Hey Sridhar, I essentially follow the general protocol on Abcam (http://www.abcam.com/index.html?pageconfig=resource&rid=11422) where you coat with a polyclonal antibody to your target protein (adiponectin) overnight, block (I use BSA), incubate samples (usually I do this overnight at 4C, but 2hr at room temp usually works too), and then use an HRP-conjugated detector antibody against the target, followed by ABTS (~100pg/ml detection limit) or TMB (higher sensitivity) as a substrate.

For you it might take some troubleshooting to get the concentration of antibodies right, but should be fairly straightforward.

Hey Sridhar, I essentially follow the general protocol on Abcam (http://www.abcam.com/index.html?pageconfig=resource&rid=11422) where you coat with a polyclonal antibody to your target protein (adiponectin) overnight, block (I use BSA), incubate samples (usually I do this overnight at 4C, but 2hr at room temp usually works too), and then use an HRP-conjugated detector antibody against the target, followed by ABTS (~100pg/ml detection limit) or TMB (higher sensitivity) as a substrate.

For you it might take some troubleshooting to get the concentration of antibodies right, but should be fairly straightforward.

You could also coat your plate with your antigen, in this case swine adiponectin, and follow afterwards with incubation with your serum or an anti-adiponectin antibody as a standard amount. Please look at the datasheet of SBA Clonotyping System/HRP Kit from Southern Biotech.

Hi Sridhar, I would just like to add (this may be really basic, but it is worth to mention) that you should be using plates that are good for ELISA (i.e. NUNC plates) and also that you should try what is the best coating buffer to use (i.e. carbonate buffer, PBS, TBS, etc...). Good luck.

David posted a link to a great protocol, similar to the one I use. I tend to incubate the plates at room temperature overnight - the polyclonal antibodies are very stable and this is fine. However, if you are coating with an unstable antigen, room temperature may not be the way to go. It probably goes without saying, but be sure to include a few wells of a suitable positive and negative control in every plate as things can change from one trial to another.

I essentially follow a general ELISA protocol as previously mentioned, but when working with a new protein for ELISA, I would have different protein concentrations (I usually start at 1ug/ml) and multiple dilutions of your (+) sample (at least 8) to obtain the best concentration to coat your ELISA plates and the right dilution for your samples (for example: if you are doing screening of samples).

I agreed with Ricardo about selection of useful plate and buffer ... the attached file may be helpful.

Article Synthesis of hapten and preparation of specific polyclonal a...

I agreed with Ricardo about selection of useful plate, buffer and find an optimum concentration of the antibody when working with a new protein for ELISA.I did myself. For Ex. :

1.Antigen was dissolved in 50 μl of sodium bicarbonate buffer (pH 9.6). 2-3 well of a 96-well plate were uncoated with 0.5 μg of the antigen for controls. Over night in refrigerator or 2 hr RT;

2.Wells were blocked with 1% BSA (v/v) in 400 μl TBS (0.025 M Tris, 137 mM NaCl, pH 7.6) for 2 h and washed with TBS-T (TBS in 0.1% Tween (v/v));

3.To find an optimum concentration, the antibody at varying concentrations of 0, 0.5, 2, 8, 32 and 128 Ag in 50 μl of TBS was added into each reactive well, respectively. After incubation at 37o C for 2 h, the excess Ab was washed out with TBS-T, 4-5 times;

4.Wells were incubated for 1 h with the HRP-conjugated detector antibody against the target, and the the excess HRP-conjugated was washed out with TBS-T, 4-5 times.

5.Added TMB (higher sensitivity) as a substrate. Good Luck.

Thanks for a;; the wonderful feedback..........I will try it out and let u know results

Coating buffer and assay diluent are very important (carbonate buffer, PBS, etc) you have to try all. This is a good protocol http://www.rndsystems.com/Resources/images/5670.pdf

I do complerely agree with Sridhar's suggestion/s.

Good luck

LS Chaturvedi

There is an old fashion way of titering the Ab, it is a heamagglutination assay. It is very sensitive, and gives you a range for your monoclonal Ab's. You can find the protocol in Ben Pernis ancient book Immunochemistry. Back in the 1980's we did the Hb agglutination assay along with the Eliza, remember this was when there was no commercial anything to do Eliza's. The important step is the blocking with BSA.

Coating: antigen or antibody az 1 - 10 µg/mL in a 0.1 mol/L carbonate buffer, 4 °C, overnight. Volume: 50 - 200 µL (depening on the price and avaiblibilty of your antibody/antigen) Remove as much as possible, store the caoted plates frozen (upside down)

Blocking: normally not required (only if you would like to store the plates at room temparature)

Washing: PBS 0.1% v/v Tween 20, 3 times x 1 min (V: 250 µL)

Incubation

Buffer: PBS Tween + 1% w/v BSA (or any innert protein to keep the protein concentration diring all incubation steps in most dilutions constant), + 0.01 mg/L phenolred ( to see much better where you are with your pipetting steps)

Volume: see coating (50 - 200 µL)

Sample concentration: ELISAs have a working range of 1 ng/ml up to 1 µg/mL, try to get the stanards in the same conditions as your samples

Incubation time: 90 min, room temprature

Revome as much as possible

Washing

Conjugate incubation

Buffer: see sample incubation

Concentration: normally 0.1 - 1 µg/mL is sufficient

Volume: see coating (50 - 200 µL)

Incubation time: 90 min, room temprature

Revome as much as possible

Washing

Substrat reaction

for HRP-labelled antbodies/antigens TMB is the best. ready for use solutions are avaibale: http://www.seramun.com/products/substrates/horseradish-peroxidase.html

for AP also at this adress

Volume: 50 µL - 200 µL (higher volumes are helpful to increase the sensitivity

Stop: 1 mol/l H2SO4 (in the case of 200 µL substrate you can use a 2 mol/L H2SO4)

Measurement: 2 wavelenght 492 nm + 690 nm

Calculation of the calibration curce & sampes.

