Colloid detection kit and detection card for nasopharynx cancer diagnosis and screening and application of colloidal detection kit and detection card

Abstract

The invention relates to a detection kit for nasopharynx cancer diagnosis and screening, which comprises an immunochromatography detection card, and the immunochromatography detection card comprises a reagent for detecting EB virus antigen and antibody through a colloidal gold labeling method. The invention also relates to application of the detection card and the EB virus antigen-antibody in preparation of a detection kit or a detection card for nasopharynx cancer diagnosis and screening.

Description

Colloidal gold detection kit for nasopharyngeal carcinoma diagnosis and screening, detection card and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of immunology, in particular to nasopharyngeal carcinoma diagnosis and screening, more particularly to a colloidal gold detection kit for nasopharyngeal carcinoma diagnosis and screening, a detection card and application thereof. BACKGROUND

[0002] Nasopharyngeal carcinoma (NPC) is a common malignant tumor in otolaryngology, which is characterized by malignant tumor originating from nasopharyngeal mucosa epithelial cells, and is a special type of head and neck malignant tumor. It is highly prevalent in Southeast Asia, Africa and the central and southern coastal areas of China, especially in Guangdong and Guangxi, with an annual incidence of about 30 / 10 million. The total number of cases accounts for more than half of the world, causing a series of serious population health problems. The pathological and anatomical characteristics of nasopharyngeal carcinoma determine its comprehensive treatment mainly by radiotherapy. Clinical staging is closely related to the prognosis of patients. The 5-year overall survival rate of early nasopharyngeal carcinoma patients is as high as more than 90%. Therefore, early intervention can greatly improve the long-term survival rate of patients. Therefore, exploring efficient, easy and cost-effective screening techniques and programs, and establishing a wide coverage and accessible early diagnosis platform are of great significance for improving the level of nasopharyngeal carcinoma management and diagnosis and treatment, prolonging the survival period of patients and improving the quality of life.

[0003] Nasopharyngeal carcinoma has a very low incidence in most parts of the world, but it is highly prevalent in the southern region of China, especially in Guangdong Province, so it is also called 'Guangdong cancer' [1] The China Cancer Center statistics report shows that in 2015, there were about 60,000 new cases of nasopharyngeal carcinoma in China, and about 34,000 deaths [2] In China, the incidence of nasopharyngeal carcinoma has a huge regional difference, which increases from north to south. In some northern regions, the male incidence is as low as 3 / 10 million [1] , but in the Xijiang and Pearl River basins, the incidence is as high as 25-40 / 10 million. Developed countries such as Europe and the United States have a very low incidence of nasopharyngeal carcinoma, lack of research subjects, and have less relevant research. Therefore, most of the research on nasopharyngeal carcinoma is carried out in China.

[0004] At present, the main screening methods for nasopharyngeal carcinoma in China are endoscopic examination, EBV-DNA detection and serological examination. Among them, endoscopic examination (nasopharyngoscopy) is widely used in clinical and physical examination. Combined with histopathological examination, it is considered as the gold standard for the diagnosis of nasopharyngeal carcinoma in China. Nasopharyngoscopy can detect early nasopharyngeal epithelial hyperplasia, which is a precancerous lesion of nasopharyngeal carcinoma. However, nasopharyngoscopy still has some problems, such as difficulty in detecting early nasopharyngeal carcinoma, unstable success rate of biopsy, and misdiagnosis risk. In addition, nasopharyngoscopy is an invasive examination with poor compliance, and it consumes a lot of manpower and medical resources, making it difficult to apply to large-scale population screening. Nasopharyngeal injury and cross infection caused by insufficient experience of the operator or improper cleaning of the equipment have also been reported.

[0005] Currently, EBV-DNA detection analyzes the EB virus DNA content in the plasma or serum of the subject to determine whether the subject is infected with EB virus and the degree of infection. The advantage of this method is that it can directly detect the presence of the virus, and it has high specificity for early screening of nasopharyngeal carcinoma [8] . However, EBV-DNA detection has poor early diagnosis effect. Some reports show that the specificity of this method for detecting NPC patients is as high as 99.3%, while the sensitivity is only 41.5% [9] . In addition, EBV-DNA detection has not yet achieved standardization, and its detection results are easily affected by experimental operation, sample quality and other factors. The detection results between different laboratories may differ, which not only affects the accuracy of the detection results, but also causes trouble for doctors in diagnosis and treatment.

[0006] For EBV antibody serological detection, the commonly used serological screening target is the VCA-IgA and EBNA1-IgA of EB virus (EBV), and the detection method is mostly enzyme-linked immunosorbent assay (ELISA). However, the reported sensitivity of this method for double targets is only 72.3%, the sensitivity of VCA-IgA alone is 68.1%, and the sensitivity of EBNA1-IgA alone is 53.3%, which does not have good screening sensitivity [3] . In addition, the ELISA detection method uses venous blood serum / plasma samples, so that the detection can only be carried out in the central laboratory of the medical institution, and cannot be carried out in large-scale risk population screening at the grassroots level. Therefore, it is of great importance to develop a nasopharyngeal carcinoma screening product with high sensitivity and high specificity for nasopharyngeal carcinoma screening and prevention and control.

[0007] The development of nasopharyngeal carcinoma is a multi-factor synergistic process. In order to achieve more convenient, accurate and high compliance screening, it is of great significance to develop a detection technology with high sensitivity and high specificity for early diagnosis of nasopharyngeal carcinoma.

