added to washing buffers. This can cause problems where excessive frothing takes place producing poor washing conditions, since air is trapped and prevents the washing

 

• Mold in incubation box and/or wash buffer bottle • Visually check incubation boxes and wash buffer bottles

• Clean any moldy containers

• Be sure all containers are free of cleaning agents before using

• Set-up routine cleaning schedule

• Too much conjugate concentrate used in • Prepare fresh working conjugate

preparing working stock • Follow conjugate preparation

• Incubation temperature too high • Check room temperature, whether at 22–28°C/Check AC

Serology/Immunology 689

Problem: Poor reproducibility or bad duplication

Possible causes Corrective action

• Bubbles in wells • Use pin or needle to burst. Use separate pin for each well

• Dispensing error • Check dispensing instrument

• Finger tips on plates • Clean bottom surface of plate with wash buffer, blot to dry.

• Misaligned wells in plate • Realign wells

• Improper washing technique • Be certain to wash the specific no. of times. Fill each well to the rim with

wash buffer. Do not allow well to overflow. Blot plate dry at end of wash

Problem: Poor sensitivity

Possible causes Corrective action

• Usage of non-human serum based calibrators • Rerun with human serum based calibrators (primary standards)

(for quantitative assays)

• Insufficient conjugate concentration added • Prepare fresh working conjugate, follow working procedure

in preparing working stock

• Error in pipetting working conjugate • Check calibration of pipettes

• First incubation time insufficient • Repeat run using proper incubation time

• Temperature more than suggested by manufacturer • Process plate continuously throughout entire assay procedure

• Plates being held too long after first incubation before

further processing

Problem: High absorbance of calibrator

Possible causes Corrective action

• Plates being held too long after first • Process plate continuously throughout entire assay procedure

incubation at a higher temperature than

what is recommended by the manufacturer

before further processing

• Insufficient sample volume added • Check calibration of pipettes

Problem: Specimen absorbance out of range of calibrators

Possible causes Corrective action

• Concentration in specimen is too high • Dilute with ‘0’ calibrator and reassay

Problem: Overall low absorbance

Possible causes Corrective action

• Temperature of room < 20°C • Increase time of reaction between enzyme/substrate (check with manufacturer)

• It is recommended to maintain 22–28°C ambient temperature in the laboratory

Problem: Controls out of range

Possible causes Corrective action

• Contamination of controls • Rerun assay with new controls

• Contamination of calibrators • Rerun assay with new calibrators

Problem: strips slip from holder

Possible causes Corrective action

• Improper handling • Grasp holder on grip marks when tapping

690 Concise Book of Medical Laboratory Technology: Methods and Interpretations Problem: Strips do not fit in holder

Possible causes Corrective action

• Improper alignment or incorrect holder • Rotate strip 180 and reinsert or use correct

 holder

Problem: Substrate A is blue

Possible causes Corrective action

• Contaminated • Obtain fresh substrate A

Problem: Substrates A and B turn blue when mixed

Possible causes Corrective action

• Contaminated • Obtain fresh substrate A and B

Problem: Stop solution yellow

Possible causes Corrective action

• Contamination • Obtain fresh stop solution

Problem: Waited over 30 minutes before measuring plate

Possible causes Corrective action

• End product of enzyme reaction may • Rerun the essay

precipitate and cause error

Problem: No color even after 30 minutes incubation with substrate

Possible causes Corrective action

• Improper mixing of substrate A and B • Remix the substrates

• Substrate not working • Contact manufacturer

Problem: Color develops very quickly

Possible causes Corrective action

• Contaminated enzymes • Common in wells, pretreatment may be necessary

• Make sure all reservoirs are clean

Problem: Color develops too slowly

Possible causes Corrective action

• Sample not at room temperature • Bring samples to room temperature before assay run

• Conjugate too weak • Check dilutions and time when diluted

• Contamination inhibits activity of enzyme, • Avoid wrong preservatives

e.g. sodium azide on peroxidase

• Low temperature of laboratory of substrate solution • Makes sure temperature of substrate is correct

TECHNICAL TIPS

Washing

The purpose of washing is to separate bound and unbound

(free/unwanted) reagents/serum components. This

involves the emptying of microwells of reagents followed

by the addition of liquid into the wells. Such a process is

performed at least 3–6 times for every well. The liquid used

to wash wells is usually buffered (PBS) in order to maintain

isotonicity, since most Ag-Ab reactions are optimal under

such conditions. Tap water is not recommended, since

tap water varies greatly in composition (pH, molarity,

and so on). Generally, the mechanical action of flooding

wells with a solution is enough to wash wells of unbound

Serology/Immunology 691

reagents. Some workers leave washing solution for a

short time (soak time) after each addition (1–5 minutes).

Sometimes detergents, notably Tween-20 (0.05%) are

added to washing buffers. This can cause problems where

excessive frothing takes place producing poor washing

conditions, since air is trapped and prevents the washing

solution from contacting the well surface. For most cases,

this addition does not contribute significantly to the

washing procedure. When using detergents, care has to be

taken that they do not affect reagents adversely (denature

Ag), and greater care is needed to prevent frothing in the

wells.