Effective Blocking Procedures

ELISA Technical Bulletin - No. 3

Life
Sciences

Judy Gibbs Introduction

Corning Incorporated
Solid phase immunoassays, such as ELISA, involve
Life Sciences
the immobilization of biomolecules, primarily
Kennebunk, ME
proteins, to the surface via passive or covalent
interactions. The ability of the surface to interact
Table of Contents
with proteins and other biomolecules is obviously
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 an essential feature; however, non-specific binding
(NSB) of other proteins or biomolecules to unoc-
Typical Problems Associated with cupied spaces on the surface during subsequent
Blocking Reagents . . . . . . . . . . . . . . . . . . . . . . . 2 steps of the assay can be detrimental to the speci-
ficity and sensitivity of the assay results. Non-spe-
Detergent Blockers. . . . . . . . . . . . . . . . . . . . . . . 2 cific binding to the surface can be minimized by
saturating these unoccupied binding sites with a
blocking reagent — a collective term for various
Protein Blockers. . . . . . . . . . . . . . . . . . . . . . . . . 3
substances that are used to reduce NSB without
taking an active part in specific assay reactions.
Miscellaneous Blockers. . . . . . . . . . . . . . . . . . . . 5 (Other factors can influence NSB, such as pro-
tein-protein interactions that are unique to each
Matching the Blocker to the Surface . . . . . . . . . 5 ELISA system, and must be considered during
assay development and optimization.) Blocking
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 reagents and methods are typically chosen in an
empirical manner, since a single standardized pro-
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 cedure has not been determined suitable for all
applications. However, for any given application
or assay, a best method usually can be found quite
 readily if one chooses a blocking advantages and disadvantages of each type
reagent/method based on: and assess how these features will affect
the assay. Some of the major problems
◗ the type of surface,
associated with blocking reagents in
◗ the type of biomolecule immobilized general are:
to the surface, and
◗ lot-to-lot inconsistencies (certain
◗ the type of detection probe/system
sources of bovine serum albumin, fish
employed.
gelatin, and normal mammalian serum
The two major classes of blocking vary in quality from lot-to-lot),
reagents are: ◗ masking of surface bound proteins by
◗ proteins, and interfering with specific protein-protein
◗ detergents (typically non-ionic). interactions (fish gelatin tends to block
protein-protein interactions more
Both classes have advantages and dis- tenaciously than protein-surface
advantages, which will be discussed in this interactions, thus reducing specific
bulletin and measured against the proper- binding more so than non-specific
ties of an ideal blocking reagent. (Keeping binding),
in mind that a universal blocking reagent
◗ lack of molecular diversity (many single
for all assays is idealistic, not realistic.)
molecule blocking reagents lack the
An ideal blocking reagent should:
diversity to block surfaces comprised
◗ inhibit non-specific binding (passive of hydrophobic, ionic and covalent
and covalent) of assay components to regions),
the surface, ◗ cross-reactivity with assay components
◗ inhibit non-specific protein-protein (i.e., Protein A will cross-react with the
interactions, non-specific IgG molecules of normal
◗ exhibit no cross-reactivity with mammalian serum),
subsequent assay components (i.e., ◗ disruption of non-covalent bonds
antibodies, protein A), between specific biomolecules and the
◗ act as a stabilizer for (or assist in surface (i.e., non-ionic detergents may
renaturing) biomolecules by minimizing displace hydrophobically attached
the effects of denaturation caused by proteins and biomolecules),
phase transitions associated with solid ◗ interference with detection due to
phase assays, endogenous enzyme activity, intrinsic
◗ exhibit low enzyme activity (or other fluorescence, etc.
activity that may interfere with the
detection method), Detergent Blockers
◗ not disrupt the bonds that immobilize One of the major classes of blocking
the specific protein or biomolecule to reagents is detergents — non-ionic and
the surface, and ionic. For solid phase immunoassays on
◗ exhibit consistent, reproducible polystyrene (or other hard plastic), ionic
performance with every lot. detergents are seldom used as the sole
blocking mechanism due to:
Blocking a surface to reduce non-specific
◗ their propensity to disrupt ionic and
binding is a compromise between low
hydrophobic biomolecule-surface
background and high sensitivity and
bonds,
specificity. The best blocking reagent
and method for any particular assay will ◗ their ability to solubilize proteins, and
be an optimized, but not absolute, choice. ◗ their tendency to inhibit (or terminate)
enzyme-substrate reactions.
Typical Problems Associated Zwitterionic detergents are simply poor
with Blocking Reagents blockers so are not even considered as
Since no blocking reagent or method is blocking reagents. Typically, detergents
ideal for all assays, one must consider the used as blocking reagents are non-ionic;

