 ug/ml.

[0087] 7. Washing Solution

[0088] TBST buffer with PH 7.4 (30 times concentrated solution)

[0089] 8. Streptavidin-Labeled Alkaline Phosphatase

[0090] Streptavidin-labeled alkaline phosphatase is prepared by purchasing Invitrogen streptavidin-labeled alkaline phosphatase and diluting 50,000 times with a diluent.

Embodiment 2

[0091] To evaluate the kit for detecting serum TK1 obtained in embodiment 1, the specific method is as follows:

[0092] 1. Sample preparation:

[0093] (1) Basic serum: 1 ml serum (concentration 2.2 pmol/L)+0.1 ml distilled water;

[0094] (2) Recovery sample 1: Serum 1 ml+0.1 ml antigen solution (concentration 11 pmol/L);

[0095] (3) Recovery sample 2: Serum 1 ml+0.1 ml antigen solution (concentration 80 pmol/L).

[0096] (4) Quality control sample 1: Take 1 ml standard product 1+5 ml standard product diluent (final concentration is 2.2 pmol/L).

[0097] (5) Quality control sample 2: Take 1 ml standard product 1+1 ml standard product diluent (final concentration is 10 pmol/L).

[0098] 2. Preparation before the experiment

[0099] 1) Taking a bottle of washing solution and dilute it 30 times with purified water;

[0100] 2) Mixing the magnetic particle reagent thoroughly until there is no visible precipitation.

[0101] 3. Experimental method, this kit is automatically completed by an automatic chemiluminescence analyzer, or can be completed manually.

[0102] 1) Adding 10 μl sample to be tested, 60 μl sample diluent and 30 μl magnetic particle reagent to the detection tube, and incubating at 37° C. for 10 minutes;

[0103] 2) Adding a magnetic field to settle the magnetic particles in the reaction system in the detection tube, removing the supernatant, and removing the magnetic field after washing for many times;

[0104] 3) Adding 100 μl of anti-reagent to the washed system in step 2) and incubating at 37° C. for 10 minutes;

[0105] 4) Adding a magnetic field to settle the magnetic particles in the reaction system in the detection tube, removing the supernatant, and removing the magnetic field after washing for many times;

[0106] 5) Adding 100 μl of streptavidin-labeled alkaline phosphatase reagent to the washed system in step 4) and incubating at 37° C. for 10 minutes;

[0107] 6) Adding a magnetic field to settle the magnetic particles in the reaction system in the detection tube, removing the supernatant, and removing the magnetic field after washing for many times;

[0108] 7) Adding 200 μl of chemiluminescent substrate, mixing well, reacting for 2 minutes at room temperature and avoid light, and detecting the relative luminescence intensity (RLU).

[0109] 4. Standard curve

[0110] 1) Preparation of standards

[0111] Dissolving the 31 peptide antigen freeze-dried powder (purchased) in the standard diluent (10 mM Na.sub.2HPO.sub.4, 10 mM NaH.sub.2PO.sub.4, 150 mM NaCl, 1% BSA, 5% glycerol, pH 7.4) to prepare a concentration of 1 mg/ml, using Kit 3 to test and confirm the concentration of the antigen solution, adjusting the concentration to 20 pmol/L (standard 1) after the test. Continue to use the standard diluent to dilute standard 1 3 times to obtain standard 2, and dilute standard 2 with standard diluent 3 times to obtain standard 3, Dilute standard 3 twice to obtain standard 4, and use standard diluent as standard 5. The theoretical concentrations of the obtained standards 1-5 are: 20 pmol/L, 6.6 pmol/L, 2.2 pmol/L, 1.1 pmol/L and 0 pmol/L.

[0112] 2) Drawing the standard curve

[0113] Using kit 1 to test 5 standard products to obtain the luminescence value, and drawing the standard curve according to the theoretical concentration combined with the luminescence value, as shown in FIG. 1, the R value is 0.999.

