IgM antibody joint detection device and method for coxsackievirus A16 and enterovirus 71
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A technology for coxsackie virus and enterovirus, which is applied in measurement devices, disease diagnosis, biological testing, etc., can solve the problems of high-level structure impact of amplified fragments, high RT-PCR requirements, and difficulty in finding universal primers. Novel, improved sensitivity, simple structure effect
Pending Publication Date: 2015-11-11
吉林双正医疗科技有限公司
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However, its sensitivity depends on the matching of the virus carried by the patient and the primers used on the one hand, and is also greatly affected by the higher-order structure of the amplified fragment on the other hand.
In addition, RT-PCR itself has high requirements on the operating environment and operators, and is expensive, so it is difficult to use in the majority of grassroots units
At present, only a few units in the market have RT-PCR capabilities, and it is difficult to find universal primers, and the primers currently used are genotype specific
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[0033] The preparation method of the IgM antibody combined detection device of coxsackievirus A16 and enterovirus 71 of the present invention comprises the high-specificity coxsackievirus A16 (CA16) antigen and enterovirus 71 (EV71) antigen of solid phase purification ) antigen (detection line T1, T2) and goat anti-mouse IgM polyclonal antibody (control line) of nitrocellulose membrane, glass fiber adsorbed with colloidal gold-labeled mouse anti-human IgM, sample pad, absorbent paper and other auxiliary materials are thus pasted production. Specific steps are as follows:
[0034] (a) Preparation of colloidal gold by trisodium citrate reduction method;
[0035] (b) using colloidal gold labeled mouse anti-human IgM prepared in step (a) to obtain immune colloidal gold;
[0036] (c) Diluting the immune colloidal gold in step (b) with a gold spraying buffer to obtain an immune colloidal gold solution, and spraying the immune colloidal gold solution on the glass fiber mat to prepa...
[0046] Quickly add gold chloride into heated 100ml purified water, and after the solution boils again, quickly add trisodium citrate, gold chloride: trisodium citrate 1: (0.5-2), continue to boil, and observe that the color of the solution changes from yellow to After the black turns purple again, and finally turns into a stable wine red, continue heating for 10 minutes by timing. The colloidal gold particles are 20-60nm. In this embodiment, 30nm colloidal gold is selected for the next experiment.
[0047] 2. Preparation of immunocolloidal gold
[0048] 1) Take 100ml of 30nm colloidal gold solution, add 140μl of pH regulator, mix well; let stand for 5min;
[0049] 2) In the 30nm colloidal gold solution, add mouse anti-human IgM at a ratio of 14 μg per milliliter of colloidal gold solution, a total of 1.4 mg, mix well; let st...
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Abstract
The invention relates to a joint detection device and a preparation method of helicobacter pylori urease antibodies IgM and IgG. The helicobacter pylori urease antibodies are prepared from a nitrocellulose membrane, a glass fiber adsorbing a colloidal gold labeled helicobacter pylori urease antigen and a mouse IgG, a sample pad, absorbent paper and other auxiliary materials, wherein the materials are adhered together, and the nitrocellulose membrane contains a purified high-specificity mouse-anti-human IgM and IgG antibodies, and a goat-anti-mouse antibody in a solid phase manner. The joint detection device has the advantages that the structure is simple, the conception is novel, the nitrocellulose membrane are coated with the anti-human IgM and IgG antibodies, so that the specificity is strong, and the helicobacter pylori urease antibodies IgM and IgG in a specimen are simultaneously detected without increasing the production operation complexity. Proper gold spray buffer and sample pad treating fluid are matched to effectively improve the reaction sensitivity on the basis of guaranteeing complete release of immunocolloidal gold, and under the same threshold, the use amount of the immunocolloidal gold can be reduced to save the cost. The detection device is high in sensitivity, strong in specificity, simple, convenient and strong in practicability and can realize the time-saving aim during operation.
Description
technical field [0001] The invention relates to the field of medical testing equipment, in particular to a joint detection device and method for IgM antibodies of Coxsackievirus A16 and Enterovirus 71, which can realize Coxsackievirus A16 (CA16) and Enterovirus 71 ( Sensitive, specific and rapid detection of EV71) IgM antibody. Background technique [0002] Hand, foot and mouth disease (HFMD) is an acute infectious disease in children with herpes on the skin of hands, feet and oral mucosa as the main symptoms. The disease has been reported abroad in 1957, and the hand, foot and mouth disease epidemic was first reported in Shanghai, China in 1981. In recent years, the prevalence of EV71 virus has been on the rise in the Asia-Pacific region. There are more than 20 enteroviruses (types) that cause HFMD, among which coxsackievirus A16 (CA16) and enterovirus 71 (EV71) are the most common. Usually, HFMD caused by CA16 infection is indistinguishable from HFMD caused by EV71 infec...
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