Frequently Asked Questions (FAQ)

STORAGE / SHELF LIFE / BATCHES

How should I store opened microplates if I do not use all the wells at once?

Opened plates are sensitive to storage conditions:

  • It is essential to immediately close the aluminium envelope after opening. 
  • Do not throw away the desiccant: the plate should be sealed with the desiccant inside the envelope. This should be carried out rapidly, as the desiccant will quickly absorb humidity and become inactivated after 10-30 minutes.
  • The end of the aluminium envelope should be cut with scissors and closed in an air-tight manner along the entire length of the envelope, with scotch tape, for example. (Simply folding-over the edges and/or stapling is insufficient to ensure sealing.). Please refer to the photos on the document Plate storage.pdf
  • Store at 5°C ± 3°C.

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 can 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 at +4°C for up to 2 days.

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

What is the shelf life/expiry date of the ID Screen® ELISA kits?

The majority of ID Screen® kits have a shelf-life of 24 months from the production date.

How long may I store each reagent before and after opening / reconstitution?

Please refer to the document Storage conditions.pdf, which indicates the storage conditions (duration and temperature) for each kit component.

APPLICATIONS (species, DIVA, sample types)

I am using an ID Screen® indirect multi-species test. Which species may I test?

ID Screen® indirect multi-species tests include a multi-species conjugate which recognizes mammalian IgG antibodies.
However, validation data does not exist for all species for all tests. When testing a species for which IDvet does not have validation data, check that the negative sample baseline remains low (indirect test), or that the distribution of S/N% results is not considerably altered with respect to the negative control value.

Is it possible to differentiate between vaccinated and infected animals with ID Screen® ELISAs?

Yes, some IDvet tests allow for DIVA applications (Differentiation Infected and Vaccincated animals).

The IBRgE and Aujeszky gE tests detect naturally-infected animals in a vaccinated population, provided that animals are vaccinated with gE-deleted vaccines.

For avian influenza, if vaccination is performed using a homologous hemagglutinin to the highly pathogenic field strain (H5), but a heterologous neuraminidase subtype, serologic surveillance can be performed by testing for antibodies against the homologous N subtype (N1) as evidence of natural infection.

Is it possible to test pooled milk or serum samples?

Some kits allow for the testing of pooled milk or serum.  Please refer to the kit insert of the product in question.

TESTING PROCEDURE

How do I prepare the 10x conjugate for use?

Please refer to the document 1x Conjugate preparation conditions
also available in French version: Conditions de préparation du conjugué 1x
and Russian version: 1x Conjugate preparation conditions in Russian

Do I need to include a reference blank on my plate?

No. A blank was included in the past in order to account for spontaneous TMB substrate colour change, or because the microplate plastic was not completely transparent.

Commercial kits no longer require the use of such a blank, as both the quality of plastic and TMB have improved over the years.

Is it possible to inter-change reagents from different batches or kits?

Yes.  Non-biological components (wash, buffers, stop, substrate) may be interchanged between products and between batches.  As much as possible, however, use the reagents delivered with the kit.

May the ID Screen® ELISAs be used with robots?

Yes, our kits have been used successfully with robots in many countries.

SERUM TESTING

Can I use hemolysed serum/plasma samples?

Yes, low to moderate hemolysis does not in general affect the ELISA performance.

May I use heat-treated serum samples?

For analysis by the complement fixation test, sera may require heat decomplementation (56°C for 30 minutes).  These heat-treated samples may also be tested by ELISA, although the decomplementation procedure may cause an increase in the background signal.

MILK TESTING

May I use heat-treated milk samples?

Yes – low heat treatment will not affect assay results.

How should I prepare milk samples for testing?

Please refer to the document Recommendations for milk testing
also available in French version Recommandations pour test sur échantillon de lait

Is it okay to use ID Screen® milk ELISAs with samples containing preservatives?

Yes. Milk samples containing preservatives, such as Bronopol and sodium azide, may be used.

How long may milk and plasma/serum samples be stored, and in what conditions? Can samples be frozen?

This will depend on how the samples have been stored.  If samples are stored at 4°C, they should not be kept any longer than 2 days. You may conserve samples at -20ºC for long-term storage (for up to about 5 years), but be sure that recipients are well sealed to avoid evaporation. Avoid repeated freezing/thawing cycles (preferably no more than three).

WASHING

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.

What type of washing system should I use?

All available washing methods are compatible with IDvet kits:

  • squeeze bottle
  • pipette
  • manual comb system
  • automatic washers

We suggest that you validate the internal washing system before testing high sample numbers.

Generally, systems which increase the contact time between the wash solution and the plate increase washing efficiency.

It is possible to include a soaking time between two steps (serum and conjugate or conjugate and substrate). Generally, the quality of the wash step is equivalent, or increased, with a soaking step. This observation is particularly true for milk tests, where the perfect dissolution of fat may require increased contact time between the wash solution and the plate.

What recommendations can you make to optimize the washing steps?

The washing system plays an important role in the quality of the final results:

  • Fill the wells completely at each wash (300µl).
  • Tap hard after the last wash to remove all fluid.
  • Avoid drying of wells between washings. 
  • Do not scrape the bottom of the wells during the washing steps, which could remove the antigen/antibody complexes.
  • If using a manual wash method, overflowing of wells is not a problem. However, after the first wash it is important to empty the wells quickly to avoid contamination between wells.
  • The cleanliness of the wash system may affect the quality of the results. Bacterial contamination of a tube or pump may arise if the system is never washed. We suggest that you pay particular attention to the cleanliness of the washing apparatus, regularly decontaminating the washer with a suitable detergent.

Will the sterility or the quality of the water used to reconstitute the wash solution affect test results?

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.
It is not necessary to use sterile water, or to store the distilled water in a sterile environment.
While the 1X wash solution will not be sterile in these conditions, its low level of bacteria will not affect results.

A high level of bacteria in the water, however, caused by the progressive contamination of the wash solution or the use of dirty glassware, can lower the quality of the test.

In this case, you will notice a reduction in the difference between the positive and negative signals, and an increase in the background signal.

Generally, it is recommended that the 1X wash solution be prepared at the beginning of the week, and not be kept for more than 5 days (ex. from Monday to Friday).
When making new 1X solution, it is important to wash the glassware and to rinse the wash system (manual or automatic).

INTERNATIONAL SERUM STANDARDS / FREEZE-DRIED POSITIVE STANDARDS

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
RUMINANTS
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
IDvet BLV AGID BLVAGID
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)
SWINE
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. sci.tech. Off int. Epiz., 13(3), 947-960.

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. Please refer to the List of positive and negative sera available from IDvet.pdf.

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.

TROUBLESHOOTING

What aspects of testing should I check if I have a problem with results?

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.

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 (info@id-vet.com) 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 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).

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.