You should coat the 96-well plate with the antibody first then add culture medium into the well to meaure it. But, you do need to put in the same culture medium (without cell) as the negative control since you usually put serum in DMEM. The protein you are interested may already in the serum. You also need a negative control which is a well coat with nonspecific IgG. The problem you have now is to have a real positive control that you can compare your absrobance, or fluorescence, to obtain the level. If you plan to use absorbance the reading should not be higher than 3 (depends on your plate reader). Also make sure your antibody does not give you a lot of nn-specific binding which is not uncommonly seen in polyclonal antibodies. Good luck,

You should coat the 96-well plate with the antibody first then add culture medium into the well to meaure it. But, you do need to put in the same culture medium (without cell) as the negative control since you usually put serum in DMEM. The protein you are interested may already in the serum. You also need a negative control which is a well coat with nonspecific IgG. The problem you have now is to have a real positive control that you can compare your absrobance, or fluorescence, to obtain the level. If you plan to use absorbance the reading should not be higher than 3 (depends on your plate reader). Also make sure your antibody does not give you a lot of nn-specific binding which is not uncommonly seen in polyclonal antibodies. Good luck,

if your protein is secreted in media, then just take media and coated into ELISA plate with 1;10 dilution and make serial dilution in successive wells. use coating buffer Tris-HCl 100 mM, pH=9.0. you can coat 100µl solution per well overnight, next day just block it with PBS+2.5% BSA, 1 h RT. add primary abs in PBS+2.5% BSA (dilution depends, 1:500 to 1:1000 may be good option), 1 h RT. 4X wash with PBS+Tween 20 solution. add secondary abs in PBS+2.5% BSA, 1 h rt. 4x wash in PBST and develop it.

If your media e.g. FCS already has that protein then it might give background, then if your expressed protein has some purification scheme such as his tag. then i wcommend to purify it first and then go for checking expression.

Dear Mohamed Alalem i hope the follow the below steps will be useful to you

1. First coat your primary anti-body to the 96-well (for sandwich ELISA) it also depends on the Kit- if your kit manufacturer provides pre-coated wells then you can start directly

Note if your protein of interest itself is antibody then you can perform either direct ELSA or sandwich ELISA

2. Now add your cell culture supernatant/ cell lysate (called samples) it also includes your cell control, your treatment with drug, positive control and negative control of your experiment, standards with different dilutions, fresh FBS media (% as used for your experiment), Non specific blank (diluents/ diluted blocking buffer) with required dilution (this you have to find out on trail basis and how intense your color also the Absorption reading whether its fall within your standards reading) (you should not dilute your FBS media and cell control since they are representing directly your basal secretion unless if the basal secretion itself is showing more Absorption reading

3. For sandwich ELISA - once your sample is added coat wash with wash buffer (usually buffer with detergent) suggested by your Kit maker and block your plate with blocking buffer has per your kit protocol (usually 1%-3% BSA in PBS/TBS/TRIS (buffers selection is based on sensitivity of your enzyme to phosphates))

4. After blocking, wash the plate once again with wash buffer and add secondary/ detection antibody and fluorescence (FITC)/Enzyme conjugate (HRP/TMB ) and incubate as suggested (usually one hour)

5. After incubation, wash your plate and read fluorescence intensity of your plate in case Fluorescence based with specific reader / In case of Enzyme based add respective substrate and wait till color development usually color develops in 20 min. once color developed After color development add 2N sulfuric acid and reaction stop solution provided in kit (this is essential to avoid further enzyme action and color normalization throughout the wells). Take the absorbance at its wave length and also with correlative wavelength in-order to avoid your error

6. Plot a linear graph with your standard reading and match your concentration with respective reading.

7. Extrapolate your unknown protein concentration using your standard linear graph.

8. For interpretation, First subtract your values from Non specific reading then from your FBS media alone value. To the resulting value do one way anova with that your cell control value and find your EC50 or IC50 of your experiment

A sandwich ELISA is definitely the way to go as this will reduce non-specific binding

Let your cells secret the protein in DMEM. If your ELISA plates are not ready immediately freeze your supernantants at -70-80C. Then coat your ELISA plates with your primary antibodies. Wash and block and then add you supernatants. Incubate appropriately, wash and then add your secondary antibodies. Wash and finally develop your ELISA and read ODs. That should measure your protein in the DMEM.

I think you got several suggestion/s as above. The answer of Your Question about first coating the wells with primary antibody is the First Step and I beleive you are aware to using the high affinity binding special 96-well plate to perform it.

