A test kit for determining prolactin (PRL)
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SICHUAN ORIENTER BIOLOGICAL TECH
- Filing Date
- 2023-11-14
- Publication Date
- 2026-06-30
Smart Images
Figure BDA0004548113460000031 
Figure BDA0004548113460000041 
Figure BDA0004548113460000042
Abstract
Description
Technical Field
[0001] This invention relates to the field of biological detection kit technology, and specifically to a detection kit for measuring prolactin (PRL). Background Technology
[0002] Prolactin (PRL) is a protein hormone secreted by the anterior cells of the pituitary gland. Structurally, it belongs to the same protein family as growth hormone (hGH) and placental prolactin (hPL), containing 198 amino acids and a molecular weight of 22,500 Daltons. Its main physiological function is to stimulate and maintain lactation in women. The biological half-life of PRL is approximately 20–50 minutes. PRL levels gradually increase after the 8th week of gestation, at which point it functions to regulate amniotic fluid volume, amniotic fluid ion concentration, and fetal extracellular fluid volume, thus protecting the fetus. With the participation of estrogen, progesterone, glucocorticoids, and insulin, PRL promotes the maturation of mammary vesicles and milk secretion, maintaining milk production during lactation. In the presence of testosterone (T), PRL promotes the development of the prostate and seminal vesicles in men and enhances the effect of LH on Leydig cells, increasing testosterone synthesis. Therefore, the measurement and dynamic monitoring of prolactin have auxiliary diagnostic value for female fertility, postpartum lactation capacity, sex hormone levels, and male sexual dysfunction.
[0003] The main detection methods for prolactin (PRL) currently include chemiluminescent immunoassay and enzyme-linked immunosorbent assay (ELISA). PRL exists in various forms in the body: biologically and immunologically active 23kD prolactin monomers; biologically inactive 50-60kD large prolactin; and non-biologically active interfering substances that accumulate in the body due to the formation of large molecular weight immune complexes from PRL binding with IgG antibodies. These interfering substances can interfere with PRL detection, leading to falsely elevated results.
[0004] Therefore, this patent application is filed. Summary of the Invention
[0005] The technical problem to be solved by this invention is that the detection of PRL is currently affected by interference from the presence of interfering substances. This invention provides a test kit for measuring PRL prolactin. Through the combined action of oxidants and surfactants, non-specific interference is prevented, the detection precision is improved, the specificity is greatly improved, and the clinical concordance rate is significantly increased.
[0006] The purpose of this invention is to provide a detection kit for determining prolactin (PRL), comprising immunomagnetic beads, an oxidant, and a surfactant, wherein the oxidant is a peroxidant and the surfactant is an anionic surfactant.
[0007] In this invention, an oxidant is used to break down interfering substances, and a surfactant is used to encapsulate the broken-down substances to prevent non-specific interference, thereby improving detection precision and avoiding false positives in the results.
[0008] In some optional embodiments, an analysis buffer solution, which is a Tris-HCl buffer solution with a pH of 8.5, is also included.
[0009] In some optional embodiments, the oxidant is a hydrogen peroxide solution with a mass fraction of 10% to 20%, and the surfactant is an aqueous solution of sodium dodecyl sulfate with a mass fraction of 0.0025% to 0.01%.
[0010] In some optional embodiments, the immunomagnetic beads are a mixture of magnetic beads coated with mouse anti-human PRL monoclonal antibody 1 and a buffer solution. This allows for specific binding to the sample. In some optional embodiments, the buffer solution is a buffer solution containing 0.1% bovine serum albumin by mass.
[0011] In some optional embodiments, the magnetic beads coated with mouse anti-human PRL monoclonal antibody are at a concentration of 0.1 mg / mL to 0.3 mg / mL in the buffer solution to ensure optimal reagent performance. In some optional embodiments, an analytical buffer, which is a Tris-HCl buffer solution, is also included.
[0012] In some optional embodiments, the pH of the Tris-HCl buffer solution is 8.5.
[0013] In some optional embodiments, an enzyme working solution is further included, which is a mixture of an enzyme labeled with PRL monoclonal antibody 2 and an enzyme working buffer. In some optional embodiments, the enzyme working buffer is a casein buffer solution containing 0.5% by mass.
[0014] In some optional embodiments, the enzyme labeled with PRL monoclonal antibody 2 is at a mass concentration of 0.01 ug / mL to 0.3 ug / mL in the buffer to ensure optimal reagent performance.
