Legipid® | FAQs

0. What is Legionnaires' disease?

0. What is Legionnaires' disease?

Legionaires' disease (Legionellosis) is a serious and sometimes fatal form of pneumonia caused by the bacteria Legionella.  Legionaires' disease is a bacterial infection of the lungs, which is fatal if not treated quickly and properly.  High conventrations (104 - 10Legionella CFU/L) in water sources are considered a risk of infection.

1. What does Legipid® Legionella Fast Detection kit detect?

1. What does Legipid® Legionella Fast Detection kit detect?

The kit detects Legionella sp in water samples. At the moment, the genusLegionella has 50 species and includes 70 different serogroups. Legionella pneumophilais the most pathogenic species, especially serogroups 1 and 3, and it is causing over 90% of the Legionnaire’s diseases in the world.

2. How does Legipid® Legionella Fast Detection kit work?

2. How does Legipid® Legionella Fast Detection kit work?

It is based on the selective capture of Legionella by immunomagnetic particles, which have fixed antibodies attached to their surface. The immunomagnetic particles react with the bacteria’s cell envelope, where its virulent properties are held. The complex formed by the Legionella and the particles are captured by a magnet, and are marked after a simple washing process, with a reading antibody. These marks produce color, when the appropriate reagents are added. 

3. How does the Legipid® Legionella Fast Detection kit capture the Legionella cells?

3. How does the Legipid® Legionella Fast Detection kit capture the Legionella cells?

The capturing reaction is an antigen-antibody reaction, in the interface of two mobile surfaces in an aqueous medium. On the one hand the antibodies fixed to the surface of a magnetic sphere, and on the other hand the antigens exposed on the bacteria’s surface. This interaction is cooperative and the strength of the union depends on the integrity of both surfaces. In this case, it depends on the integrity of the bacteria’s surface, which is affected by its viability. 

4. Has the Legipid® Legionella Fast Detection kit  an international validation recognition?

Yes, the Legipid® kit has been validated against the ISO 11731 standard (reference method) and has been certified by AOAC, validation organism with international recognition.

5. How has the Legipid® Legionella Fast Detection kit been validated?

5. How has the Legipid® Legionella Fast Detection kit been validated?

The Legipid® Legionella Fast Detection test has been validated against the reference method (culture) and has been acknowledged as equivalent to the culture method by an internationally renowned technical organization, AOAC. The manufacturer of the product can provide all the technical documents for the implementation of this technique in the lab. These documents are:

  • Certification by AOAC-RI PTM 
  • Validation study report published in the scientific magazine Journal of AOAC International 
  • Validation report published in the scientific magazine BMC Microbiology

Please remember that the document EA-4/10 G: 2002, Accreditation for microbiological laboratories, covers the validation subject which is contained in the ISO 17025 standard. This document specifies that “Laboratories should keep the validation data of the commercial test systems (kits) they use. This data can be obtained from inter-comparison exercises or from validation data provided by the manufacturers which are subject to being evaluated by a third party (AOAC, for example)".

6. What is its expiration date?

Its expiry date is 6 month and it is indicated in the package insert. 

7. Which are the storage conditions?

The kit should be stored in the refrigerator (between 2 ºC and 8 ºC). When it is time to use, the kit should be left at room temperature (20 ºC-25 ºC) for at least 30 minutes. 

8. What precautions should I take in the preparation of the L4 reagent?

It is important to note that each vial of reagent L4 can be used for a maximum of 4 assays, discarding the excess if it's used for less analysis.
The L4 reagent  is composed by a solid part, contained in the plunger cap, and a portion of liquid in the glass compartment. Remove the security seal and the protective cap. Press the piston to insert it  fully into the shutter. Shake the mixture vigorously for 10 seconds. Once the L4 reagent  is prepared, it should be used IMMEDIATELY and it should never be reserved.

9. How should the samples be filtered to be tested with the Legipid® kit? 

9. How should the samples be filtered to be tested with the Legipid® kit?

Generally by filtration. The final elution obtained with the appropriate eluent (specified) by the manufacturer is the prepared sample to be analyzed with the Legipid® kit.

We can filter the volume of the original sample using the product Filterpid, or acquiring a portable filtration system. Of course, you can use a conventional filtration laboratory system. The filtered volume from the original sample is called examined volume and the test result is always referred to it. The probability of detection improves as the examined volume increases. For this reason, it is recommended to filtrate at least 1 liter of the original water sample, although sometimes, smaller volumes are filtered due to operational or regulatory reasons. 

