CN111458510A - Early esophageal cancer and high risk group screening marker and related joint inspection card - Google Patents
Early esophageal cancer and high risk group screening marker and related joint inspection card Download PDFInfo
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Abstract
The invention belongs to the technical field of bioengineering and biomedicine, particularly relates to the field of autoantibody engineering and oncology, and more particularly relates to an application of a group of early esophageal squamous cell carcinoma screening markers and marker specificity detection reagents and a joint inspection card prepared from related specificity detection reagents. The molecular marker comprises an HNMT autoantibody, a CARD18 autoantibody and a PCMTD1 autoantibody, and the invention also provides application of a specific detection reagent of the molecular marker in preparation of an early esophageal squamous cell carcinoma screening kit, a test strip or a detection CARD. The detection object of the prepared early esophageal squamous cell carcinoma screening joint inspection card is a serum sample, the joint inspection card can be used for rapidly screening early esophageal squamous cell carcinoma of high risk groups by collecting patient serum, and the joint inspection card has higher detection sensitivity and specificity on the early esophageal squamous cell carcinoma.
Description
Technical Field
The invention belongs to the technical field of bioengineering and biomedicine, particularly relates to the field of autoantibody engineering and oncology, and more particularly relates to an application of a group of early esophageal squamous cell carcinoma screening markers and marker specificity detection reagents and a joint inspection card prepared from related specificity detection reagents.
Background
Esophageal squamous cell carcinoma is one of the most common malignant tumors worldwide, the death rate is the sixth, more than half of about 50 ten thousand esophageal squamous cell carcinoma patients occur in China every year worldwide, and the incidence rate is 100 times higher than that of esophageal squamous cell carcinoma in western countries. The histological type and the epidemic characteristics of the esophageal squamous cell carcinoma in the Chinese and western countries are obviously different, so that the scientific problems and the research ideas concerned by the Chinese and western countries are obviously different, and the research results are difficult to refer to and share mutually. The prognosis of esophageal squamous cell carcinoma is very poor, the 5-year survival rate of patients with middle and late esophageal squamous cell carcinoma is only about 15%, and the 5-year survival rate of patients with early esophageal squamous cell carcinoma is about 90%. Although the 5-year survival rate of early esophageal squamous cell carcinoma is obviously higher than that of middle and advanced stages, the early cancer accounts for about 5 percent of patients who are clinically diagnosed for the first time. Therefore, it is extremely important to improve the diagnosis rate of early esophageal squamous cell carcinoma. Because patients with esophageal squamous cell carcinoma lack obvious specific symptoms in early stage and lack economical, efficient and sensitive biomarkers suitable for screening of a large range of high risk groups, the patients with esophageal squamous cell carcinoma who are clinically diagnosed at present are mostly in middle and late stage at the time of diagnosis. The early detection of the esophageal squamous cell carcinoma is an effective means for improving the survival rate of patients with the esophageal squamous cell carcinoma, so that the screening of high-efficiency and specific molecular markers of the esophageal squamous cell carcinoma is particularly important for the early detection and the early screening of patients with the esophageal squamous cell carcinoma, and the method is also an urgent problem to be solved.
The inventor collects the related information of more than 30 ten thousand cases of esophageal squamous cell carcinoma patients in 35 years, utilizes a molecular mechanism to screen an esophageal squamous cell carcinoma specific marker, establishes a molecular marker screening system for the early discovery of high risk groups and esophageal squamous cell carcinoma, screens the molecular marker on the basis, simultaneously adopts a protein microarray chip technology to carry out pairing research on peripheral blood of more than ten thousand cases of esophageal squamous cell carcinoma patients and healthy volunteers in combination with a research recently carried out by the inventor, discovers a plurality of autoantibodies related to the occurrence of esophageal squamous cell carcinoma, screens 3 autoantibodies related to early lesions of esophageal squamous cell carcinoma from the autoantibodies, CARD18 autoantibodies and PCMTD1 autoantibodies as early diagnosis indexes, and improves the discovery rate of early esophageal squamous cell carcinoma, the method has important significance for reducing the death rate of the esophageal squamous cell carcinoma.
Disclosure of Invention
Aiming at the problems and the defects in the prior art, the invention aims to provide an application of a group of specific detection reagents for early esophageal squamous cell carcinoma screening markers and a joint inspection card prepared from the related specific detection reagents.
Based on the purpose, the invention adopts the following technical scheme:
the invention provides an application of a detection reagent capable of specifically detecting HNMT, CARD18 and PCMTD1 or the group of gene autoantibodies in an early esophageal squamous cell carcinoma screening kit, test paper or detection CARD.