[0114] 5. Recovery rate experiment

[0115] The basic serum, the recovery sample 1 and the recovery sample 2, each sample were repeatedly tested 3 times according to the method of the present disclosure, and the results are as follows:

TABLE-US-00001 Measurement Recovery Added Recovery Recovery Acceptable value mean value concentration concentration rate range Basic 2.239 2.245 sample 2.269 2.228 Recovery 3.321 3.336 1.100 1.091 99.18% 95-105% sample 1 3.374 3.314 Recovery 10.224 10.272 8.000 8.026 100.33% 95-105% sample 2 10.31 10.281

[0116] 6. Precision experiment

[0117] Quality control sample 1 (2.2 pmol/L) and quality control sample 2 (10 pmol/L) were repeated 20 times according to the method of the present disclosure. The results are as follows:

TABLE-US-00002 quality control sample 1 quality control sample 2 (2.2 pmol/L) (10 pmol/L) measurment measurment value CV value CV 2.279 1.01% 10.304 0.44% 2.270 10.363 2.279 10.290 2.233 10.199 2.224 10.208 2.246 10.192 2.252 10.199 2.258 10.182 2.264 10.214 2.270 10.209 2.276 10.219 2.282 10.214 2.288 10.205 2.294 10.216 2.300 10.193 2.306 10.187 2.296 10.227 2.299 10.194 2.302 10.215 2.278 10.219

[0118] The experimental results show that the kit of the embodiment of the present disclosure has a higher recovery rate and precision.

Embodiment 3

[0119] In this embodiment, three kits were used to detect TK1 in the sera of 148 cases of physical examination. These three kits used 3 different antibodies and methods respectively, as follows:

[0120] Kit 1: The kit is obtained in embodiment 1, and matches with an automatic chemiluminescence immunoassay analyzer for testing;

[0121] Kit 2: The kit contains the hTK1-IgY pAb obtained from the 31 peptide of human TK1 immunization of embodiment 1 and the sandwich IgY+IgG formed by the mouse anti-hTK IgG monoclonal antibody obtained from the 31 peptide of hTK1 immunization of embodiment 1, and matches the kit test of the automatic chemiluminescence immunoassay analyzer;

[0122] Kit 3: Dot blot enhanced chemiluminescence (ECL) immunoassay kit based on hTK1-IgY pAb (purchased from SINO-SWED TONGKANG BIO-TECH (SHENZHEN) LIMITED, trade name: thymidine kinase 1 (TK1) cell cycle analysis kit).

[0123] The test found that the coincidence rate of the test results of kit 1 and kit 3 was 73%, while the coincidence rate of the test results of kit 2 and kit 3 was only 54%. The specific experimental results are shown in FIG. 2. The distribution of serum TK1 concentration values from low to high values of individuals in the human population is analyzed by using Excel to make a graph. The characteristics are the same as those published in the current routine physical examination screening population survey research. FIG. 3 shows that the distribution characteristics of kit 1 and kit 3 are approximately normal distribution, with the main peak between 0.2-0.3 pmol/L. When the concentration of STK1p gradually increased from 0.6 pmol/L to 2 pmol/L, a small continuous decrease tail peak was found. The observation that the serum TK1 concentration distribution of the physical examination population presents an approximate normal distribution is an important finding. It is pointed out that the distribution of serum TK1p concentration conforms to the natural law distribution, and its determination sensitivity can reach 0.1-0.2 pmol/L. But it is observed that the distribution of serum TK1 concentration was between 0.6 pmol/L-2.0 pmol/L−>2.0 pmol/L, there is a continuous small tail peak that rises. The continuous small tail peaks of this increase reflect that individuals in this segment may have an increased risk of developing precancerous diseases/malignant tumors. However, in kit 2, the serum TK1 value is 83% negative, and the serum TK1 value does not show a main peak between 0.2-0.3 pmol/L, which is an approximate normal distribution characteristic, indicating that the sensitivity is not high.