Good luck

LS Chaturvedi

If you have FCS in your medium it will be hardly possible to coat the secreted proteins directly on ELISA plates. Thus you may coat your antibodies block and then apply your detection abs. Ideally you may use monoclonal sandwich pairs.

It depends on what you have. If this is a protein (not antibody) and you have two antibodies for that then do a capture ELISA (but be sure that those antibodies should have different epitopes). Coat your one antibody, then use your growth medium and serial dilute. As a control if you have this protein from other source (like immunized serum etc and you know the concentration). As a negative control use only media (DMEM + serum but no protein), then use another antibody for capture elisa followed by secondary conjugated Ab. If you have only one Ab for your protein, then coat directly with the media having protein and use medium without cells as negative control. You might get background so use specific secondary (like if it is IgG2a then use secondary conjugated IgG2a to reduce the background). Good luck.

I would like to thank you all for your care and assistance. I have polyclonal antibodies and I would like to know please which method is better to use. If I coat the wells with the primary antibody and block them before applying the growth medium (to test whether it contains the antigene of interest), should I use the primary antibody again to bind to the (anticipated ) antigene before using the tagged secondary antibody or else should I use just the secondary antibody? and how can I guarantee that the secondary antibody would not bind to the caoing primary antibody regardless of the presence or abscence of the antigene? Instead, I was thinkng to first coat the wells with antigene (presumably it is secreted into the growht medium) so I was planning to grow cell culture in a serum-free-DMEM for 24 hrs prior to harvest (to avoid serum interference with the protein of interest during coating of the plates with the DMEM. then I would like to block the wells and proceed with primary and secondary antibodies incubation and detection.

Please if any body has an idea to share with me about the feasibility of such approach.

Best Regards

Usually, coating plate with supernatant is not reliable and the data is often very hard to be interpreted. The ELISA with the prcedure like coating antibody------blocking-------samples(supernatant)-------detection antibody-------, as others suggested, will give you clear result, no matter if it is what you expected.

good luck.

Do you want to quantitate or to check whether cells are secreting proteins or not. If you want to check then my suggestion is to coat plate with different dilution of medium containing protein and as negative control coat with the same medium. block it, wash it in ELISA washer with soak. So that there will be minimal chances of non specific binding. then use your polyclonal Ab. wash again and do with conjugated secondary. If the results shows that you well containing protein has 3 fold higher binding then negative control, means your cells are secreting the protein.

Do you have two different antibody for your protein??

Dear Mohamed,

it is not advisable to use the same antibody (especially if monoclonal) for both coating and detection. Your coated antibodies will bind to antigen epitopes and hence the same antibodies cannot bind again at the detection step. Even if polyclonal, it is advisable to have the antibodies raised in different hosts to minimize cross-reactivity. Your best chance of specifically detecting your protein of interest would be by a sandwich ELISA, and for that you primary and detector antibodies need to be different and not same. If you only have one antibody (monoclonal or polyclonal), then your only choice will be to coat with the culture supernatant, but remember you will not get very optimal output from that.

I hope this helps.

All the best

The answer to your question depends on what type of antibodies you are using. Is your primary antibody a monoclonal or a polyclonal? Is your secondary antibody raised against your antigen, or is it against the primary antibody?

If you use the primary antibody to capture the antigen, you should use a different antibody - one that binds the antigen, not the antibody - to detect the antigen. If you use a secondary antibody that binds to the primary antibody, not the antigen, you will get binding to the capture antibody you bound to the plate, not based on antigen binding.

Coating an ELISA plate with an impure antigen prep [such as conditioned medium, even if it is serum-free] is sub-optimal at best, and useless at worst. The problem is there are likely to be many proteins in that supernatant, not just the one you are interested in. They may bind the plate better than your protein and outcompete it for binding sites. Then you may get little or no signal at all, which will be a waste of time and reagents.

If your primary antibody is polyclonal and is purified, you could tag an aliquot of it with NHS-biotin [Pierce makes a kit for this] and use it to bind the captured antigen, and detect it with a streptavidin-enzyme conjugate.

However you coat your plates, be sure to block the wells with a solution of PBS containing 3% BSA for at least 1 hour. Wash solutions should contain .1% Tween 20 to reduce non-specific binding.

Gaood luck!

If you have antigen (Ag), then first coat with this. Best for this is over night at 4 C. Then blocking with BSA in tween-PBS buffer usually for 45 minutes. Incubate in supernatant (with possible present of Ab), over night. You can grow your cells in serum free medium(not DMEM)-it is avalible on the market.Then follow with secondary Ab coated with peroxidase HRP, then detect with ABTS kit for ELISA. We use this procedure for detecting Ab in supernatant of monoclonal and policlonal cells got from spleen of immunized mouse.

