Quality Control: In-house Controls for ELISA tests Dr. John

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Quality Control In-house Controls for ELISA tests
Dr. John Ridderhof Chief, Office of Laboratory
Systems Development Division of Laboratory
Systems/PHPPO
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Who should make and use In-House HIV Controls?
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Who should make and use In-House HIV Controls?

• Any lab which is conducting HIV EIA testing

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Criteria for Developing
Quality Controls for HIV

• Low Positive
• Between the cut off and positive control
• At a level where variability can be followed
• Generally 2 times the cut off

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Process for Preparing In-house Controls

• Serial Dilution of High Positive Stock Sample
• Select Suitable Dilution
• (pooled HIV- plasma)
• Produce Large Batch
• Test Stability
• Test Batch Variation
• Dispense, Label, Store

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Making Suitable Dilutions
100 ul serum in tube 1
Mix and Transfer
Discard
100ul diluent in each tube
Each tube is a 12 dilution of the previous tube
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This titration will result in the following
dilutions
12 14 18 116 132 164 1128 1256 .
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Selecting a Suitable Sample Dilution
Serial Dilutions on Abbott AxSYM HIV-1/HIV-2
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Calculation for control
Dilution 18192 gave us our desired value (about
2 times the cut off) for this assay. To create a
larger batch you would combine the
following 1ul of pooled positive serum 8191
ul of pooled negative serum Error in measuring
1 ul of serum is high, So the following volumes
would be better 10ul of pooled positive serum
81.91 ml of pooled negative serum
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Batch Production

• Prepare positive sample
• centrifuge
• heat inactivate (56ºC for 30 min.)
• Mix positive sample in diluent
• magnetic stirrer
• Identify each batch with a unique ID number

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Stability Testing

• Assess the rate of deterioration

QC Sample
Day 7
Day 14
Day 21
Day 28
Storage
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-20c
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4c
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16-25C
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Batch Validation

• Dispense aliquots
• Test aliquots
• Confirm desired titre level
• compare against target value
• Confirm minimal batch variation
• acceptable if CV lt20
• aim for lt10

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Storage of QC Samples

• Validated batch aliquoted into smaller user
  friendly volumes for storage
• Establish a storage protocol
• store at -20oC
• in use vials stored at 4oC
• use 1ml vial maximum of one week
• freeze-dried
• requires accurate reconstitution)
• chemically preserved

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When should you use your in-house control?

• Any time you run an EIA.
• On each plate in your EIA run.

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Use the results of your in-house control as a
part of testing validation.Track the OD of your
control over time.