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Method for rapidly identifying human parathyroid glands

A parathyroid, rapid technology, applied in the field of immunoassay, can solve the problems of low contrast between color bands and background, limited detection sensitivity, lack of high specificity detection, etc., to reduce the incidence of hypoparathyroidism, Improvement of fluorescent signal-to-background ratio and reduction of medical expenses

Active Publication Date: 2017-11-28
无锡市江原实业技贸有限公司
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

[0006] (1) The colloidal gold labeling process is an electrostatic adsorption process, which is a physical adsorption, so the stability in the liquid phase is poor, often causing the labeled protein molecules to fall off again
[0007] (2) The test result is judged by displaying a single purple-red strip, and the color is single, so it is difficult to realize multiple inspection and joint inspection
[0008] (3) Only when the gold particles gather to a certain amount, the human naked eye can observe the purple-red band, and the color band has little contrast with the background, thus limiting the detection sensitivity
[0009] (4) The matrix effect of different materials is obvious, and the background interference is very large
[0010] (5) The detection sensitivity is low
[0011] (6) Quantitative detection cannot be realized
[0012] At present, there is also a detection method that uses quantum dots to label rapid immunochromatographic test strips, but this method cannot achieve quantitative detection of the test substance.
[0013] In addition, in the immunoassay methods for detecting PTH levels, there is still a lack of combinations that can be detected with high sensitivity and specificity in double-antibody sandwich assays. Finding more suitable immunogenic PTH epitope peptides and preparing specific Sexual PTH antigen and antibody are also the key points to be solved

Method used

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  • Method for rapidly identifying human parathyroid glands
  • Method for rapidly identifying human parathyroid glands
  • Method for rapidly identifying human parathyroid glands

Examples

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Embodiment 1

[0055] Embodiment 1: Human PTH epitope peptide

[0056] The human PTH described herein is known in the art, and the complete PTH is composed of a single polypeptide chain containing 84 amino acids, with a molecular weight of about 9500 Daltons. Its amino acid sequence is known in the art and can be found in professional databases such as NCBI. The specific sequence is as follows:

[0057] Human PTH(1-84):

[0058] SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ (SEQ ID NO: 1)

[0059] The inventors of the present invention finally screened and obtained two antigenic epitope peptides with good antigenicity after a lot of theoretical research and experimental exploration.

[0060] PTH epitope peptide (1) is a peptide segment containing 11 amino acids at the 19th-29th position of the N-terminal of the human PTH polypeptide, thereby constituting an antigenic epitope peptide (1) containing 11 amino acids: ERVEWLRKKLQ (SEQ ID NO :2)

[0061] ...

Embodiment 2

[0064] Example 2: Preparation of PTH Antibody

[0065] The PTH epitope peptides (1) and (2) obtained in Example 1 were linked to carrier proteins to prepare antigens (1) and (2) for immunization, and the animals were immunized with the obtained antigens (1) and (2), respectively, Antigen (1) is thus used to prepare specific monoclonal antibodies and polyclonal antibodies, and antigen (2) is used to prepare specific monoclonal antibodies and polyclonal antibodies.

[0066] 1. Antigen preparation: PTH peptides were linked with carrier protein BSA to prepare PTH antigen. Take 0.25 mL each of PBS with pH 6.00.01 mol / L and dimethyl sulfoxide (DMSO) to dissolve 5 mg of BSA, and take 1.2 mg of MBS to dissolve in 100 mL of DMSO. Add MBS to the BSA solution and stir at room temperature for 30 minutes, then centrifuge at 5000 r / min at 4°C to collect the supernatant. Dissolve PTH (19-29) and PTH (51-71) in 0.01mol / L pH7.2 PBS and DMSO respectively in about 500 μL. Mix BSA-MBS with eac...

Embodiment 3

[0080] Example 3: Specific identification of human PTH antibodies (1) and (2)

[0081] Detection was performed by ELISA. The ELISA plates were coated with human PTH, Actin protein, and neuron-specific enolase NSE as detection antigens, and the specific reactions of the prepared PTH monoclonal antibodies (1) and (2) with different proteins were detected by ELISA, respectively. Normal BALB / c mouse serum was used as negative control, and PBS solution was used as blank control.

[0082] Result: PTH monoclonal antibody (1) and (2) only react positively (P / N>2.1) with PTH respectively, and are negative with Actin protein, neuron-specific enolase NSE reaction, illustrate and utilize the present invention The monoclonal antibodies (1) and (2) prepared from the PTH epitope peptide have specificity respectively.

[0083] Identification of polyclonal antibodies was performed using the same method as described above for identifying the specificity of monoclonal antibodies.

[0084] The...

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Abstract

The invention discloses a method for rapidly identifying human parathyroid glands. The method is realized by using parathyroid hormone (PTH) fluorescence immunochromatography test paper to determine the content of PTH in liquid to be detected; the test paper is used for detecting human PTH by using a double-antibody sandwich method and a fluorescence immunochromatography; the antibodies are prepared by using specific antigenic epitope peptide. The method can be used for accurately detecting the human PTH in a matter to be detected, and is simple and convenient to operate, wide in detection range, high in specificity and good in sensitivity.

Description

technical field [0001] The invention belongs to the field of immune detection, and in particular relates to a method for quickly identifying human parathyroid glands. Background technique [0002] Thyroid cancer is the most common malignant tumor of the head and neck, and it is also one of the malignant tumors with the fastest growing incidence rate in recent years. Thyroid cancer has become the fifth most common malignant tumor in women. The treatment of thyroid cancer is mainly based on surgical treatment. After total thyroidectomy, the function of parathyroid glands should be determined as soon as possible, and targeted measures should be taken to prevent and treat hypoparathyroidism. If the parathyroid gland is accidentally removed during the operation, the blood calcium concentration of the patient will be significantly reduced, causing tetany, and even life-threatening. It is reported in the literature that the incidence of transient hypocalcemia after thyroidectomy i...

Claims

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Application Information

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IPC IPC(8): G01N33/558G01N33/533G01N33/577
CPCG01N33/533G01N33/558G01N33/577
Inventor 邹贤朱利国程晓青范俊朱国华戴军包建东高芸王国瑞孙志强李秀龙周彬曹亚琴杨克勤
Owner 无锡市江原实业技贸有限公司
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