Diagnostic reagent kit (enzyme-linked immunosorbent assay (ELISA)) for enterovirus (EV) 71-type antibody (immune globulin M (IgM))
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An enterovirus and enzyme-linked immunosorbent technology, applied in the field of biomedicine, can solve the problems of kit specificity, insufficient sensitivity and stability, inability to supply the market in large quantities, and high cost of virus culture, so as to achieve improved sensitivity and specificity, practical Strong, avoiding the effect of interfering factors
Inactive Publication Date: 2011-11-16
BEIJING BEIER BIOENG
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[0005] The detection of enterovirus 71 antibody (IgM) has been reported in the relevant literature. Basically, the virus culture of enterovirus 71 is used as the antigen as the indirect ELISA of the coated antigen. The kit has the advantages of specificity, sensitivity and stability. There are still many deficiencies in this aspect; and, due to the high cost of virus culture and low efficiency, it is impossible to supply the market in large quantities.
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example 1
[0056] Example 1. Preparation of anti-human-IgM (u chain) monoclonal antibody pre-coated microwell plate
[0057] 1.1 Coating
[0058] Na 2 CO 3 1.6g
[0059] NaHCO 3 2.9g
[0060] Deionized water 1000ml,
[0061] Adjust the pH to 9.6, add an appropriate amount of anti-human-IgM (μ chain) monoclonal antibody, mix well and add 100ul (microliter) per well into the microwell plate, place at 37°C for 2 hours, room temperature overnight, then shake off the well Inner liquid, pat dry;
[0062] 1.2 Washing
[0063] Na 2 HPO 4 .12H 2 O 3.72g
[0064] NaH 2 PO 4 0.4g
[0065] NaCl 6.8g
[0066] Tween-20 0.5ml
[0067] Deionized water 1000ml,
[0068] Adjust the pH to 7.2 and add 150ul per well into the microwell plate, then shake off, repeat washing once, and pat dry.
[0069] 1.3 Closed
[0070] Na 2 HPO 4 .12H 2 O 3.72g
[0071] NaH 2 PO 4 0.4g
[0072] NaCl 6.8g
[0073] Bovine Serum 100.0ml
[0074] Deionized water 1000ml,
...
example 2
[0079] Example 2. Preparation of enzyme-labeled antigen working solution
[0080] Na 2 HPO 4 .12H 2 O 3.72g
[0081] NaH 2 PO 4 0.4g
[0082] NaCl 6.8g
[0083] Bovine Serum 100.0ml
[0084] Casein 2.5g
[0085] Calf Serum 100ml
[0086] Proclin300 1ml
[0087] Deionized water 900ml,
[0088] Adjust the pH to 7.2, add an appropriate amount of recombinant enterovirus 71 VP1 antigen labeled with horseradish peroxidase, and store at 2-8°C.
example 3
[0089] Example 3. Preparation of Positive Control
[0090] take A 450 Mix multiple human sera positive for anti-EV71-IgM with a value ≥ 2.0, heat in a water bath at 56°C for 120 minutes, cool naturally, and adjust to A450 value > 1.0 with 0.01mol / L PBS, 20% BSA, pH 7.2 buffer , adding five ten thousandths of sodium thimerosal, stored at 2 ~ 8 ℃ environment.
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Abstract
The invention relates to the field of biomedicine, in particular to an enzyme-linked immunization diagnostic reagent kit for detecting an enterovirus (EV) 71-type antibody (immune globulin M (IgM)), and a preparation method and application of the diagnostic reagent kit. The probability of hand-foot-and-mouth disease and severe infection (viral encephalitis, viral cerebrospinal meningitis and pulmonary edema) caused by EV71 type is relatively higher, and case fatality rate is relatively higher and can be 10 to 25 percent. The enzyme-linked immunization diagnostic reagent kit of the EV71-IgM antibody can be used for diagnosing the infection of the EV71 type. According to related documents about the detection of the EV71-IgM, EV71 virus cultures serving as indirect enzyme-linked immuno sorbent assay (ELISA) of envelope antigens has defects in such aspects as specificity, sensitivity and stability, and due to high cultivation cost and low efficiency, a large amount of virus cannot be supplied to the market. In order to overcome the defects, the invention provides the reagent kit which is used for detecting the EV71-IgM in human blood serum, required by clinical examination, simple and convenient to operate and applicable to all medical disease control departments, and the preparation method and the application of the reagent kit. The invention has the technical scheme that: firstly, the human blood serum is added into a micro-pore plate, wherein the IgM antibody is obtained by an anti-mu chain which is pre-enveloped on the micro-pore plate, and other uncombined components are washed and removed; secondly, an enzyme labeling object is added, the EV71-IgM in the obtained IgM can be combined with the specificity of an EV71 recombinant antigen which is labeled by horse radish peroxidase (HRP), and after washing, the HRP can react with substrates which are added subsequently; and finally, the aim of detecting the EV71-IgM antibody is fulfilled.
Description
Technical field: [0001] The invention relates to the field of biomedicine, in particular to an enzyme-linked immunoassay kit for detecting enterovirus 71 antibody (IgM) and a preparation method and application thereof. technical background: [0002] Enterovirus EV71 infection is a general term for diseases caused by human enterovirus 71 (EV71). Clinically, it mainly manifests as hand, foot and mouth disease. Hand, foot and mouth disease often occurs in infants and young children. Some children with EV71 infection manifest Herpetic angina, viral encephalitis, viral meningitis, pulmonary edema, pulmonary hemorrhage, etc. in severe patients. The most common pathogens causing hand, foot and mouth disease are Coxsackievirus A16 and enterovirus 71; diseases caused by enterovirus 71 infection have a large proportion of severe infections, and the fatality rate is also high. The rate can reach 10%-25%, so the etiological diagnosis is of great significance. [0003] Enterovirus type...
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