CN-121978356-A - Parathyroid hormone detection probe and preparation method and application thereof

CN121978356ACN 121978356 ACN121978356 ACN 121978356ACN-121978356-A

Abstract

The invention belongs to the technical field of biomedical detection and immunoassay, and particularly discloses a parathyroid hormone detection probe, a preparation method and application thereof. The parathyroid hormone detection probe provided by the invention comprises a capture probe and a signal probe, wherein the capture probe is a magnetic particle with a PTH specific antibody 1 fixed on the surface, and the signal probe is a PTH specific antibody 2 with a photosensitizer marked on the surface. The immunodetection probe provided by the invention can solve the problems of unstable enzyme activity, high detection cost, complex operation and the like in the existing enzyme-labeled immunodetection technology. Unlike available chemiluminescent or enzyme catalyzed signal amplifying mechanism, the present invention combines photosensitive oxidation reaction with double antibody sandwich immunological recognition, and utilizes the characteristic of photosensitive agent to produce active oxygen effectively under irradiation of visible light to catalyze color change of chromogenic substrate, so as to realize high sensitivity and high specificity detection of PTH.

Inventors

YI JIANING
LAN MINHUAN
ZENG JIE
LEI TINGTING
LIU LUYAO
YU JIE
YANG XIAOCHEN
LIU SIHAN
Zhao Shaojing
XU YATING

Assignees

湖南省人民医院(湖南师范大学附属第一医院)

Dates

Publication Date: 20260505
Application Date: 20260207

Claims (10)

1. A parathyroid hormone detection probe is characterized by comprising a capture probe and a signal probe, wherein the capture probe is a magnetic particle with a PTH specific antibody 1 immobilized on the surface, and the signal probe is a PTH specific antibody 2 with a photosensitizer marked on the surface.
2. The parathyroid hormone detection probe of claim 1 wherein the PTH-specific antibody 1 is a C-terminal recognition antibody of PTH and the PTH-specific antibody 2 is an N-terminal recognition antibody of PTH.
3. The parathyroid hormone detection probe of claim 1 wherein the magnetic particles are surface modified with functional groups, the functional groups comprise at least one of carboxyl, amino or streptavidin, and the magnetic particles are selected from at least one of magnetic ferroferric oxide microspheres, nickel magnetic microspheres or cobalt magnetic microspheres; The photosensitizer is at least one selected from phthalocyanine compounds, porphyrin compounds, phenothiazine dyes, xanthene dyes or natural photosensitizers.
4. A method for preparing a parathyroid hormone detection probe as claimed in any one of claims 1 to 3, comprising the steps of: s1, mixing magnetic particles with a PTH specific antibody 1 in a buffer solution in the presence of an activating agent for reaction, collecting a product through magnetic separation, and closing to obtain a capture probe; S2, mixing the PTH specific antibody 2 with a photosensitizer in a buffer solution in the presence of an activating agent for reaction, and purifying to obtain a signal probe; in the step S2, the molar ratio of the PTH specific antibody 2 to the photosensitizer is1 (4-7).
5. The preparation method of claim 4, wherein in the step S1, the activating agent comprises at least one of carbodiimide cross-linking agent, glutaraldehyde and genipin, the buffer solution comprises at least one of phosphate buffer solution, tris-HCl buffer solution, HEPES buffer solution, MES buffer solution and borate buffer solution, the pH value of the buffer solution is 5.0-9.0, the temperature of the mixing reaction is 4-37 ℃ and the time is 0.5-4 h.
6. The preparation method according to claim 4, wherein in the step S2, the activating agent comprises at least one of a carbodiimide/succinimide system, glutaraldehyde and sulfo-SMCC, the buffer solution comprises at least one of a carbonate buffer solution, a phosphate buffer solution and a borate buffer solution, the pH value of the buffer solution is 7.0-9.5, the mixing reaction is carried out under a dark condition, and the reaction temperature is 4-25 ℃ for 1-6 h.
7. The use of the parathyroid hormone detection probe of any one of claims 1-3 for detecting parathyroid hormone in a sample, wherein the method for detecting parathyroid hormone in the sample comprises the following steps: s10, performing immune reaction, namely mixing and incubating a capture probe with a sample solution to combine PTH with a specific antibody 1, and adding a signal probe to form a sandwich immune complex; s20, separating and washing, namely performing magnetic separation, washing and removing unbound substances; S30, photosensitive color development, namely adding a color development substrate into the washed compound, and carrying out photosensitive oxidation reaction under visible light irradiation; S40, detecting and analyzing, namely measuring the absorbance value of the reaction system, and calculating the concentration of PTH in the sample to be detected according to a standard curve.
8. The use according to claim 7, wherein the chromogenic substrate is selected from at least one of 3,3', 5' -Tetramethylbenzidine (TMB), 2 '-biazo-bis (3-ethylbenzothiazoline-6-sulfonic Acid) (ABTS), o-phenylenediamine (OPD), 3-amino-9-ethylcarbazole (AEC) or 3,3' -Diaminobenzidine (DAB).
9. The use according to claim 7, wherein in step S10, the incubation temperature is 28-32 ℃ and the incubation time is 10-60 min, the buffer is phosphate buffer or Tris-HCl buffer, and the pH is 7.2-7.6; In the step S30, the irradiation wavelength of the visible light is 400-700 nm, the illumination intensity is 50-90 mW/cm 2 , the irradiation time is 20-25 min, and the temperature of the photo-sensitive oxidation reaction is 15-40 ℃.
10. A parathyroid hormone detection kit comprising the parathyroid hormone detection probe of any one of claims 1-3.