Hope this helps.

Everybody's answers are good. Just also pay attention to the plate you use. I remember an old paper in which one investigator described ELISA results using 7 different ELISA plates and you cannot imagine how different were the results. Not any high binding plate is good, so if you have doubts for your assay (meaning you are not using a pre-established kit for your protein but you are trying to develop the assay yourself), try different plates (2-3 recognized brands).

Also, if you develop your own ELISA and coat the plate first with an antibody for capturing, please be sure that

1) The antibody doesn't contain glycerol. It will not be coated.

2) the two antibodies you use in case of sandwich ELISA (capture and developing), should recognize different epitopes of your protein and preferable being from different sources (mouse-Rabbit) unless the developing antibody is already HRP conjugated. If you have to use a secondary-HRP that will bind to your developing antibody it should not recognize the capture antibody.

Good luck

Hi guys, this may be out of topic. But I have similar question. In Sandwich ELISA, Is it possible that the blocker (such as BSA) can prevent the binding of the antigen to the coated antibody? Providing that the antigen is very big (~300-400KDa). I tried a few times with various dilution of antigen and I get similar reading to the blank (wells without antigen).

It should not happen. Reduce the amount of BSA, check if your protein contains a putative inhibitor that prevents or the binding to your capture antibody or inhibit the color reaction by HRP. Do you have a standard control?

I agree that this should not happen, but one thing that can occur is that the BSA preparation [some are purer than others] may contain trace proteins which cross-react with your antibody, thereby preventing binding. This can be problematic if you are assaying antibodies, since bovine IgG is a common contaminant of BSA.

You can try ultrapure BSA, or drop the concentration - I routinely use 3% BSA, but many kits use 1% BSA. Alternately, try human serum albumin instead of BSA.

I performed normal ELISA and it worked. The only problem is the result is not consistent from one assay to the other. What i want to try is negative ELISA which read less absorbance as more protein bind to the primary antibody, then the 2nd conjugated antibody will bind to free primary antibody to give signal. This may work only, if the free well surface is blocked by a blocker. Theoretically...I will try by decreasing the blocking concentration next time. The one I used now is 3% with PBS. I'm just wondering if there is any possibility, that the primary antibody is situated in the middle of large BSA, and when the antigen is bigger than the space provided between the BSA, cause the unsuccessful binding.

If the IgG contamination is the case, would it be possible to obtain any reading with classic ELISA? The thing is, I got reading with classic ELISA but not so consistent. Using human albumin, that may worth investigating. Thank you :)

Previous responders have addressed most of the issues. However, when you coat with an antibody it is important that as much of the material present is antibody specific to your protein of interest - i.e. affinity purified polyclonal antibody or a monoclonal. If you try to coat with a non purified antiserum then most of the proteins that coat the well will NOT be specific to your protein of interest and your ability to capture the antigen will be very limited and your assay will lack any sensitivity. Ideally it is best to coat with a monoclonal and use a polyclonal from a different species as your detection antibody. Coating with antigen from cell culture medium is too inefficient and too inconsistent to be useful in a direct ELISA.

I support both the replies: of Ru-Jeng Teng and that of Felix Toka. However, I would comment upon that-in RU-Jeng Teng's method non specificity is expected and in Felix Toka's method more specifity is expected. A.M.Jana

Fareed - I'm confused about what you are trying to accomplish,. Are you trying to measure unbound primary antibody? This would be lost in the wash - unless you transfer the supernatants to another plate with some form of antibody to capture the unbound primary antibody . . .

Bovine IgG in the BSA used for blocking is only a problem if the secondary antibody cross reacts with it. But if [for example] you were measuring TGF-beta, which is sometimes linked to IgG, that could be a significant problem if the capture antibody cross-reacts with bovine TGF-beta.

Gary - No, I'm not trying to measure the unbound primary antibody. There is a method in ELISA that work the other way around. Rather than detect the number of primary antibody bound to the antigen, we detect the number of antibody that is not bound by the antigen. The more antigen bound to the primary antibody, the lower absorbance you read. If the antigen is in lower concentration, then, the number of unbound primary antibody exposed to 2nd antibody increased and gave a higher reading.

I was thinking this method because we have limited fund to develop new antibody for my antigen, as sandwich ELISA need 2 different source of antibody. No commercial one. So, I thought, rather than detect how many antibody bind to antigen, what if we detect how many antigen can bind to the antibody? We can measure this by having constant amount of antibody (saturated the well) and serial dilution of the antigen. This way, we may almost treat a polyclonal antibody as a monoclonal antibody.

