ELISA Troubleshooting Forum

IL-4 ELISA detection problems

Tue, 24 Jan 2012 22:14:42 +0000

Hello everyone,

I work as a tech (in training at the moment) at a research lab looking at the CRTh2 gene and it's role in asthma and other inflammatory diseases. At the moment we are running into a few problems in our efforts to optimize some experiments. Please take a look and contribute any advice or thoughts. If you need more info/details please just ask as I will attempt to be brief, and am no expert. I am excited to see what online discussion can bring to the research table!

For now I'm wondering if anyone has any experience working with the IL-4 cytokine. We are having trouble detecting IL-4 in PGD2 treated CD4+ CRTh2+ cell supernatants using ELISAs. At the moment we are using Diaclone ELISA IL-4 Eli-pair Kits and getting very low detections even though standards go down to 1.1pg/ml. Does anyone know any good protocols/kits for IL-4 that have worked well? Any that have accurate detection at very low concentrations? We are trying different dilutions, new serum (we were using FBS and are testing if Human Serum makes a difference). Supernatants are from 2million cells/ml cultures, we are trying 4million cells/ml culture to see if IL-4 production gets bumped well into detectable ranges for our kits.

Please share your thoughts and experiences. We would greatly appreciate your insight.
Sincerely,
BJoseph

Trying to design an ELISA

Mon, 23 Jan 2012 00:09:44 +0000

Three days ago i'd barely heard of an ELISA, now i've been asked to come up with a protocol for one. I'm really confused about some of the basics and would appreciate any help.

Previously i have purified a particular GST-fusion protein, collected a series of elution fractions which i then ran on an SDS-PAGE gel and the results matched pretty much perfectly with what i was expecting. Also ran a BCA which indicated high protein concentrations.

But now i have to design an ELISA to demonstrate my fusion proteins "superiority" to the fusion proteins that are currently in use. I don't know what exactly i'm trying to prove! From my reading of it, i presume the ELISA will give me a bunch of OD absorbance values which i can slot into a calibration curve to obtain concentrations? Am i simply to demonstrate that my purification protocol resulted in a higher yield - by relating the concs from the ELISA back to the original weight of the cell pellet i began with?

As for designing the ELISA, i think i have an idea of the overall steps but one thing is really confusing me - which samples do i actually include in it? I know that i have to include a standard control, and positive and negative controls - but what about my elution fractions? I'm a complete beginner when it comes to ELISAs, but it seems to me that i am not trying to indicate any kind of trend (i.e. by running all my elution fractions and coming up with a nice curve) - but trying to obtain results that are as high as possible. For some reason i have something in my head about mixing my 3 or 4 strongest samples, but when i think about it i've no idea what the purpose of this would be. Maybe i'm completely misunderstanding it but i would really appreciate if someone would enlighten me here! Do i pool all my samples together and plate a series of dilutions? Do i pool my 3 or 4 strongest samples and plate a series of dilutions? Do i plate each fraction i obtained at the same dilution?

If the purpose of the ELISA was just to prove my protein was present then it would have been clearer but this trying to prove that my purification was better/leads to a higher protein concentration has confused me completely about what i'm trying to do.

Sorry that this post is long and rambling, it's just the more i read about ELISAs the more i realise i don't understand! My general plan (washing/blocking etc steps aside) is:

1. Coat wells with antigen (diluted samples)
2. Apply primary antibody
3. Apply secondary antibody linked to HRP
4. Add substrate TMB
5. Read results from ELISA plate reader

But i can't really think about the later steps until i know what i'm starting with/understand what results i'm looking for.

One last question, one protocol i was reading mentioned the preparation of a citrate-acetate buffer but this doesn't seem to be mentioned anywhere else. Is it just something that is used in the preparation of the TMB solution, or does it have some other purpose in the ELISA?

Anyway - if you read this, thanks, and i'd be hugely grateful for any help in relation to any of those questions, in particular the choice of samples i am assaying...

Anyone familiar with ELISA of cytokines in mouse plasma?

Thu, 19 Jan 2012 12:48:05 +0000

I am new to ELISA and I am going to measure TNF-alpha in mouse plasma (from heparinized blood).

The blood was collected from terminal bleeding and I got about 300~400ul plasma. I am not sure about what diluent I should use for diluting the samples.

Also, does anyone know about the basal level of TNF-alpha (in pg/ml) in mouse plasma when doing ELISA?

Many thanks!

Possible to calculate avidity/affinity from an ELISA?

Fri, 06 Jan 2012 15:35:47 +0000

I'm comparing 2 different sets of antibodies to see which is more effective at binding a particular protein.

I've done an ELISA and got 2 curves showing one IS more effective than the other at both high and low concentrations of antibody by measuring optical density, but I'm having difficulty quantifying the difference. Is it possible to calculate avidity/affinity/some proper measure directly from optical density results? Neither curve has reached a plateau so I have no max value.

If not, is the best I can calculate simply a relative % difference between optical densities of the 2 antibodies? I would really like to quantify them in a way not necessarily relative to each other if possible.