Description

Parathyroid hormone detection probe and preparation method and application thereof Technical Field The invention relates to the technical field of biomedical detection and immunoassay, in particular to a parathyroid hormone detection probe and a preparation method and application thereof. Background Parathyroid hormone (Parathyroid Hormone, PTH) is a single-chain polypeptide hormone synthesized and secreted by parathyroid main cells, which plays a central role in the regulation of calcium and phosphorus metabolic homeostasis in humans. Abnormality in PTH levels is closely associated with various diseases such as primary hyperparathyroidism, secondary hyperparathyroidism (common in chronic renal patients), hypoparathyroidism, and osteoporosis. Therefore, the PTH concentration in blood is rapidly and accurately detected, and the method has important clinical significance for diagnosis, differential diagnosis, treatment effect evaluation and prognosis judgment of the diseases. Currently, the main methods for detecting PTH in clinical laboratories are mainly based on the principle of immunoassay, including enzyme-linked immunosorbent assay, chemiluminescent immunoassay, electrochemiluminescent immunoassay and the like. These methods typically rely on enzyme-labeled (e.g., horseradish peroxidase, HRP) or chemiluminescent-labeled secondary antibodies to effect detection by catalyzing the production of color, light, or electrical signals from the substrate. Although these techniques are well established, there are some inherent limitations in that, firstly, the activity of the enzyme labels is susceptible to environmental temperature, pH and storage conditions, stability is relatively poor, possibly resulting in batch-to-batch differences, and secondly, the chemiluminescent method, while having high sensitivity, generally requires expensive instruments and special reagents, has high detection cost, and in addition, some methods have complicated operation steps, have long detection time, and are difficult to meet the requirements of rapid detection in clinical emergency or surgery. In recent years, researchers have begun to explore non-enzyme dependent signal amplification strategies in an effort to find more stable, convenient detection schemes. Wherein, the nanometer material which simulates enzyme catalysis, in particular has peroxidase activity, has shown good application prospect. However, there is still room for improvement in the catalytic efficiency, stability and biocompatibility of these mimic enzymes. The photosensitizing oxidation reaction is an emerging signal generation mechanism, which utilizes a photosensitizer to generate active oxygen under specific wavelength illumination, and further oxidizes a colorless chromogenic substrate (such as TMB) to generate color change. The process does not depend on biological enzyme, and has the potential advantages of mild reaction conditions, easiness in control, stable signals and the like. If the photosensitive oxidation reaction and the high-specificity immune recognition reaction can be combined, a novel PTH detection probe is constructed, partial defects of the traditional enzyme labeling technology are hopeful to be overcome, and a novel PTH detection method with better performance is provided for clinic. Therefore, development of a novel PTH immunodetection method based on a photo-oxidative reaction, which has high sensitivity, high specificity, good stability and simple operation, is a research-worthy direction in the field. Disclosure of Invention The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the invention provides a parathyroid hormone (PTH) immunodetection probe based on a photosensitive oxidation reaction, a preparation method and detection application thereof. The immunodetection probe provided by the invention can solve the problems of unstable enzyme activity, high detection cost, complex operation and the like in the existing enzyme-labeled immunodetection technology. Unlike available chemiluminescent or enzyme catalyzed signal amplifying mechanism, the present invention combines photosensitive oxidation reaction with double antibody sandwich immunological recognition, and utilizes the characteristic of photosensitive agent to produce active oxygen effectively under irradiation of visible light to catalyze color change of chromogenic substrate, so as to realize high sensitivity and high specificity detection of PTH. The invention also provides a preparation method of the PTH detection probe. The invention also provides application of the PTH detection probe. In a first aspect of the present invention, there is provided a parathyroid hormone detection probe comprising a capture probe which is a magnetic particle having a PTH-specific antibody 1 immobilized on the surface and a signal probe which is a PTH-specific antibody 2 having a photosensitizer labeled on the surface. According