Zymeds Recommended ELISA Methods

The following ELISA procedures are recommended for use with multi-well, plastic (polystyrene or polyvinyl) microtiter plates with capacities of 0.25 ml per well. Reagents may be coated onto other types of solid-phase matrices, however the amount should be adjusted according to their size and nature.

Designing a DetectionSystem
Different detection systems can be used in ELISAs to achievesimilar results. The system chosen will depend on prioritizingseveral factors, including; sensitivity, specificity, protocoltime, reliability, and cost among others. However, most ELISAs canbe done using one of the two following systems. Direct method - fornormal sensitivity - use a secondary antibody directly conjugatedto HRP or AP. LAB-SA - for high sensitivity - use a secondaryantibody directly conjugated to biotin, and then add streptavidinconjugated to HRP or AP.

Coating thePlate
Use 100-200 µl of antigen at a concentration of 1-5 µg/ml in PBS,pH 7.4 (buffer 1) or 0.1M NaHCO₃, pH 9.6 (buffer 2). Addto each well of a polyvinyl or polystyrene microtiter plate.Incubate at 4^oC overnight. For a capture assay, use100-200 µl at an antibody concentration or 5-10 µg/ml.

Blocking Extraneous BindingSites
Shake off excess antigen coating solution. Block remaining bindingsites in each well by incubating with 200 µl of 1 % BSA in 10 mMPBS (buffer 1) at 25^oC for one hour.

Endogenous PeroxidaseActivity
If the antigen of interest contains endogenous peroxidase activity,pretreat antigen-coated plates with 0.1% phenylhydrazine (200 µlper well) in PBS (buffer 1) for 1 hour at 37^oC. In somecases, phenylhydrazine may not completely inhibit such activity. Ifnecessary, perform an additional treatment with a 0.3% solution ofH₂O₂ in methanol (200 µ l/well). Incubate atroom temperature for 30 min.

Choosing a conjugate forELISA
Horseradish peroxidase and alkaline phosphatase are the most widelyemployed enzyme conjugates in enzyme immunoassay (EIA). Both arecomparable in sensitivity, and selecting one over the other isoften no more than a personal preference. If an assay samplecontains interfering reagents, such as preservatives oroxidizing/reducing compounds, peroxidase conjugates should beavoided. Likewise, a high level of alkaline phosphatase activity inthe assay sample precludes the use of this enzyme as a signalgenerator. In rare cases, neither peroxidase nor alkalinephosphatase is appropriate, and an alternative enzyme, such asbeta-galactosidase, must be selected. In general however,conjugates of peroxidase and alkaline phosphatase give faster colordevelopment than beta-galactosidase, and are used much morefrequently. It may be useful to note that the dose-response curveof alkaline phosphatase is often superior to that of peroxidase,though both enzymes are equally sensitive in ELISA systems.

BiotinConjugates
Biotin conjugates are generally diluted from 1:5,000 to 1:10,000with PBS-TWEEN (buffer 3) as recommended. Each well is filled with200 µl, and incubated for 1 hour at 37^oC. After 3 washeswith PBS-TWEEN, 200 µ l of enzyme-conjugated streptavidin is added(also diluted 1:5,000 to 1:10,000). The appropriatesubstrate/chromogen mixture is used for color development (seebelow). The plate is incubated at room temperature for 15-30 minand the absorbance read at the appropriate wavelength.

Alkaline PhosphataseConjugates
Dilute from 1:1,000 to 1:2,000 with Tris-buffered saline (buffer5), or as recommended. Each well is filled with 200 µl of theenzyme-conjugate and incubated at 37^oC for one hour.After the plate is washed 3 times with PBS-TWEEN (buffer 3), 200 µlof substrate is then added to each well (see below).

APSubstrates
For most applications, PNPP (p-Nitrophenyl phosphate) is thesubstrate of choice. It is dissolved 1 mg/ml in 0.1 M2-amino-2-methyl-1,3-propanediol buffer, pH 10.3 (buffer 6) or 0.1M diethanolamine, pH 10.3 (buffer 8). The yellow color ofnitrophenol can be measured at 405 nm, after a 15-30 min incubationat room temperature. This reaction may be stopped by the additionof an equal volume of 0.75 N NaOH. Keep the substrate solutionfrozen in working aliquots. Zymed's liquid pNPP is stable insolution for 12 months. Discard when yellow.

Horseradish PeroxidaseConjugates
Dilute from 1:2,000 to 1:4,000 with PBS-TWEEN (buffer 3), or asrecommended. Each antigen coated well is filled with 200 µl ofconjugate. The plate is incubated at 37^oC for one hour,then washed 3 times with PBS-T (buffer 1). Substrate/chromogensolution (200 µl) containing ABTS 0.5 mg/ml in 0.1 M citratebuffer, pH 4.2 and 0.03% hydrogen peroxide (buffer 4) is added toeach well. After an incubation at room temperature for 15-30 minthe absorbance at 416 nm is measured by a microtiter plate EIAreader (405 nm is also acceptable).

HRPSubstrate
The substrate for peroxidase (HRP) is hydrogen peroxide. In HRPassays, cleavage of H₂O₂ is coupled to theoxidation of a hydrogen donor which changes color during thereaction.

HRPChromogens
ABTS (2,2'-Azino-di-(3-ethylbenz-thiazoline Sulfonic Acid)):Dissolve 0.5 mg/ml in 0.1 M citrate buffer, pH 4.2 containing 0.03%hydrogen peroxide (buffer 4). The green color is measured at 416 nm(405 nm is also acceptable).

OPD (o-Phenylenediamine):Dissolve 1 mg/ml in 0.1 M citrate-phosphate buffer, pH 5.0containing 0.03% hydrogen peroxide (buffer 7). Enzyme activity maybe stopped by adding an equal volume of 2NH₂SO₄. The tangerine color is measured at 492nm. This substrate is light sensitive.

TMB (Tetramethylbenzidine):Add TMB (ready-to-use) solution to each well. Incubate for 15-30min. Add an equal volume of stopping solution (2 NH₂SO₄) and read absorbance at 450nm.