Also, if one antibody fails to bind at low concentrations, but the other antibody DOES bind at low concentrations - is it possible to distinguish between avidity and affinity and say that this reflects AFFINITY because concentrations are so low that that is the sort of interaction which is most likely? Or would it still be impossible to distinguish avidity/affinity?

I've never had to analyse this sort of data in any depth before except for the obvious "one is better than the other", so I'm a bit shakey on how the data correlates with concepts I know already (affinity, avidity etc.).

Any advice would be appreciated!

Thanks.

How to determine the optimal concentration of a primary antibody solution

Thu, 05 Jan 2012 05:20:29 +0000

I have looked around the ELISA and Immunoassay board a little bit, but I have not been able to find a post that addresses my problem. However, I am very new to all of this, and my ability to even recognize the answers to my questions is not very good. So if my question has been dealt with elsewhere, I apologize for creating a new topic.

I am currently trying to create an indirect ELISA protocol for measuring the competitive adsorption of human albumin, gamma globulin, and fibrinogen onto various polyethylene samples. The samples are rather large (20X15X3 mm) and, in the first step, require incubation with 5-10 ml of protein solution (Albumin - 4000 ppm; gamma globulin - 1000 ppm; and fibrinogen - 300 ppm). In the second step, the samples (after washing) will be incubated with goat anti-human antibodies for albumin, gamma globulin, or fibrinogen. However, I do not have very much experience with ELISA (I have performed ELISA kit experiments before, but nothing more), and I am unsure in what concentrations I should prepare the primary antibody solutions. In references I have seen people quote concentrations ranging fro 1/400 to 1/5000. However, if I want the antibodies to be in excess of the attached protein, wouldn't I need to use a concentration greater than 1/400? Especially if my initial protein solution has a concentration of 4000 ppm (using albumin as an example)? Any help or guidance that you can provide would be very much appreciated. Thank you.

serum vs plasma - hormone ELISA

Wed, 04 Jan 2012 14:40:29 +0000

Hello,
Quick question - I have a set of plasma samples which we have access to to look at thyroid hormones using ELISA.
The kit specifies that serum should be used, not plasma.

I am not quite sure how / why plasma would interfere with the assay - any idea?
Is there a reasonable protocol out there to prepare plasma derived serum? I haven't come accross any and given that bloods were collected on anti-coagulant, this may not be feasible...

Hope someone can advise.

Many thanks
Ecatharina

SDS and/or NaOH's affect on ELISA results

Fri, 16 Dec 2011 19:41:42 +0000

Hi Bioforum community,

I would certainly appreciate your input, be it literature supported fact or opinion, regarding my problem. As a bit of background, I am in a field that is a blend between polymer science and biochemistry so I often run into very unusual experiments such as the following: I have to "melt" my polymer/protein matrix in one of the following mixtures; .1N NaOH and 5% SDS, 13.5N NaOH, or almost any water miscible organic solvent (that doesn't precipitate proteins). In order to quantify the minute amount of protein I need to use an ELISA assay (no a simple mBCA assay will not do the trick). My question to you is: will any of these systems not destroy any hope of reliable results from my experiment? If not, does anyone have suggestions?

Many thanks,
Cheers,
Feldman

cytokine levels in brain tissue

Tue, 13 Dec 2011 10:37:11 +0000

I want to qantify one cytokine level in striatum protein extract with elisa and western blot.
I dont have signal in western blot.
I got weak signal in elisa in 150ug of protein extract, but no signal in lower contentrations of extracts.
I want to ask if there is any eventual way to improve my sandwich elisa, a part from using high protein extracts to optimize the conditions of elisa.
If there is another tool more efficient and easy to qantifiy my cytokine levels.
thank you for help

ELISA test - set up and data analysis

Fri, 09 Dec 2011 16:20:59 +0000

Hello!
I'm a new member of forum and a new user of ELISA tes. I have some questions for you.

O.D. values of a reader is the same of another reader or not? Are they similar? Can I compare O.D. valuese of a reader with the values obtained with other readers?

I will use a commercial kit for dosage a protein.

I will follow the instructions on the data shee of the kit, but how do I know if the values obtained of the calibrators are correct (O.D. values?) or not?
Can I understand this thing with data analysis? I will use the 4 parameter logistic model. There is a parameter usefull for undestand if the calibration curve is ok?

Thank you very much for your help.

Paolo

a protocol for ELISA without FCS

Tue, 29 Nov 2011 20:43:26 +0000

hello,
I'm looking for an ELISA procedure i can coduute without usinf FCS (fetal calf serum- yeah they kill the calf and the cow just to extract a s small amount...). any suggestion? I'm using ovalbumin as the antigen.
thank you

p.s.- i found this article but they worked with insulin- i wonder how well will it work on ovalbumin... what do you think?
http://www.pcrm.org/research/animaltestalt/animaltesting/development-of-a-novel-diagnostic-elisa-for-human

ELISA BKG

Tue, 29 Nov 2011 10:01:57 +0000

Hello,

I am trying to set up a sandwich ELISA, using two affinity purified rabbit polyclonal antibodies against the same antigen (the two antibodies are against two different petides of the same antigen). One of the antibodies is used for coating and the second for capturing. The capturing antibody is biotinylated and the final detection is made with streptavidin HRP.

