Moving away from Mouse Bioassay (MBA). Time to implement the new legistation for lipophilic toxins will end on 31 December 2014

Escrito por admin el . Posteado en Marine biotoxins, Okatest

3 months left to stop mouse bioassay for determination of lipophilic marine toxinsThe liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the reference method for lipophilic toxins since July 2011. According to the European Commission EU 15/2011 this method has to be used as matter of routine, both for the purposes of official controls, at any stage of the food chain, and own-checks by food business operators. This legislation applies to European countries and any other exporting to Europe.

For many years the MBA has been widely applied by experience laboratories or small producers. Minimum training on handling animals and laboratory skills were required to identify positive samples to diarrheic toxins. However, the implementation of a mass spectrometry LC/MS method is quite a challenging task that some may fail to achieve. Small to medium processors would have to subcontract analyses by this method and many private laboratories offering testing by MBA may have to stop this service due to the high investment and expertise required.

Official and private laboratories, producers and any other stakeholders were given up to the 31st of December 2014 to change methodologies. Reference laboratories from the main producer countries such as France, UK, Ireland, Spain or Italy accredited their LC-MS/MS methods well before the implementation date. However, other laboratories are still using the MBA until the last days.

At an early stage of the approved legislation some opinion leaders raised concerns about the capacity of the validated LC-MS/MS method to protect consumers (Otero et al, Analytical Chemistry 2011). The effect of uncontrolled factors in the method, together with the lack of information on toxicity made some to believe that the decision of this new method was made with haste. Although other experts disagreed with those conclusions, the controversy is still opened. A time extension to implement the new reference method was also requested by Spain to the EU commission. Thus, many laboratories in or outside the EU have still long to go, are undeceive or are even looking for other alternatives.

The European legislation (2074/2005 and 15/2011) also allows other analytical methods to monitor lipophilic toxins, providing they fulfil the method performance criteria stipulated by the EU-RL (European Reference laboratory) and offer the same level of public health. Two other methods, a phosphatase inhibition assay -PPIA- (Smienk et al, Toxins 2012) and LC-MS/MS (Van den Top et al, Food & Contaminants 2011) comply with these requisites.

The phosphatase inhibition assay OkaTest is the only commercial kit that has been inter and intra-laboratory validated; as stated by the EU-RL, it fulfils the requirements of Regulation (EC) 2074/2005 and can be used for the detection of Okadaic Acid group toxins (OA, DTX1, DTX2, DTX3 including their esters).

OkaTest: colorimetric phosphatase inhibition assay

Okatest-colorimetric-phosphatase-inhibitor-assay

OkaTest requires basic laboratory skills and minimum equipment. So, it can be easily implemented by shellfish processors or laboratories without high investment.

The presence of Okadaic Acid toxins determined by OkaTest in few hours could help make faster decisions as per not to harvest or to further product processing. Using the kit for own checks prior to harvest, when ropes or rafts are near to a closed area, or in areas with toxins levels below the legal limit, could save a lot of money. The use of this kit is especially interested in those areas where Okadaic Acid toxins blooms are common.

To fully monitor lipophilic toxins OkaTest must be complemented with other methodology able to detect yessotoxins (YTX), pectenotoxins (PTX) and azaspirazids (AZA).

The combination of OkaTest and LC-MS/MS can be a good system to monitor lipophilic toxins in molluscs bivalves.

OkaTest and LC-MS/MS are both reliable analytical tools which advantages can be combined to bring important benefits to the users. LC-MS/MS is able to identify individual toxins from various groups of lipophilic toxins. However, it requires specialised personnel and large investment. OkaTest detects the potential sample toxicity, quantifying the Okadaic Acid toxins by PPIA, and does not need either specific equipment or qualified staff. Laboratories with large number of samples or limited access to LC-MS equipment could benefit from combining both methodologies. A shorter time-to-results and lower cost per sample can be achieved implementing the two methods together for routine monitoring of lipophilic toxins. LC-MS/MS requires 2 injections per sample. One of them is used to determine YTXs, AZAs, free OA and PTXs and a second injection to identify the Okadaic Acid derivatives. The second injection could be diverted to OkaTest, as this kit is able to determine total OA-toxins and can analyse more than 43 samples within the same day. However, it takes up to 20 hours to process 25 samples with LC-MS/MS. So, results could not be reported until the following day (Blanco et al, Revista Galega dos Recursos Mariños 2013).

The combination of both methodologies could also be used in events of OA-toxins blooms, previously identified by LC-MS/MS. The frequency and number of points of sampling increase under an event of toxic bloom; OkaTest can be used to analyse the extra number of samples and so to follow up the Okadaic Acid bloom.

The analysis of antibiotics in meat has never been easier

Escrito por admin el . Posteado en Antibiotics

The presence of antibiotics in food has been a concern for years because of technological and analytical reasons but mostly due to their side-effects in human health. Besides the toxicity and allergenicity of some antibiotics, the main risk for humans comes from the misuse of antibiotics in animal husbandry that has been linked to antibiotic resistances. According to WHO antimicrobial resistances are one of the most serious threats to global public health over the world.