[0008] It is found that nasopharyngeal carcinoma is closely related to Epstein-Barr virus (EBV). When EBV is in the lytic replication state from the latent stage, it can cause abnormal proliferation of nasopharyngeal epithelial cells and lead to cancer [4] . Patients with nasopharyngeal carcinoma have antibody profiles against various EBV antigens in their serum. Generally, the risk of nasopharyngeal carcinoma is evaluated by detecting serum EBV antibodies, which provides an effective detection index for the diagnosis of nasopharyngeal carcinoma. The early antigen (EA) of EBV is an early protein expressed during the transition from the latent stage to the lytic infection stage, which is a marker of EBV entering the lytic stage. EBNA (EBV nuclear antigen) protein is an important protein expressed by EBV in the latent stage, which can cause a strong antibody response in the body. Zta protein is a protein expressed by EBV early gene BZLF1, which initiates the cascade effect of EBV lytic replication, allowing EBV to enter the lytic replication cycle from the latent stage [11-12] . Multiple studies have found that in patients with nasopharyngeal carcinoma, EA-IgG antibodies, Zta-IgG antibodies, and EBNA-IgA antibodies are highly expressed. Hu [5][4] et al. reported that in 60 healthy controls and 100 patients with nasopharyngeal carcinoma, using enzyme-linked immunosorbent assay (ELISA) method, the detection sensitivity of EA-IgG was 89%, and the specificity was 96.7%, both higher than other EBV indicators such as VCA-IgA and EBNA-IgA. The results showed that R. Dardari [6] et al. used ELISA method to retrospectively analyze the serum of 100 patients with nasopharyngeal carcinoma and 77 healthy controls as well as 28 patients with other non-NPC tumors (such as laryngeal cancer, tongue cancer, Hodgkin's lymphoma, etc.). In subjects aged less than 30 years, the sensitivity of VCA-IgA was 19%, and the specificity was 94%. The sensitivity of EA-IgG was 70%, and the specificity was 90%. The sensitivity of EBNA-IgA was 35%, and the specificity was 100%. In subjects aged more than 30 years, the sensitivity of VCA-IgA was 52%, and the specificity was 89%. The sensitivity of EA-IgG was 81%, and the specificity was 97%. The sensitivity of EBNA-IgA was 61%, and the specificity was 97%. Similarly, there are documents [7] reporting that under the ELISA methodology, the plasma of 232 patients with nasopharyngeal carcinoma and non-nasopharyngeal carcinoma patients in the hospital was detected, the sensitivity of EA-IgA was 91.0%, and the specificity was 90.6%. The sensitivity of EA-IgG was 97.4%, and the specificity was 94.3%. Chen et al.

[0010] The EBV antibody detection was performed on the serum of 51 nasopharyngeal carcinoma patients and 167 non-nasopharyngeal carcinoma patients, and it was found that the single target EBNA-IgA sensitivity was 86.5%, and the specificity was 83.6%; the Zta-IgG sensitivity was 82.8%, and the specificity was 74.5%, and the double target EBNA-IgA and Zta-IgG combined sensitivity was increased to 99.1%, and the specificity was increased to 86.8%. This shows that the double target joint detection improves the serum diagnosis result of nasopharyngeal carcinoma.

[0011] Li et al. performed ELISA detection on 126 nasopharyngeal carcinoma patients and 789 healthy control groups, and the results also found that the sensitivity of VCA-IgA and EBNA1-IgA double target detection was 72.3%, which was significantly higher than that of VCA-IgA and EBNA1-IgA single target detection (68.1% and 53.3% respectively). The above reports and data show that the EA-IgG antibody, Zta-IgG antibody and EBNA-IgA antibody detection of EB virus all have potential application value for in vitro diagnosis of nasopharyngeal carcinoma. However, the above reports all use ELISA method, which needs to be matched with related instruments and equipment, and cannot provide ordinary people and home detection. SUMMARY

[0009] The present application provides an EB virus early antigen (EA) antibody detection kit (colloidal gold method) for nasopharyngeal carcinoma screening to solve the problems of complicated operation, low portability of EBV antibody detection in the prior art.

[0010] The first aspect of the present application relates to a detection kit for nasopharyngeal carcinoma diagnosis and screening, which comprises an immunochromatographic detection card comprising a reagent for detecting EB virus early antigen antibody by colloidal gold labeling method.

[0011] The early antigen is preferably EAD-P47 / 54, and more preferably has the sequence shown in SEQ ID No. 1.

[0012] The antibody can be IgG antibody, IgA antibody, and preferably IgG antibody.

[0013] In a further embodiment, the detection card comprises a sample pad, a colloidal gold pad, a detection line T, a quality control line C and a water absorption paper according to the sample flow direction, the colloidal gold pad is coated with colloidal gold labeled EB virus early antigen, the detection line T is coated with a secondary antibody of the antibody to be detected, and the secondary antibody is preferably anti-human IgG antibody.

[0014] In a further embodiment, the immunochromatographic detection card further comprises a reagent for quality control.

[0015] The reagent for control is:

[0016] a colloidal gold-labeled antigen coated in a colloidal gold pad, and an antibody against said antigen coated in a quality control line C, or

[0017] a colloidal gold-labeled antibody coated in a colloidal gold pad, and a secondary antibody against said antibody coated in a quality control line C, or an antigen against which said antibody is directed.

[0018] Said antibody is preferably chicken IgY, and said secondary antibody is preferably a goat anti-chicken IgY antibody.

[0019] The second aspect of the present application relates to the use of an Epstein-Barr virus early antigen in the preparation of an immunochromatographic test card or test kit for the diagnosis and screening of nasopharyngeal carcinoma, wherein:

[0020] Said test kit comprises said chromatographic test card, and said immunochromatographic test card is the immunochromatographic test card of the first aspect described above.

[0021] The third aspect of the present application relates to an immunochromatographic test card for the diagnosis and screening of nasopharyngeal carcinoma, which is the immunochromatographic test card of the first aspect described above.

[0022] In addition, the present application also provides an antibody test kit (colloidal gold method) for Epstein-Barr virus antigens to solve the problems of complicated operation and low portability of EBV antibody detection in the prior art.

[0023] The fourth aspect of the present application relates to a test kit for the diagnosis and screening of nasopharyngeal carcinoma, which comprises an immunochromatographic test card comprising reagents for detecting Epstein-Barr virus antigen antibodies by colloidal gold labeling method. Said immunochromatographic test card includes an immunochromatographic test card for detecting Zta-IgG antibodies and / or an immunochromatographic test card for detecting EBNA-IgA antibodies, and can further include an immunochromatographic test card for detecting EA-IgG antibodies. Said EA (early antigen) is preferably EAD-P47 / 54, more preferably having the sequence shown in SEQ ID No. 1. Said Zta preferably has the sequence shown in SEQ ID No. 2. Said EBNA is preferably Epstein-Barr virus nuclear antigen 1 (EBNA1), more preferably having the sequence shown in SEQ ID No. 3.