I have a very serious problem of cross-reaction between the two antibodies, that is when I do NOT add any antigen I get a BKG signal of OD >1.5.
All other controls (no coating, no detecting antibody) are negative.
The use of different concetrations of coating and detection antibody reduced the overall signal (without changing the signal-to-noise ratio).
Different blocking buffers also did not help solve the problem.
Can someone please help me understand from where this BKG derives?

Thank you very much

Veatriki

plotting ELISA results

Sat, 26 Nov 2011 07:57:30 +0000

Hi there,

I recently carried out my first ELISA, and to each well I added varying concentrations of my antibody to my protein. I started with a 1:200 dilution and then titrated 2 fold so ended up having 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:102400, 1:204800, 1:409600 and then added each conc. to a well, and read plate.

I got readings and now have to plot them on a graph..just wondering what do I write on the X-axis...Y would be absorbance and X would would be concentration of antibody but how do I write concentration in terms of an X-axis...hope that makes sense!?

Thanks,
biology_06er

ELISA plate longevity

Tue, 22 Nov 2011 18:12:43 +0000

Has anyone done a longevity study on their in house ELISA plates?

I have tried protecting with sucrose, trehalose, storing at 4c and 37c,desiccating vs. non desiccating and working with different type of preservatives in my diluent buffer.

after one week with these variables, the results at 450nm dropped from 1.2 to 7.0.

Has anyone tried anything that I haven't mention?

ELISA theory and saturation

Mon, 14 Nov 2011 19:47:17 +0000

I have a question about the theory of ELISA assay quantification.

It is my understanding that, using the TMB substrate (or any other HRP substrate), the analyte is actually a product of the HRP which is directly bonded to your secondary antibody. So, in a sense, you don't really read "quantity of secondary antibody" you read "quantity of analyte that is directly proportional to quantity of secondary (in the linear range of the assay).

So, what exactly causes the saturiation (end of the linear range) in an ELISA assay? Is it the capabilities of your spectrophotometer? Is that all your substrate ends up converted into the analyte by the HRP?

Indirect ELISA - Interpreting results

Tue, 08 Nov 2011 15:07:33 +0000

Hi,

I'm a sophomore from Ohio.

For my labs involving indirect ELISA, I was given various sera immunised with different peptides to see which peptide was better at eliciting an immune response. I made titration curves for each serum (optical density on y-axis and serial dilution factor (i.e. neat, 1/2, 1/4, 1/8, 1/6 etc) on x-axis). I was then told to determine the titre by reading the end-point from each curve i.e. obtain the value of the serial dilution factor (from the x-axis) where the curve from serum crosses the baseline provided by the negative control.

So now that I've obtained a value for titre in the form of the highest serial dilution factor, what do I do with this? I know these values can help tell me which sera have worked and have got more antibodies but how can I quantify this further? Some people talk about drawing standard curves or reference curves but does that fit in with titration curves? Do I need to know the values of the original concentrations? I don't understand beyond this point at all.

Help is greatly appreciated, thanks.

phosphoprotein detection in unstimulated samples

Thu, 20 Oct 2011 23:18:42 +0000

Hi,
I want to examine the insulin signaling pathway for any changes in the levels of phosphoproteins in response to my treatment. I plan on using multiplex/Westerns. However, the tissue samples that I have are from mice that were not stimulated by insulin or any other growth factor. in addition, these mice were fasted overnight before killing so presumably any physiological response is muted as well. Does anyone out there have any experience with detecting phosphoproteins in unstimulated samples?
Any input is appreciated.
Thanks

CXCR3 rat immunohistochemistry

Mon, 10 Oct 2011 09:54:52 +0000

I need to develop an IHC of CXCR3 in rat tissue. I am using the one from Santa Cruz, and even I found references with positive staining, any of them explain the protocol, and wich I did from the datasheet didn't work. my question is if anibody have done in rat and could explain me if there is any trick. thanks a lot

ELISA Kits for proteins in brain homogenates

Wed, 05 Oct 2011 09:38:13 +0000

Hello everybody,

I need to assess some proteins in mouse brain homogenates. I never did ELISA before, so I would like to ask for some tips for purchase ELISA Kits. Moreover I think that our candidate proteins are not very common to assess (BDNF - this one is quite common, IGF-II, Sonic hedgehog). I've already found few kits for them, but usually they are recommended only for cell culture suspension or plasma and other liquids. Can you please give me an advice where and how to look for proper Kit for less common proteins? Or if somebody has an experience with brain homogenates I would be happy for any advice!

Thank you in advance.

Jana

High Background help

Wed, 28 Sep 2011 15:13:49 +0000

I'm running a Sandwich ELISA with OPD as my substrate. All of the sudden I've been getting high background (high blank wells). Doesn't seem to be any particular changes in reagents between the two runs when the problem started. Any suggestions on things to check or possible causes? Thanks.

Best,
Grad

Serum reacting with HRP substrate

Fri, 23 Sep 2011 14:19:51 +0000

Anyone know of any serum protein that might be reacting with an HRP substrate, such as TMB, to give off signal?