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 the most common being Tween 20. Our recommendation for using a non-
Detergents are considered temporary ionic detergent as a blocking reagent for
blockers; they do not provide a perma- hard plastic assays (i.e., 96 well plates or
nent barrier to biomolecule attachment to strips) is to include it in the wash buffer
the surface because their blocking ability and not use it as the sole blocking reagent
can be removed by washing with water or for the assay. The preferred non-ionic
aqueous buffer. To be useful as the sole detergent for this purpose is Tween 20,
blocking reagent in an assay, detergents which is also the most commonly used
must be present in all the diluents/buffers at concentrations ranging from 0.01 to
subsequent to coating the surface with a 0.1%. Some non-ionic detergents, such as
capture molecule. However; when used in Triton X-100, although excellent blockers
conjunction with a protein blocker, deter- of non-specific binding to the surface, can
gents provide added convenient and inex- cause a high loss of specific binding, result-
pensive blocking ability during wash ing in false negative results. By using non-
steps, etc. by blocking areas on the sur- ionic detergents at low concentrations in
face that may become exposed due to wash buffers, the negative aspects can be
protein/biomolecule desorption. avoided, while the benefit of added block-
ing ability can still be exploited.
Non-ionic detergents are advantageous
for the following reasons: They are:
Protein Blockers
◗ inexpensive, even though they must
Protein blockers can serve two purposes:
be used at a concentration equal to or
greater than their Critical Micelle ◗ block non-occupied sites on the surface
Concentration (CMC) value (typical and
concentrations for Tween 20 are 0.01% ◗ space out and stabilize biomolecules
to 0.10%), bound to the surface to reduce steric
◗ extremely stable and can be stored in hindrance and denaturation problems
diluted form (i.e., wash buffers) at room associated with solid phase assays.
temperature for extended periods of Unlike non-ionic detergents, proteins
time without experiencing any loss of are permanent blockers and only need to
blocking activity, be added once after the surface is coated
◗ useful in washing solutions because with the capture molecule. However, it is
their presence blocks areas on the sur- common practice to add protein blockers
face that may be physically stripped of to diluents used for subsequent assay
specifically bound biomolecules during reactants to further reduce background
the wash step and helps dislodge loosely and stabilize surface bound biomolecules.
bound biomolecules that are physically Some of the most commonly used protein
trapped in corners. blockers are:
Major disadvantages associated with ◗ bovine serum albumin,
non-ionic detergents are: ◗ non-fat dry milk or casein,
◗ they may disrupt non-covalent ◗ whole normal serum, and
biomolecule-surface bonds,
◗ fish gelatin.
◗ they block hydrophobic interactions
only, Each of these blockers has its own
advantages and disadvantages.
◗ residual detergent left in wells following
the immobilization of a peroxidase Bovine Serum Albumin
conjugate can interfere with its Bovine serum albumin (BSA) is typically
enzymatic activity, used at a 1 to 3% concentration. BSA is
◗ they are not permanent blockers, and inexpensive and can be stored dry or as a
◗ they cannot be used with sterile solution at 4°C. The use of BSA
lipopolysaccharides due to their ability as a blocking reagent is well documented
to successfully compete against these and has been proven to be a good blocker
biomolecules for surface space. of non-specific protein-surface binding