[0024] Original sequences of EAD-P47 / 54, Zta and EBNA1 proteins:

[0025] > SEQ ID NO. 1 (EAD-P47 / 54 protein sequence)

[0026] 1 mettqtlrfk tkalavlskc ydhaqthlkg gvlqvnllsv nyggprlaav anagtaglis

[0027] 61 fevspdavae wqnhqspeea paavsfrnla ygrtcvlgke lfgsaveqas lqfykrpqgg

[0028] 121 srpefvkltm eyddkvsksh htcalmpymp pasdrlrneq migqvllmpk tasslqkwar

[0029] 181 qqgsggvkvt lnpdlyvtty tsgeacltld ykplsvgpye aftgpvakaq dvgaveahvv

[0030] 241 csvaadslaa alslcripav svpilrfyrs giiavvagll tsagdlpldl svilfnhase

[0031] 301 eaaastasep edksprvqpl gtglqqrprh tvspspsppp pprtptwesp arpetpspai

[0032] 361 pshssntale rplavqlark rtssearqkq khpkkvkqaf npli

[0033] >SEQ ID NO. 2 (Zta protein sequence)

[0034] 1 mmdpnstsed vkftpdpyqv pfvqafdqat rvyqdlggps qaplpcvlwp vlpeplpqgq

[0035] 61 ltayhvstap tgswfsapqp apenayqaya apqlfpvsdi tqnqqtnqag geapqpgdns

[0036] 121 tvqtaaavvf acpganqgqq ladigvpqpa pvaaparrtr kpqqpeslee cdseleikry

[0037] 181 knrvasrkcr akfkqllqhy revaaaksse ndrlrlllkq mcpsldvdsi iprtpdvlhe

[0038] 241 dllnf

[0039] >SEQ ID NO. 3 (EBNA protein sequence)

[0040] 1 msdegpgtgp gnglgekgdt sgpegsggsg pqrrggdnhg rgrgrgrgrg ggrpgapggs

[0041] 61 gsgprhrdgv rrpqkrpsci gckgthggtg agagaggaga ggagagggag agggaggagg

[0042] 121 aggagaggga gagggaggag gagagggaga gggaggagag ggaggaggag agggagaggg

[0043] 181 aggagaggga ggaggagagg gagaggagga ggagaggaga gggaggagga gaggagagga

[0044] 241 gaggagagga ggagaggagg agaggaggag agggaggaga gggaggagag gaggagagga

[0045] 301 ggagaggagg agagggagag gagaggggrg rggsggrgrg gsggrgrggs ggrrgrgrer

[0046] 361 arggsrerar grgrgrgekr prspssqsss sgspprrppp grrpffhpvg eadyfeyhqe

[0047] 421 ggpdgepdvp pgaieqgpad dpgegpstgp rgqgdggrrk kggwfgkhrg qggsnpkfen

[0048] 481 iaeglralla rshverttde gtwvagvfvy ggsktslynl rrgtalaipq crltplsrlp

[0049] 541 fgmapgpgpq pgplresivc yfmvflqthi faevlkdaik dlvmtkpapt cnirvtvcsf

[0050] 601 ddgvdlppwf ppmvegaaae gddgddgdeg gdgdegeegq e

[0051] In a further embodiment, the test card comprises, in the direction of sample flow, a sample pad, a colloidal gold pad coated with colloidal gold-labeled EB virus early antigen, a test line T coated with a secondary antibody of the antibody to be detected, preferably an anti-human IgG antibody, a control line C, and a blotting paper.

[0052] In a further embodiment, the immunochromatographic test card further comprises a reagent for quality control.

[0053] The reagent for control is:

[0054] colloidal gold-labeled antigen coated in the colloidal gold pad, and an antibody of the antigen coated in the control line C, or

[0055] colloidal gold-labeled antibody coated in the colloidal gold pad, and a secondary antibody of the antibody coated in the control line C or an antigen against which the antibody is directed.

[0056] The antibody is preferably chicken IgY, and the secondary antibody is preferably goat anti-chicken IgY antibody.

[0057] The fifth aspect of the present application relates to the use of EB virus early antigen in the preparation of an immunochromatographic test card or test kit for the diagnosis and screening of nasopharyngeal carcinoma, wherein:

[0058] The test kit comprises the chromatographic test card, and the immunochromatographic test card is the immunochromatographic test card described in the fourth aspect above.

[0059] The sixth aspect of the present application relates to an immunochromatographic test card for the diagnosis and screening of nasopharyngeal carcinoma, which is the immunochromatographic test card described in the fourth aspect above. BRIEF DESCRIPTION OF DRAWINGS

[0060] Figure 1 is the EAD-P47 / 54 protein sequence shown in SEQ ID NO. 1;

[0061] Figure 2 is a schematic diagram of the test card structure; A: colloidal gold pad, the upper part overlaps with the NC membrane; B: sample pad, the lower part overlaps with the edge of the PVC bottom plate; C: blotting paper, the lower part overlaps with the NC membrane;

[0062] Figure 3 shows the colloidal gold signal value results of 61 subjects (33 nasopharyngeal carcinoma patients and 28 healthy people);

[0063] Figure 4 is a receiver operating characteristic curve of the colloidal gold signal value of the plasma samples of 61 subjects (33 nasopharyngeal carcinoma patients and 28 healthy people);

[0064] Figure 5 is the EA-IgG detection result of 27 normal fingertip blood (capillary blood) samples under colloidal gold methodology;

[0065] Figure 6 is the consistency comparison result of the EA-IgG detection result of 15 normal fingertip blood samples and venous blood plasma samples under colloidal gold methodology;

[0066] Figure 7 is the result of the ELISA signal value of the plasma samples of 61 subjects (33 nasopharyngeal carcinoma patients and 28 healthy people);

[0067] Figure 8 is a receiver operating characteristic curve of the ELISA signal value of the plasma samples of 61 subjects (33 nasopharyngeal carcinoma patients and 28 healthy people);

[0068] Figure 9 is the result of the EA-IgG colloidal gold signal value of the plasma samples of 250 subjects (73 nasopharyngeal carcinoma patients and 177 healthy people) described in Example 5;

[0069] Figure 10 is a receiver operating characteristic curve of the EA-IgG colloidal gold signal value of the plasma samples of 250 subjects (73 nasopharyngeal carcinoma patients and 177 healthy people) described in Example 5;

[0070] Figure 11 is the result of the Zta-IgG colloidal gold signal value of the plasma samples of 250 subjects (73 nasopharyngeal carcinoma patients and 177 healthy people) described in Example 5;

[0071] Figure 12 is a receiver operating characteristic curve of the Zta-IgG colloidal gold signal value of the plasma samples of 250 subjects (73 nasopharyngeal carcinoma patients and 177 healthy people) described in Example 5;

[0072] Figure 13 is the result of the EBNA1-IgA colloidal gold signal value of the plasma samples of 250 subjects (73 nasopharyngeal carcinoma patients and 177 healthy people) described in Example 5;

[0073] Figure 14 is a receiver operating characteristic curve of the EBNA1-IgA colloidal gold signal value of the plasma samples of 250 subjects (73 nasopharyngeal carcinoma patients and 177 healthy people) described in Example 5;

[0074] Figure 15 is a receiver operating characteristic curve of the colloidal gold signal value of the three-target joint analysis of EA-IgG, Zta-IgG and EBNA1-IgA antibodies in the plasma samples of 250 subjects (73 nasopharyngeal carcinoma patients and 177 healthy people) described in Example 5. Embodiments of the present application

[0075] The first aspect of the present application relates to a detection kit for nasopharyngeal carcinoma diagnosis and screening, comprising an immunochromatographic detection card, which comprises reagents for detecting early antigen antibodies of Epstein-Barr virus by colloidal gold labeling method.