According to the above use, preferably, the detection reagent comprises a molecule for detecting the expression of the HNMT autoantibody, a molecule for detecting the expression of the CARD18 autoantibody and a molecule for detecting the expression of the PCMTD1 autoantibody.
According to the above use, preferably, the molecule for detecting the expression or non-expression of the HNMT autoantibody is an antibody of the HNMT autoantibody capable of specifically binding to the HNMT autoantibody; the molecule for detecting whether the CARD18 autoantibody is expressed is an antibody of the CARD18 autoantibody capable of specifically binding with the CARD18 autoantibody; the molecule for detecting whether the PCMTD1 autoantibody is expressed is an antibody of the PCMTD1 autoantibody specifically binding with the PCMTD1 autoantibody.
According to the application, preferably, the detection object of the early esophageal squamous cell carcinoma screening kit, the test strip or the test card is a serum sample.
The invention also provides a triple detection CARD for screening early esophageal squamous cell carcinoma, which contains specific detection reagents for detecting HNMT autoantibodies, CARD18 autoantibodies and PCMTD1 autoantibodies.
According to the above triple test CARD for early esophageal squamous cell carcinoma screening, preferably, the detection reagent comprises a molecule for detecting whether the HNMT autoantibody is expressed, a molecule for detecting whether the CARD18 autoantibody is expressed and a molecule for detecting whether the PCMTD1 autoantibody is expressed.
According to the above triple test card for early esophageal squamous cell carcinoma screening, preferably, the molecule for detecting whether the HNMT autoantibody is expressed is an antibody of the HNMT autoantibody capable of specifically binding with the HNMT autoantibody; the molecule for detecting whether the CARD18 autoantibody is expressed is an antibody of the CARD18 autoantibody capable of specifically binding with the CARD18 autoantibody; the molecule for detecting whether the PCMTD1 autoantibody is expressed is an antibody of the PCMTD1 autoantibody capable of specifically binding with the PCMTD1 autoantibody.
According to the triple detection card for screening early esophageal squamous cell carcinoma, preferably, the three colloidal gold test strips have the same structure and comprise a sample pad, a colloidal gold pad, a nitrocellulose membrane and an absorption pad which are sequentially laid on a bottom plate along the length direction, wherein the nitrocellulose membrane is provided with a detection line and a quality detection line, and the quality detection line is arranged between the detection line and the absorption pad; the colloidal gold pad is adsorbed with a colloidal gold-labeled antibody A which can be specifically combined with the autoantibody to be detected, the detection line position is fixed with an antibody B which can be specifically combined with the autoantibody to be detected, and the antibody A and the antibody B respectively perform binding reaction with different sites of the autoantibody; a secondary antibody capable of specifically binding to the antibody a is fixed to the position of the quality control line.
According to the triple test CARD for early esophageal squamous cell carcinoma screening, preferably, the antibody A comprises a monoclonal antibody of an anti-HNMT autoantibody, a monoclonal antibody of an anti-CARD 18 autoantibody and a monoclonal antibody of an anti-PCMTD 1 autoantibody; antibody B includes polyclonal antibodies against HNMT autoantibodies, polyclonal antibodies against CARD18 autoantibodies and polyclonal antibodies against PCMTD1 autoantibodies; the secondary antibody is a polyclonal antibody against antibody a.
The invention provides an application of a detection reagent capable of specifically detecting an HNMT autoantibody in an early esophageal squamous cell carcinoma screening kit, a test strip or a detection card.
The invention provides an application of a detection reagent capable of specifically detecting CARD18 autoantibody in an early esophageal squamous cell carcinoma screening kit, test paper or a detection CARD.
The invention provides an application of a detection reagent capable of specifically detecting a PCMTD1 autoantibody in an early esophageal squamous cell carcinoma screening kit, a test strip or a detection card.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention provides a detection reagent capable of specifically detecting HNMT autoantibody, CARD18 autoantibody and PCMTD1 autoantibody, and provides a triple detection CARD for rapidly detecting the autoantibody, and rapid screening of early esophageal squamous cell carcinoma of high risk people can be rapidly carried out through the triple detection CARD.
(2) The triple detection card adopts the combination of the specific detection reagents of three autoantibodies to rapidly screen early esophageal squamous cell carcinoma, and compared with the screening of the specific detection reagent of a single autoantibody to early esophageal squamous cell carcinoma, the triple detection card adopts the combination of the specific detection reagents of three autoantibodies to screen esophageal squamous cell carcinoma, and has higher sensitivity and specificity, the detection sensitivity to early esophageal squamous cell carcinoma reaches 74.39%, and the specificity reaches 86.18%.
