ELISA Troubleshooting Forum

Primary antibody quantitation

Mon, 09 Nov 2009 09:01:32 -0800

I am in the process of developing an ELISA method, and wish to quantitate the concentration of primary antibody that has successfully bound to the well of the microplate during the initial plate coating. I want to do this to confirm that I have actually managed to bind antibody to the plate, and to determine the optimal concentration of antibody to coat with. Does anyone have a protocol or a link to such a method? Can the Bradford or Lowry assay be modified to assay solid-phase protein?

Thanks for your help in advance!

Protein present in Western, not ELISA

Fri, 06 Nov 2009 07:17:38 -0800

Hi!

I haven't done very many ELISAs and since we have no postdocs in our lab and my PI is strictly DNA/molecular-based, I'm pretty much trying to learn how to do them from mat/meths in papers (ha!).

My first problem is that I'm trying to detect (and quantify) one protein out of a mixture of proteins (plant soluble proteins). The protein is definitely there in high quantities (confirmed by many Westerns) but my ELISAs are not picking it up. I'm using alkaline phosphatase and I get some colour change after many hours' incubation but not much. My positive controls (purified protein of interest) always come up without any problems.

To further complicate things, I did some work on this protein a year or so ago and never had any problem picking it up. The difference now is that the tissue I'm working with has been freeze-dried and ground into a powder, as opposed to a TSP extraction from fresh tissue with carb/bicarb buffer.

Could it be that the freeze drying has altered my protein somehow and that it makes no difference for Western because my samples are denatured anyway before loading?

My second question is, sometimes my standard curve is a bit backwards, as in the lower concentrations actually give a higher OD than the higher concentrations. Could this mean I haven't properly optimised the Ab concentrations for my ELISA?

Thank you for any help you can give me - I'm stranded in an ELISA-knowledge-free world and I'll be grateful for any tips you can give me, however small.

ELISA results

Mon, 02 Nov 2009 01:38:07 -0800

Hi..
please tell me why my ELISA plate directly gives yellow color after adding the substrate

How to denaturate protein for ELISA

Tue, 27 Oct 2009 09:47:47 -0700

Hi, I have overexpressed my protein in a cell line and have collected the media to study the secretion of my protein. How could i use Urea or guanidine hydrocloride to denaturate my samples before ELISA? Or what other method could I use to denaturate my protein?
Thank you

Automated elisa work

Tue, 20 Oct 2009 17:43:42 -0700

Hi I am helping my group to look at automated elisa in a high throughput format. Anyone know any good products that do that?

I have heard of the no wash Elisa called Surefire made by PerkinElmer�s and TGR BioSciences, has anyone tried that yet?

http://las.perkinelmer.com/Content/Related...SureFireERK.pdf

Let me knowif it is of any good. Thanks

How does one develop a 5-point Calibrator for Cardiolipin???

Thu, 08 Oct 2009 11:22:51 -0700

Cardiolipin's Calibrators are not very stable in a 32 degree or oven, so it has been hard to develop a 5 point calibrator vs. a single point (which is already in place). The only thing I can come up with is developing a monoclonal Ab. Can anyone give some insight on how to tackle this problem since there are 5-point Cardiolipin test kits out there on the market???

Problem with ELISA false positives and noise

Wed, 07 Oct 2009 03:25:05 -0700

Help please kind experts.
We have an ELISA with the following protocol for a 30kD protein in unconcentrated urine.

Two monoclonal mouse antibodies were raised using the synthetically produced C-terminal 100 amino acids (Biosynthesis Inc, USA) as an antigen (Antibody Production Services Ltd, UK). One of these, APS1, was conjugated to Alkaline Phosphatase using the Lightning Link Alkaline Phosphatase Conjugation kit (Innova Biosciences, UK), whilst the other, APS2, was conjugated to biotin using the Lightning Link Biotin Conjugation kit (Innova Biosciences, UK). APS2-biotin was captured onto a 96-well streptavidin-coated plate (Nunc 436014, USA) at a concentration of 4μg /ml.

After washing, 100μl of urine or a dilution of the fragment in
buffer was incubated in each well for 1 hour at room temperature.

The plate was then washed 8 times in buffer and the secondary detection antibody � APS1-Alkaline phosphatase was added to each well at a concentration of 4μg/ml (1 hour at room temperature).

After a final wash step a colourmetric agent � pNPP (Sigma, USA) was added and the absorption of light at 405nm was measured after one hour. The dilution series was used to generate a standard curve by which the concentration of EN2 in each sample was measured.

To generate a decent looking standard curve we are having to make 5 measurements at each point and then take the median measurement.
We are also having to use 5 wells per sample and use a median to obtain a sensible output as individual wells vary by as much as a factor of 2.http://www.protocol-online.org/forums/style_emoticons/default/sad.gif

What are the most likely sources of our woes? All help gladly accepted

The protein we are measuring is sticky but that doesn't explain highish readings at 0 concentration.

Other ELISAs work fine on the equipment.

coating meat tissue to ELISA plates

Mon, 21 Sep 2009 05:42:28 -0700

Hi,

Does anyone have any idea on how to coat ELISA plates with meat tissues?

