Judy Gibbs Corning IncorporatedLife SciencesKennebunk, ME

Table of Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Typical Problems Associated with Blocking Reagents . . . . . . . . . . . . . . . . . . . . . . . 2

Detergent Blockers. . . . . . . . . . . . . . . . . . . . . . . 2

Protein Blockers. . . . . . . . . . . . . . . . . . . . . . . . . 3

Miscellaneous Blockers. . . . . . . . . . . . . . . . . . . . 5

Matching the Blocker to the Surface . . . . . . . . . 5

Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

IntroductionSolid phase immunoassays, such as ELISA, involvethe immobilization of biomolecules, primarilyproteins, to the surface via passive or covalentinteractions. The ability of the surface to interactwith proteins and other biomolecules is obviouslyan essential feature; however, non-specific binding(NSB) of other proteins or biomolecules to unoc-cupied spaces on the surface during subsequentsteps of the assay can be detrimental to the speci-ficity and sensitivity of the assay results. Non-spe-cific binding to the surface can be minimized bysaturating these unoccupied binding sites with ablocking reagent — a collective term for varioussubstances that are used to reduce NSB withouttaking an active part in specific assay reactions.(Other factors can influence NSB, such as pro-tein-protein interactions that are unique to eachELISA system, and must be considered duringassay development and optimization.) Blockingreagents and methods are typically chosen in anempirical manner, since a single standardized pro-cedure has not been determined suitable for allapplications. However, for any given applicationor assay, a best method usually can be found quite

Effective Blocking Procedures ELISA Technical Bulletin - No. 3

Life Sciences

readily if one chooses a blockingreagent/method based on:

◗ the type of surface, ◗ the type of biomolecule immobilized

to the surface, and ◗ the type of detection probe/system

employed.

The two major classes of blockingreagents are:

◗ proteins, and ◗ detergents (typically non-ionic).

Both classes have advantages and dis-advantages, which will be discussed in thisbulletin and measured against the proper-ties of an ideal blocking reagent. (Keepingin mind that a universal blocking reagentfor all assays is idealistic, not realistic.) An ideal blocking reagent should:

◗ inhibit non-specific binding (passiveand covalent) of assay components tothe surface,

◗ inhibit non-specific protein-proteininteractions,

◗ exhibit no cross-reactivity withsubsequent assay components (i.e.,antibodies, protein A),

◗ act as a stabilizer for (or assist inrenaturing) biomolecules by minimizingthe effects of denaturation caused byphase transitions associated with solidphase assays,

◗ exhibit low enzyme activity (or otheractivity that may interfere with thedetection method),

◗ not disrupt the bonds that immobilizethe specific protein or biomolecule tothe surface, and

◗ exhibit consistent, reproducibleperformance with every lot.

Blocking a surface to reduce non-specificbinding is a compromise between lowbackground and high sensitivity andspecificity. The best blocking reagent and method for any particular assay willbe an optimized, but not absolute, choice.

Typical Problems Associated with Blocking ReagentsSince no blocking reagent or method isideal for all assays, one must consider the

advantages and disadvantages of each typeand assess how these features will affectthe assay. Some of the major problemsassociated with blocking reagents ingeneral are:

◗ lot-to-lot inconsistencies (certainsources of bovine serum albumin, fishgelatin, and normal mammalian serumvary in quality from lot-to-lot),

◗ masking of surface bound proteins byinterfering with specific protein-proteininteractions (fish gelatin tends to blockprotein-protein interactions moretenaciously than protein-surfaceinteractions, thus reducing specificbinding more so than non-specificbinding),

◗ lack of molecular diversity (many singlemolecule blocking reagents lack thediversity to block surfaces comprised of hydrophobic, ionic and covalentregions),

◗ cross-reactivity with assay components(i.e., Protein A will cross-react with thenon-specific IgG molecules of normalmammalian serum),

◗ disruption of non-covalent bondsbetween specific biomolecules and thesurface (i.e., non-ionic detergents maydisplace hydrophobically attachedproteins and biomolecules),

◗ interference with detection due toendogenous enzyme activity, intrinsicfluorescence, etc.