(3) The triple detection CARD has the advantages of being minimally invasive, easy to detect and easy to judge the result, improves the sensitivity and specificity of screening early esophageal squamous cell carcinoma by carrying out combined detection on the HNMT autoantibody, the CARD18 autoantibody and the PCMTD1 autoantibody in the serum of an object to be detected, is suitable for large-scale screening of asymptomatic people in high incidence areas of esophageal squamous cell carcinoma, and therefore, the triple detection CARD can carry out early evaluation and diagnosis on early esophageal squamous cell carcinoma of high risk people, and is convenient and easy to implement.
Drawings
Fig. 1 is a schematic structural diagram of a triple-link detection card in embodiment 1 of the present invention;
FIG. 2 is a schematic diagram of a layout structure and operation of a colloidal gold test strip according to embodiment 1 of the present invention;
FIG. 3 is a ROC graph of specific detection reagents of HNMT autoantibody, CARD18 autoantibody and PCMTD1 autoantibody which are separately used for screening and diagnosing patients with early esophageal squamous cell carcinoma;
FIG. 4 is a ROC graph showing the combination of specific detection reagents of HNMT autoantibody, CARD18 autoantibody and PCMTD1 autoantibody for screening and diagnosing patients with early esophageal squamous cell carcinoma.
Detailed Description
In order to make the description of the present disclosure more complete and complete, the following description is given for illustrative purposes with respect to the embodiments and specific examples of the present invention; it is not intended to be the only form in which the embodiments of the invention may be practiced or utilized. The embodiments are intended to cover the features of the various embodiments as well as the method steps and sequences for constructing and operating the embodiments. However, other embodiments may be utilized to achieve the same or equivalent functions and step sequences.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the reagents used are commercially available.
The experimental procedures, for which specific conditions are not indicated in the examples, are generally conventional in the art, e.g. according to conventional conditions such as those in Sambrook et al, molecular cloning, A laboratory Manual (third edition) (scientific Press, 2002), or according to conditions recommended by the reagent manufacturers; the reagents, materials and instruments used are not indicated by manufacturers, and are all conventional products commercially available.
Example 1 triple test card for early esophageal squamous cell carcinoma screening
1. Structure of triple detection card
A triple detection card for screening early esophageal squamous cell carcinoma, as shown in figure 1, comprises a PVC base plate and three colloidal gold test strips laid on the base plate in parallel, wherein the three colloidal gold test strips are respectively as follows: the test paper for detecting the HNMT autoantibody, the CARD18 autoantibody and the PCMTD1 autoantibody. Three colloidal gold test paper strip structures are the same, as shown in fig. 2, include along length direction lay sample pad, colloidal gold pad, nitrocellulose membrane and the absorption pad on the PVC bottom plate in proper order, the colloidal gold pad is pressed and is established between sample pad and nitrocellulose membrane, promptly: one end is overlapped below the sample pad, and the other end is overlapped above the nitrocellulose membrane; the nitrocellulose membrane is provided with a detection line and a quality detection line, and the quality detection line is arranged between the detection line and the absorption pad. The width of the PVC flat plate is 50mm, and the length of the PVC flat plate is 75 mm; the width of a single colloidal gold test strip is 10mm, and the length is 65 mm. The lengths of the sample pad, the colloidal gold pad, the nitrocellulose membrane and the absorption pad are 18mm, 29mm and 18mm in sequence; the overlapping length of the sample pad and the colloidal gold pad is 9mm, and the overlapping length of the colloidal gold pad and the nitrocellulose membrane is 9 mm; the distance between the colloidal gold pad and the detection line is 5mm, and the distance between the detection line and the quality inspection line is 10 mm.
The detection line is fixedly provided with an antibody B which can be specifically combined with the autoantibody to be detected, and the antibody A and the antibody B respectively perform binding reaction at different sites of the autoantibody; a secondary antibody capable of specifically binding to the antibody a is fixed to the position of the quality control line.
Reagent sources or methods of preparation
2.1 antibody Source (method for producing antibody)
2.1.1 preparation of antibody A
Separating serum from blood with positive HNMT autoantibody, CARD18 autoantibody and PCMTD1 autoantibody, adding 37% ammonium sulfate aqueous solution into the separated serum to precipitate the three autoantibodies to obtain crude globulin, adding 0.1 mol/L glycine 0.1ml into the crude globulin, adding sodium bicarbonate to adjust ph to 7.2, adding distilled water to the total volume of the crude globulin to be 15m L, eluting with sephadex filler chromatographic column with the inner diameter of 1cm and the outer diameter of 1.5cm, wherein the eluent is Tris-HCl buffer saline solution with the concentration of 0.1 mol/L pH8.0, NaCl aqueous solution with the concentration of 0.14 mol/L, the elution flow rate of 0.47m L/min, collecting the HNMT autoantibody in 8-14min, collecting the small and large autoantibodies in 17-22min, collecting the PCMTD 2 autoantibody in 23-27min, collecting the small and large and small molecular autoantibodies in 8-14min, respectively, and collecting the small and small antibodies of the HNMT 18 and PCMTD 4934 autoantibodies.
