Hi, this is an international platform. Please write your comments in English down. Cheers, Nadine

Hey Mostafa,

Here I am explaining a classical manual method for an indirect ELISA. You dont need any special setup for this. All you need is to start your experiment with coating your sample (protein or conjugated sugar; antibodies in case of sandwich ELISA) either overnight at 4 degree centigrade or 2 to 3 hours at room temperature. This is something you need to optimise as it varies from sample to sample. For antibodies dilution, you need to optimise the concentration and for that you need to go for a antibody titration step before doing the real experiment. Meaning, coat the ELISA plate at least in 5 triplicates (15 wells in total) with a fixed concentration of your sample ( e.g. lets say 20nM) . Try an increasing molar concentration of primary antibody (no need to dilute if you are using serum) and see at which concentration, you are getting best signal. Do not forget to include another 15 wells for negative control.

These are the most important things you need to remember. (1) use of negative control (2) do not forget blocking step (3) A stringent wash after primary and secondary antibody incubation.

Good luck!

Thank you so much Mishra

Your answer is very helpful.

Does it cheaper if I buy a kit or go for manual method?

I have got 50 serum samples of mice which It would be a lot of money to run assay for all of them.

I have 10 mice in each group, I am afraid if I decrease numbers of mice, the data wont goes significant, so what do you usually do in this situations? has it ever happened to you?

And also do I have to work triplicate? would it be OK if do duplicate?

Thank you Mishra again!

Have a good time

Mostafa

Hi,

I think it would be easy if you could buy a kit from company and use the company provided manual. It is quite simple and easy method. Just you have to careful about your pipetteing because CV would be very important one to look for. Higher CV will create problem to differentiate from one group to another one.

If you use 96 well plate, then you can use 40 samples with duplicate. So 40+40+ 2(blank)+14 (7 standard )= 96. If you use 5 or 6 standard then you will get more well to use for your sample.

Duplicate works fine, in the same time you can save money.

Hope it will work for you!

We run our ELISAs with duplicates.

Thank you very very much friends, Dear Halim and Gund.

I wish you all luck in your research

Sincerely

Mostafa

مصطفی جان سلام

روش تکراری 40+40+2 و 14 تا روش خوبیه و میتونی از اون استفاده کنی.

کارشناس ازمایشگاه میکروبیولوژی لیستر

سلام آقا حسن ممنون

منظورتون از "روش تکراری 40+40+2 و 14" چیه میشه توضیح بفرماین؟

یه سوال دیگه: اگه با سمپلر معمولی الایز را انجام بدهیم اشکالی پیش می یاد؟ بیشتر طول میکشه تا همه ول ها آماده بشن آیا این خطا ایجاد می کنه؟

همچنین می تونیم از یک ظرف برای تمام محلولها استفاده کنیم؟ (جهت استفاده از سمپلر 8 کاناله)

حسن آقا خیلی خیلی مخلصیم

با آرزوی موفقیت برای ششما در هر کجای جهان که هستین.

قربان شما

مصطفی

همچنین آیا یکبار استاندارد برای چندین بار الایزا قابل تعمیمه یا باید حتما هر بار استاندارد براشون بذاریم

با تشکر

مصطفی عزیز با سلام

میتونی با ایمیل من در ارتباط باش.

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ارادتمند رسولی

Hi, this is an international platform. Please write your comments in English down. Cheers, Nadine

hi Nadine

you are completely right, I am sorry.

A good suggestion is also to start a basis on other literature using ELISA techniques- there are so many available on PubMed. It often depends on what you are measuring/targeting, but for common cytokines you can often commercially buy stock of capture antibody, and secondary antibody, both of which give suggestions on dilutions (of course it's a great idea to test variations on their suggestion to find what is best for you).

I agree with Bibhudatta, blocking and washing are very important steps to ensure minimal background signal so that your assay can be highly sensitive.

As for using serum, it depends on what you are measuring. Some cytokines which can be low you can add multiple dilutions of neat, 1:2, 1:4. In contrast, measuring IgG is often high so you may need to start dilutions around 1:100, 500 or even 1:1000.

Another thing to think about is whether you can include a standard curve. Sometimes this is difficult if you are measuring something novel, but with cytokines and certain antibodies you can buy stock with a determined concentration, and this you can serially dilute to then calculate how much is actually in your samples.

Hopefully this is helpful, good luck with ELISAs!