There is two things:

a) You need to coat your plate with enough proteins. Either your antigen ( or antibody ) is at a sufficient concentration to do the job or another overlaying material should be added. Otherwise, anything added at the next elisa step, can still stick to the plate.

b) some unwanted reaction between your serum, your second antibody, or your revelation system must be suppressed. A blocking agent in the liquid phase is there needed. Either in your dilution buffer or in your washing buffer.

Skim-milk, BSA, fish serum when available, are usually suitable.

And don't also forget to add a little bit detergent ( tween 20 10^4 generally 10^3 sometimes ) in your washing buffer!

Good luck!

J.

Yes, serum molecules may bind to your blocking agent. This is highly variable between sera, as well as between blocking agents.

I have used the indirect ELISA technique to characterize various blocking buffers, including skim milk, BlockAce(TM), cell culture grade BSA, premium grade BSA & BSA fraction V. Some antibodies were shown to bind to proteins present in some blocking agents. Premium grade BSA and BSA fraction V were best suited to the assays I have developed, giving lowest background & non-specific binding in sandwich ELISA applications.

Thank you so much Karl,

May I ask one more question? Can I exclude skimmilk antibodies from the serum by using blocking buffer (containing skimmilk) as a dilution buffer for serum?

Blocking is a kind of modern voodoo. There is a limited binding capacity of the polystyrene surface you will find an optimal concentration between 1 - 5 (10) µg/mL for the coating protein. Mostly at basic conditions because you need most of the free charges in the amino acids neutralized. The binding to polystyrene will be by hydrophobic interactions. The affinity of those reaction is very high (at 10Ex15 mol/L). But is will take it's time due to the multipoint reaction.

What is blocking? If you believe that there are some free binding sites at your plates, do it, but if you compare the detection limits or the imprecision you will see either no change or only some worsening. From a coated plate the coating solution should be removed as complete as possible and the plates can be stores (upside down) frozen until use. Those plates should never be washed with a detergent containing buffer. The hydrophobic end of the detergent molecules will replace the hydrophobic amino acid residues from their interaction with the polystyrene, slowly but surely.

Blocking is only useful in commercial Here is a blocking helpful to reach a shelf life of 1 – 3 years at room temperature. Normally an inert (and well defined) protein is used (BSA, kappa-casein, hydrolyzed gelatin). The normal concentration is at 5 - 20 µg/mL and some sugar is used. This is to bind some water which keeps the coated protein alive (in function). Otherwise the risk for denaturation is present. Normally 1 - 3% sugar (glucose, mannose or a mixture of both) is used.

In my experience, adding a blocking agent to the serum diluent may not prevent nonspecific binding. A cell culture grade BSA solution was used to dilute calibrators, as well as a serum used for detection. Very high background was observed in calibrator wells, but not in sample wells, though the same detection solution was applied to the whole plate. If the anti-blocking antibodies in the serum were saturated by the blocker in the diluent, they should not have bound to the blocker in my calibrators.

Good quality blocking agents are not particularly expensive when bought in bulk, and will last quite a long time if stored correctly. In addition, since serum production may yield a variable product, you may get more consistent results in the long term by using a nonreactive blocker, rather than by controlling for the serum's nonspecific binding to a lower quality blocker.

Yes you can, more simple and cheap.

I have been using skim milk in almost all the ELISA I have performed. For antibody determination in serum and urine (from mice) this blocking reagent was fine. However for cytokine quantitation I use BSA solution.

Before to consider a blocking step by putting a few skimmilk in the sample dilution buffer, you've to check your background level. Most of the time, you don't need it.

J.

Normally milk works well but if you have a serum sample from someone with a casein allergy you'll have background. BSA works well and I've actually found the 5-10% FBS works best.

Unorthodox blockers. Use 2 to 5% Irish Cream in buffer. Alcohol keeps cream in solution while blocking. Also, as alternative blocker, try diluted fish serum (if you can get it). One of the big companies use it as a blocker. Has less non-specific proteins to interact.

Sir. Jacques Cohen,

Thank you. That makes sense to me. May I ask you one more question? If Blocking buffer can increase the background, and sometimes blocking step is not necessary, why scientists suggest us to block the plate by protein? When we need blocking step and why?

I greatly appreciate any answer you could give me.

Thank you again.

There is two things:

a) You need to coat your plate with enough proteins. Either your antigen ( or antibody ) is at a sufficient concentration to do the job or another overlaying material should be added. Otherwise, anything added at the next elisa step, can still stick to the plate.

b) some unwanted reaction between your serum, your second antibody, or your revelation system must be suppressed. A blocking agent in the liquid phase is there needed. Either in your dilution buffer or in your washing buffer.

Skim-milk, BSA, fish serum when available, are usually suitable.

And don't also forget to add a little bit detergent ( tween 20 10^4 generally 10^3 sometimes ) in your washing buffer!

Good luck!

J.

Thank you very much Sir Jacques Cohen

The point of using blockers is what Jacques mentioned. There are two types of blockers one that you use for surface (in order to close any open surface which is not occupied by antibody and where effectively your analyte or detection antibodies can bind) and others that you use to block the cross reactivity of your antibodies toward serum proteins (as they are used an carrier during antibody generation).

Thus to block surfaces the wise choice of blocker is protein coming from either non animal source or from irrelevant animal source which is a distant species than the animal in which your antibodies are raised (though most of the serum.albumin proteins may be homologues). As you suggested casein is a good choice but for further improving your blocking experience people usually compromise over cross reactivity and use a mixture of casein and bsa. Hope this will be helpful information.

Thank you Chandra,

May I ask one more question?

Should the protein in blocking buffer and carrier protein in diluent buffer be the same? Could we use different proteins for blocking and diluent buffers?

As I know, scientists usually use the same protein for both buffers, is there any reason?

Thank you again.

Usually BSA conjugates are used as carrier proteins for developing antibody. Thus in most cases people use bsa in both buffers without even knowing what was the conjugate used.

So it matters to know what conjugate was used to choose and design an appropriate diluent buffer. As I mentioned before people usually compromise on crossreactivity and use casein and bsa in blocking buffer.....

Thank you for your reply, Chandra.

If you want to completely eliminate blocking issues, you need to shift to a carbohydrate or better yet, one of the synthetic blockers on the market. Polyvinyl alcohol and Ficoll for example.

Using proteins as a "block" in an antibody assay has always struck me as a potential problem.