Embodiment 4

[0124] In this embodiment, Kit 1 and Kit 2 in embodiment 3 are used to perform TK1 test on the cell lysates of TK1 positive cell lines and negative cell lines, as follows:

[0125] The TK1 positive cell line (human colon tumor TK1.sup.+: ht29) and TK1 negative cell line (human colon tumor: 143bTK−, TK1 gene knockout cells) were cultured to the logarithmic growth phase, and the concentration is 1*10.sup.7/ml. After centrifuging 1 ml of cell suspension, e the supernatant was removed and 1 ml of cell lysate (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% NP40) was added, and treated at 4° C. for 20 min, centrifuged at 15,000 rpm for 10 min, and the supernatant was taken. The negative cell line lysate and positive cell line lysate were diluted with PBS respectively, and after the negative cell line lysate was diluted 10 times, the positive cell line lysate was diluted 10 times, 50 times, and 100 times, respectively, the cell lysate was tested using kit 1 and kit 2 respectively.

[0126] The test results of kit 1 are shown in FIG. 3. The dilution concentration of TK1 positive cell line lysate is related to the expression of TK1. However, the results of kit 2 were abnormal, and there was no correlation between the dilution concentration of TK1 positive cell line lysate and the expression of TK1 (not shown). According to the data of the TK1 value of the positive cell line given in the figure, it is calculated that a tumor cell growing in the logarithmic phase contains approximately 0.021 pg of TK1 protein. If the total protein of a tumor cell is 200 pg, it is only 0.01% (0.021 pg/200 pg total protein=0.01%). Therefore, the content of TK1 in a growing tumor cell is very low, and only a highly sensitive detection system can perform accurate detection. When the TK1 in the tumor cells is released into the blood, it is calculated according to the volume of about 5000 ml of human blood, 52 million tumor-growing cells are needed to detect the TK1 value in the serum. However, the existing imaging system requires 1 billion tumor cells to reach a small tumor with a diameter of about 1 mm before it can be detected by imaging.

[0127] The kit 1 detection system of the present disclosure has high sensitivity. In the case of micro malignant tumors that are not detectable on images, an increase in the serum TK1 value is detected, warning the patient to have precancerous diseases with abnormal proliferating cells/micro malignant tumors that are not detectable on images. According to FIG. 2 and FIG. 3, the correlation diagram 4 between the value of STK1p and the number of tumor cells (proliferation rate) and time is drawn. Below the image detection threshold line indicates precancerous basic or small malignant tumors that are not detectable or untouchable in the image, and above the image detection threshold line indicates malignant tumors that are detectable or touchable in the image. The kit of the embodiment of the present disclosure can detect precancerous diseases of small malignant tumors/abnormally proliferating cells with STK1>2 pmol/L and undetectable images. As shown in the Figure, serum TK1 expression is closely related to the number of tumor cells in the early and middle phases of tumor growth (less than 1 billion tumor cells). However, in many cases, serum TK1 expression decreases in the late phase of tumor growth. This is because although the tumor tissue increases and reaches a larger volume in the later phase of tumor growth, it can be detected by imaging. However, different degrees of tissue necrosis will occur in the center of such large tumor tissues, which leads to a decrease in the proliferation rate and a correspondingly low concentration of STK1p. Furthermore, the kits of the embodiments of the present disclosure can be used for early detection of small latent malignant tumors that are not detectable on images by detecting the elevated value of serum TK1. At the same time, the risk assessment of the precancerous disease process of abnormally proliferating cells can also be used.

[0128] In the description of this Specification, description with reference to the terms “an embodiment”, “some embodiments”, “examples”, “specific examples”, or “some examples” etc. mean that the specific features, structures, materials or characteristics described in combination with the embodiment or example are included in at least one embodiment or example of the present disclosure. In this Specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the described specific features, structures, materials or characteristics may be combined in any one or more embodiments or examples in a suitable manner.

[0129] Although the embodiments of the present disclosure have been shown and described, those of ordinary skill in the art can understand various changes, modifications, substitutions and modifications can be made to these embodiments without departing from the principle and purpose of the present disclosure. The scope of the present disclosure is defined by the claims and their equivalents.