elisa technology 2012

IDEXX Livestock and Poultry Diagnostics

© 2012 IDEXX Laboratories, Inc. All rights reserved.

ELISA Technology, Best Practices & Troubleshooting


2 © 2012 IDEXX Laboratories, Inc. All rights reserved.

We can divided this presentation in 3 part

1. Introduction : general presentation of the ELISA test kit

2. Good Laboratory Practices

3. Troubleshooting



Introduction

• Rapidly detects and quantifies antigens or antibodies against bacteria, viruses and other pathogens

• Robust and user-friendlyRobust and user-friendly

• One of the most sensitive and reproducible test methods available

ELISA Test Kits



Introduction

• Test a large number of samples at the same time (up to 94)

• Automate the procedure using robotics or other types of automated equipment

• Use software to calculate and report results (xChek Software)

ELISA Test Kits



Antigen and Antibody Basics



Introduction

• Antigen (Ag) - “any foreign substance that elicits an immune response (i.e. the production of specific antibody molecules) when introduced into the tissues of a susceptible animal and is capable of combining with the specific antibodies formed.”

Antigen Definition



Introduction

• Antigens are generally of high molecular weight and commonly are proteins or polysaccharides

• Antigens that give strong immune responses are strongly immunogenic

• The small site on an antigen to which an antibody binds is called an epitope

• Most common antigens are viruses and bacteria

Antigen



Introduction

• Antibody (Ab) - “an immunoglobulin capable of specific combination with the antigen that caused its production in a susceptible animal.”

Antibody Definition Basics



Introduction

• Antibodies are produced in response to the invasion of foreign molecules (antigens) in the body

• Antibodies exist as one or more copies of a Y-shaped unit

Antibody Basics



Indirect Format

Steps in an Indirect ELISA



Blocking Format

Steps in a Blocking ELISA



The difference between Blocking and Indirect ELISA tests

• A broad range of Ab variants will bind to the plate

• The conjugate is an anti-species IgG conjugate which will bind to all already bound Ab

• Highly sensitive but a small possibility to have non-specific Ab binding to the plate resulting in false positive results

• Can only be used for specific species depending upon which conjugate is used in the test

• The amount of bound conjugate is proportional to the Ab level in the sample

• Quantitative results can be calculated (S/P)

Indirect ELISA



The difference between Blocking and Indirect ELISA tests

• A broad range of Ab variants will bind to the plate

• The conjugate is a Monoclonal Ab (specific Ab) which will bind to only a specific epitope on the coated plate if this space is not already occupied by an Ab from the sample

• Highly specific, it doesn’t matter if any non-specific binding has happened to other parts of the plate as this will not be detected by the conjugate

• Indirectly proportional to the Ab level in the sample, which is not as easy to use quantitatively

• Not species dependent as the conjugate binds directly to the Ag on the plate

• Important to select an epitope which is common for all strains or variants that should be detected

Blocking ELISA



IDEXX ELISA Test Kit Components

• Coated Plates

• Positive and Negative Control

• Sample Diluent

• Conjugate

• Substrate (TMB)

• Stop

• Wash Solution



ELISA Test Usage

• Highly sensitive screening tool

• ELISA’s give a very low number of false positive results

• Unexpected positives should be confirmed by another test system (HI, Culture, IPMA, Western Blot, Complement Fixation, PCR)

• The test will not tell you if the detected Ab are maternal Ab, from vaccination, or from infection (i.e. DIVA* test)

• *DIVA – ”Differentiating Infected from Vaccinated Animals”

• It will not tell you if the Ab level is on it’s way up or down in the animals

• To see the trend, it’s necessary to take new samples, compare the results and establish baselines



Specificity of an ELISA Test

Diagnostic Specificity

- The % of correctly scored negative results the test is giving on a true negative population

- Balanced with the sensitivity of a test

Analytical Specificity

- The degree to which the assay doesn’t cross react with other analytes



Sensitivity of an ELISA Test

Diagnostic Sensitivity

- The % of positive results in a true positive population

Relative Sensitivity

- The % of positive results compared to a reference method



Some ELISA Calculations

S/P, S/N and Titer

• S/P (For Indirect tests))

Sample O.D. – Negative Control O.D. --------------------------------------------------- Positive O.D. – Negative Control O.D.

• S/N (For Blocking tests)

Sample O.D. ----------------------------- Negative Control O.D.



Good Laboratory Practice



Good Laboratory Practices

• Receiving kits - you need to check:- Inspect for damage- Record date received and when used- Store properly per kit insert instructions- Note kit expiration date

• General reagent handling- ALWAYS Follow kit insert (check often for revisions)- Warm up reagents (2–3 hours)- Mix reagents ( by inversion or with vortex)- Avoid contamination (no “pour backs”, use designated reagent reservoirs)

Reagent Handling




• Check equipment performance routinely

• Follow manufacturers’ cleaning guidelines for washers

• Check reader calibration – use a calibration plate

• Calibrate pipettes in house or send out for calibration or repair

• Keep a record of cleaning/maintenance/repair for all equipment

Maintenance and Calibration




Quality Control: Temperature

• Maintain temperature at 18–25°C (check everyday)

• Monitor and track temperature over time

• Temperatures too high can elevate optical density (OD) values

• Temperatures too low can decrease OD values

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Temperature Tracking February 2012

Day

Degr

ee C

Tracking laboratory temperature over time




Quality Control: Environmental Conditions

• Sun exposure can affect OD values - keep reagents away from windows

• Air conditioning and heating can cause OD fluctuations - use an incubator to keep temperatures consistent