ZEULAB has developed a new procedure to analyze antibiotics in meat that combines an inhibitor tube test (Explorer 2.0) and the new device e-Reader. This device coupled to Explorer 2.0 incubates the assay at the selected temperature, determines the endpoint of the assay and interprets the results. Qualitative and numeric results are displayed in the screen and are also stored.

This system has been validated for meat samples according to the European Commission Decision 2002/657/EC and the results have been published in Food Additives & Contaminants: Part A, a peer-reviewed journal.

Sensitivity towards 25 substances from several groups of antimicrobials was investigated in a first step. Detection capabilities for six substances representing the six major antimicrobial groups were also determined in bovine muscle. The detection capabilities for amoxicillin (10 µg l-1), cefalexin (200 µg l-1), doxycyclin (100 µg l-1), sulfamethazine (100 µg l-1), tylosin (100 µg l-1) and neomycin (200 µg l-1) were in all cases at or below the maximum residue limit (MRL). Specificity and applicability of the test were demonstrated with muscle samples from four animal species (bovine, porcine, ovine and poultry) and results were found to be very satisfactory. Ruggedness was evaluated on negative and spiked samples with sulfamethazine as a representative antimicrobial. Neither false-positives nor false-negatives were detected when varying the sample volume, the time of pre-incubation, the temperature of incubation and the batch of the test.

These results proved that Explorer 2.0 coupled toe-Reader is a valuable tool for the screening of a broad range of antimicrobials in muscle. This new methodology simplifies the analysis and increases the accuracy of interpretation of the test results since the endpoint of the assay is automatically determined and results are interpreted objectively.

More information about the validation of the new procedure may be found in: http://www.tandfonline.com/doi/full/10.1080/19440049.2014.934303

More information about Explorer 2.0 and e-Reader may be found in our web site and in our Youtube chanel

ZEULAB has developed a new procedure to analyze antibiotics in meat that combines an inhibitor tube test (Explorer 2.0) and the new device e-Reader. This device coupled to Explorer 2.0 incubates de assay at the selected temperature, determines the endpoint of the assay and interprets the results. Qualitative and numeric results are displayed in the screen and are also stored.

This system has been validated for meat samples according to the European Commission Decision 2002/657/EC and the results have been published in Food Additives & Contaminants: Part A[Zeu1] , a peer-reviewed journal.

Sensitivity towards 25 substances from several groups of antimicrobials was investigated in a first step. Detection capabilities for six substances representing the six major antimicrobial groups were also determined in bovine muscle. The detection capabilities for amoxicillin (10 µg l-1), cefalexin (200 µg l-1), doxycyclin (100 µg l-1), sulfamethazine (100 µg l-1), tylosin (100 µg l-1) and neomycin (200 µg l-1) were in all cases at or below the maximum residue limit (MRL). Specificity and applicability of the test were demonstrated with muscle samples from four animal species (bovine, porcine, ovine and poultry) and results were found to be very satisfactory. Ruggedness was evaluated on negative and spiked samples with sulfamethazine as a representative antimicrobial. Neither false-positives nor false-negatives were detected when varying the sample volume, the time of pre-incubation, the temperature of incubation and the batch of the test.

These results proved that Explorer 2.0 coupled toe-Reader is a valuable tool for the screening of a broad range of antimicrobials in muscle. This new methodology simplifies the analysis and increases the accuracy of interpretation of the test results since the endpoint of the assay is automatically determined and results are interpreted objectively.

More information about the validation of the new procedure may be found in:

http://www.tandfonline.com/doi/full/10.1080/19440049.2014.934303

More information about Explorer 2.0 and e-Reader may be found in our web site (www.zeulab.com) and in our Youtube chanel https://www.youtube.com/watch?v=4PwmPkfID2w

EGG ALLERGY: NEW ELISA TESTS FOR EGG DETECTION

Escrito por admin el . Posteado en Allergens

Food allergy involves adverse reaction of immunity system to determined foods (mostly proteins). Food allergies represent an important health problem affecting mainly children (6-8%). Symptoms can vary form mild (hives) to severe reactions such as anaphylaxis. The amount of food allergen that can trigger an allergic reaction is different in each patient and trace of allergens (little microgram) can be enough to induce a reaction. Hence, rigorous information about foods that contain allergens is very important to allow allergic people manage their diet.

egg, egg allergy, food allergies, zeulabEgg allergy is one of the most common in children, generally aged less than 5 years old. Egg is an ingredient widely used food industry to manufacture products such as tortillas, bread and bakery. Egg is not only added in food as a basic ingredient, but also as additive or supplementary condiment. Moreover, white egg is used as clarification agent in wine industry. Altogether, egg can be a hidden allergen and it can be present in a non declared way in food. According to European legislation (Annex II of Regulation (EU) 1169/2011) egg and thereof require mandatory mention in food labels. In order to claim correct information label in food, it is important to perform assays to verify egg presence/absence in food, especially when egg is used as additive. Moreover, analytical controls are needed to verify measures to avoid cross contamination in facilities where food manufacturing is realized with and without egg.

