Bench Tips

Bench Tips

Tips to obtain desired results for a ELISA test

Washing of ELISA Plates

  • Follow procedures for the preperation of wash buffer
  • Check washers before use to determine they are working properly. Perform routine maitainace.
  • Completely fill the wells
  • When washing do not allow wells to verflow
  • Be certain to wash the specified number of times.
  • Completely aspirate contents from wells
  • After washing, blot the wells to remove residual fluid


Pipetting of liquid regents

  • Regular calibration of pipettes is required as per manufacturer’s instructions
  • During pipetting, avoid touching the wall of the wells
  • Avoid splashing of reagents & solutions
  • Use new pipette tip for each sample, control, standard and reagent addition.
  • New tips are advised on the multichannel pipettes for each reagent to be added.
  • Long tips are recommended to avoid space between tip top and barrel.
  • A continuous check & removal of residual fluid from pipette barrel is required
  • Tight fitting of tips to pipettes should be ensured before use.


Use of Microplate

  • Microplate must be used at room temperature.
  • Level individual breakaway wells evenly in microplate frame to avoid bowing off wells and to avoid poor washing.
  • Light sensitive substrate must be added & allowed to react in dark
  • Pouches must be dated on day of opening.
  • Plate bottom can be washed with wash buffer to remove finger prints.
  • Seal unused wells in pouches along with the desiccant.
  • Microwells must be kept at horizontal level for washing, reagent addition and plate reading.
  • The wells must not be allowed to become dry at any time during assay.


Temperature control in ELISA

  • All reagents must be brought to room temperature (22-28 oC) 30 min prior to use.
  • Appropriate incubation temperature must be maintained during assay. A low incubation temperature yield OD values, while higher temperatures produce OD values and evaporation in wells can cause edging effect.
  • A calibrated thermometer must be used to check temperature at regular intervals.
  • Assay steps must be followed and executed as per manual specified time.
  • Time & temperature of incubation must be recorded properly.
  • OD reading must be taken immediately or as specified after adding stop solution.


Incubation in ELISA

  • Its good practice to rotate plates during incubation for better antigen antibody reaction.
  • The effect of rotating plates is to mix the reactants completely during the incubation step.
  • The mixing ensures that potentially reactive molecules are continuously coming in contact with the solid-phase for the better reactivity.
  • The process of diffusion allows limited mixing of the reagents in stationary phase.


Substrate in ELISA

  • Use freshly prepared substrate
  • Do not hold substrate solution longer than 1 hour
  • Follow procedure of working substrate solution
  • The temperature of solution is important because it effects the rate of color development.
  • Do not add fresh substrate to reagent bottle containing old substrate


Conjugate in ELISA

  • All conjugate and its diluents must be store at recommended temperatures.
  • Diluted standards must be stored at recommended temperature and should not be used after specified time.
  • Working solution of secondary conjugates must be used in specified dilution and within specified time.


Stop Solution in ELISA

  • Stop reagents is added to all test well to stop all enzymatic reactions in ELISA.
  • After addition of stop solutions, OD values of the test samples are read immediately.
  • Stop solutions are strong acids or strong bases must of right molar concentration to bring denaturation of the enzymes.
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