[0015] Compared with the prior art, the present invention has the following advantages and beneficial effects:
[0016] During the detection of PRL, PRL binds to its IgG antibody to form a large molecular weight immune complex, which accumulates in the body and forms interfering substances, interfering with the detection of PRL and leading to falsely elevated results. The detection kit for PRL prolactin provided in this embodiment of the invention uses an oxidant to cleave the interfering substances and a surfactant to encapsulate the cleaved substances, thereby improving the detection precision, specificity, and clinical concordance rate. Detailed Implementation
[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the embodiments. The illustrative embodiments and descriptions of this invention are only used to explain this invention and are not intended to limit this invention.
[0018] Example 1:
[0019] Preparation of a kit containing an oxidant but without a surfactant:
[0020] A. Take 40 mL of magnetic beads coated with mouse anti-human PRL monoclonal antibody 1. Dilute these immunomagnetic beads to 0.2 mg / mL with a buffer solution containing 0.1% bovine serum albumin and divide them into 4 equal portions.
[0021] B. Take 40 mL of Tris-HCl analytical buffer solution with pH = 8.5 and divide it into 4 equal parts. Then, one part is not added with oxidant (as a control), and the other three parts are added with hydrogen peroxide solution with a mass fraction of 10%, 15%, and 20% respectively as oxidants. Then, prepare the kits with enzyme working solution and label them as kits C1, A1, A2, and A3 respectively.
[0022] The enzyme working solution is a mixture of enzyme labeled with PRL monoclonal antibody 2 and enzyme working buffer. The enzyme working buffer is a casein buffer solution containing 0.5% by mass. The concentration of enzyme labeled with PRL monoclonal antibody 2 in the buffer is 0.1 μg / mL.
[0023] Example 2:
[0024] Preparation of kits containing oxidants and surfactants:
[0025] Take 40 mL of magnetic beads coated with mouse anti-human PRL monoclonal antibody 1 and divide them into 4 equal portions;
[0026] Take 40 mL of Tris-HCl analysis buffer, then add 15% hydrogen peroxide solution as an oxidant according to the ratio, and then divide it into 4 equal parts; one part is without surfactant SDS (sodium dodecyl sulfate aqueous solution, as a control), and the other three parts are added with 0.0025%, 0.005%, and 0.01% SDS according to the ratio, respectively. Then, prepare the kit with enzyme working solution, and label them as kit C2, B1, B2, and B3 respectively.
[0027] The enzyme working solution is a mixture of the enzyme labeled with PRL monoclonal antibody 2 and the enzyme working buffer, which is a casein buffer solution containing 0.5% by mass. The concentration of the enzyme labeled with PRL monoclonal antibody 2 in the buffer is 0.1 μg / mL.
[0028] The reagent kits A1, A2, A3, B1, B2, B3, and C1 and C2 (as controls) were tested.
[0029] Experiment 1:
[0030] A kit specificity comparison experiment was conducted between groups A1, A2, and A3 and group C1.
[0031] Experimental materials: Immunomagnetic beads coated with mouse anti-human PRL monoclonal antibody 1, Tris-HCl buffer solution at pH 8.5, hydrogen peroxide stock solution (30%), enzyme working solution labeled with PRL monoclonal antibody 2, substrate solution for the Wovent Bio fully automated immunoassay system, Wovent Bio cleaning solution, Wovent LA2000 fully automated chemiluminescence immunoassay analyzer, PRL calibrators (S0-S5), 40 fresh clinical PRL samples, and a third-party clinical test report (Roche).
[0032] The PRL calibrators S0-S5 were measured using kits from groups A1, A2, A3, and C1. The differences in the measurement curves under different oxidant concentrations were observed, and samples S1-S40 were measured and compared with Roche measurements.
[0033] The experimental results are shown in Tables 1-3.
[0034] Table 1. Calibration data of reagent kits in groups A1, A2, A3, and C1.
[0035]
[0036]
[0037] Table 2. Measurement of Roche samples after calibration of kits in groups A1, A2, A3, and C1.
[0038]
[0039]
[0040] Table 3. Correlation between the calibrated kits of groups A1, A2, A3, and C1 and Roche measurements.
[0041] Grouping Linear equations Correlation coefficient r Group C1 <![CDATA[y=1.5716x-7.6657 R 2 =0.7790]]> 0.78 Group A1 <![CDATA[y=1.2059x-2.8093 R 2 =0.8317]]> 0.83 Group A2 <![CDATA[y=1.0586x+1.7065 R 2 =0.9125]]> 0.91 Group A3 <![CDATA[y=1.0259x-0.4056 R 2 =0.7717]]> 0.77
[0042] Experimental Conclusions: Based on the test data in Tables 1-3, compared to group C1, the kits in groups A1, A2, and A3 showed improved signals and better sensitivity after the addition of the oxidant. When testing samples, the kit in group A2 with 15% oxidant solution showed better correlation with Roche's comparative measurements, but some abnormal measurements still existed, and there were samples that did not match the clinical diagnosis.