The three collected autoantibodies are respectively used as antigens to immunize a mouse, a mouse immune spleen cell and a human myeloma cell are extracted to be fused under the action of polyethylene glycol to form a hybridoma cell, the hybridoma cell is cultured in a selective culture medium (HAT culture medium), the unfused cell dies, the hybridoma cell survives and is screened for positive cloning to obtain a plurality of different monoclonal antibodies, the monoclonal antibodies are screened by enzyme-linked immunosorbent assay (E L ISA) according to different antigenic determinants of the monoclonal antibodies, the hybridoma cell of the monoclonal antibody of the anti-HNMT autoantibody, the hybridoma cell of the monoclonal antibody of the anti-CARD 18 autoantibody and the hybridoma cell of the monoclonal antibody of the anti-PCMTD 1 autoantibody are respectively selected for cloning and amplification, and the monoclonal antibody of the anti-HNMT autoantibody, the monoclonal antibody of the anti-CARD 18 autoantibody and the monoclonal antibody of the anti-PCMTD 1 autoantibody are respectively collected, so that the antibody A is prepared.
2.1.2 preparation of antibody B
Separating serum from blood with positive HNMT autoantibodies, CARD18 autoantibodies and PCMTD1 autoantibodies, and adding a 37% ammonium sulfate aqueous solution into the separated serum to promote the precipitation of the three autoantibodies to obtain crude globulin; the method of 2.1.1 is adopted, crude globulin is eluted and separated by a sephadex filler chromatographic column, globulin with different molecular sizes is eluted and separated, and three autoantibodies, namely an HNMT autoantibody, a CARD18 autoantibody and a PCMTD1 autoantibody, are respectively collected.
Injecting the three collected autoantibodies serving as antigens into an abdominal cavity of a guinea pig, taking blood from the heart after 72 hours to obtain whole blood, naturally coagulating at room temperature, placing the whole blood in an ice-snow environment at 4 ℃ until the blood coagulates, separating serum, inactivating at 56 ℃ for 30min, and roughly extracting the polyclonal antibody by using DEAE-cellulose (DE 32). The method comprises the following specific steps of weighing 50g of DEAE-cellulose, placing the DEAE-cellulose in a 1000m L beaker, washing with distilled water to remove floating fine particles, adjusting the pH to 7.4 by using 0.05 mol/L phosphate buffer solution, mixing the serum with the DEAE-cellulose after the pH adjustment, placing the mixture in a 4 ℃ for standing and adsorption for 1 hour, performing suction filtration by using a Brinell filter funnel, and collecting filtrate to obtain the roughly-extracted polyclonal antibody.
The crude polyclonal antibody is eluted and separated by a sephadex filler chromatographic column, the specific process is that 0.1 mol/L glycine 0.1m L is added into the crude polyclonal antibody, sodium bicarbonate is added to adjust the pH value to 7.4, distilled water is added until the total volume of the crude polyclonal antibody is 15m L, the sephadex filler chromatographic column with the inner diameter of 1cm and the outer diameter of 1.5cm is adopted for elution and separation, wherein the eluent is 0.1 mol/L Tris-HCl buffer salt solution with the pH value of 8.0, the salt solution is 0.14 mol/L NaCl water solution, the elution flow rate is 0.47m L/min, the polyclonal antibody of the anti-HNMT autoantibody is collected in a period of 5-11min, the polyclonal antibody of the anti-CARD 18 autoantibody is collected in a period of 13-18min, the polyclonal antibody of the anti-PCMTD 1 autoantibody is collected in a period of 21-25min, and the polyclonal antibody, namely the anti-HNMT autoantibody, the anti-PCD 18 polyclonal antibody, the anti-PCD 35 1 polyclonal antibody, namely the polyclonal antibody, is prepared by the separation and purification.
2.1.3 preparation of the second antibody of antibody A
Respectively taking the monoclonal antibody of the anti-HNMT autoantibody, the monoclonal antibody of the anti-CARD 18 autoantibody and the monoclonal antibody of the anti-PCMTD 1 autoantibody prepared by 2.1.1 as antigens, injecting the antigens into the abdominal cavity of a guinea pig, obtaining whole blood by adopting a heart blood sampling method after 72 hours, naturally coagulating the whole blood at room temperature, then placing the whole blood on ice and snow at 4 ℃ for blood coagulation, separating serum, performing inactivation treatment at 56 ℃ for 30min, and performing crude extraction of the polyclonal antibody by using DEAE-cellulose (DE 32).
