CN111175521A - Preparation method of fluorescence immunochromatographic test paper for bedside rapid identification of medullary thyroid carcinoma and lymph node metastasis - Google Patents
Preparation method of fluorescence immunochromatographic test paper for bedside rapid identification of medullary thyroid carcinoma and lymph node metastasis Download PDFInfo
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Abstract
本发明公开了一种用于甲状腺髓样癌及淋巴结转移床旁快速鉴别的荧光免疫层析试纸的制备方法，其包括，底板、层析膜、吸水纸、样品垫、结合垫；所述结合垫上涂有偶联了降钙素抗体1或抗体5的荧光微球；所述层析膜上设有检测线和质控线，所述质控线上涂有抗抗体1或5同源IgG的抗体；所述检测线上涂有所述降钙素抗体5或抗体1。本发明优选降钙素作为甲状腺髓样癌及淋巴结转移床旁检测指标，从众多抗体中优选出特异性良好的抗体，并进行组合，荧光微球偶联抗体与检测线划膜抗体相互配合，检测灵敏度和特异性得了显著提高，偏差小，实现了快速精准检测。
The invention discloses a preparation method of a fluorescent immunochromatographic test paper for bedside rapid identification of medullary thyroid carcinoma and lymph node metastasis, which comprises a bottom plate, a chromatography film, an absorbent paper, a sample pad and a binding pad; The pad is coated with fluorescent microspheres coupled with calcitonin antibody 1 or antibody 5; the chromatography membrane is provided with a detection line and a quality control line, and the quality control line is coated with anti-antibody 1 or 5 homologous IgG the antibody; the detection line is coated with the calcitonin antibody 5 or antibody 1. In the present invention, calcitonin is preferred as a bedside detection index for medullary thyroid cancer and lymph node metastasis, and an antibody with good specificity is selected from many antibodies and combined, and the fluorescent microsphere-conjugated antibody and the detection line scribing antibody cooperate with each other. The detection sensitivity and specificity have been significantly improved, and the deviation is small, enabling fast and accurate detection.
Description
Technical Field
The invention relates to a fluorescence chromatography test paper and a preparation method and a product thereof, in particular to a preparation method of fluorescence immunochromatographic test paper for rapid identification of medullary thyroid carcinoma and lymph node metastasis bedside; belongs to the technical field of biological medicine.
Background
Medullary Thyroid Carcinoma (MTC) is a malignant tumor that originates in C-cells of the parafollicular thyroid cell. The C cell is a neuroendocrine cell, synthesizes and secretes hormones such as calcitonin and calcitonin gene-related peptide, and belongs to cells which take amine precursors and carry out decarboxylation reaction, so the C cell is one of neuroendocrine system tumors. MTC is less common clinically, accounting for about 5% -10% of thyroid cancer in China. The accurate preoperative diagnosis can help to select proper operation and treatment mode, avoid secondary operation and is very important for clinic and patients. At present, Fine Needle Aspiration Cytology (FNAC) is the first choice method for primarily diagnosing medullary thyroid cancer, but histologic and cytological morphological characteristics of medullary thyroid cancer are very complex, and the method is a difficulty in cytological diagnosis of thyroid cancer. At present, the sensitivity of FNAC initial diagnosis of medullary thyroid carcinoma is far from meeting the clinical requirement.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
As one aspect of the invention, the invention provides a preparation method of a fluorescence immunochromatographic test paper for rapid identification of medullary thyroid carcinoma and lymph node metastasis bedside.
In order to solve the technical problems, the invention provides the following technical scheme: a preparation method of fluorescence immunochromatographic test paper for rapid identification of medullary thyroid carcinoma and lymph node metastasis bedside comprises a bottom plate, a chromatographic membrane, absorbent paper, a sample pad and a combination pad; the combination pad is coated with a fluorescent microsphere coupled with calcitonin antibody 1 or antibody 5;
the chromatographic membrane is provided with a detection line and a quality control line, and the quality control line is coated with an antibody of an anti-antibody 1 or 5 homologous IgG; the detection line is coated with the calcitonin antibody 5 or the antibody 1.