ZEULAB releases a new ELISA Sandwich test to detect egg, “PROTEON Ovalbumin, in an effort to offer analytical tools to the food sector in this issue. Ovalbumin is the major protein in white egg (54%) and together ovomucoid, lysozyme and ovotransferrin are the main allergens in egg. “PROTEON Ovalbumin” allows quantifying egg quantity in whole egg powder ppm in an accurate way. This test is available for a wide range of matrix in sectors such as bakery and wine industry. Besides, ZEULAB has improved “PROTEON Ovomucoid”. Ovomucoid is a heat stable glycoprotein that represents 11% of white egg proteins. So this ELISA Sandwich is designed to detect egg in food matrix treated at high temperatures where proteins could be denaturalized. Both tests are available in 48 and 96 well plate format and results are traceable with the NIST 8445 reference material.

 

Mouse bioassay for determination of lipophilic marine toxins in shellfish comes to its end

Escrito por admin el . Posteado en Marine biotoxins, Okatest

The presence of marine toxins in shellfish products could lead to fatal food poisoning. Monitoring programs to control levels of toxins establish monthly or even weekly sampling in production areas. Opening or closure of harvest areas would depend upon  the results obtained. Post-harvest and end product testing is also carried out in some countries. Currently, paralytic (saxitoxins), ammesic  (domoic acid) and lipophilic toxins (okadaic acid and derivatives, yessotoxins, pectenotoxins and azaspirazids) are legislated.

Mouse bioassay (MBA) has been used for long time for monitoring lipophilic marine toxins. However, concerns about the ethical problems and analytical results obtained were raised some years ago among different stakeholders.

Three mice per sample are injected with a solvent solution and the death of 2 out of 3 mice in 24 h determines the presence of lipophilic toxins above the regulatory limits (160 micrograms per kilogram of okadaic acid, derivatives and pectenotoxins together).

Social awareness and legal requirements to avoid animal experiments together with the proven unreliable results produced by mouse assay have lead to ban this method for monitoring of lipophilic marine toxins. In 2011 Commission Regulation (EU) No. 15/2011 established the liquid chromatography mass spectrometry as reference method. Member Estates must replace the mouse assay by the current reference method or any other method complying with the legislation before the 31st of December 2014.

The European legislation allows several analytical methods for monitoring of lipophilic toxins providing they fulfil the method performance criteria stipulated by the EU-RL (European Reference laboratory) and offer the same level of public health.

OkaTest, a phosphatase inhibition assay, fulfils the requirements of Chapter III A (4) a, b, and c of Annex III to Regulation (EC) 2074/2005 and can be used for the detection of Okadaic Acid group toxins OA, DTX1, DTX2, DTX3 including their esters. METHOD FOR DETECTION OF OKADAIC ACID TOXINS GROUP IN EDIBLE MOLLUSCS – click here.

OkaTest is an enzymatic kit for quantitative determination of Okadaic Acid (OA) and other carboxylic toxins of the OA group including DTX1, DTX2 and DTX3 by a colorimetric phosphatase inhibition assay. This method is applicable to shellfish species such as mussels, clams, oysters and scallops. More info here.

The toxicity of Okadaic Acid and DTXs is directly related to their inhibitory activity against a family of structurally related serine/threonine protein phosphatases (PP), in particular PP1 and PP2A. This strong inhibitory property is used to determine Okadaic Acid content in shellfish by means of a microplate assay, using the enzyme PP2A and a chromogenic substrate for this enzyme. The enzyme hydrolyses the substrate and the product can be measured by an absorbance measurement at 405 nm using a microplate reader. As the ability of the PP2A to hydrolyse the substrate depends on the presence of Okadaic Acid and analogues in the samples, the toxin concentration can be calculated by using a standard curve.

The kit includes five standards and has a working range from 63 µg/kg to 352 µg/kg. The LOD and LOQ are 44 µg/kg and 56 µg/kg, respectively.

OkaTest has been single and inter-laboratory validated (Smienk et al. Toxins 2012 and  Smienk. et al. 2013, JAOAC,  96,1,77-85).

ZEULAB Collaborates with the Food Bank

Escrito por admin el . Posteado en Antibiotics

zeulab-+-food-bank-donation-explorer-2.0

Zeulab has collaborated with the Food Bank after finishing the social campaign that took place during 2013.  Zeulab has donated 1% of Explorer 2.0 to the Food Bank, helping people to have access to food every day.

Last February CEO Dr. Pedro Razquin gave out the donation to Mr. José Ignacio Alfaro Director of the Food Bank from Zaragoza.

Zeulab wants to thank you all that helped us to collaborate with this cause and we invite you to keep cooperating with us in future campaigns in where Zeulab will be taking part of.

For more information about how to donate in Spain and in Europe please contact Food Bank from Zaragoza, Spanish Federation of food Banks and European Federation of Food Banks.

food bank + zeulab donation explorer 2.0