The crude polyclonal antibody is eluted, separated and purified by a sephadex filler chromatographic column, and the specific process comprises the steps of adding 0.1 mol/L glycine 0.1m L into the crude polyclonal antibody, adding sodium bicarbonate to adjust the pH to 7.3, adding distilled water until the total volume of the crude polyclonal antibody is 15m L, eluting and separating by using the sephadex filler chromatographic column with the inner diameter of 1cm and the outer diameter of 1.5cm, wherein the eluent is a Tris-HCl buffer salt solution with the inner diameter of 0.1 mol/L pH8.0, wherein the salt solution is a NaCl solution with the concentration of 0.14 mol/L, the elution flow rate is 0.47m L/min, collecting the polyclonal antibody of the monoclonal antibody against the HNMT autoantibody in a time period of 9-14min, collecting the polyclonal antibody of the monoclonal antibody against CARD18 autoantibody in a time period of 17-21min, collecting the polyclonal antibody of the monoclonal antibody against the HNMT autoantibody in a time period, and purifying by the monoclonal antibody against the CARD18 autoantibody, namely the monoclonal antibody against the PCMT 1 autoantibody, and the polyclonal antibody against the monoclonal antibody against the HNMT, namely the polyclonal antibody, and the polyclonal antibody against the CHD 18, and the monoclonal antibody are respectively prepared, and the polyclonal antibody against the anti-HCA polyclonal antibody, and the anti-HCD antibody, and the polyclonal antibody are prepared by.
2.2 preparation of colloidal gold labeled antibody A:
(1) preparing a colloidal gold solution, namely adding a 50m L1.2 mM chloroauric acid solution into a 200m L round-bottom flask, heating and stirring the solution while heating, adding 5m L1 wt% of sodium citrate into the boiling solution after the solution is boiled, continuing to boil the solution for 15min after the solution turns to wine red, stopping heating, continuing to stir the solution, naturally cooling the solution to obtain a colloidal gold solution, and storing the colloidal gold solution at 4 ℃ in a dark place, wherein the particle size of the colloidal gold is 10-15 nm.
(2) Taking 3 centrifuge tubes, respectively adding 10m L of the colloidal gold solution prepared in the step (1), adjusting the pH value to 5-9, respectively adding antibody A of antibody A, CARD18 of HNMT autoantibody and antibody A, PCMTD1 of autoantibody into the 3 centrifuge tubes, uniformly stirring for lh, respectively adding 3m L10 wt% of PEG20000 solution into each centrifuge tube, stirring for 30min, respectively adding 1m L1 wt% of BSA solution into each centrifuge tube, sealing, continuously stirring overnight, centrifuging the sealed solution, adding a buffer solution, and re-suspending to respectively prepare three colloidal gold labeled antibodies A aiming at different autoantibodies.
Detection principle of triple detection card/colloidal gold test strip
The three colloidal gold test strips on the triple detection card respectively and independently complete the detection work, and the detection principle is the same. The following takes an HNMT autoantibody detection colloidal gold test strip as an example to explain the detection principle of the colloidal gold test strip: dropping the diluted serum to-be-detected liquid on a sample pad, moving the to-be-detected liquid to the direction of an absorption pad under the capillary siphon effect, forming a conjugate with the colloidal gold-labeled antibody A when the to-be-detected liquid passes through the colloidal gold pad, continuously carrying out electrophoresis on the conjugate to a detection line position, and combining an antibody B fixed at the detection line position with other sites on the autoantibody to form an antibody A-autoantibody-antibody B conjugate, so that the detection line is colored (red); and if the liquid to be detected does not contain the autoantibody to be detected, the detection line does not develop color.
Meanwhile, the colloidal gold labeled antibody A flows to the position of the quality control line along with the liquid, and is captured by a second antibody which is fixed at the position of the quality control line and can be specifically combined with the antibody A, so that the quality control line is colored (red); if the quality inspection line is colored, the result is valid; if the quality control line is colored, the result is invalid, and the quality control line needs to be detected again.
Use method of joint inspection card
Taking 0.3m L serum of an individual to be detected, mixing and diluting the serum and a sample diluent according to the volume ratio of 1:50 to obtain a solution to be detected for later use, wherein the sample diluent is PBS buffer solution containing 1wt% BSA and 0.05% (v/v) Tween 20.