[0076] The early antigen (EA) of Epstein-Barr virus includes EA-D (including EAD-p138, EAD-P47 / 54) and EA-R (including EAR-p30, EAR-p85). The EA detected in the present application can be EA-D, preferably EAD-P47 / 54, and more preferably the EA shown in SEQ ID NO. 1. Site mutations or improvements can be made within the region of SEQ ID NO. 1, which can also be used for the detection of EAD-P47 / 54 protein antibodies and applied to the diagnosis or screening of nasopharyngeal carcinoma and its early precancerous lesions.

[0077] The EA antibody detected in the present application can be IgG antibody, IgA antibody, etc., preferably IgG antibody.

[0078] The detection of the EA antibody of Epstein-Barr virus in the present application can be carried out by colloidal gold detection. Colloidal gold detection is a new type of immunolabeling technology that uses colloidal gold as a tracer marker for antigen-antibody.

[0079] Colloidal gold immunochromatography

[0080] Specific antigens or antibodies are fixed on the membrane in strip form, and colloidal gold labeled reagents (antibodies or monoclonal antibodies) are adsorbed / coated on a colloidal gold pad (also known as "binding pad"). When the sample to be tested is added to the sample pad at one end of the test strip / detection card, it moves forward by capillary action, dissolves the colloidal gold labeled reagents on the colloidal gold pad, and then reacts with each other. When the sample to be tested moves to the region of the fixed antigen or antibody, the combination of the sample to be tested and the gold labeled reagents specifically binds to it and is trapped, and accumulates on the detection band / line, and the color development result can be observed by the naked eye. This method has now developed into a diagnostic test strip, which is very convenient to use.

[0081] In the present application, the detection of EA antibody can be performed by sandwich method. The colloidal gold-labeled early antigen and colloidal gold-labeled antibody for quality control can be immobilized on the colloidal gold pad, the secondary antibody of the antibody to be detected can be immobilized on the test line (T line), and the secondary antibody of the antibody for control or the antigen against which the antibody is directed can be immobilized on the control line (C line). Alternatively, the colloidal gold-labeled early antigen and colloidal gold-labeled antigen for quality control can be immobilized on the colloidal gold pad, the secondary antibody of the antibody to be detected can be immobilized on the test line (T line), and the antibody against the antigen for control can be immobilized on the control line (C line).

[0082] The term "secondary antibody" used herein is an antibody that specifically binds to a certain antibody, for example, an antibody that specifically binds to human IgG - a mouse anti-human IgG antibody is a secondary antibody of human IgG.

[0083] When the sample to be detected is added to the test card (sample pad), the liquid will move to the colloidal gold pad, T line and C line in turn by chromatography.

[0084] When the sample contains the antibody to be detected, the antibody to be detected binds to the colloidal gold-labeled EA antigen on the colloidal gold pad. The antibody to be detected (which has bound to the colloidal gold-labeled antigen) is captured by the secondary antibody coated on the T line, and the colloidal gold aggregates and develops color. At the same time, the antibody for quality control or the antigen on the colloidal gold pad is captured by the secondary antibody of the antibody or the antigen against which the antibody is directed, or the antibody against the antigen coated on the C line, and the colloidal gold aggregates and develops color. Therefore, when two red lines appear on the test card, it indicates that the sample contains the antibody to be detected, which is positive.

[0085] When the sample does not contain the antibody to be detected, only the antibody for quality control or the antigen on the colloidal gold pad is captured by the secondary antibody of the antibody or the antigen against which the antibody is directed, or the antibody against the antigen coated on the C line, and one red line appears on the test card, which is negative.

[0086] In further embodiments, the antibody coated on the colloidal gold pad and labeled with colloidal gold for quality control can be chicken IgY, and accordingly the secondary antibody of chicken IgY is coated on the control line.

[0087] The second aspect of the present application relates to the use of an early antigen of Epstein-Barr virus in the preparation of an immunochromatographic test card or test kit for the diagnosis and screening of nasopharyngeal carcinoma, wherein

[0088] The test kit comprises the chromatographic test card, and the immunochromatographic test card is the immunochromatographic test card described in the first aspect above.

[0089] The third aspect of the present application relates to an immunochromatographic test card for the diagnosis and screening of nasopharyngeal carcinoma, which is the immunochromatographic test card described in the first aspect above.

[0090] The sample source for detection using the detection kit or detection card for nasopharyngeal carcinoma diagnosis and screening of the present application can be whole blood, plasma, serum, peripheral blood and the like, preferably peripheral blood sample and plasma, and more preferably peripheral blood.

[0091] The antibody used in the present application can be prepared by antibody preparation techniques known in the art (for example, hybridoma method, genetic engineering method and the like), or can be a commercially available antibody.

[0092] The present application is based on the detection of EAD-P47 / 54 protein specific to nasopharyngeal carcinoma, and adopts the colloidal gold method to achieve specific and sensitive detection of nasopharyngeal carcinoma patients from a fingertip blood sample. In group 1, fingertip blood plasma samples of 61 subjects (33 nasopharyngeal carcinoma patients and 28 healthy people) were detected, and the receiver operating characteristic curve analysis showed that the area under the curve (AUC) of EA-IgG was 0.9957, and the sensitivity of the detection was 100.00% and the specificity was 92.86% at a suitable positive threshold. In group 2, peripheral blood samples of 27 subjects (27 samples collected from normal people) were detected, and the positive rate was 11.11% and the specificity was 88.89%. In group 3, plasma samples of 15 subjects (15 samples collected from normal people) were detected simultaneously, and the coincidence rate of fingertip blood and venous blood plasma was 100%.

[0093] The detection kit of the present application has the same detection performance as the approved EA-IgG ELISA detection kit.

[0094] EMBODIMENT

[0095] The present application will be further described in detail below in combination with the drawings and specific embodiments of the present application, and the embodiments are only used to explain the present application and are not used to limit the scope of the present application. In the following examples, the test methods used are conventional methods unless otherwise specified; and the materials, reagents and the like used are commercially available reagents and materials unless otherwise specified.