TQ

plasma and serum

Wed, 16 Sep 2009 17:39:53 -0700

will it make any difference if i use serum/edta plasma for hscrp (the kit sample requirement is citrated plasma)? but in my writing, i used serum for other biomarker.

fixing bacterial cells in ELISA

Mon, 14 Sep 2009 00:16:29 -0700

Hi,
i am about to do ELISA for my Phd projects. In the assay i have to coat the plates with E.coli. i have read that some researchers fixed their bacterial cell with 1% formaldehyde prior to coating them onto the plate and dry them out overnight in 37C. can somebody explain to me why do the need to fix the cell first? wouldn't it will effect the result as compare to non fix-cell? and what are the effects of drying them out?

TQ!!!

ELISA - calculate diluted concentration

Sun, 13 Sep 2009 08:39:00 -0700

Hi, when the standard curve is already plotted, how to calculate the concentration from a 1:10 diluted samples? is it just multiplied by 10 as final concentration? but i cannot get the consistent result when i'm doing 1:2, 1:4, 1:8. Please suggest. Thank you.

serum storage temperature

Tue, 08 Sep 2009 22:30:33 -0700

is it if put serum and plasma in -80 degree will affect the elisa result? if i used to put my serum in -80, now if i want to put in -20, can? thanks

Acid stop solution for HRP/TMB reaction

Sun, 06 Sep 2009 01:29:07 -0700

HRP speeds up an electron transfer reaction. TMB is the donor and gets oxidized, and H2O2 is reduced to O2 and H2O.
H2SO4 denatures the enzyme as proteins dont survive under an extreme pH.

If the acid is just to denature the HRP, then why the blue colour of oxidized TMB changes yellow?

weird elisa result

Sat, 05 Sep 2009 07:14:39 -0700

i ran another commerical kit this morning, but the results were so weird. i did all my samples in triplicate, and some samples have very big standard deviation. for example, for same sample, one got 0.600 absorbance, the other two got 0.100 absorbance only. What happened? btw, my blank has higher absorbance than most of my samples, end up most of my samples have concentration of lower detection limit (< o ng/ml). Does this make sense?

What antibodies to use for ELISA??

Tue, 01 Sep 2009 16:15:52 -0700

Hello,
What types of antibodies does everyone use for sandwich ELISA??
Coat with a monoclonal, then detect with a polyclonal or visa versa?
Can you use abs from the same animal? For example, rabbit Pab for coating AND for detection?
Any thoughts or suggestions and what has worked for you would be most appreciated!
Thank you!

ELISA Standard curves and sample concentration

Sun, 30 Aug 2009 10:14:25 -0700

Hi all, I using ELISA commercial kit. At first, i ran my first ELISA run using duplicate samples, but later on i found some big standard variation so thought might run another plate using same sample (i thought i can use my second plate result to replace one of the first plate's results). However, because the kit required us to run Standard everytime we run the assay, and ended up my second plate result of same sample had different concentration. The absorbance value for the sample in Run 1 and Run 2 are almost the same, but the standard curve is different. Do you think i allow to check my Run 2 sample concentration using the Run 1 standard curve? Or what would you advice? Thanks

coloring Dye

Sat, 29 Aug 2009 02:59:38 -0700

Hi,

Can someone tell me what is the blue, green and red-dye usually use in making ELISA sample buffer?

thanks

calculation for antibodies

Thu, 20 Aug 2009 02:41:53 -0700

hi
i have a x antibody need to dilute to 1 in 10000
so i need of 42ml in total , so 1/10000x42ml =42ul of antibody is that right
please can cananybody answer me

Major ELISA Issue

Wed, 19 Aug 2009 13:15:30 -0700

hello

i am having trouble with my sandwich elisa with streptavidin hrp, 1 step tmb substrate.

6/10 plates i have run have presented the same issue and I have been observed by a collegue who confirmed its not my trchnique causing the odd results.

So, my plate develops perfect except for rows A,B, and C down the entire plate including std curve (500ng). its not a plate washer issue as we rotate our plates 180 and perform a second wash each step to overcome washer variability. all my incubations are at 37 for one hour. originally i thought the issue was stemming from stacked plates but I stopped that habit and results were the same.

so again, everything to the right of row B/C develops perfect but everything to the left doesnt develop at all. any ideas?

ELISA kit detection B. cereus toxins

Thu, 13 Aug 2009 05:37:07 -0700

I am looking for a way to detect a low quantity of Bacillus cereus (contamination) in a vaccine batch. The toxin which I want to detect is the Bacillus diarrhoeal enteretoxin (BDE). I have found some ELISA kits online that can detect this toxin in food with a sensitivity of 1ng/ml (how does this compare to the average ELISA sensitivity?) sample but can this kit also be used for vaccine samples? I also came across the reverse passive latex agglutination (RPLA) method for detecting the toxin. Does anyone know how this works and its suitabilty for vaccine samples? Any help would be appreciated, thanks!