The preferable scheme of the preparation method of the fluorescence immunochromatographic test paper for rapid identification of medullary thyroid carcinoma and lymph node metastasis bedside provided by the invention is as follows: the adding amount of the antibody on the bonding pad is 200-400 mug antibody/mg fluorescent microsphere.
AsThe invention relates to a preferable scheme of a preparation method of fluorescence immunochromatographic test paper for rapid identification of medullary thyroid carcinoma and lymph node metastasis bedside, which comprises the following steps: the content of the fluorescent microspheres on the bonding pad is 1-10 mu g/cm2。
The preferable scheme of the preparation method of the fluorescence immunochromatographic test paper for rapid identification of medullary thyroid carcinoma and lymph node metastasis bedside provided by the invention is as follows: the content of the antibody on the quality control line is 0.5-2 mug/cm.
The preferable scheme of the preparation method of the fluorescence immunochromatographic test paper for rapid identification of medullary thyroid carcinoma and lymph node metastasis bedside provided by the invention is as follows: the content of the antibody on the detection line is 0.5-2 mug/cm.
The preferable scheme of the preparation method of the fluorescence immunochromatographic test paper for rapid identification of medullary thyroid carcinoma and lymph node metastasis bedside provided by the invention is as follows: the calcitonin has an amino acid sequence of CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP.
The preferable scheme of the preparation method of the fluorescence immunochromatographic test paper for rapid identification of medullary thyroid carcinoma and lymph node metastasis bedside provided by the invention is as follows: the fluorescent material of the fluorescent microsphere is europium.
The preferable scheme of the preparation method of the fluorescence immunochromatographic test paper for rapid identification of medullary thyroid carcinoma and lymph node metastasis bedside provided by the invention is as follows: the chromatographic membrane is a nitrocellulose membrane.
The preferable scheme of the preparation method of the fluorescence immunochromatographic test paper for rapid identification of medullary thyroid carcinoma and lymph node metastasis bedside provided by the invention is as follows: the medullary thyroid carcinoma and lymph node metastasis bedside rapid identification is carried out, and a sample to be detected is a puncture tissue sample.
The invention has the beneficial effects that: the calcitonin is preferably selected as a detection index beside a thyroid medullary carcinoma and lymph node metastasis bed, an antibody with good specificity is preferably selected from a plurality of antibodies and combined, the fluorescent microsphere coupled antibody is matched with a detection line membrane scratching antibody, the detection sensitivity and specificity are obviously improved, the deviation is small, and the rapid and accurate detection is realized.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 is a schematic cross-sectional view of a fluorescence immunochromatographic test strip along the length direction;
FIG. 2 is a schematic plan view of the fluorescence immunochromatographic test strip.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example 1:
the CT (calcitonin) is known in the art, and the complete CT (calcitonin) is a single polypeptide chain containing 32 amino acids, and the amino acid sequence thereof is known in the art and can be found in professional databases such as NCBI, and the specific sequences are as follows:
human CT (1-32):
CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP(SEQ ID NO：1)
the CT antibody (calcitonin) related to the invention comprises a monoclonal antibody, and is shown in table 1 in detail:
TABLE 1
Specificity identification of the CT antibody:
and (3) performing detection by ELISA, respectively coating an ELISA plate by using CT, Procalcitonin (PCT), thyroglobulin (Tg) and Thyroid Stimulating Hormone (TSH) as detection antigens, respectively detecting specific reactions of selected CT antibodies 1-5 and different proteins by ELISA, taking the serum of a normal BALB/c mouse as a negative control, and taking PBS as a blank control.
As a result: the antibodies 1-5 respectively only react with CT to be positive (P/N > 2.1), and the Procalcitonin (PCT), Neuron Specific Enolase (NSE), thyroglobulin (Tg) and Thyroid Stimulating Hormone (TSH) to be negative, which shows that the antibodies 1-5 screened by the invention respectively have specificity.
The results show that: the CT antibodies 1-5 respectively react with CT to be positive (P/N > 2.1) and react with Procalcitonin (PCT), neuron-specific enolase NSE thyroglobulin (Tg) and Thyroid Stimulating Hormone (TSH) to be negative, which shows that the antibodies 1-5 respectively have specificity.