[0096] Example 1 Preparation of colloidal gold immunochromatographic detection card

[0097] 1. Materials and methods

[0098] EBV-EA recombinant antigen is the EB virus early antigen EAD-P47 / 54 shown in SEQ ID NO. 1.

[0099] Murine anti-human IgG antibody was purchased from Guangdong Feipeng Biological Co., Ltd.

[0100] Chicken IgY and goat anti-chicken IgY antibody were purchased from Wuhan Aokebotai Biological Technology Co., Ltd.

[0101] 2. This embodiment relates to the preparation of an EA-IgG antibody detection card.

[0102] In the EB virus early antigen (EA) IgG antibody detection kit (colloidal gold method), the detection card (also referred to as "reagent strip") structure is shown in Figure 1, and the detection card solid carrier (test strip) includes four parts of sample pad, colloidal gold pad, NC (nitrocellulose) membrane, and absorbent paper.

[0103] Detection card preparation process:

[0104] 2.1 Coating of nitrocellulose membrane (coating of detection line and quality control line)

[0105] Spray 1 μL / cm of goat anti-chicken IgY antibody (concentration 1 mg / ml) on the quality control line with quality control line buffer (containing 0.3% trehalose and 10 mM PBS (pH 7.0)), and spray 1 μL / cm of mouse anti-human IgG antibody (concentration 1 mg / ml) on the detection line with detection line buffer (containing 0.3% trehalose and 10 mM PBS (pH 7.0)), and control the drying temperature at 45±1°C, and the drying time is 12-24 hours.

[0106] 2.2 Pretreatment and coating of colloidal gold pad

[0107] Uniformly add 6% sucrose, 0.3% polyethylene glycol 6000, and 10 mM PBS (pH 7.0) to the colloidal gold pad, control the drying temperature at 45±1°C, and the drying time is 12-24 hours. Prepare the colloidal gold solution (containing EBV-EA recombinant antigen, chicken IgY is 1 mg / ml) with colloidal gold diluent (containing 5% sucrose, 5% trehalose, 0.3% polyethylene glycol 6000, and 10 mM PBS (pH 7.0)) to the working concentration, and uniformly coat the pretreated colloidal gold pad, control the drying temperature at 45±1°C, and the drying time is 12-24 hours.

[0108] 2.3 Treatment of sample pad

[0109] Uniformly add 0.3% sucrose, 0.2% HIER-M-004, and 10 mM PBS (pH 7.0) to the sample pad, control the drying temperature at 45±1°C, and the drying time is 12-24 hours.

[0110] 2.4 Pasting of components

[0111] In a clean room with relative humidity below 30%, paste the intermediate products (coated nitrocellulose membrane, colloidal gold pad, absorbent paper, treated sample pad) on the PVC bottom plate in the order required.

[0112] 2.5 Cutting, loading, and bagging

[0113] In the clean workshop, the pasted PVC base plate is cut into 4.0±0.1 mm wide semi-finished test paper strips with a slitting machine, the semi-finished test paper strips are assembled in a plastic test card, and the test card is pressed tightly with a card pressing machine. The corresponding position of the closed test card rear bottom cover is pasted with a bar code corresponding to the product batch number, and the assembled test card and desiccant are loaded into an aluminum foil bag and sealed with a heat sealing machine.

[0114] Example 2 Detection performance of EA-IgG target in plasma samples for nasopharyngeal carcinoma under colloidal gold methodology

[0115] 1. Main reagents and materials

[0116] (1) EB virus early antigen (EA) IgG antibody detection kit (colloidal gold method), including the test card prepared in Example 1, sample diluent (Tris-HCl solution containing surfactant Tween);

[0117] (2) 20 μL of plasma sample;

[0118] (3) Colloidal gold card reader;

[0119] 2. Specific implementation steps

[0120] (1) When stored at low temperature, the kit should be restored to room temperature (18-26°C) before use;

[0121] (2) Tear open the outer packaging, take out the test card, and place it flat on the operation table;

[0122] (3) Add 20 μL of plasma sample to the reaction well;

[0123] (4) Immediately add 2 drops of sample diluent (about 80 μL);

[0124] (5) Observe the results for 10-15 minutes, and do not read the results after 15 minutes.

[0125] 3. Result analysis

[0126] 61 cases of subjects (33 cases of nasopharyngeal carcinoma (NPC) patients, 28 cases of healthy people (as control, Control) plasma sample test results show that the kit has 100% detection rate for 33 cases of nasopharyngeal carcinoma patients, and can specifically distinguish 26 cases of healthy people, realizing the specific and sensitive detection of nasopharyngeal carcinoma. Receiver operating characteristic curve analysis (software: GraphPad Prism9.0) shows that the area under the curve (AUC) is 0.9957, and under the appropriate positive threshold (Cut off), the sensitivity of the detection is 100.00%, and the specificity is 92.86%. See Figures 3-4 and Tables 1-2.

[0127] This embodiment is the performance verification of EA-IgG target detection, which can have better diagnostic analysis performance for nasopharyngeal carcinoma.

[0128] Example 3 Consistency comparison of detection performance of EA-IgG target in fingertip blood (peripheral blood) sample and venous blood plasma sample under colloidal gold methodology

[0129] 1. Main reagents and materials

[0130] (1) EB virus early antigen (EA) IgG antibody detection kit (colloidal gold method) described in Example 2;

[0131] (2) 20 μL of venous blood plasma sample and 20 μL of fingertip blood sample from the same person;

[0132] (3) Colloidal gold card reader.

[0133] 2. Specific implementation steps

[0134] (1) When stored at low temperature, the kit should be restored to room temperature (18-26°C) before use;

[0135] (2) Open the outer package, take out the detection card, and place it flat on the operation table;

[0136] (3) Add 20 μL of plasma sample or 20 μL of peripheral blood, respectively;

[0137] (4) Immediately add 2 drops of sample diluent (about 80 μL);

[0138] (5) Observe the results for 10-15 minutes, and do not read the results after 15 minutes.

[0139] 3. Results analysis

[0140] The fingertip blood (peripheral blood) samples and plasma samples of the above 61 subjects (33 nasopharyngeal carcinoma (NPC) patients, 28 healthy people) were detected by the EB virus early antigen (EA) IgG antibody detection kit. The detection results show that the kit can detect peripheral blood samples with fingertip blood samples, and the detection performance is consistent with that of venous blood plasma samples. The fingertip blood samples of 15 normal people and the venous blood plasma samples have the same detection rate, and the coincidence rate is 100%. See Figures 5-6.