The test result is always referred to the examined volume. For example, if 1 liter of sample is filtered, the result will be expressed in cfu/l, and if 500 ml of sample is filtered, then the result will be expressed in cfu/0.5l; in general, cfu/examined volume.

When filtering with a conventional laboratory filtration system, two types of filter are used. A glass fiber pre-filter of 2.7 µm diameter placed over a nitrocellulose or polycarbonate filter (typically used in laboratories). After the filtration, the glass fiber pre-filter is removed carefully, and next, the nitrocellulose or polycarbonate filter is eluted as usual. This pre-filter helps filtration, protects the filter and avoids interfering substances in the reaction.

10. Can several samples be analyzed at the same time?

Yes, and you can contact us so we can tell you which is the best solution to suit your needs. In order to satisfy your needs, we have two types of devices or magnetic racks (MP2, MP4), and different packs for a variable number of assays. (10, 40, 100 and 500).
You can see our recommendations in the following table:

RECOMMENDATIONS

(ASK US)

RACK

10 or less assays per batch

More than 10 assays per batch

PACK

MP2

MP4

10

X

40

X

X

100

X

X

500

X

11. Does the kit contain dangerous substances?

No, all the reagents are harmless as it is specified in the Material Safety Data Sheet.

12. How are results interpreted?

12. How are results interpreted?

A test (T) is considered positive if: 

1. The test (T) has higher color than the control (C) 2 minutes after the beginning of the colorimetric reaction. In such case, stop the reaction following the instructions of the package insert. The general estimation of the level of Legionella can be obtained by comparing the test color (T) with the color chart (appendix of the package insert).

2. If there is no color difference 2 minutes after the beginning of the reaction, let the reaction progress for 8 more minutes (10 minutes in total). A positive test (T) for Legionella must have a stronger color than the control (C) color 10 minutes after the beginning of colorimetric reaction. The estimated level of Legionella is low, up to two orders of magnitude (102 CFU/test portion).

A test (T) is considered negative if: 

3. The test (T) doesn’t have color difference with the control (C) 10 minutes after the beginning of the colorimetric reaction.

13. How are the results of the kit compared with culture?

Ask us and we will provide you with an appropriate validation protocol. Avoid the preparation of artificial samples with biomass grown by culture (colonies or growing grass) because the growth in an artificial environment introduces a mechanism of aggregation of the bacteria. This is due to the stimulated production of extracellular material, which acts as a glue that aggregates cells, covering and hiding the antigens, making this material much less detectable.

For this reason, it is strongly recommended the use of certified reference material, (CRM, Legionella pneumophilaATCC 33152) to prepare samples for verification. In this context, all the biological material isn’t equally detectable by immunosensing, due to the fact that during its manufacturing process they might include certain components (for example, alginates or binding agents) that interfere with the accessibility of the antigens. Antigens must be recognized in order to capture the target bacteria.

Specifically, Biótica recommends the use of CRM del Laboratoire BioRéférence (bioreference@eurofins.com). It is not recommended the use of “HPA lenticules” material, which presents very weak immune-detectability.

Starting from the concentrated sample, we will use a small part of it for culture (for example 0.1-0.2 milliliters from each sample) and 9 ml for the kit.

Please do not hesitate to contact Biotica for any related questions (info@iristech.net).

14. What precautions should I take with the negative control?

Only those specified in the protocol. Be especially careful in the correct execution of the washing steps, to avoid leaving an unspecified mark which can cause a residual coloration.

15. What is Filterpid?

Filterpid (Cat. Nº 211-10-00) is a complete and easy solution to prepare a sample, to be analyzed by the fast test Legipid®. It has excellent separating performance of the Legionella microorganisms, present in the original water sample. By “original water” we mean pure water, rivers, potable and industrial water. The assembled system includes a 60 ml syringe connected to a pre-filter, which is connected to a micro-filtration compact unit. Filterpid doesn’t require a pump, it can be operated manually. The original water sample is forced manually through the device by pushing the syringe (positive pressure). Bacteria are kept inside the micro-filtration unit. For the elution, two syringes of 10 ml are connected; one to each end of the unit, and a simple protocol is followed.

An on-site filtration is also possible in the facilities, by using a pipe adaptor.

16. How can the technique be accredited in my laboratory?

In Spain, the accreditation entity is ENAC. Talk to your auditor and provide him with all the documentation on the validation and the AOAC Certificate. In accordance with technical note 32, he will show you the next steps to take.

17. Where can I buy these products?

Contact us by email info@iristech.net 
North America_USA:  +1.404.348.4130 or by fax +1.404.806.9525 
Europe_Germany:  +49.36705.21634 or by fax +1.404.806.9525