Preparing a fluorescence immunochromatographic test paper for detecting human CT in an object to be detected:
1. study subjects: the specimens are from 100 patients (including MTC patients) with surgical operation in the hospital, and thyroid gland (including lesion of suspected MTC patient) and lymph node are punctured by using a syringe.
2. Reagents and instrumentation:
the fluorescent microsphere labeled antibody of the immunochromatographic test paper is selected from 1 calcitonin antibody in table 1, the membrane scratching antibody on the detection line is selected from another calcitonin antibody in table 1, an antibody combination which has no cross reaction and is suitable for fluorescent immunochromatographic detection and has high specificity and sensitivity is screened, the quality control antibody on the quality control line in immunochromatographic is an antibody of antibody homologous IgG in antibodies 1-5, a carboxylated polystyrene time-resolved fluorescent microsphere, a fluorescent detector is from the original real industry trades of Yangtze river in Wuxi city, a nitrocellulose membrane (Merck-Millipore, USA), a sample pad, a combination pad, a bottom plate and water absorption paper are purchased from Shanghainejei company, and other reagents are domestic analytical pure.
3. The preparation method of the fluorescence immunochromatographic test strip comprises the following steps:
3.1 treatment of the sample pad
The sample was soaked in 0.05M pH7.4 phosphate buffer containing 1% BSA, 0.1% Triton100 for 5 hours and then oven dried.
3.2 preparation of fluorescent microsphere binding pad:
washing the fluorescent microspheres by using MES (methyl methacrylate) activation buffer solution with the pH value of 4.0; re-dissolving the mixture to the original volume by using MES buffer solution, adding 1mg/ml carbodiimide (EDC) and 1mg/ml N-hydroxysuccinimide (NHS) of the same volume, and reacting for 20 minutes at room temperature; washing the fluorescent microspheres, redissolving the fluorescent microspheres by using 0.05M phosphate buffer solution with pH7.4, then adding one of antibodies 1-5 according to the time resolution of 20 mu g antibody/mg, reacting for 2 hours at room temperature, adding 0.05M phosphate buffer solution with pH7.4 containing 10% BSA to 1% BSA, and reacting for 30 minutes at room temperature; washing fluorescent microspheres, re-dissolving to 2 times of original volume with phosphate buffer containing 1% BSA, 0.1% Tween-20, 0.4% S9, 5% sucrose, 0.1% Proclin-300, 0.05M pH7.4, and mixing with one of antibodies 1-5 according to (0.6 × 30) cm2Spraying 56 mu g of fluorescent microspheres on the bonding pad, drying for 2 hours at 37 ℃ in the dark to prepare a fluorescent microsphere bonding pad, and adding a drying agent for sealing and storing for later use;
3.3 Cross-membrane antibodies to T and C lines
Sticking a nitrocellulose membrane 2 on a PVC (polyvinyl chloride) bottom plate 1, respectively diluting one of CT (computed tomography) antibodies 1-5 and an anti-antibody 1-5 homologous IgG antibody to 1mg/ml by using a 0.05M phosphate buffer solution with the pH value of 7.4 and containing 5% of sucrose, spraying the two on the nitrocellulose membrane at the interval of 0.5cm, drying at 37 ℃ for 6h, and adding a drying agent for sealing for later use;
3.4 Assembly of fluorescent immunochromatographic test strip
Under the environment that the humidity is less than 35% and the temperature is 20-25 ℃, the nitrocellulose membrane base plates (1 and 2) marked with the C-line antibody and the T-line antibody are combined with the marked antibody combination pad 5 marked with the fluorescent microsphere, the sample pad 4 and the absorbent paper 3 to form a microfiltration system, the microfiltration system is cut into a width of 0.4cm, and the microfiltration system is filled into a card shell to prepare the test strip (figure 1 and figure 2). FIG. 1 is a schematic cross-sectional view of a fluorescence immunochromatographic test strip along the length direction; FIG. 2 is a schematic plan view of the fluorescence immunochromatographic test strip; the reference numbers in the figures denote: 1-bottom plate, 2-nitrocellulose membrane, 3-absorbent paper, 4-sample pad, 5-combination pad, 6-quality control band, and 7-detection band.