[0141] The embodiment case shows that the kit can use fingertip blood and venous blood as detection samples, and the detection performance of the two sample types is consistent.

[0142] Example 4: Under different methodologies (colloidal gold method and ELISA method), EA-IgG target achieves specific and sensitive detection of nasopharyngeal carcinoma in plasma samples

[0143] 1. Main reagents and materials

[0144] (1) EB virus early antigen (EA) IgG antibody detection kit (colloidal gold method) described in Example 2;

[0145] (2) 40 μL of plasma sample;

[0146] (3) Colloidal gold card reader;

[0147] (4) Enzyme reader;

[0148] (5) EB virus early antigen (EA) IgG antibody detection kit (enzyme-linked immunoassay (ELISA)), purchased from Beijing Bell Bioengineering Co., Ltd.

[0149] 2. Specific implementation steps

[0150] (1) The colloidal gold detection EB virus early antigen (EA) IgG antibody detection kit (colloidal gold method) detection method refers to the specific implementation steps in Example 2;

[0151] (2) ELISA detection is performed according to its instructions.

[0152] 3. Results analysis

[0153] The plasma samples of the above 61 subjects (33 nasopharyngeal carcinoma (NPC) patients and 28 healthy people) were detected by the EB virus early antigen (EA) IgG antibody detection kit and the ELISA kit. The detection results of the plasma samples of the above 61 subjects showed that under the colloidal gold methodology, the receiver operating characteristic curve analysis (software: GraphPad Prism 9.0) showed that the area under the curve (AUC) was 0.9957, and under the appropriate positive threshold, the sensitivity of the detection was 100.00%, and the specificity was 92.86%; under the ELISA methodology, the receiver operating characteristic curve analysis (software: GraphPad Prism 9.0) showed that the area under the curve (AUC) was 0.9911, and under the appropriate positive threshold, the sensitivity of the detection was 84.85%, and the specificity was 96.43%; at the same time, under the colloidal gold methodology, the detection rate of nasopharyngeal carcinoma in the early stage (stage I and stage II) was 100%, and under the ELISA methodology, the detection rate of nasopharyngeal carcinoma in the early stage (stage I and stage II) was 80%. See FIGS. 3-4, 57-8 and Tables 1-2.

[0154] Tables 1 and 2 are a comparison of the sensitivity and specificity of 33 nasopharyngeal carcinoma patients and 28 normal people under the ELISA method and the colloidal gold methodology.

[0155] Table 1

[0156]

[0157] Table 2

[0158]

[0159] The performance verification of the EA-IgG plasma method in this embodiment example can be expected that under the colloidal gold methodology, the EA-IgG target has better diagnostic analysis performance for nasopharyngeal carcinoma.

[0160] Example 5 Detection performance of EA-IgG, Zta-IgG and EBNA1-IgA three targets on nasopharyngeal carcinoma plasma samples

[0161] 1. Main reagents and materials

[0162] (1) EB antigen antibody detection kit (colloidal gold immunochromatography method), including EA-IgG antibody detection card, Zta antigen IgG antibody detection card and EBNA1-IgA antibody detection card prepared according to the method described in Example 1, sample diluent (Tris-HCl solution containing surfactant Tween and physiological saline solution); wherein:

[0163] EBV-EA, Zta antigen and EBNA1 (i.e. Epstein-Barr virus nuclear antigen 1, EBNA1) antigen, respectively, are the EB virus early antigen EADP47 / 54 shown as SEQ ID NO. 1, the EB virus Zta antigen shown as SEQ ID NO. 2 and the EB virus EBNA1 antigen shown as SEQ ID NO. 3;

[0164] The EA-IgG antibody detection card is prepared according to the method described in Example 1, the Zta antigen IgG antibody detection card is prepared according to the method described in Example 1 by replacing EA with Zta antigen, and the EBNA1-IgA antibody detection card is prepared according to the method described in Example 1 by replacing EA with EBNA1 antigen.

[0165] Murine anti-human IgG antibody, purchased from Guangdong Feipeng Biological Co., Ltd.; murine anti-human IgA antibody, purchased from Ningbo Maiyue Biological Co., Ltd.

[0166] Chicken IgY and goat anti-chicken IgY antibody, purchased from Wuhan Aokebotai Biological Technology Co., Ltd.

[0167] (2) 30 μL of plasma sample;

[0168] (3) Colloidal gold card reader.

[0169] 2. Specific implementation steps

[0170] (1) When stored at low temperature, the kit should be restored to room temperature (18-26℃) before use;

[0171] (2) Tear open the outer packaging, take out the detection card, and place it flat on the operation table;

[0172] (3) Add 20 μL of plasma sample to the reaction well;

[0173] (4) Immediately add 2 drops of sample diluent (about 80 μL);

[0174] (5) Observe the results for 10-15 minutes, and do not read the results after 15 minutes

[0175] 3. Result analysis

[0176] Referring to Table 3, the detection results of the plasma samples of 250 subjects (73 nasopharyngeal carcinoma patients and 177 healthy people) showed that the target EA-IgG had a detection rate of 89.04% (65 / 73) for 73 nasopharyngeal carcinoma patients, and could specifically distinguish 177 healthy people, achieving specific and sensitive detection of nasopharyngeal carcinoma. Receiver operating characteristic curve analysis (software: GraphPad Prism 9.0) showed that the area under the curve (AUC) was 0.950, and under the appropriate positive threshold, the sensitivity of the detection was 89.04% and the specificity was 91.53%. The target Zta-IgG had a detection rate of 87.67% (64 / 73) for 73 nasopharyngeal carcinoma patients, and could specifically distinguish 177 healthy people, achieving specific and sensitive detection of nasopharyngeal carcinoma. Receiver operating characteristic curve analysis (software: GraphPad Prism 9.0) showed that the area under the curve (AUC) was 0.891, and under the appropriate positive threshold, the sensitivity of the detection was 87.67% (64 / 73) and the specificity was 85.31%. The target EBNA1-IgA had a detection rate of 87.67% (64 / 73) for 73 nasopharyngeal carcinoma patients, and could specifically distinguish 177 healthy people, achieving specific and sensitive detection of nasopharyngeal carcinoma. Receiver operating characteristic curve analysis (software: GraphPad Prism 9.0) showed that the area under the curve (AUC) was 0.922, and under the appropriate positive threshold, the sensitivity of the detection was 87.67% and the specificity was 89.27%.