I would recommend to use horseradish peroxidase (HRP) as label and luminol as luminescence enhancer (if you have an access to luminometer). HRP-luminol luminescence can be further enhanced with certain phenolic compounds (see e.g. Journal ofLuminescence132(2012)1021–1024). I recommend also to use a couple of days by reading for instance the "Techniques for Immune Function Analysis. Application Handbook 2nd Edition - Immunology and Host-Pathogen Interactions". Especially Chapter 9 could be usefull in planning the test system. You'll get it for free from web page http://www.molecularstation.com/forum/immunology-host-pathogen-interactions/74251-techniques-immune-function-analysis-application-handbook-2nd-edition.html .

Luminescence labels have the advantage that HRP anf luminol are not expensive and there is not bacground from excitation light as in fluorescence systems..

• Coat microtiter plates with target protein diluted in 50 mM carbonate buffer at pH 9.0. Optimal concentrations should be determined by titration, but for purified antigens 50 µl per well at 1 µg/ml is usually sufficient. Incubate overnight at 4º C covered with parafilm.

• Remove antigen solution. Add 200 μl/well of blocking buffer (PBS containing 1% BSA and 0.02% azide) to block non-specific protein binding. Incubate for 1–2 hours at room temperature, or overnight at 4º C.

• Remove blocking buffer. Wash once with PBS with 0.02% azide. Damp strip wells or plates are usually stable in resealable plastic storage bags for 4 weeks at 4º C. Before using, remove excess liquid.

• Add test antibody samples and controls at 50 µl/well diluted in blocking buffer. Antibodies may be serially diluted for determining titer or diluted to previously determined working concentration for screening assays or antigen quantitation. Incubate 1 hour at room temperature.

• Wash plates three times with PBS containing 0.05% Tween-20 (Tween-20: sc-29113), removing excess liquid as above.

• Add 50 µl/well of alkaline phosphatase conjugated secondary antibody (Conventional Secondary Antibodies for Western Blotting) diluted to 1:100–1:1000 in blocking buffer. Optimal antibody concentration is determined by titration. Incubate 1 hour at room temperature.

• Remove liquid in wells. Wash three times with PBS containing 0.05% Tween-20 and slap plate dry.

• Wash wells once with diethanolamine buffer (10 mM diethanolamine, 0.5 mM MgCl2 (pH 9.5) and remove liquid.

• Dilute substrate (PNPP, sc-3720) in diethanolamine buffer to a final concentration of 1 mg/ml. Add 50 µl/well. Allow to develop for 10–20 minutes or until positive control reaches an OD 405/490 of about 1.0. Stop reaction by adding 50 µl of 0.1 M EDTA (EDTA: sc-29092), pH 7.5. Read plates on microtiter plate reader at OD 405/490.

This is the protocol Santacrux suggested me....an indirect ELISA method where you coat your samples first......appreciate your feedback

If I have understood your problem,my suggestion is like this.Raise polyclonal antibodies as well as prepare monoclonal antibodies and perform Sandwich ELISA for better specificity with your samples.A.M.Jana

I designed and developed my own competitive ELISA. It can be done! I tried different plates and decided on Nunc Maxisorp Flat bottom plates (VWR, Mississauga, ON). These plates maximize antibody coating. Maybe you want to read their description as to the chemistry behind it on their site. These plates were coated with 100µl of goat anti-rabbit IgG Fc fragment (Pierce, Rockford, IL) at 10µg/ml in PBS overnight at 4°C. The remaining binding sites were blocked with 1% (BSA) in PBS 0.05% Tween-20 (PBST) for 2-24h and the wells then washed with PBST to remove excess unbound BSA. The reason that I coated them with Fc fragment is because it orients the antibody with it's binding spots (light chains) facing outwards for maximum interaction.

We have implemented a sandwich method for any rabbit primary antibody (but you could set it up for other species). The deal is to coat the plate with a purified anti-Fc IgG on the plate. You can make a bunch of plates and store them in the frig. So after a wash the first step in the assay is incubating in primary ab against the substance you want to measure. We have found this method to be universal and with better sensitivity, presumably because the Fc domaine is bound to the coated antibody leaving the antigen binding Fab domain "waving in the breeze". The methods for coating given by others are good.

How to remove bound antibodies (where the target proteins are bound) from ELISA plate ?

The above suggests are exceptional and should be very helpful for those needing trouble shooting. Speaking as the ancient mariner reading the above young colleagues you are all excellent chemist, congratulations how how you have all progressed these techniques. Well done. Burt

Dear follow the principle of the kits you want to produce and you will succeed.

this week have project of design sandwich ELIZA so after complete and valid my work i sent the procedure as homemade kit

with best regard to u