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 on medium and high binding surfaces, as ◗ some preparations of NFDM may
well as many of the pre-activated covalent contain histones that interfere with
surfaces. An advantage associated with anti-DNA determinations, and
using BSA is its compatibility with Protein ◗ alkaline phosphatase activity can be
A. Disadvantages associated with BSA inhibited by some preparations of
include: NFDM.
◗ lot-to-lot variability — primarily related Overall, these are minor issues. NFDM
to the fatty acid content (BSA used as a is an excellent blocking reagent. Due to
blocking reagent should be fatty acid its molecular diversity and amphipathic
free), characteristics, NFDM is the preferred
◗ presence of phosphotyrosine in blocking reagent for many covalent
Fraction V preparations that cross- surfaces.
reacts with anti-phosphotyrosine
antibodies, Fish Gelatin
◗ cross-reactions with antibodies prepared Although fairly popular as a blocking
against BSA-hapten conjugates (BSA is reagent, fish gelatin has some major dis-
typically linked to small haptens that advantages. Typically, gelatin is not an
lack the ability to elicit an immune adequate blocker when used alone and
response as individual molecules), and is actually the least effective biomolecule-
surface blocker discussed in this bulletin.
◗ lack of diversity required to block some It blocks mainly protein-protein interac-
covalent surfaces (surfaces that feature tions, sometimes masking specific sur-
hydrophobic, ionic and covalent face bound proteins and interfering with
characteristics). immunoreactivity. The inferior surface
Despite its disadvantages, BSA is probably blocking ability and the protein-masking
the most widely used blocking reagent for characteristic of gelatin results in higher
solid phase immunoassays. background and decreased sensitivity.
Gelatin also tends to vary in quality from
Non-Fat Dry Milk lot-to-lot. The greatest advantage associ-
Non-fat dry milk (NFDM) is typically ated with fish gelatin is its lack of cross-
used at 0.1 to 0.5% concentrations and is reactivity with mammalian antibodies
relatively inexpensive; however, prepara- and Protein A.
tions vary in quality. We have found only
one source of NFDM (a 2% solution) Whole Sera
that exhibits acceptable lot-to-lot consis- For extremely difficult blocking prob-
tency and stability. NFDM, either home- lems, the use of normal whole sera at a
made or commercial, has a tendency to 10% concentration is recommended.
deteriorate rapidly if not properly prepared Due to its molecular diversity, whole
and stored. Although casein, a non-fat sera effectively blocks non-specific:
dry milk component, can be used as a
◗ biomolecule-surface (passive
stable blocking reagent (primarily for
adsorption) interactions,
DNA blots), NFDM tends to be more
dispersible in aqueous buffers than pure ◗ biomolecule-covalent surface
casein. This may explain why it is the interactions, and
better blocker of the two on hard plastic ◗ protein-protein interactions, while
surfaces. Although NFDM is compatible acting as a protein stabilizer as well.
with Protein A and exhibits little cross- The disadvantages of using normal whole
reactivity with typical immunoassay com- sera as a blocking reagent center around
ponents, it does express the following its cross-reactivity with Protein A and
reactivity related problems: anti-IgG antibodies. Since many immuno-
◗ milk contains phosphotyrosine which assays rely on a system that utilizes a
reacts with anti-phosphotyrosine labeled (enzyme, radiolabel, etc.) second-
antibodies, ary anti-IgG antibody, blocking with nor-

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 mal whole sera can lead to false positive protein blocker (1% BSA, 0.2% NFDM,
reactions and high non-specific binding 10% normal sera, etc.) is suggested to
due to this cross-reactivity issue. Alter- effectively minimize non-specific binding.
natives to normal mammalian sera are The choice of protein blocker is more
fish or chicken sera. Both lack the cross- dependent on the assay’s reactive bio-
reactivity problems associated with their molecules than on the surface itself.
mammalian equivalents, yet retain the Surfaces that are highly charged and
positive aspects of being molecularly exhibit little to no hydrophobic character
diverse in order to block surfaces with must be blocked with a protein blocker.
mixed characteristics (hydrophobic, hydro- Since an ionic surface is typically only
philic and covalent functional groups). used for the immobilization of small,
ionic molecules, the chosen blocker must
Miscellaneous Blockers be both relatively small to prevent the
As assays become more sensitive and eclipsing of the specific capture molecule
surfaces become more diverse, there is and express the appropriate ionic species
a need for alternative blocking reagents in order to interact with the surface
that perform a variety of functions charge. BSA (1 to 3%) or non-fat dry
beyond reducing non-specific back- milk (0.2 to 2%) can be used for most
ground. Examples of alternative blockers assays; however, a smaller molecule such
include polymers such as polyethylene as ethanolamine (10%) may be necessary
glycol (PEG), polyvinyl alcohol (PVA), when very small biomolecules are specifi-
and polyvinylpyrrolidone (PVP). These cally bound. Non-ionic detergents are
blocking reagents are known for their useless in terms of blocking an ionic
ability to coat hydrophobic surfaces and surface.
render them both non-binding as well as
hydrophilic. This hydrophilicity-produc- Covalent Surfaces
ing characteristic has been exploited for (See the Corning Surface Selection Guide on
assays designed as one-step on lateral the Corning web site for additional infor-
flow matrices (i.e. over-the-counter mation on ELISA Plates with Covalent
pregnancy tests). Surfaces.)
An amine surface used with bifunctional
Matching the Blocker crosslinkers must be blocked with a pro-
to the Surface tein blocker capable of interacting with
unreacted hydrophobic sites, ionic sites
Passive Surfaces and covalent sites. We suggest using
Hydrophobic surfaces consist of those non-fat dry milk (02 to 2%) if possible.
typically referred to as medium binding. Another option is to use 10% normal
These surfaces can be effectively blocked serum as a primary blocking reagent or
with either non-ionic detergents or pro- as a constituent of the post-coating assay
tein blockers. In our experience, the buffer(s). Non-ionic detergents are in-
combined use of 0.02% Tween 20 and efficient as blockers for this surface, but
1% BSA has been ideal for most assays including Tween 20 in the wash buffer
on medium binding surfaces. can enhance the removal of non-bound,
Surfaces that are comprised of hydrophobic physically trapped biomolecules.
and ionic binding sites are typically termed Pre-activated covalent surfaces (N-
high binding. Due to the ability of IgG oxysuccinimide, Maleimide, Hydrazide,
and its conjugates to displace detergents, Universal) most always consist of hydro-
high binding surfaces are slightly more phobic and covalent regions. Amphipathic
difficult to block than medium binding proteins tend to be the most efficient
surfaces. The combined use of a non- blockers of covalent surfaces. Non-ionic
ionic detergent (0.02% Tween 20) and a detergents will not block covalent inter-