[0177] Using a binary logistic regression statistical method to analyze the three targets EA-IgG, Zta-IgG and EBNA1-IgA, the logistic regression formula Logit (P) = -2.991 + 1.216 *[EA-IgG] assignment + 0.488 *[Zta-IgG] assignment + 2.223 *[NA1-IgA] assignment was obtained, and the corresponding colloidal gold signal value was substituted. The joint three targets had a detection rate of 94.52% (69 / 73) for 73 nasopharyngeal carcinoma patients, and could specifically distinguish 177 healthy people, achieving specific and sensitive detection of nasopharyngeal carcinoma. Receiver operating characteristic curve analysis (software: IBM SPSS Statistics 24) showed that the area under the curve (AUC) was 0.959, and under the appropriate positive threshold, the sensitivity of the detection was 94.52% and the specificity was 90.40%. See Figures 9-15 and Table 3.

[0178] From the above data, the detection performance of the joint three targets is the best, which can significantly improve the detection performance of nasopharyngeal carcinoma.

[0179] Table 3

[0180]

[0181] Case 6 Detection performance of EA-IgG target, Zta-IgG target and EBNA1-IgA target on the staging of nasopharyngeal carcinoma

[0182] 1. Main reagents and materials

[0183] (1) The EB antigen antibody detection kit (colloidal gold immunochromatography) described in Example 5, comprising the detection card prepared in Example 5, sample diluent (Tris-HCl solution containing surfactant Tween and physiological saline solution);

[0184] (2) 30 μL of plasma sample;

[0185] (3) Colloidal gold card reader.

[0186] 2. Specific implementation steps

[0187] (1) When stored at low temperature, the kit should be restored to room temperature (18-26°C) before use;

[0188] (2) Tear open the outer packaging, take out the detection card, and place it flat on the operation table;

[0189] (3) Add 20 μL of plasma sample to the reaction well;

[0190] (4) Immediately add 2 drops of sample diluent (about 80 μL);

[0191] (5) Observe the results for 10-15 minutes, and do not read the results after 15 minutes.

[0192] 3. Result analysis

[0193] 73 plasma samples of nasopharyngeal carcinoma patients, including 2 samples of stage I nasopharyngeal carcinoma, 4 samples of stage II nasopharyngeal carcinoma, 26 samples of stage III nasopharyngeal carcinoma, 39 samples of stage IV nasopharyngeal carcinoma, and 2 samples of un-staged nasopharyngeal carcinoma.

[0194] As shown in Table 4 and Table 5, the detection results show that the detection rate of target point EA-IgG for I stage nasopharyngeal carcinoma sample is 50%, the detection rate of II stage sample is 100%, the detection rate of III stage sample is 88.46%, the detection rate of IV stage sample is 89.74%, the detection rate of early stage nasopharyngeal carcinoma (I+II stage) is 83.33%, and the detection rate of late stage (III+IV stage) is 89.23%; the detection rate of target point Zta-IgG for I stage nasopharyngeal carcinoma sample is 50%, the detection rate of II stage sample is 75%, the detection rate of III stage sample is 80.77%, the detection rate of IV stage sample is 94.87%, the detection rate of early stage nasopharyngeal carcinoma (I+II stage) is 66.67%, and the detection rate of late stage (III+IV stage) is 89.23%; the detection rate of target point EBNA1-IgA for I stage nasopharyngeal carcinoma sample is 50%, the detection rate of II stage sample is 75%, the detection rate of III stage sample is 84.62%, the detection rate of IV stage sample is 92.31%, the detection rate of early stage nasopharyngeal carcinoma (I+II stage) is 66.67%, and the detection rate of late stage (III+IV stage) is 89.23%.

[0195] The combined three target points have a detection rate of 94.52% for 73 nasopharyngeal carcinoma patients (see Table 3), the detection rate for I stage nasopharyngeal carcinoma sample is 100%, the detection rate for II stage sample is 75%, the detection rate for III stage sample is 92.31%, the detection rate for IV stage sample is 97.44%, the detection rate for early stage nasopharyngeal carcinoma (I+II stage) is 83.33%, and the detection rate for late stage (III+IV stage) is 95.38%.

[0196] Table 4

[0197]

[0198] Table 5

[0199]

[0200] From the above data, it can be seen that the detection performance of the combined three target points is the best, and the detection performance for early stage nasopharyngeal carcinoma is significantly improved.

[0201] The application is based on the detection of serum antibodies of EAD-P47 / 54 protein, Zta protein and EBNA1 protein specific to nasopharyngeal carcinoma, and can realize specific and sensitive detection of nasopharyngeal carcinoma patients by using a colloidal gold method. Plasma samples of 250 subjects (73 nasopharyngeal carcinoma patients and 177 healthy people) in group 1 were detected, and binary logistic regression statistical method was used to analyze EA-IgG, Zta-IgG and EBNA1-IgA three targets, and the logistic regression formula Logit (P) =-2.991+1.216*[EA-IgG] assignment+0.488*[Zta-IgG] assignment+2.223*[NA1-IgA] assignment was obtained, and the corresponding colloidal gold signal value was assigned and calculated; the receiver operating characteristic curve analysis (software: IBM SPSS Statistics 24) showed that the area under the curve (AUC) was 0.968, and under the appropriate positive threshold, the sensitivity of the detection was 94.52% and the specificity was 90.40%. In group 2 (73 nasopharyngeal carcinoma patients), the combined three targets had a detection rate of 94.52% for 73 nasopharyngeal carcinoma patients, a detection rate of 100% for stage I nasopharyngeal carcinoma samples, a detection rate of 75% for stage II samples, a detection rate of 92.31% for stage III samples, and a detection rate of 97.44% for stage IV samples. The detection rate of early nasopharyngeal carcinoma (I+II) was 83.33%, and the detection rate of advanced nasopharyngeal carcinoma (III+IV) was 95.38%.

[0202] The present application shows that the single target detection and three target joint detection using EA-IgG target, Zta-IgG target and EBNA1-IgA target can realize specific and sensitive detection of nasopharyngeal carcinoma patients by using colloidal gold method, and the effect is at least equivalent to the detection result of ELISA.

[0203] According to the disclosure of the present application, all the detection cards and detection kits disclosed and claimed herein can be prepared and used without undue experimentation. Although the detection cards and detection kits and detection methods have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes can be made to the reagents in the detection cards and detection kits described herein without departing from the concept, spirit and scope of the present application. More specifically, certain reagents described herein can be replaced with chemically and physiologically related reagents, while achieving the same or similar results. All such similar substitutions and modifications that are obvious to those skilled in the art are considered to be within the spirit, scope and concept of the present application as defined by the appended claims.