And (3) dropwise adding the solution to be detected on the sample pad of the HNMT detection colloidal gold test strip, the CARD18 detection colloidal gold test strip and the CARD18 detection colloidal gold test strip, standing for 5min, and observing the color development result.
Determination of detection result
5.1 the result judgment standard of a single colloidal gold test strip is as follows:
negative results: the detection line is not colored, and the quality detection line is red;
positive results: the detection line is red, and the quality detection line is red;
invalid result: the quality detection line does not develop color, and the result is invalid whether the detection line develops color or not.
5.2 the result judgment standard of the triple test card is as follows:
at least one of the three colloidal gold test strips, namely the HNMT autoantibody detection colloidal gold test strip, the CARD18 autoantibody detection colloidal gold test strip and the CARD18 autoantibody detection colloidal gold test strip, is positive, and the result of the three-joint detection CARD is judged to be positive; and if the detection results of the three colloidal gold test strips are negative, the result of the triple joint detection card is judged to be negative.
Example 2 validity analysis of triple test card for early esophageal squamous cell carcinoma screening
1. Serum sample collection
The research institution of the inventor carries out a large-scale early esophageal squamous cell carcinoma general investigation activity in villages and towns of Linzhou city, Anyang city, Henan province during the 10 th month to the 11 th month of 2019, and finds 215 patients with early esophageal squamous cell carcinoma confirmed and the same number of healthy volunteers in the same region in a local search.
Among these, the criteria for patient inclusion into the early esophageal squamous cell carcinoma patient group are as follows:
(1) cases receiving surgical resection;
(2) the postoperative pathology is confirmed to be early esophageal squamous cell carcinoma;
(3) the patients do not receive the new adjuvant therapy before the operation.
And selecting the same number of healthy volunteers as a healthy volunteer group. "healthy volunteers" are defined as volunteers who have not found any pathological changes in the upper digestive tract by gastroscopy.
Serum samples were collected from patient groups: blood of patient group is extracted, serum is separated by centrifugation, 0.1wt% sodium azide and 0.05wt% glycerol are added after sterilization, and the mixture is stored in a common refrigerator at 4 ℃.
Serum specimen collection from healthy volunteer groups: blood of healthy volunteers was collected, serum was separated by centrifugation, sterilized, added with 0.1wt% sodium azide and 0.05wt% glycerin, and stored in a common refrigerator at 4 ℃.
Test strip effectiveness evaluation for early esophageal squamous cell carcinoma screening
The test strip for screening early esophageal squamous cell carcinoma is used for detecting early esophageal squamous cell carcinoma of 215 serum specimens in a collected patient group and 215 serum specimens in a healthy volunteer group, and is compared with a diagnosis result obtained by pathological biopsy of the patient group, and the result is shown in table 1, table 2, figure 3, table 3 and figure 4, wherein the table 1 is used for analyzing the expression difference of three autoantibodies in the serum of an early esophageal squamous cell carcinoma patient and a healthy volunteer, and the table 2 and the figure 3 are used for analyzing the detection result of the early esophageal squamous cell carcinoma patient and a healthy volunteer by using the specific detection reagent of the autoantibody; table 3 and FIG. 4 show the sensitivity and specificity of specific detection reagents for different combinations of autoantibodies for early esophageal squamous cell carcinoma detection.
Oddsratio (hereinafter referred to as OR value) in Table 1 represents here the ratio of the probability of esophageal squamous cell carcinoma to the probability of non-esophageal squamous cell carcinoma, and the specific calculation formula of OR is illustrated by the HNMT + group and the HNMT-group as examples: OR = ratio of esophageal squamous cell carcinoma in HNMT + group/ratio of esophageal squamous cell carcinoma in HNMT-group, i.e., OR = (59/14)/(156/201) =5.43, also indicating that HNMT positive persons are at a 5.43-fold greater risk of esophageal squamous cell carcinoma than HNMT negative persons.
When the OR value is less than OR equal to 1, the gene has no indication effect on the occurrence of esophageal squamous cell carcinoma; when the OR value is more than 1, the gene is a risk factor for generating esophageal squamous cell carcinoma and has an important indication effect on the occurrence of the esophageal squamous cell carcinoma.
As can be seen from Table 1, the OR values calculated from the serum detection results of the negative group and the positive group of the HNMT autoantibody, the CARD18 autoantibody and the PCMTD1 autoantibody markers of the early esophageal squamous cell carcinoma patients and the healthy volunteers are 5.43, 7.37 and 7.821 respectively, which indicates that the three autoantibody markers have different expressions between the sera of the early esophageal squamous cell carcinoma patients and the healthy volunteers.
