Product Name:	Mouse Monkeypox Virus (MPXV) IgM ELISA Kit
SKU:	AEFI01133
Size:	96T
Reactivity:	Mouse
Range:	1.563-100ng/ml
Sensitivity:	0.938ng/ml
Detection Method:	Indirect ELISA
Detection Wavelength:	OD450
Reaction Duration:	1.5 hours
Synonyms:	MPXV IgM ELISA Kit, Monkeypox virus IgM ELISA Kit

This kit is based on indirect ELISA detection method and takes 3h assay time. The microplate provided in this kit has been precoated with antigen. HRP-antibody is used as detection antibody. Add standard and properly diluted sample into relevant well respectively. After incubation, wash unbound components. Add HRP-detection antibody, then it binds with Anti-MPV IgM bound to precoated antibody. Wash unbound components and add TMB substrate solution. Then, TMB was catalyzed by HRP to produce a blue color product that turned yellow after adding a stop solution. Read the O.D. absorbance at 450nm in a microplate reader. Calculate the concentration of Anti-MPV IgM in the sample by plotting standard curve. The concentration of the target substance is proportional to the OD450 value.

Recovery:

Add a certain amount of Anti-MPV IgM into the sample. Calculate the recovery by comparing the measured value with the expected amount of Anti-MPV IgM in the sample.

Sample Type	Recovery Range(%)	Average(%)
Serum(n=5)	96-102	100
EDTA Plasma(n=5)	86-103	97
Heparin Plasma(n=5)	85-102	96

Linearity:

Dilute the sample with a certain amount of Anti-MPV IgM at 1:2, 1:4 and 1:8 to get the recovery range.

Sample Type	1:2	1:4	1:8
Serum(n=5)	88-102%	85-92%	91-100%
EDTA Plasma(n=5)	85-104%	89-100%	85-97%
Heparin Plasma(n=5)	86-95%	87-100%	88-99%

Precision:

Intra-assay Precision: samples with low, medium and high concentration are tested 20 times on the same plate.

Inter-assay Precision: samples with low, medium and high concentration are tested 20 times on three different plates.

Item	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (ng/ml)	3.07	12.18	47.85	3.11	11.87	46.35
Standard deviation	0.14	0.63	2.19	0.17	0.7	1.97
CV(%)	4.58	5.16	4.58	5.32	5.86	4.26

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	Place whole blood sample at room temperature for 2 hours or at 2-8°C overnight. Centrifuge for 20min at 1000xg and collect the supernatant to detect immediately. Or you can aliquot the supernatant and store it at -20°C or -80°C for future’s assay..
Plasma	EDTA-Na2/K2 is recommended as the anticoagulant. Centrifuge samples for 15 minutes at 1000×g 2-8°C within 30 minutes after collection. Collect the supernatant to detect immediately. Or you can aliquot the supernatant and store it at -20°C or -80°C for future’s assay. For other anticoagulant types and uses, please refer to the sample preparation guideline..
Tissue Sample	Generally tissue samples are required to be made into homogenization. Protocol is as below: 3.1. Place the target tissue on the ice. Remove residual blood by washing tissue with pre-cooling PBS buffer (0.01M, pH=7.4). Then weigh for usage. 3.2. Use lysate to grind tissue homogenates on the ice. The adding volume of lysate depends on the weight of the tissue. Usually, 9mL PBS would be appropriate to 1 gram tissue pieces. Some protease inhibitors are recommended to add into the PBS (e.g. 1mM PMSF). 3.3. Do further process using ultrasonic disruption or freeze-thaw cycles (Ice bath for cooling is required during ultrasonic disruption; Freeze-thaw cycles can be repeated twice.) to get the homogenates. 3.4. Homogenates are then centrifuged for 5 minutes at 5000×g. Collect the supernatant to detect immediately. Or you can aliquot the supernatant and store it at -20°C or -80°C for future’s assay. 3.5. Determine total protein concentration by BCA kit for further data analysis. Usually, total protein concentration for Elisa assay should be within 1-3mg/ml. Some tissue samples such as liver, kidney, pancreas which containing a higher endogenous peroxidase concentration may react with TMB substrate causing false positivity. In that case, try to use 1% H2O2 for 15min inactivation and perform the assay again. Notes:PBS buffer or the mild RIPA lysis can be used as lysates While using RIPA lysis, make the PH=7.3. Avoid using any reagents containing NP-40 lysis buffer, Triton X-100 surfactant, or DTT due to their severe inhibition for kits’ working. We recommend using 50mM Tris+0.9%NaCL+0.1%SDS, PH7.3. You can prepare by yourself or contact us for purchasing.
Cell Culture Supernatant	Collect the supernatant: Centrifuge at 2500 rpm at 2-8℃ for 5 minutes, then collect clarified cell culture supernatant to detect immediately. Or you can aliquot the supernatant and store it at -80°C for future’s assay..
Cell Lysate	5.1. Suspension Cell Lysate: Centrifuge at 2500 rpm at 2-8℃ for 5 minutes and collect cells. Then add pre- cooling PBS into collected cell and mix gently. Recollect cell by repeating centrifugation. Add 0.5-1ml cell lysate and appropriate protease inhibitor (e.g. PMSF, working concentration: 1mmol/L). Lyse the cell on ice for 30min-1h or disrupt the cell by ultrasonic disruption. 5.2. Adherent Cell Lysate: Absorb supernatant and add pre-cooling PBS to wash three times. Add 0.5-1ml cell lysate and appropriate protease inhibitor (e.g. PMSF, working concentration: 1mmol/L). Scrape the adherent cell with cell scraper. Lyse the cell suspension added in the centrifuge tube on ice for 30min-1h or disrupt the cell by ultrasonic disruption. 5.3. During lysate process, use the tip for pipetting or intermittently shake the centrifugal tube to completely lyse the protein. Mucilaginous product is DNA which can be disrupted by ultrasonic cell disruptor on ice. (3~5mm probe, 150-300W, 3~5 s/time, 30s intervals for 1~2s working). 5.4. At the end of lysate or ultrasonic disruption, centrifuge at 10000rpm at 2-8℃ for 10 minutes. Then, the supernatant is added into EP tube to detect immediately. Or you can aliquot the supernatant and store it at - 80°C for future’s assay. Notes:Read notes in tissue sample.
Other Biological Sample	Centrifuge samples for 15 minutes at 1000×g at 2-8℃. Collect the supernatant to detect immediately. Or you can aliquot the supernatant and store it at -80°C for future’s assay..

Sample Type:	Serum, Plasma, Cell culture supernatant, Cell or tissue lysate, Other liquid samples
Storage:	2-8°C(Sealed), Don't cryopreserve.
Specificity:	Specifically binds with Anti-MPV IgM , no obvious cross reaction with other analogues.