Detergent BlockersOne of the major classes of blockingreagents is detergents — non-ionic andionic. For solid phase immunoassays onpolystyrene (or other hard plastic), ionicdetergents are seldom used as the soleblocking mechanism due to:

◗ their propensity to disrupt ionic andhydrophobic biomolecule-surfacebonds,

◗ their ability to solubilize proteins, and ◗ their tendency to inhibit (or terminate)

enzyme-substrate reactions. Zwitterionic detergents are simply poorblockers so are not even considered asblocking reagents. Typically, detergentsused as blocking reagents are non-ionic;

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the most common being Tween 20.Detergents are considered temporaryblockers; they do not provide a perma-nent barrier to biomolecule attachment tothe surface because their blocking abilitycan be removed by washing with water oraqueous buffer. To be useful as the soleblocking reagent in an assay, detergentsmust be present in all the diluents/bufferssubsequent to coating the surface with acapture molecule. However; when used inconjunction with a protein blocker, deter-gents provide added convenient and inex-pensive blocking ability during washsteps, etc. by blocking areas on the sur-face that may become exposed due toprotein/biomolecule desorption.

Non-ionic detergents are advantageousfor the following reasons: They are:

◗ inexpensive, even though they must be used at a concentration equal to orgreater than their Critical MicelleConcentration (CMC) value (typicalconcentrations for Tween 20 are 0.01%to 0.10%),

◗ extremely stable and can be stored indiluted form (i.e., wash buffers) at roomtemperature for extended periods oftime without experiencing any loss ofblocking activity,

◗ useful in washing solutions becausetheir presence blocks areas on the sur-face that may be physically stripped ofspecifically bound biomolecules duringthe wash step and helps dislodge looselybound biomolecules that are physicallytrapped in corners.

Major disadvantages associated with non-ionic detergents are:

◗ they may disrupt non-covalentbiomolecule-surface bonds,

◗ they block hydrophobic interactionsonly,

◗ residual detergent left in wells followingthe immobilization of a peroxidaseconjugate can interfere with itsenzymatic activity,

◗ they are not permanent blockers, and ◗ they cannot be used with

lipopolysaccharides due to their abilityto successfully compete against thesebiomolecules for surface space.

Our recommendation for using a non-ionic detergent as a blocking reagent forhard plastic assays (i.e., 96 well plates orstrips) is to include it in the wash bufferand not use it as the sole blocking reagentfor the assay. The preferred non-ionicdetergent for this purpose is Tween 20,which is also the most commonly used at concentrations ranging from 0.01 to0.1%. Some non-ionic detergents, such asTriton X-100, although excellent blockersof non-specific binding to the surface, cancause a high loss of specific binding, result-ing in false negative results. By using non-ionic detergents at low concentrations inwash buffers, the negative aspects can beavoided, while the benefit of added block-ing ability can still be exploited.

Protein BlockersProtein blockers can serve two purposes:

◗ block non-occupied sites on the surfaceand

◗ space out and stabilize biomoleculesbound to the surface to reduce sterichindrance and denaturation problemsassociated with solid phase assays.

Unlike non-ionic detergents, proteins are permanent blockers and only need tobe added once after the surface is coatedwith the capture molecule. However, it iscommon practice to add protein blockersto diluents used for subsequent assayreactants to further reduce backgroundand stabilize surface bound biomolecules.Some of the most commonly used proteinblockers are:

◗ bovine serum albumin, ◗ non-fat dry milk or casein, ◗ whole normal serum, and ◗ fish gelatin.

Each of these blockers has its ownadvantages and disadvantages.

Bovine Serum AlbuminBovine serum albumin (BSA) is typicallyused at a 1 to 3% concentration. BSA isinexpensive and can be stored dry or as asterile solution at 4°C. The use of BSAas a blocking reagent is well documentedand has been proven to be a good blockerof non-specific protein-surface binding

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on medium and high binding surfaces, aswell as many of the pre-activated covalentsurfaces. An advantage associated withusing BSA is its compatibility with ProteinA. Disadvantages associated with BSAinclude:

◗ lot-to-lot variability — primarily relatedto the fatty acid content (BSA used as ablocking reagent should be fatty acidfree),

◗ presence of phosphotyrosine inFraction V preparations that cross-reacts with anti-phosphotyrosineantibodies,

◗ cross-reactions with antibodies preparedagainst BSA-hapten conjugates (BSA istypically linked to small haptens thatlack the ability to elicit an immuneresponse as individual molecules), and

◗ lack of diversity required to block somecovalent surfaces (surfaces that featurehydrophobic, ionic and covalentcharacteristics).

Despite its disadvantages, BSA is probablythe most widely used blocking reagent forsolid phase immunoassays.