In addition, as can be seen from table 2, when the specific detection reagents of three autoantibody markers, namely, the HNMT autoantibody, the CARD18 autoantibody and the PCMTD1 autoantibody, are used alone, the positive rate of diagnosis on early esophageal squamous cell carcinoma is between 27.44% and 38.6%, the detection sensitivity is between 27.44% and 38.6%, and the detection specificity is between 92.56% and 93.49%; wherein, the jotan index is the sum of the sensitivity and specificity minus 1 in statistics, the numerical range is 0-1, the more the jotan index is close to 1, the higher the diagnostic value is, the higher the application value of the method is, the specific detection reagent of the three autoantibody markers of the invention is used alone, the jotan index is between 0.2093 and 0.3116, and as can be seen from fig. 3, the area under the ROC curve of the detection of early esophageal squamous cell carcinoma is between 0.6047 and 0.6558 when the specific detection reagent of the three autoantibody markers is used alone. It can be seen that the specific detection reagents for the three autoantibody markers, namely, the HNMT autoantibody, the CARD18 autoantibody and the PCMTD1 autoantibody, have good specificity when used alone, but have insignificant sensitivity and overall diagnostic efficacy, so that the specific detection reagents for the three autoantibody markers, namely, the HNMT autoantibody, the CARD18 autoantibody and the PCMTD1 autoantibody, have limited diagnostic value for early esophageal squamous cell carcinoma when used alone.
In addition, the results of the detection of early esophageal squamous cell carcinoma using a combination of specific detection reagents for different autoantibody markers are shown in table 3 and fig. 4. As can be seen from table 3, as the number of specific detection reagents of the autoantibody marker increases, the positive rate of early esophageal squamous cell carcinoma diagnosis increases from 27.44% to 74.39%, and the detection sensitivity increases from 27.44% to 74.39%, when the specific detection reagents of the three autoantibody markers are used in combination, the detection sensitivity of early esophageal squamous cell carcinoma reaches 74.39%, that is, when the test strip for screening early esophageal squamous cell carcinoma of early esophageal is applied to a patient with esophageal squamous cell carcinoma, the percentage of esophageal squamous cell carcinoma that can be correctly diagnosed is 74.39%. Although the detection specificity is gradually reduced along with the increase of the number of the specific detection reagents of the autoantibody markers, when the specific detection reagents of the three autoantibody markers are used in combination, the specificity can still reach 86.18%, and the result shows that the percentage of esophageal squamous cell carcinoma which is correctly diagnosed as not suffering from esophageal squamous cell carcinoma is 86.18% when the patients suffering from early esophageal squamous cell carcinoma are detected by the combination of the specific detection reagents of the three autoantibodies; therefore, the specific detection reagent combination corresponding to the three autoantibody markers of the HNMT autoantibody, the CARD18 autoantibody and the PCMTD1 autoantibody is used for early esophageal squamous cell carcinoma diagnosis, so that the diagnosis sensitivity can be greatly improved on the premise of ensuring the diagnosis specificity. In addition, with the increase of the number of corresponding specific detection reagents of the autoantibody markers, the john index is continuously increased and gradually tends to 1, and the fact that the specific detection reagents of the three autoantibody markers are jointly used has higher diagnostic value for early esophageal squamous cell carcinoma is shown.
Moreover, as can be seen from fig. 4, when the specific detection reagents of the three autoantibodies jointly detect the expression level of the autoantibody in the serum of the early esophageal squamous cell carcinoma patient, the area under the ROC curve is 0.8028, which indicates that the diagnosis of the early esophageal squamous cell carcinoma using the early esophageal squamous cell carcinoma screening test strip of the present invention has high sensitivity and specificity at the same time.
In conclusion, the method for detecting the expression level of the autoantibody in serum by combining the specific detection reagents of the HNMT autoantibody, the CARD18 autoantibody and the PCMTD1 autoantibody can keep higher specificity and improve the diagnostic sensitivity, and has good diagnostic value on early esophageal squamous cell carcinoma.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the present invention, but rather as the following description is intended to cover all modifications, equivalents and improvements falling within the spirit and scope of the present invention.
Claims (10)
1. The application of the detection reagent capable of specifically detecting HNMT, CARD18 and PCMTD1 or the group of gene autoantibodies in an early esophageal squamous cell carcinoma screening kit, test strip or test CARD.
2. The use of claim 1 wherein said detection reagents comprise a molecule that detects the expression of HNMT autoantibodies, a molecule that detects the expression of CARD18 autoantibodies and a molecule that detects the expression of PCMTD1 autoantibodies.