4. The detection method comprises the following steps:
4.1 sampling: the suspected thyroid medullary carcinoma tissue in vitro is punctured with a 26-gag syringe needle fine needle for 5 times in each position of the same tissue, and a 1ml syringe is connected behind the needle after sampling.
4.2 sample pretreatment: a tube containing 200. mu.l PBS buffer was taken and allowed to equilibrate to room temperature, ensuring that all liquid was at the bottom of the tube before use. And (3) inserting the syringe punctured by the fine needle into the buffer solution, sucking and beating the buffer solution to fully wash and uniformly mix the buffer solution and the buffer solution to prepare 200 mu l of a sample to be detected.
4.3 sample adding: 60 mul of the sample to be detected is added into the sample adding area of the CT fluorescence immunochromatographic test paper, and the thin film chromatography reaction is carried out for 5 minutes in an incubator.
4.4 detection: and inserting the reacted fluorescence immunochromatographic test paper and the calibration card into a card inserting port of a portable fluorescence immunity quantitative analyzer, operating the analyzer, and automatically reading the card by the analyzer to obtain a quality control band C value and a detection band T value in the fluorescence immunochromatographic test paper. 7 minutes were required to complete the entire assay.
4.5 judging the result:
when the T/C is more than or equal to 0.5, the result is positive, which indicates that the CT concentration in the tissue fluid of the puncture object is higher, and the tissue fluid can be identified as MTC or lymph node metastasis;
when T/C <0.5, the result is negative, indicating that the concentration of CT in the puncture tissue fluid is low, and it can be identified as non-MTC or no lymph node metastasis.
5. Drawing a standard curve: the CT standard was prepared in 6 different concentrations, 0pg/ml, 10pg/ml, 50pg/ml, 100pg/ml, 500pg/ml, 1000pg/ml, each concentration being 5 replicates.
6. And (3) testing the performance of the fluorescence immunochromatographic test strip: (1) sensitivity: the 10 blanks were measured, the mean (x) and standard deviation(s) were taken, and x + -2 s was calculated, from which the corresponding dose was looked up on the standard curve. (2) After the test strip is stored for 6 months at 4 ℃ in a dark place, the fluorescence immunochromatographic test strips of the CT of the same batch and different batches are respectively extracted, a standard substance with the concentration of 200pg/ml is used for testing, and the difference CV between batches is calculated. (3) And preparing the standard substance into 6 concentrations corresponding to the CT standard curve, and performing specificity detection.
7. And (3) detecting the CT content in the eluents of different tissues: and respectively taking the MTC focus puncture solution and the normal thyroid puncture solution to detect the CT content.
8. Statistical treatment: data were analyzed using SPSS 19.0 statistical software and Chi between groups2The test shows that the difference is statistically significant when P is less than 0.05. The pairwise t test was used for correlation and difference comparisons.
In the research example 1, the antibodies 1-5 are combined in pairs to be used as a membrane scratching antibody and a labeled antibody respectively, and the detection sensitivity, the detection group difference and the group difference of each antibody combination are researched.
Note: those below cut off value are negative and those above cut off value are positive
The calcitonin is preferably selected as a detection index beside a thyroid medullary carcinoma and lymph node metastasis bed, an antibody with good specificity is preferably selected from a plurality of antibodies and combined, the fluorescent microsphere coupled antibody is matched with a detection line membrane scratching antibody, the detection sensitivity and specificity are obviously improved, the deviation is small, and the rapid and accurate detection is realized.
Comparative example:
based on the results of example 1 above, the microsphere conjugate pad spray buffer was changed to 1% BSA, 0.5% Tween-20, 5% sucrose, 0.1% Proclin-300, 0.05M phosphate buffer pH 7.2-7.6. The other reagents and methods are the same as example 1, and the performance of the test paper is analyzed by the same method as example 1.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
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