Diagnostic reagent kit (enzyme-linked immunosorbent assay (ELISA)) for enterovirus (EV) 71-type antibody (immune globulin M (IgM))
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
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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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