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Mr.Cao

HOW TO SETUP ELISA KITS

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Dear Mabtech.

I'm a new member. I'm starts setup a new Elisa kit

Mabtech can you suggest how to setup a kit elisa to detetion pesticides, antibiotic... I dont' know where is material like antigen, antibodies to coat in plate ? And where are conjugate, and substrate to develope a new elisa kit.

Some body can suggest me!

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Dear Mr. Cao,

Welcome to our forum! 

You write that you want to detect pesticides and antibiotics using ELISA, we don’t have any ELISAs or antibodies that can be used for this. Our focus is cytokines, immunoglobulins and apolipoproteins. 

I can give you some general ELISA information. First I think you should visit our assay principle section on our website, it is very informative and can guide you in how an ELISA assay is working: https://www.mabtech.com/knowledge-center/assay-principles/elisa-assay-principle

ELISA is an immunoassay that enables sensitive quantification of analytes in solution e.g. cell supernatants, plasma, serum, CSF, saliva etc. The assay relies on the antibodies that capture and detect the analyte of target, and the antibodies’ affinity, avidity and antigen interactions are essential for the quality of the assay. There are different types of ELISA’s, but we at Mabtech utilized Sandwich ELISA as this is the most sensitive ELISA assay.

To set up a Sandwich ELISA you need:

·       ELISA plates, high protein binding plates, there are several different available, Corning and Nunc are examples of two brands that have this kind of plates. 

·       An antibody pair with specificity against analyte you are interested in. Preferably, the antibody pair should have been validated for ELISA.
The antibody pair should contain one capture antibody and one detection antibody. If the detection antibody is biotinylated you will also need an enzyme conjugate like Streptavidin-HRP. Streptavidin binds to the biotin part on the antibody. We have SA-HRP conjugates (link here). Some detection antibodies are directly conjugated with an enzyme e.g. HRP, then the SA-HRP conjugate is not needed.

 ·       A substrate that reacts with the enzyme. In the above examples, I have mentioned HRP, and a substrate that reacts with HRP is the TMB ELISA substrate (link here).

 ·       TMB substrate needs to be stopped with e.g. H2SO4 (0.2M).

 ·       An ELISA reader that can measure the absorbance.

·       Apart from this you also need several buffers, what buffers that should be used is dependent on the analyte and the antibodies, therefore I cannot recommend any specific. In our ELISAs these are the most common buffers: Coating buffer (PBS pH 7.4). Washing buffer (PBS pH 7.4 with 0.05% Tween), blocking/incubation buffer ( PBS pH 7.4 with 0.1% BSA and 0.05%Tween20) for blocking and dilution of antibodies and samples.

I hope this can give you some advice! Good luck!

Lena Beckman

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Dear Lena Beckman

Thank you for your reply. it's so helpful for me. Thank you so much.

About 2 year ago, I was used to ELISA to test some animal disease. But they just teached to me how to used the Elisa kits. But i don't know to setup a elisa kit. I know for the elisa kit we need a:

1. Plate 96 well. 

2. We will coat angtigen or antibody dependent on the types of ELISA, incubation and wash. 

3. We will add our sample into plate,  incubation, wash

4. Add conjugate, incubation,  wash, 

5. Add substrate, incubation.

6. Add stop solution. And read result assay.

I know that plate, conjugate, substrate, stopsolution will by from suppliers. But i don't know where is antigens or antibody in step 2.

Can you suggest to me, where i can buy the antigen i need or the ways to have it.

This is the firstly time i join to forum, and the first time i setup a elisa kit so there are many thing i don't know! Hope you don't fell annoying.

Thank you!

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Dear Mr. Cao,

Thank you for using our forum! I'm sorry that I haven't responded to you earlier, or that no one else has been able to give you an answer.

I've no experience of detecting antibiotics or pesticides. I made a search on the internet and I could find several different suppliers, but I'm no able to recommend any special to you as I'm not familiar with them. I'll ask around in our organization and I hope someone knows, but since our focus is on cytokines, immunoglobulins and apolipoproteins, we are not experts on the detection of antibiotics.

I really hope you get the ELISA to work! And please add a reply here on how you solved it, it might help some other researcher.

Best regards,

Lena

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