• Contamination of wells by conjugate • Carefully add conjugate to wells. Pipette reagent to center bottom of microwell

 

ELISA TROUBLESHOOTING ASPECTS

Introduction

ELISAs have emerged as the mainstay for diagnosis of

various human diseases in a modern clinical laboratory.

Despite being an extremely sensitive and specific assay

format, several pre-analytical and analytical factors may

affect the performance of ELISAs. Thus, it is imperative

that laboratory professionals be aware of the problems so

that he/she has more control over the final assay results.

Many errors can be avoided if the protocol is read and fully

understood before starting the assay.

¾ On identifying assay failure, check the expiration

dates of the individual reagents and ensure that all the

reagents have been stored as indicated on the product

label

¾ Once this has been established, check for signs of

instability or deterioration in reagent solutions, (e.g.

precipitation or discoloration)

¾ All substrate solutions should be colorless

¾ Use clean plastic disposable pipettes, tips, and

containers for reagent preparation and storage

¾ Avoid cross-contamination of kit reagents by changing

pipette tips between addition of each calibrator, sample

and reagent

¾ Ensure that specified incubation times and temperatures

have been adhered to and that no substitution of kit

reagents has occurred

¾ To improve accuracy, it is recommended that samples

and standards be run in duplicate.

In this technical series, we present various problems

commonly encountered in ELISAs and the necessary

corrective action.

Serology/Immunology 687

Problem: High negative control value or high background

Possible causes Corrective action

• Contamination of negative control wells by • When washing, do not allow wells to overflow

positive control

• Contamination of negative control vial • Check pipette barrel for residual fluid of dried material. Remove if present

• Always format negative control wells before positive control

• Use new pipette tip for each sample

• Check the pipette tips are long enough to provide air space between top of

tip and the barrel

• Rerun with fresh reagents

• Insufficient washing or contamination of negative • Make sure wells are completely filled. While washing ensure residual

control by conjugate conjugate is removed from well

• Pipette all specimen and reagents in the center bottom of the microwell

Avoid contact with inner wall and rim

• Rewash

• Non-specific attachment of antibodies • Unsuitable blocking buffer or omission of blocking buffer

• Wells no preprocessed to prevent non specific attachment of antibodies

• Antispecies conjugate reacts with reagent coated • Set-up controls to assess weather any reagent binds unexpectedly to any

on plate reagent

Problem: Low positive control value or low absorbance

Possible causes Corrective action

• Reagent not at room temperature • Make certain all kit components are at RT (22–28°C).

• Test volume low • Ensure pipette tips are fitted correctly/tightly

• Check pipette barrels for obstructions

• Check calibration of pipettes

• Substrate A and B not freshly combined or incorrectly • Prepare substrate immediately before use

prepared (in case of 2-reagent substrate system) • Follow working reagent preparation

• Contamination of substrate with or bacterial • Rerun the assay with fresh reagents

contamination of positive control

• Incubation time too short • Check calibration of timers

• Record time of incubation

• Moisture in pouches • Check whether desiccant in pouch is in working condition

• Seal unused wells in pouches

• Date pouches when first opened

• Improper incubation temperature • Check incubator temperature/room temperature (22–28°C)

• Room temperature too low for substrate incubation • Check temperature of the working area

• Washing step too vigorous • Reduce pressure in wash system

• Reagent not mixed before using • Mix the reagents before use

• Wells allowed to dry after assay has started • Complete all assay steps without interruption

• Failure to add stop solution • Addition of stop solution increases intensity of color reaction and stabilizes

final color reaction

• Insufficient conjugate concentrate added in • Prepare conjugate accurately

preparing working stock • Follow working reagent preparation as described by the manufacture

688 Concise Book of Medical Laboratory Technology: Methods and Interpretations Problem: Entire plate gives positive OD or color all over plate

Possible causes Corrective action

• Inadequate was volume or contamination • When washing, fill the wells to the rim and ensure no overflow

of substrate by residual conjugate left in well

• Too strong conjugate • Check dilution

• Antispecies antibodies react with absorbed antigen • Check suitable controls

• Serum factors in heated sera • Do not heat sera

• Substrate solution contaminated by conjugate • Check pipette barrels for residual fluids or dried material, remove if present

• Pipette tips should be long enough to provide air space between top of tip

and pipette barrel

• For automated system, make sure reagent lines are in proper position. Do

not switch lines

• Substrate solution is not fresh • Do not hold substrate solution longer than manufacturer claims

• Failure to stop reaction • Check bottle before use

• Acid not added • Check assay procedure

• Plate sat idle too long before reading • Read within 30 minutes of adding stop solution

• Chromogen may not be working • Use fresh chromogen

• Substrate solution container is dirty • Do not add fresh substrate to reagent bottle containing old substrate

• Clean old solution bottle with acid and thoroughly rinse with distilled water

• Plate exposed to light during substrate incubation • Place plates in dark immediately after addition of substrate solution

(Check Product Insert)

Problem: False positive reactions

Possible causes Corrective action

• Inadequate washing • Check washer before use to determine they are working properly

• Clogged cannulas in washer • Perform routine maintenance

• Contamination of wells by conjugate • Carefully add conjugate to wells. Pipette reagent to center bottom of microwell

• Splashing of conjugate on rims of wells • Avoid contact with sides and rims of wells

during conjugate addition • Check alignment and delivery of automated systems

• Contamination of substrate solution by conjugate • Check pipette barrels for residual fluid or dried material. Remove if present

• Check pipette tips are long enough to provide air space between top of tip

and pipette barrel

• RBCs in test sample • Centrifuge before use

• Evaporation of sample of conjugate during the 37°C • Place the covered test plate in a prewarmed (37°C) moist incubation

incubation (if 37°C is a must, not applicable in RT box inside the incubator (dry or humidified)

incubation)