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Protocols

ELISA FAQs

Can I use other ELISA buffers (ex. PBS) and substrates (ex. ABTS or OPD) with Bethyl’s ELISA kits?

Yes, our ELISA kits contain antibodies and a reference standard to establish a curve. We test and qualify our kits using the reagents that are listed in the product specification sheet. However, other standard ELISA reagents will work with our antibodies.

Do Bethyl’s ELISA kits contain polyclonal or monoclonal antibodies?

Polyclonal

What type of samples can I use with Bethyl’s ELISA kits?

Samples can be of biological (i.e. serum, plasma, urine, feces) or cell culture origin.

Are there any stopping points during the ELISA?

Yes, after adding the coating antibody or blocking solution to the wells, the covered plates can be incubated overnight at 4 C. The coating antibody or blocking solution must be left on the plate during the incubation period. We do not recommend leaving these solutions on the plate for more than 24 hours.

How should I store my samples before use?

We recommend that you store your samples at 4 C. However, biological or cell culture samples can be initially frozen until use but should not be repeatedly freeze-thawed. Once the sample is thawed, it should be stored at 4 C.

How do I dilute my samples?

Since all experimental designs are different, it is difficult to determine an overall dilution for all samples. If you have a general idea of the concentration of your samples, you will want to make dilutions so that your samples will fall in the middle range (or linear range) of the standard curve. Otherwise, you may want to take a few of your samples and make several dilutions (ex. 1:100, 1:1000, and 1:10,000) to determine a dilution range for your samples. Each investigator must determine the best dilution for their samples.

What if my raw OD values are too low or too high?

If the raw OD values are too low, then the dilution of the HRP conjugate should be decreased (ex. Used a 1:50,000 dilution, then try a 1:25,000 dilution). If the raw OD values are too high, then the dilution of the HRP conjugate should be increased (ex. Used a 1:50,000 dilution, then try a 1:100,000 dilution).

What can I do to decrease my background?

Usually background is caused by cross contamination of samples or reagents. Make sure to always use a plate washer or wash by hand with a multi-channel pipette. Do not use the “squirt and dump” method for washing. You may also add another wash to each step. Make new reagents if background problems continue and make sure that you are not using a BSA product with an ELISA kit that may cross react (Bovine Albumin ELISA, Bovine Transferrin ELISA, Bovine IgG ELISA, Goat IgG ELISA, and others)

The quantitation of my sample is not consistent or seems to be incorrect. What can I do to correct this problem?

Make sure that you are quantitating your samples in the linear region of the curve. Samples that quantitate too close to the extreme ends of the curve may be inaccurate and difficult to reproduce.

Where can I buy the stop solution (2 M Sulfuric Acid)?

Sulfuric Acid is sold by chemical companies such as Sigma or VWR. The stock solution is 18 M and should be diluted to 2 M with distilled water.

What type of software is needed to graph a 4-parameter curve?

Some of the software choices are SoftMax Pro by Molecular Devices, KC Jr., SigmaPlot, or others. You cannot directly plot a 4-parameter curve with Microsoft EXCEL.

I do not have software that will perform a 4 parameter standard curve. What should I use to analyze my data?

You can use a linear regression curve in Microsoft EXCEL. If you use this type of curve, only use a maximum of 5 points on the curve. We recommend that you discard the upper and lower points and plot your standard curve with the 5 middle points.