Human ECHO-IgM (Enteric Cytopathic Human Orphan virus-Immunoglobulin M) ELISA Kit (HUFI04723)

Product Name:	Human Echovirus (ECHO) IgM ELISA Kit
Product Code:	HUFI04723
Size:	96 Assays
Alias:	ECHO-IgM ELISA Kit
Detection method:	Trapping ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human ECHO-IgM (Enteric Cytopathic Human Orphan virus-Immunoglobulin M) concentrations in serum plasma and other biological fluids.
Sensitivity:	Qualitative
Range:	Qualitative
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:	Matrices listed below were spiked with certain level of Human ECHO-IgM (Enteric Cytopathic Human Orphan virus-Immunoglobulin M) and the recovery rates were calculated by comparing the measured value to the expected amount of Human ECHO-IgM (Enteric Cytopathic Human Orphan virus-Immunoglobulin M) in samples. Enquire for more information.
Linearity:	The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human ECHO-IgM (Enteric Cytopathic Human Orphan virus-Immunoglobulin M) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. Enquire for more information.
CV(%):	Intra-Assay: CV<8% Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate(Dismountable)	8×12 strips	2-8°C
Negative Control	1 vial	2-8°C/-20°C
Positive Control	1 vial	2-8°C
Sample Dilution Buffer	12ml	4°C (Protect from light)
Human ECHO-IgM (Enteric Cytopathic Human Orphan virus-Immunoglobulin M) antigen	6ml	2-8°C
HRP-Conjugate	12ml	2-8°C
TMB Substrate A	6ml	2-8°C
TMB Substrate B	6ml	2-8°C (Protect from Light)
Stop solution	6ml	-
Wash Buffer (20X)	25ml	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step	Protocol
1.	Label the sample wells, 1-3 Negative Controls, 1-2 Positive Controls and 1 blank wells.
2.	Add 100uL Negative Controls and Positive Controls to each wells.
3.	Add 100uL sample dilution buffer to sample wells and then add 10uL sample serum or plasma. Gently tap the plate to ensure thorough mixing. Seal the plate with a cover and incubate at 37°C for 60 min.
4.	Remove the cover, and wash plate 5 times with Wash buffer and let the wash buffer stay in the wells for 1- 2 minute each time.
5.	Add 50 uL Human ECHO-IgM (Enteric Cytopathic Human Orphan virus-Immunoglobulin M)-antigen to each well and gently tap the plate to ensure thorough mixing. Then add 50 µL HRP- Conjugates to each well, except blank well.
6.	Seal the plate with a cover and incubate at 37°C for 30 min.
7.	Remove the cover, and wash plate 5 times with Wash buffer and let the wash buffer stay in the wells for 1- 2 minute each time.
8.	Add 50 ul of TMB substrate A and 50 ul of TMB substrate B into each well. Gently tap the plate to ensure thorough mixing. Cover the plate and incubate at 37°C in dark within 15 min. And the shades of blue can be seen in the Positive Controls. Negative Controls wells show no obvious color.
9.	Add 50 ul of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
10.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution. (Use the blank well to set zero)

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	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.