[0043] Experiment 2:
[0044] A kit specificity comparison experiment was conducted between groups B1, B2, and B3 and group C2.
[0045] Using the kits from groups B1, B2, B3, and C2 obtained in Example 2, PRL calibrators S0-S5 were measured. The differences in the measurement curves when oxidants and surfactants were added simultaneously were observed, and samples S1-S40 were measured and compared with Roche values.
[0046] The experimental results are shown in Tables 4-6.
[0047] Table 4. Calibration data of reagent kits for groups B1, B2, B3, and C2.
[0048]
[0049] Table 5. Measurement of Roche samples after calibration of kits for groups B1, B2, B3, and C2.
[0050]
[0051]
[0052] Table 6 shows the correlation between the calibrated kits and Roche measurements for groups B1, B2, B3, and C2.
[0053] Grouping Linear equations Correlation coefficient r Group C2 <![CDATA[y=1.5716x-7.6657 R 2 =0.7790]]> 0.78 Group B1 <![CDATA[y=1.0485x-1.2454 R 2 =0.9397]]> 0.94 Group B2 <![CDATA[y=1.0188x+1.8451 R 2 =0.9895]]> 0.99 Group B3 <![CDATA[y=1.0606x-1.7477 R 2 =0.9592]]> 0.959
[0054] Experimental Conclusions: As shown in Tables 4-6, compared with group C2, the correlation between the kits in groups B1, B2, and B3 and Roche measurements was significantly improved after calibration when surfactants were added. The kit in group B2, which added 0.005% SDS aqueous solution, showed better anti-interference effect on abnormal samples compared to groups B1 and B3, and also performed better in comparison with Roche measurements. In conclusion, the method used in group B2 was the most effective in resolving interference from interfering substances.
[0055] In summary, the two experiments demonstrate that adding both an oxidant and a surfactant to the analytical buffer of the PRL assay kit improves sensitivity. The oxidant cleaves interfering substances, and the surfactant encapsulates these cleaved substances, resulting in enhanced anti-interference capabilities, significantly improved specificity, and a higher clinical concordance rate when testing clinical samples. This approach offers better anti-interference performance compared to adding only an oxidant, which merely cleaves interfering substances.
[0056] Anything not mentioned or described in detail in the embodiments of this invention is something that can be obtained using existing technology.
[0057] The above specific embodiments further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above are merely specific embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. The use of oxidizing agents and surfactants in the preparation of a detection kit for determining prolactin (PRL), characterized in that, The detection kit includes immunomagnetic beads, an oxidant, and a surfactant. The oxidant is a hydrogen peroxide solution with a mass fraction of 10% to 20%, and the surfactant is an aqueous solution of sodium dodecyl sulfate with a mass fraction of 0.0025% to 0.01%. The oxidant cleaves the interfering substance, and the surfactant encapsulates the cleaved substance to prevent non-specific interference. The interfering substance is an immune complex formed by the binding of PRL and its IgG antibody.
2. The application according to claim 1, characterized in that, The test kit also includes an analysis buffer solution, which is a Tris-HCl buffer solution with a pH of 8.
5.
3. The application according to claim 1, characterized in that, The immunomagnetic beads are a mixture of magnetic beads coated with mouse anti-human PRL monoclonal antibody 1 and buffer solution.
4. The application according to claim 3, characterized in that, The immunomagnetic bead buffer solution uses a buffer solution containing 0.1% bovine serum albumin by mass.
5. The application according to claim 3, characterized in that, The magnetic beads coated with mouse anti-human PRL monoclonal antibody have a mass concentration of 0.1 mg / mL to 0.3 mg / mL in the buffer solution.
6. The application according to claim 1, characterized in that, The test kit also includes an enzyme working solution, which is a mixture of an enzyme labeled with PRL monoclonal antibody 2 and a buffer solution.
7. The application according to claim 6, characterized in that, The enzyme working buffer solution uses a casein buffer solution containing 0.5% by mass.
8. The application according to claim 6 or 7, characterized in that, The enzyme labeled with PRL monoclonal antibody 2 has a mass concentration of 0.01 ug / mL to 0.3 ug / mL in the buffer solution.