Non-Fat Dry MilkNon-fat dry milk (NFDM) is typicallyused at 0.1 to 0.5% concentrations and isrelatively inexpensive; however, prepara-tions vary in quality. We have found onlyone source of NFDM (a 2% solution)that exhibits acceptable lot-to-lot consis-tency and stability. NFDM, either home-made or commercial, has a tendency todeteriorate rapidly if not properly preparedand stored. Although casein, a non-fatdry milk component, can be used as astable blocking reagent (primarily forDNA blots), NFDM tends to be moredispersible in aqueous buffers than purecasein. This may explain why it is thebetter blocker of the two on hard plasticsurfaces. Although NFDM is compatiblewith Protein A and exhibits little cross-reactivity with typical immunoassay com-ponents, it does express the followingreactivity related problems:

◗ milk contains phosphotyrosine whichreacts with anti-phosphotyrosineantibodies,

◗ some preparations of NFDM maycontain histones that interfere withanti-DNA determinations, and

◗ alkaline phosphatase activity can beinhibited by some preparations ofNFDM.

Overall, these are minor issues. NFDM is an excellent blocking reagent. Due toits molecular diversity and amphipathiccharacteristics, NFDM is the preferredblocking reagent for many covalentsurfaces.

Fish GelatinAlthough fairly popular as a blockingreagent, fish gelatin has some major dis-advantages. Typically, gelatin is not anadequate blocker when used alone and is actually the least effective biomolecule-surface blocker discussed in this bulletin.It blocks mainly protein-protein interac-tions, sometimes masking specific sur-face bound proteins and interfering withimmunoreactivity. The inferior surfaceblocking ability and the protein-maskingcharacteristic of gelatin results in higherbackground and decreased sensitivity.Gelatin also tends to vary in quality fromlot-to-lot. The greatest advantage associ-ated with fish gelatin is its lack of cross-reactivity with mammalian antibodies and Protein A.

Whole SeraFor extremely difficult blocking prob-lems, the use of normal whole sera at a10% concentration is recommended. Due to its molecular diversity, whole sera effectively blocks non-specific:

◗ biomolecule-surface (passiveadsorption) interactions,

◗ biomolecule-covalent surfaceinteractions, and

◗ protein-protein interactions, whileacting as a protein stabilizer as well.

The disadvantages of using normal wholesera as a blocking reagent center aroundits cross-reactivity with Protein A andanti-IgG antibodies. Since many immuno-assays rely on a system that utilizes alabeled (enzyme, radiolabel, etc.) second-ary anti-IgG antibody, blocking with nor-

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mal whole sera can lead to false positivereactions and high non-specific bindingdue to this cross-reactivity issue. Alter-natives to normal mammalian sera arefish or chicken sera. Both lack the cross-reactivity problems associated with theirmammalian equivalents, yet retain thepositive aspects of being molecularlydiverse in order to block surfaces withmixed characteristics (hydrophobic, hydro-philic and covalent functional groups).

Miscellaneous BlockersAs assays become more sensitive andsurfaces become more diverse, there is a need for alternative blocking reagentsthat perform a variety of functionsbeyond reducing non-specific back-ground. Examples of alternative blockersinclude polymers such as polyethyleneglycol (PEG), polyvinyl alcohol (PVA),and polyvinylpyrrolidone (PVP). Theseblocking reagents are known for theirability to coat hydrophobic surfaces andrender them both non-binding as well ashydrophilic. This hydrophilicity-produc-ing characteristic has been exploited forassays designed as one-step on lateralflow matrices (i.e. over-the-counterpregnancy tests).

Matching the Blocker to the Surface

Passive SurfacesHydrophobic surfaces consist of thosetypically referred to as medium binding.These surfaces can be effectively blockedwith either non-ionic detergents or pro-tein blockers. In our experience, thecombined use of 0.02% Tween 20 and1% BSA has been ideal for most assays on medium binding surfaces.

Surfaces that are comprised of hydrophobicand ionic binding sites are typically termedhigh binding. Due to the ability of IgGand its conjugates to displace detergents,high binding surfaces are slightly moredifficult to block than medium bindingsurfaces. The combined use of a non-ionic detergent (0.02% Tween 20) and a

protein blocker (1% BSA, 0.2% NFDM,10% normal sera, etc.) is suggested toeffectively minimize non-specific binding.The choice of protein blocker is moredependent on the assay’s reactive bio-molecules than on the surface itself.