• Prevent evaporation - use plate covers and/or plate sealers




ELISA Plate Timing

• Adding samples and controls - Timing is critical- Elapsed time between the addition of the first and last sample to the

plate must be as short as possible- Check kit insert for correct incubation times- Use a multichannel pipette to minimize

pipetting time




ELISA Plate Timing

• Multiple plate runs- Allow a time interval between plates, so you can perform each assay step

carefully- Use small batch sizes to ensure proper handling and technique- Use a separate timer for each plate to avoid confusion between plate

incubation times




ELISA Plate Washing

• Check equipment performance- Keep a record of repairs- Keep a record of cleaning and maintenance

• Check kit insert for correct number of washes !!!!• Tap plate out after final aspiration- Look for excessive moisture or color on absorbent

material

• Add next reagent right after washing- Do not let the plate dry out




Remember…

• Warm up reagents before start

• Mix reagents

• Mix samples before adding them to the plate, pay attention to avoid bubble (background issue and cross contamination)

• Do not pour reagents back into bottles !!!!

• Use disposable or designated reagent reservoirs

• Carefully time incubations

• Wash the right number of cycles with the correct wash solution




Remember…

• Use good quality water (deionized or distilled)

• Do not mix reagents from different kits or lots

• Pipette carefully

• Use a multichannel pipette to minimize time between controls and samples

• If using part of a solid plate, cover the rest of the plate with an adhesive cover

• After finishing the test, aspirate, dry and put in a sealed bag with dessicants



Troubleshooting



Troubleshooting

Remember…

• Reproducibility and reliability depend on proper technique and attention to detail

• Some laboratory conditions can cause poor ELISA performance



Troubleshooting

OD Values Too High

Possible causes:• Poor quality water used to wash plates

• Insufficient washing or poor washer performance

• Contaminated washer system0.000

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High OD Values

Data Point

OD

Valu

e

Values out of range



Troubleshooting

OD Values Too High

Possible causes (continued):• Wrong wash formulation

• Reader malfunctioned or was not blanked properly (if the OD readings are high and the color is not dark)

• Lab temperature too high

• Reagents intermixed/contaminated



Troubleshooting

OD Values Too Low

Possible causes:• Lab temperature too low

• Wash solution prepared incorrectly

• Contaminated washer system

• Wrong wash formulation

• Too many wash cycles

• Incubation period too short

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0.200

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Low OD Values

Data Point

OD

Valu

eValues out of range



Troubleshooting

OD Values Too Low

Possible causes (continued):• Reagents intermixed or expired

• Reagents and plates too cold

• Plate read at wrong wavelength or malfunctioning reader

• Kit controls diluted

• Assay plates compromised or previously used



Troubleshooting

No Color Development

Possible causes:• Reagents used in wrong order

• Assay steps ommitted

• Samples not added to wells

• Wrong conjugate used

• Kit components contaminated



Troubleshooting

Replicates within Plate Show Poor Reproducibility

Possible causes:• Poor mixing of the sample dilutions

• Too much time taken to add samples, controls or reagents to plate

• Malfunctioning multichannel pipette

• Inconsistent washing or malfunctioning washer system

• Poor distribution of antibody in sample

• Samples not added to the diluent

Poorly reproduced duplicates



Troubleshooting

Poor Reproducibility Plate to Plate

Possible causes:• Sample identification mixed up

• Inconsistent incubation time from plate to plate

• Inconsistent washing from plate to plate

• Malfunctioning pipette

• Reagents taken from different kit lots



Sample Handling



Sample Handling

• Sample quality can have a significant impact on final assay results .Most labs have no choice regarding the quality of incoming samples. In many cases, the sample diluent formulation compensates for variations in sample quality.

• If sample quality is highly questionable, obtaining a fresh sample is strongly advised, when possible.

• Avoid numerous freeze-thaw cycles, as this may damage the antibodies or antigens in the sample. We recommend no more than 3–5 cycles.



Sample Handling

Serum/Plasma Samples:

- With trace hemolysis (light red color)- Moderate lipemia (milky appearance)

- heavily hemolyzed (dark-red color)- grossly lipemic

- Bacterially contaminated (smelly)

Little or no effect on ELISA results

Bad effect on ELISA results



Sample Handling

Meat Juice Samples:should be as clean as possible. Remove debris and lipids from the sample when pipetting.

Milk Samples:Whole milk samples can be used after centrifugation for 15 minutes at 2000 x g, or left overnight if refrigerated (2–8°C). The sample intended for the assay should be drawn from below the cream layer

Take sample from the area indicated

Take sample from the area indicated



Sample Handling

Egg Yolk Samples:Collect samples with a clean tuberculin syringe and mix the diluted samples thoroughly by vortexing.

Other Sample Types:Refer to your package insert for sample handling, preparation and storage of other sample types (e.g., albumin, cloacal swabs).

Frozen samplecan be thawed at room temperature or in a refrigerator. All thawed samples need to be thoroughly mixed prior to dilution to ensure that the proteins are dispersed throughout the sample. Mix by gentle vortexing or inverting at least five times. Frothing or over-mixing of samples will cause denaturation of serum proteins.



IDEXX Technical Service Department

• ELISA Support, Kit Inquiries, Technical Questions/Support

• 1-800-548-9997 (Option 2)

• Questions we may ask when you call:

- Your name, phone #, email address, facility address

- Kit information (kit name, lot number, expiration date)

- Kit observations

- Flock Information (flock health, biosecurity measures)

- Data (Excel, xChek databases)



Thank you for your attention!

elisa technology 2012

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