5
 actions, but their presence in wash buffers It is advisable that during the develop-
is recommended regardless of the surface ment of a blocking procedure, each of
used. The following is a recommended the proposed blockers and blocking con-
method for blocking the four covalent ditions (buffers, incubation times, etc.)
surfaces listed above: be evaluated for cross-reactivity with all
other assay reactants. The ideal blocker
1. After covalently immobilizing a specific
and blocking procedure will effectively
biomolecule to the surface, block the
and reproducibly eliminate non-specific
plate with 2% BSA for approximately
surface attachment and improve assay
30 minutes. The BSA diluent should be
sensitivity and specificity — resulting
compatible with the surface and pH
in a high signal/low noise assay.
adjusted to allow the covalent inter-
action between the blocker and the
surface to occur. If a protein blocker Technical Assistance
other than BSA is used, it must possess For additional ELISA technical support
an appropriate functional group that and bulletins or product information,
can interact with the covalent sites on please visit the Corning Life Sciences
the surface. website at www.corning.com/lifesciences
2. Due to the complexity of the surface or call 1-800-492-1110.
chemistry, the addition of 10% normal
sera (such as fetal bovine, goat, fish or References
chicken sera) to all reactant diluents is 1. Bjerrum, O.J. and Heegaard, N.H.H.
recommended and necessary for most Handbook of Immunoblotting of Proteins,
assays. Normal sera have the molecular Volume 1. Boca Raton, Florida: CRC
diversity necessary to block non-specific Press, Inc.: 1988.
binding due to hydrophobic, ionic, and 2. Craig, J.C. et. al. Journal of Biological
covalent interactions. Standardization. 17; 1989; 125-135.
3. Engvall, E. and Perlmann, P.
Conclusion Immunochemistry. 8:871; 1971.
4. Gardas, A. and Lewartowska, A. Journal
In summary, the selection of an approp-
of Immunological Methods. 106; 1988;
riate blocking system is essential to the 251-255.
development of a specific and sensitive
5. Graves, H.C.B. Journal of Immunological
assay. Most often the choice is based on Methods. 111; 1988; 157-166.
convenience, literature and “what has tra-
6. Kenny, G.E. and Dunsmoor, C.L. Israel
ditionally worked.” In reality, empirical Journal of Medical Sciences. 23; 1987;
testing is required to both choose the best 732-734.
blocker(s) and optimize the blocking pro- 7. Maggio, E.T. Enzyme Immunoassay.
cedure. This testing is heavily influenced Boca Raton, Florida: CRC Press, Inc.; 1980.
by the surface chemistry as well as inter- 8. Stenberg, M. and Nygren, H. Journal of
actions unique to the specific assay reac- Immunological Methods. 113; 1988; 3-15.
tants, primarily cross-reactivity. A blocker 9. Vogt, R.F. et. al. Journal of Immunological
can totally inhibit non-specific reactions Methods. 101; 1987; 43-50.
with the surface and not reduce signal-
to-noise due to cross-reactivity issues.

Revised 7/01

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