troubleshooting: ELISA & rapidmay 2005

troubleshooting: ELISAmay 2005

Specification sheet• Specification sheet is provided with all Panbio ELISA

kits• Kit should perform as per the specification sheet

Example of specification sheet found in all Panbio ELISA kits

Common ELISA problems• High absorbances• Low absorbances *• Poor duplicates*• All wells yellow• All wells negative

* Most common problems

High absorbances• Cross contamination

– Repeat assay taking care when washing & pipetting.

• Insufficient or inefficient washing or reading– Check washer efficiency - use Panbio ELISA Chek Plus kit.

• Wavelength of filter not correct– Check wavelength is 450 nm. If dual wavelength reader available,

use ref filter 600-650 nm.

• High assay background– Repeat assay & include a well that contains only Serum Diluent or

absorbent (i.e. a blank well).

• Contaminated TMB– Check that TMB is colourless or faint blue.

• Incubation too long or incubation temperature too high– Check incubation time and temp.

• Incorrect dilution of serum– Repeat assay, ensuring correct serum dilution is used.

Low absorbances• Incubation time too short or temperature too low

– Ensure time and temperature of assay inc are correct– Check incubator is set at the correct temperature

• Incorrect dilution or pipetting of sera– Repeat assay ensuring correct dilutions and volumes are used– Ensure controls are sufficiently mixed

• Incorrect filter wavelength– Check wavelength is set at 450 nm. If dual wavelength reader

available, use ref filter 600-650 nm.

• Contaminated conjugate solution – COMMON– Dispense conjugate directly from bottle using clean pipette tip;

avoid transferring conjugate to another container if possible.– Do not return unused conjugate to bottle– Ensure all pipettes and probes used to dispense the conjugate are

clean and free from serum, detergent and bleach.

• Kit has expired– Check expiration date of kit and do not use if expired

Low absorbances cont.• Air blank reading high

– Investigate caused of high background absorbance– Check performance of reader using PanbIO ELIS Chek Plus kit

• Incorrect storage of kit– Ensure kit is stored at 2-8°C, plate is sealed in foil pouch and

desiccant sachet is blue/purple

• Kit reagents not equilibrated to room temperature– Allow sufficient time for reagents to equilibrate to room temperature

prior to performing assay

• Incorrect reagents used– Check reagents used match those listed on the specification sheet

• Over-washing of plate (e.g. inclusion of a long soak step)– Repeat assay using the recommended wash procedure (washx6,

no soak)

• Under-washing of plate following serum incubation step– Repeat assay using recommended wash procedure)

Poor duplicates• Poor mixing of samples

– Mix reagents gently and equilibrate to room temp

• Poor pipette technique– Calibration may need to be checked– Check pipetting technique

• Addition of reagents at inconsistent timing intervals; reagent addition taking too long; air bubbles when adding reagents

– Use consistent time when adding reagents

• Inefficient washing - wash buffer left in wells, inconsistent washing, inadequate washing - COMMON

– Tap out wash buffer after washing– Check wells are sufficiently and uniformly filled & aspirated when

washing

• Reader not calibrated or warmed up before plate is read– Check reader precision using Panbio ELISA Chek Plus kit– Check reader manual to ascertain warm-up time of instrument

Poor duplicates cont.• Optical pathway not clean

– Gently wipe bottom of plate

– Check reader light source and detector are clean

• Spillage of liquid from wells– Repeat assay, taking care not to knock the plate or splash

liquid

• Serum samples exhibit microbial growth, haemolysis or lipaemia– It is not recommended to use serum samples exhibiting

microbial growth, haemolysis or lipaemia

• Uneven well volumes due to evaporation– Cover plate with lid or plate sealer (not provided)during

incubation

All wells yellow• Contaminated TMB

– Check TMB is colourless

• Contaminated reagents– Check reagents for turbidity

• Incorrect dilution of serum– Repeat assay, ensuring correct serum dilution is used

• Incorrect storage of kit– Ensure kit is stored at 2 – 8°C, plate is sealed in foil pouch

and desiccant sachet is blue/purple

• Inefficient washing– Tap out wash buffer after washing

– Check wells are sufficiently and uniformly filled & aspirated when washing

All wells negative• Test not performed correctly - reagents not added or not

added in the correct sequence (stop solution added at any stage will knock out ODs)

– Check procedure and check for unused reagents– Ensure that Stop Solution was not added before conjugate or TMB– Ensure that serum was diluted in correct diluent; e.g. do not use

Serum Absorbent for an IgG ELISA

• Contaminated conjugate solution– Dispense conjugate directly from bottle using clean pipette tip;

avoid transferring conjugate to another container if possible.– Do not return unused conjugate to bottle– Ensure all pipettes and probes used to dispense the conjugate are

clean and free from serum, detergent and bleach.

• Over washing of plate– Repeat assay using recommended wash procedure

All wells negative cont.• Incorrect storage of kit

– Ensure kit is stored at 2-8°C, plate is sealed in foil pouch and desiccant sachet is blue/purple

• Wash buffer made up with Stop Solution instead of Wash Buffer– Ensure wash buffer is made up correctly

troubleshooting: rapid testsmay 2005

Common problems• Lines appearing after test has been read• Control line not appearing• Positive IgM line, no IgG line• Faint lines

Lines appearing after test has been read• The Dengue Duo Cassette should be read at 15

minutes while the Dengue Rapid Strip test should be read at 30 minutes.

• Bands may appear after these times due to lower levels of specific IgG or cross-reactive antibody.

• It is important not to read the Panbio rapid tests after the specified incubation times (i.e. 15 minutes for the cassette test and 30 minutes for the strip test)

Control line not appearing• Invalid result• Check that you have performed the test correctly• Repeat test

Positive IgM line, no IgG line• This is a correct result if a patient has an acute primary

dengue infection.

• The IgG cutoff for the Panbio Dengue rapid products is set above the level found in past and primary dengue infection.

Faint lines• Any sign of a line should be interpreted as a positive• If using the Dengue Duo cassette, check that the

buffer has been added correctly. • Bottle should be held vertically and 1 cm away from

the sample well.