3. The use according to claim 2, wherein the molecule for detecting the expression of the HNMT autoantibody is an antibody to an HNMT autoantibody capable of specifically binding to the HNMT autoantibody; the molecule for detecting whether the CARD18 autoantibody is expressed is an antibody of the CARD18 autoantibody capable of being specifically bound with the CARD18 autoantibody; the molecule for detecting whether the PCMTD1 autoantibody is expressed is an antibody of the PCMTD1 autoantibody capable of being specifically bound with the PCMTD1 autoantibody.
4. The use of claim 3, wherein the test object of the early esophageal squamous cell carcinoma screening kit, the test strip or the test card is a serum sample.
5. A triple-junction test CARD for screening early esophageal squamous cell carcinoma, which comprises specific detection reagents capable of detecting HNMT autoantibodies, CARD18 autoantibodies and PCMTD1 autoantibodies.
6. The triple-link detection CARD for screening early esophageal squamous cell carcinoma according to claim 5, wherein the detection reagents comprise a molecule for detecting the expression of HNMT autoantibody, a molecule for detecting the expression of CARD18 autoantibody and a molecule for detecting the expression of PCMTD1 autoantibody.
7. The triple-link detection card for screening early esophageal squamous cell carcinoma according to claim 6, wherein the molecule for detecting the expression or non-expression of HNMT autoantibody is an HNMT autoantibody antibody capable of specifically binding with the HNMT autoantibody; the molecule for detecting whether the CARD18 autoantibody is expressed is an antibody of the CARD18 autoantibody capable of being specifically bound with the CARD18 autoantibody; the molecule for detecting whether the PCMTD1 autoantibody is expressed is an antibody of the PCMTD1 autoantibody capable of being specifically bound with the PCMTD1 autoantibody.
8. The triple-junction detection card for screening early esophageal squamous cell carcinoma according to claim 7, wherein the triple-junction detection card comprises a bottom plate and three colloidal gold test strips laid on the bottom plate in parallel, wherein the three colloidal gold test strips are respectively: the test paper comprises a HNMT autoantibody detection colloidal gold test strip, a CARD18 autoantibody detection colloidal gold test strip and a PCMTD1 autoantibody detection colloidal gold test strip.
9. The triple-junction detection card for screening early esophageal squamous cell carcinoma according to claim 8, wherein the three colloidal gold test strips have the same structure and comprise a sample pad, a colloidal gold pad, a nitrocellulose membrane and an absorption pad which are sequentially laid on a bottom plate along the length direction, wherein the nitrocellulose membrane is provided with a detection line and a quality detection line, and the quality detection line is arranged between the detection line and the absorption pad; the detection line is fixedly provided with an antibody B which can be specifically combined with the autoantibody to be detected, and the antibody A and the antibody B are respectively combined with different sites of the autoantibody for reaction; and a secondary antibody capable of being specifically combined with the antibody A is fixed on the position of the quality control line.
10. The triple-link test CARD for screening early esophageal squamous cell carcinoma according to claim 9, wherein said antibody a comprises a monoclonal antibody against HNMT autoantibody, a monoclonal antibody against CARD18 autoantibody and a monoclonal antibody against PCMTD1 autoantibody; the antibody B comprises polyclonal antibodies of anti-HNMT autoantibodies, CARD18 autoantibodies and PCMTD1 autoantibodies; the secondary antibody is a polyclonal antibody against antibody A.
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| CN102585000A (en) * | 2012-02-22 | 2012-07-18 | 尉军 | Tumor marker CD25 autoantibody and application thereof |
| US20180305689A1 (en) * | 2015-04-22 | 2018-10-25 | Mina Therapeutics Limited | Sarna compositions and methods of use |
| CN109142755A (en) * | 2018-10-09 | 2019-01-04 | 福建省立医院 | It is a kind of diagnose early stage esophageal squamous cell carcinoma four kinds of autoantibody combined detection kits and application |
| CN110187108A (en) * | 2019-05-31 | 2019-08-30 | 郑州大学第一附属医院 | A kind of autoantibody joint-detection ELISA kit for early stage cancer of the esophagus screening |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102585000A (en) * | 2012-02-22 | 2012-07-18 | 尉军 | Tumor marker CD25 autoantibody and application thereof |
| US20180305689A1 (en) * | 2015-04-22 | 2018-10-25 | Mina Therapeutics Limited | Sarna compositions and methods of use |
| CN109142755A (en) * | 2018-10-09 | 2019-01-04 | 福建省立医院 | It is a kind of diagnose early stage esophageal squamous cell carcinoma four kinds of autoantibody combined detection kits and application |
| CN110187108A (en) * | 2019-05-31 | 2019-08-30 | 郑州大学第一附属医院 | A kind of autoantibody joint-detection ELISA kit for early stage cancer of the esophagus screening |
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