Just a crazy idea by the way. I test it, it did not work well. I assume that it may have to do with the problem I mention above or maybe the binding is too weak that the antigen is washed away rather than remain bound. The best way to test is, if we have different antigen with varied size. We may also be able to predict the space provided by blocker for antigen to bind to coated antibody.

Just be aware of your antibodies... for the indirect ELISA (coating with antibody and detecting with antibody) you either need polyclonal antisera or two different monoclonal antibodies. If you have just one monoclonal you have to do the direct ELISA, i.e. coat your antigen or the culture supernatant containing your antigen.

The terms of primary and secondary antibodies have been confused a bit. Usually a primary antibody is an unlabeled antibody used for detection of teh antigen and the secondary antibody is a labeled (polyclonal) antibody from another species used to detect the primary detection antibody. The coating antibody is another category.

If you are coating your plate with antibody, then adding antigen, it is vital that you use a different antibody for detection of the bound antigen. Otherwise, your secondary antibody is detecting the capture antibody, and your signal will have little or nothing to do with your antigen. An option you might want to consider is competition ELISA, where you use a set amount of labelled antigen, mix with dilutions of your culture medium ( or appropriate controls) and determine the reduction in binding of the labelled antigen due to specific competition in your samples.

You will need a second antibody that recognizes your secreted proteins, but does not interfer with your primary antibody's binding to the secreted proteins when it is coated to the plate. Otherwise, your secondary antibody to your primary antibody will recognize the coated primary antibody and give pretty much the same OD on your plate reader as your coated primary antibody should have the same concentration in the blocked wells. I had a similar problem using monoclonal antibodies that recognized the same surface cell antigen. I got lucky and found a commercially available antibody that did not block my first primary antibody's binding. Good luck.

I agree with Laura Hanson and Gary Mirick. Perhaps you should try using the secondary antibody against your antigen, provided it does not recognize by your antibody of interest. All the best!

Glad to learn so many response. I have to agree that another antibody recognizing different epitope of the protein of interest, but not binding to the coating antibody, may help. Apparently the availability of anibodies depends upon what kind of animal cells you are dealing with. I deal with sheep cells and God forbid if I can find one antibody can identify my protein of interest I will have a very happen several monhs. I really see no reason why human serum will be better since it will be very expnesive and infection is always a concern. Competition ELISA is a better method but you do need to find the pure protein of interest to coat and you will need software to analyze the data. I will suggest the 1st antibody as polyclonal whereas the second one as monoclonal that can decrease the headache of non-pecific binding.

Since you have the antibodies to those proteins secreted into DMEM medium, you must have known the proteins as well the numbers of proteins in the medium. You can test your proteins in two ways.

1. The Direct Technique.

In this technique, you have to have the antibodies conjugated to the detector enzyme such as Alkaline Phosphatase or Horse Radish Peroxidase. If you hjave more than one proteins then you have to have specific conjugated antibodies.

Coat the 96 wells with the growth medium, incubate and block, and finally run the assay. Remember you have to optimize at least the concentration of proteins in the media and also the specific conjugated antibodies.

2. Sandwich assay.

Coat the 96 wells with the optimum concentration of antibodies followed by the incubation and blocking. When running the test you have to test at least 2 dilutions of the DMEM media (depending on the concentration of the proteins in the media), one maybe 1:10 and another 1:100. With these you may be able to get rid of the masking.

Standard sandwich ELISA would be the best way to detect antigens in supernatants. Pre coat 96 well plate with primary antibody against the antigen, wash and use stabilcoat for blocking and use HRP conjugated secondary against antigen, substrate and read.

I wouldn't recommend coating with your DMEM medium containing your antigen of interest for two main reasons; First, your antigen may be at relatively lower concentrations and may be out-competed for binding to the pate by other molecules/antigens in the medium, leading to very weak specific signals. Second, there is the possibility of high degrees of non-specific binding, thereby resulting in "over-estimation" of specific analyte levels. A sandwich ELISA with two different mAbs, or polyclonal Abs will be the best approach.

@Mohamed Alalem. You have probably done your protein determination in DMEM by now. However, for those who have and will follow this thread, I would like to add that I recently tried the Cytometric Bead Array assay offered by BDBiosciences. As long as you have access to a flow cytometer (FACS Calibur, Fortessa etc. but NOT FACScan) this assay is simple and very good for protein determination in many different specimens. You can even measure phosphorylated proteins.

Perform the test in the pattern of Sandwich Elisa along with all possible controls.You can contemplate specific reaction.