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Fluorescence immunochromatography test paper for quantitatively detecting human parathyroid hormone and preparation method of fluorescence immunochromatography test paper

A fluorescence immunochromatography, parathyroid hormone technology, applied in the field of immunodetection, can solve the problems of low contrast between color bands and background, lack of high specificity detection, inability to achieve quantitative detection, etc., to reduce parathyroid function. Reduced incidence, improved detection sensitivity and confidence in results, reduced effects of external conditions and background

Active Publication Date: 2017-10-17
JIANGSU INST OF NUCLEAR MEDICINE +1
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Problems solved by technology

[0004] (1) The colloidal gold labeling process is an electrostatic adsorption process, which is a kind of physical adsorption, so the stability in the liquid phase is poor, often causing the labeled protein molecules to fall off again
[0005] (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
[0006] (3) Only when the gold particles gather to a certain amount, the naked eye can observe the purple-red band, and the color band has little contrast with the background, thus limiting the detection sensitivity
[0007] (4) The matrix effect of different materials is obvious, and the background interference is very large
[0008] (5) The detection sensitivity is low
[0009] (6) Quantitative detection cannot be realized
[0010] 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.
[0011] 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 high specificity in double-antibody sandwich assays. Finding more suitable immunogenic PTH epitope peptides, preparing specific Sexual PTH antigen and antibody are also the key points to be solved

Method used

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  • Fluorescence immunochromatography test paper for quantitatively detecting human parathyroid hormone and preparation method of fluorescence immunochromatography test paper
  • Fluorescence immunochromatography test paper for quantitatively detecting human parathyroid hormone and preparation method of fluorescence immunochromatography test paper
  • Fluorescence immunochromatography test paper for quantitatively detecting human parathyroid hormone and preparation method of fluorescence immunochromatography test paper

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Embodiment 1

[0041] Embodiment 1: Human PTH epitope peptide

[0042] 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:

[0043] Human PTH(1-84):

[0044] SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ (SEQ ID NO: 1)

[0045] 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.

[0046] 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)

[0047] ...

Embodiment 2

[0050] Example 2: Preparation of PTH Antibody

[0051] 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.

[0052] 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

[0066] Example 3: Specific identification of human PTH antibodies (1) and (2)

[0067] 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.

[0068] 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.

[0069] Identification of polyclonal antibodies was performed using the same method as described above for identifying the specificity of monoclonal antibodies.

[0070] The...

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Abstract

The invention discloses fluorescence immunochromatography test paper for achieving intraoperative judgment of parathyroid tissues through quantitatively detecting a human parathyroid hormone (PTH) and a preparation method of the fluorescence immunochromatography test paper. The test paper capable of detecting the human PTH through a double-antibody sandwich method and a fluorescence immunochromatography; the antibody is prepared by using a specific antigen peptide. The human PTH in a to-be-detected matter can be quickly and accurately detected, so that the fluorescence immunochromatography test paper is simple, convenient and fast 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 fluorescent immunochromatographic test paper for quantitative detection of human parathyroid hormone and a preparation method thereof. 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...

Claims

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Application Information

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IPC IPC(8): G01N33/78G01N33/577G01N33/574G01N33/558G01N33/543
CPCG01N33/533G01N33/54313G01N33/558G01N33/574G01N33/577G01N33/78
Inventor 范俊黄飚戴军邹贤朱利国周彬张珏张艺谢敏浩施龙顺
Owner JIANGSU INST OF NUCLEAR MEDICINE
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