This MPOX human IgG/M antibody ELISA kit employs an indirect ELISA method. In this kit, standard 96-well plates (12 strips with 8 wells/strip) are coated with the MPOXA29 protein, which combines with the corresponding antibody present in a sample and Positive Control, which is used as a calibration curve for interpretation purposes. When a biotinylated secondary anti-human IgG/M antibody is added, a complex form between biotinylated secondary antibody and human IgG/M antibody bound to the protein coated plate. A TMB substrate is added, and a blue color is generated. The depth of color is relative to the amount of the anti-A29 IgG/M antibody present. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

The Positive Control consists of an anti-A29 antibody. A biotinylated anti-Positive Control antibody is added to the Positive Control wells and treated identically to the samples. The intensity of the resulting color (measured at 450 nm) is used as a calibration curve for interpretation purposes in the different assays.

• Microplate reader capable of measuring absorbance at 450 nm
• Precision pipettes to deliver 2 µL to 1 mL volumes
• Adjustable 1-25 mL pipettes for reagent preparation
• 100 mL and 1 liter graduated cylinders
• Absorbent paper
• Distilled or deionized water

1. Prepare all reagents, samples and standards as instructed.
2. Add 100 µL positive control, or sample to each well. Incubate 1 hour at room temperature.
3. Add 100 µL prepared Biotinylated Anti-Positive Control and Biotinylated Anti-Human IgG/M Antibody into respective wells. Incubate 1 hour at room temperature.
4. Add 100 µL prepared HRP-Streptavidin solution to each well. Incubate 30 minutes at room temperature.
5. Add 100 µL TMB One-Step Substrate Reagent to each well. Incubate 10 to 30 minutes at room temperature.
6. Add 50 µL Stop Solution to each well. Read at 450 nm immediately.