¾ Use freshly prepared substrate A and substrate B (in 2-reagent substrate systems) ¾ Do not hold substrate solution longer then 1 hour

 

Normal Washing

In washing plate manually, the most important factor is

that each well receives the washing solution so that, no air

bubbles are trapped in the well or a thumb is not placed

over corner wells.

Strip/Plate Washers

¾ Various washing cycles can be programed

¾ Careful maintenance is essential, since they are prone

to machine errors, such as having a particular nozzle

being blocked.

Washing Tips

¾ Follow procedure for preparation of wash buffer

¾ Check washer alignment daily as part of routine

instrument start-up procedures

¾ Ensure that the plate is leveled

¾ Make certain will is completely filled, when washing, to

ensure residual conjugate is removed

¾ Examine the fill volume (a slight dome should be

observed at the top of the well)

¾ When washing do not allow wells to overflow

¾ Reduce pressure in wash system

¾ Check washers before use to determine they are

working properly. Perform routine maintenance

¾ Be certain to wash the specified number of times

¾ Allow approximately 20 seconds soak-time between the

addition of wash solution and subsequent aspiration (if

soak-time is not indicated in the assay pack insert)

¾ Examine the wells for complete aspiration of contents

¾ Upon completion of wash cycle, blot to remove residual

fluid.

Pipetting Tips

¾ Calibrate pipettes regularly according to manufacture’s

instructions

¾ Avoid touching side wall of well with tips

¾ Avoid splashing of sample and reagents

¾ Avoid blowing out tip contents

¾ Use a new tip for each sample/control/reagent addition

¾ New tips should be used on the multichannel pipettes

for each reagent to be added

¾ Reverse pipette when using the multichannel pipettes

to add conjugate and substrate solution

¾ Forward pipette when using the multichannel pipettes

to add stop solution

¾ Check pipette tips are long enough to provide air space

between top of tip and pipette barrel

¾ Check pipette barrel for residual fluid of dried material,

remove if present

¾ Ensure pipettes tips are fitted tightly.

Microplates

¾ Bring microplate pouches to room temperature before

opening

¾ Level microwells evenly in microplate frame as the

individual breakaway wells have very flexible plate

frames leading to bowing off wells and yield poor washes

¾ Place plates in dark immediately after addition of

substrate solution, provided the substrate is sensitive

to light

¾ Grasp holder on grip marks when tapping to avoid

strips slipping from holder

¾ Rotate strips 180° and reinsert or use correct holder if

strips do not fit in holder

¾ Seal unused wells in purchase along with the desiccant

¾ Date the pouches when first opened

¾ Clean bottom surface of plates with wash buffer to

remove fingerprints

¾ Make sure microwells are at level during washing,

reagent addition and plate/strip reading

¾ Wipe the bottom the plate with a lint-free cloth/towel

before reading

¾ Do not allow microwells to become dry once the assay

has begun.

Substrate Preparation

¾ Use freshly prepared substrate A and substrate B (in

2-reagent substrate systems)

¾ Do not hold substrate solution longer then 1 hour

¾ Follow procedure of working substrate solution

¾ The temperature of solution is important because it

effects rate of color reaction

¾ Do not add fresh substrate to reagent bottle containing

old substrate

692 Concise Book of Medical Laboratory Technology: Methods and Interpretations ¾ Clean old substrate solution bottle with H2SO4 and

thoroughly rinse with distilled water.