Surfaces that are highly charged andexhibit little to no hydrophobic charactermust be blocked with a protein blocker.Since an ionic surface is typically onlyused for the immobilization of small,ionic molecules, the chosen blocker mustbe both relatively small to prevent theeclipsing of the specific capture moleculeand express the appropriate ionic speciesin order to interact with the surfacecharge. BSA (1 to 3%) or non-fat drymilk (0.2 to 2%) can be used for mostassays; however, a smaller molecule suchas ethanolamine (10%) may be necessarywhen very small biomolecules are specifi-cally bound. Non-ionic detergents areuseless in terms of blocking an ionicsurface.

Covalent Surfaces(See the Corning Surface Selection Guide onthe Corning web site for additional infor-mation on ELISA Plates with CovalentSurfaces.)

An amine surface used with bifunctionalcrosslinkers must be blocked with a pro-tein blocker capable of interacting withunreacted hydrophobic sites, ionic sitesand covalent sites. We suggest using non-fat dry milk (02 to 2%) if possible.Another option is to use 10% normalserum as a primary blocking reagent or as a constituent of the post-coating assaybuffer(s). Non-ionic detergents are in-efficient as blockers for this surface, butincluding Tween 20 in the wash buffercan enhance the removal of non-bound,physically trapped biomolecules.

Pre-activated covalent surfaces (N-oxysuccinimide, Maleimide, Hydrazide,Universal) most always consist of hydro-phobic and covalent regions. Amphipathicproteins tend to be the most efficientblockers of covalent surfaces. Non-ionicdetergents will not block covalent inter-

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actions, but their presence in wash buffersis recommended regardless of the surfaceused. The following is a recommendedmethod for blocking the four covalentsurfaces listed above:

1. After covalently immobilizing a specificbiomolecule to the surface, block theplate with 2% BSA for approximately30 minutes. The BSA diluent should becompatible with the surface and pHadjusted to allow the covalent inter-action between the blocker and thesurface to occur. If a protein blockerother than BSA is used, it must possessan appropriate functional group thatcan interact with the covalent sites onthe surface.

2. Due to the complexity of the surfacechemistry, the addition of 10% normalsera (such as fetal bovine, goat, fish orchicken sera) to all reactant diluents isrecommended and necessary for mostassays. Normal sera have the moleculardiversity necessary to block non-specificbinding due to hydrophobic, ionic, andcovalent interactions.

ConclusionIn summary, the selection of an approp-riate blocking system is essential to thedevelopment of a specific and sensitiveassay. Most often the choice is based onconvenience, literature and “what has tra-ditionally worked.” In reality, empiricaltesting is required to both choose the bestblocker(s) and optimize the blocking pro-cedure. This testing is heavily influencedby the surface chemistry as well as inter-actions unique to the specific assay reac-tants, primarily cross-reactivity. A blockercan totally inhibit non-specific reactionswith the surface and not reduce signal-to-noise due to cross-reactivity issues.

It is advisable that during the develop-ment of a blocking procedure, each of the proposed blockers and blocking con-ditions (buffers, incubation times, etc.) be evaluated for cross-reactivity with allother assay reactants. The ideal blockerand blocking procedure will effectivelyand reproducibly eliminate non-specificsurface attachment and improve assaysensitivity and specificity — resulting in a high signal/low noise assay.

Technical AssistanceFor additional ELISA technical supportand bulletins or product information,please visit the Corning Life Scienceswebsite at www.corning.com/lifesciencesor call 1-800-492-1110.

References 1. Bjerrum, O.J. and Heegaard, N.H.H.

Handbook of Immunoblotting of Proteins,Volume 1. Boca Raton, Florida: CRCPress, Inc.: 1988.

2. Craig, J.C. et. al. Journal of BiologicalStandardization. 17; 1989; 125-135.

3. Engvall, E. and Perlmann, P.Immunochemistry. 8:871; 1971.

4. Gardas, A. and Lewartowska, A. Journal of Immunological Methods. 106; 1988;251-255.

5. Graves, H.C.B. Journal of ImmunologicalMethods. 111; 1988; 157-166.

6. Kenny, G.E. and Dunsmoor, C.L. IsraelJournal of Medical Sciences. 23; 1987; 732-734.

7. Maggio, E.T. Enzyme Immunoassay. Boca Raton, Florida: CRC Press, Inc.; 1980.

8. Stenberg, M. and Nygren, H. Journal ofImmunological Methods. 113; 1988; 3-15.

9. Vogt, R.F. et. al. Journal of ImmunologicalMethods. 101; 1987; 43-50.

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