What aspects of testing should I check if I have a problem with results? ( 4 July 2013 )

Here is a trouble-shooting check-list:

 Did you correctly follow the instructions for use (incubation times, dilutions, number of washes, preparation of 1X solutions)?

 Has the washing system been properly maintained and regularly decontaminated?

 What water was used to prepare the wash solution? We recommend using water which is distilled or purified by reverse osmosis or a system such as MilliQ or Elga, which reduces the amount of bacteria in the water.

 Were any kit reagents inter-mixed or contaminated?

 Was the dishware clean?

 Was the substrate solution transparent before use?

 Has the kit expired?

 Were all reagents and components brought up to room temperature 18-25ºC before running the assay?

 What is the room temperature in the lab? It should be between 18-25ºC. As ELISA tests are sensitive to extreme temperature, avoid running the assay close to heating sources.

 Have you ensured that the pipettes are dispensing the correct volumes, and that the pipette tips are always tightly on before use?

 Did you read the plates at the correct wavelength?

 Were previously opened plates correctly stored? Please refer to Plate storage.pdf

 Were the storage conditions respected for all components, including any 1X solutions made from concentrated solutions? Please refer to Storage conditions.pdf.

Why do I obtain discordant results when I compare ELISA results with another serological technique? ( )

When comparing results obtained by methods based on different principles (ex. VNT vs. ELISA, IFI vs. ELISA, CFT vs. ELISA), it is practically impossible to obtain perfect test agreement, unless very strongly positive and perfectly negative samples are used.

This is also true when comparing cELISA and iELISA results. The polyclonal nature of serum means that it contains populations of IgGs with different affinities for different antigens, each antigen with many epitopes. Each technique detects these populations to a different extent. Sera close to the cut-off will show more discordant results, as they contain more limited quantities of each type of antibody.

What should I do if I have a problem with test results? ( )

Please contact IDvet or your sales representative immediately. Be sure to provide the following information:

  • Contact details of your lab
  • Kit product code and batch number, ex PARAS-4P, batch 344
  • Plate number, indicated on the short side of the microplate
  • Order date or purchase order number
  • Frequency of the problem:
    – Every run? From time to time?
    Since yesterday? Last week? Last month? Last batch?
  • OD results of the plate(s), indicating the position of the positive and negative controls
  • The clinical history of the animals tested (ex. clinical signs; from an infected or disease-free herd).

What should I do if I run out of liquid reagents (controls, conjugate, wash, stop or buffers)? ( )

  • IDvet provides additional liquid reagents free-of-charge, so that you may test all the plates you purchase.
  • Simply send your request to IDvet by email ( or fax (+33 4 67 45 36 95). 
  • Indicate the reference of the required component, which can be found on the label on the top of the box, and the number of vials you wish to receive. For example, for the wash solution batch 003, you should request the reference 15-003.
  • The material will be sent to you free-of-charge, while you will be asked to pay any freight charges.

What is the shelf-life of the freeze-dried serum standards (product codes beginning with “MRI”)? ( )

We do not indicate an exact expiry date because these sera are very stable over time thanks to their freeze-dried form.

You may consider that they may be kept for up to 10 years from the production date (indicated in the upper right-hand corner of the product sheet). Store freeze-dried at +2° – 26°C.

For information regarding storage in the reconstituted (liquid) form, please refer to the product sheet. 

In general, liquid sera should be stored as follows:

  • Store at -20°C in aliquots after reconstitution.
  • Never freeze/thaw more than 3 times.
  • If reconstituted, store +4°C for up to 2 days.

Once reconstituted, the sera should be aliquoted and stored in air-tight vials (preferably with a joint, such as a cryotube). Otherwise, auto-lyophilisation may occur after 6-12 months at -20°C.

What is the purpose of the freeze-dried positive and negative sera (product codes beginning with “MRI”)? ( )

As it is often difficult for routine laboratories to obtain reference standards, and as international standards do not exist for all veterinary diseases, IDvet offers freeze-dried serum standards which may be used to check that the test’s analytical sensitivity does not vary between runs, operators and batches. Please contact IDvet for more information.

Are the IDvet ELISAs calibrated against international and national serum standards? ( )

Yes. IDvet tests meet international requirements for analytical sensitivity, as outlined in the table below:

Product name Product code Directive or Standard
ID Screen® Brucellosis Serum Indirect Multi-species BRUS-MS Reference serum OIEELISASPSS according to Commission Decision of 10 December 2008, 2008/984/CE, Annex C
ID Screen® Brucellosis Milk Indirect BRUMILK
Rose Bengal RSA-RB Reference sera OIEISS according to Commission Decision of 10 December 2008, 2008/984/CE, Annex C
Brucellosis Antigen for Complement Fixation Test AG-BRU
ID Screen® BLV Competition BLVC Reference serum E05 according to  Commission Decision of 15 December 2009, 2009/976/EU, Chapter II Annex D
ID Screen® BLV Indirect BLVS
ID Screen IBR Indirect IBRS EU reference sera EU1, EU2 and EU3(1)
ID Screen® IBR gB Competition IBRGBC
ID Screen® IBR Milk Indirect IBRMILK French Reference Milk according to the OIE Terrestrial Manual 2010, Chapter 1.4.13
ID Screen® IBR gE Competition IBRGEC EU reference sera EU1, EU2 and EU3(1)
ID Screen® Aujeszky gB Competition AUJESZKYGB Reference serum ADV 1 according to Commission Decision of 21 February 2008, 2008/184/EC, Annex III
ID Screen® Aujeszky gE Competition AUJESZKYGE
ID Screen® Classical Swine Fever E2 Competition CSFE2C Panel reference sera according to Commission Decision of 1 February 2002, 2002/106/EC, Chapter VII
ID Screen® Swine Vesicular Disease Competition SVDC Reference serum RS 01-04-94 according to Commission Decision of 4 July 2000, 200/428/EC, Chapter X

(1) Perrin B., et al (1994). Selection of European Union standard reference sera for use in the serological diagnosis of infectious bovine rhinotracheitis. Rev. Off int. Epiz., 13(3), 947-960.

What should I do if there are crystals in the concentrated wash solution? ( )

Warm and agitate the solution until all crystals are dissolved, and then prepare the 1X solution.