[0204] References

[0205] [1] Parkin D M, Whelan S L, Ferlay J, et al. Cancer incidence in five continents volume VIII[J]. IARC Sci. Publications, 2002, 155.

[0206] [2] Chen W, Zheng R, Baade P D, et al. Cancer statistics in China, 2015[J]. CA: a cancer journal for clinicians, 2016, 66(2): 115-132.

[0207] [3] Li T, Li F, Guo X, et al. Anti–Epstein–Barr Virus BNLF2b for Mass Screening for Nasopharyngeal Cancer[J]. The New England journal of medicine, 2023(9):389.

[0208] [4] Damania B, Kenney SC, Raab-Traub N. Epstein-Barr virus: Biology and clinical disease. Cell. 2022 Sep 29;185(20):3652-3670. doi:10.1016 / j.cell.2022.08.026. Epub 2022 Sep 15.

[0209] [5] Ai P, Wang T, Zhang H, et al. Determination of antibodies directed at EBV proteins expressed in both latent and lytic cycles in nasopharyngeal carcinoma[J]. Oral Oncology, 2013, 49(4):326-331.

[0210] [6] Dardari R, Hinderer W, Lang D, et al. antibody responses to recombinant epstein-barr virus antigens in nasopharyngeal carcinoma patients: complementary test of zebra protein and early antigens p54 and p138 downloaded from [J]. Journal of clinical microbiology, 39(9):3164-70.

[0211] [7] Tan L P, Tan G W, Sivanesan V M, et al. Systematic comparison of plasma EBV DNA, anti-EBV antibodies and miRNA levels for early detection and prognosis of nasopharyngeal carcinoma. [J]. Wiley, 2020(8).

[0212] [8] Chen Y, Chan ATC, Le Q, Blanchard P, Sun Y, Ma J. Nasopharyngeal carcinoma. Lancet. 2019;394:64-80. https: / / doi.org / 10.1016 / S0140 -6736(19)30956-0..

[0213] [9] Liu W, Chen G, Gong X, Wang Y, Zheng Y, Liao X, Liao W, Song L, Xu J, Zhang X. The diagnostic value of EBV-DNA and EBV-related antibodies detection for nasopharyngeal carcinoma: a meta-analysis. Cancer Cell Int. 2021 Mar 10;21(1):164. doi: 10.1186 / s12935-021-01862-7. PMID: 33691680; PMCID: PMC7944913.

[0214]

[0010] Chan, K. H., Gu, Y.et al. EBV specific antibody‐based and DNA‐based assays in serologic diagnosis of nasopharyngeal carcinoma. International journal of cancer, 105(5), 706-709.

[0215]

[0011] Li T, Li F, Guo X,et al.Anti–Epstein–Barr Virus BNLF2b for Mass Screening for Nasopharyngeal Cancer[J].The New England journal of medicine, 2023(9):389.

Claims

1. A test kit for diagnosing and screening nasopharyngeal carcinoma, comprising an immunochromatographic test card, wherein the immunochromatographic test card comprises a reagent for detecting Epstein-Barr virus early antigen antibodies by colloidal gold labeling. The early antigen is preferably EAD-P47 / 54, more preferably having the sequence shown in SEQ ID No. 1, and / or The antibody is preferably an IgG antibody.

2. The detection kit according to claim 1, wherein the detection card comprises a sample pad, a colloidal gold pad, a detection line T, a quality control line C and absorbent paper according to the sample flow direction, the colloidal gold pad is coated with the EB virus early antigen labeled with colloidal gold, and the detection line T is coated with a secondary antibody of the antibody to be tested, and the secondary antibody is preferably an anti-human IgG antibody.

3. The detection kit according to claim 1 or 2, wherein the detection card further comprises a reagent for quality control.

4. The detection kit according to claim 3, wherein the reagent for quality control is: The colloidal gold-labeled antigen coated in the colloidal gold pad, and the antibody against the antigen coated on the control line C, or The colloidal gold-labeled antibody coated in the colloidal gold pad, and the secondary antibody of the antibody coated on the quality control line C or the antigen targeted by the antibody, Preferably, the colloidal gold-labeled antibody is chicken IgY, and correspondingly, the secondary antibody of chicken IgY is coated on the quality control line.

5. Use of Epstein-Barr virus early antigen in the preparation of an immunochromatographic test card or test kit for the diagnosis and screening of nasopharyngeal carcinoma, wherein: The detection kit comprises the immunochromatographic detection card, and the immunochromatographic detection card is the immunochromatographic detection card according to any one of claims 1 to 4.

6. An immunochromatographic test card, which is the immunochromatographic test card according to any one of claims 1 to 4.

7. A test kit for diagnosing and screening nasopharyngeal carcinoma, comprising an immunochromatographic test card comprising a reagent for detecting Epstein-Barr virus antigens and antibodies by colloidal gold labeling, wherein: The immunochromatographic detection card includes an immunochromatographic detection card for detecting Zta-IgG antibodies and / or an immunochromatographic detection card for detecting EBNA-IgA antibodies, and may further include an immunochromatographic detection card for detecting early antigen (EA)-IgG antibodies; wherein: The EA is preferably EAD-P47 / 54, more preferably it has the sequence shown in SEQ ID No. 1, the Zta preferably has the sequence shown in SEQ ID No. 2, and the EBNA is preferably Epstein-Barr virus nuclear antigen 1 (EBNA1), more preferably it has the sequence shown in SEQ ID No.

3.

8. The detection kit according to claim 1, wherein the detection card comprises a sample pad, a colloidal gold pad, a detection line T, a quality control line C and absorbent paper according to the sample flow direction, the colloidal gold pad is coated with the EB virus early antigen labeled with colloidal gold, and the detection line T is coated with a secondary antibody of the antibody to be tested, and the secondary antibody is preferably an anti-human IgG antibody.

9. The detection kit according to claim 7 or 8, wherein the detection card further comprises a reagent for quality control.

10. The detection kit according to claim 9, wherein the reagent for quality control is: The colloidal gold-labeled antigen coated in the colloidal gold pad, and the antibody against the antigen coated on the control line C, or The colloidal gold-labeled antibody coated in the colloidal gold pad, and the secondary antibody of the antibody coated on the quality control line C or the antigen targeted by the antibody, Preferably, the colloidal gold-labeled antibody is chicken IgY, and correspondingly, the secondary antibody of chicken IgY is coated on the quality control line.

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