ASSAY-SPECIFIC QUESTIONS

CATECHOLAMINES

For in-vitro diagnosis the kits of the serial 6xxx must be used (ELISA and RIA are available). Namely:

Adrenaline ELISA Fast Track

BA E-6100

Noradrenaline ELISA Fast Track

BA E-6200

Dopamine ELISA Fast Track

BA E-6300

2-CAT (Adrenaline /Noradrenaline) ELISA Fast Track

BA E-6500

3-CAT (Adrenaline/Noradrenaline/Dopamine) ELISA Fast Track

BA E-6600

 

2-CAT (Adrenaline/Noradrenaline) RIA Fast Track

BA R-6500

3-CAT (Adrenaline/Noradrenaline/Dopamine) RIA Fast Track

BA R-6600

 

 

The assays of the serial 6xxx for IVD-applications had been validated for plasma (EDTA) and urine samples. Any other kind of sample – including serum – have not been validated. Serum samples might be used with these kits in research applications.

This depends on the sample volume and concentration of the analyte. In most cases the research kits of the serial 5xxxR should be used. Namely:                                      

Adrenaline Research ELISA

BA E-5100R

Noradrenaline Research ELISA

BA E-5200R

Dopamine Research ELISA

BA E-5300R

2-CAT AN (Adrenaline/Noradrenaline) Research ELISA

BA E-5400R

2-CAT ND (Noradrenaline/Dopamine) Research ELISA

BA E-5500R

3-CAT (Adrenaline/Noradrenaline/Dopamine) Research ELISA

BA E-5600R

But in some cases, it is advantageous to use the kits of the serial BA E-6XXX. Therefore, you are advised  to contact our product specialists, who have a lot of experience concerning different types of studies and protocols (support@ldn.de).

 

Different buffers can be used for tissue homogenization. Buffer type depends on the research project and if other parameters need to be measured in the same sample.
LDN recommends that tissues be homogenized in 0.01 M HCl with 1 mM EDTA and 4 mM Sodium metabisulfite. Sample dilution is dependent on expected sample concentrations. If this is unknown, we recommend a serial dilution of differing sample volumes to determine the best dilution for your particular sample type

For this procedure we offer an elaborate protocol – please  inquire (support@ldn.de).

For a period up to 6 months the samples can be stored at -20 ⁰C. For the storage at -80 ⁰C we currently have no data.

If you are running the samples for IVD-purposes, you have to accomplish the assay once started without any break (in accordance with the protocol). If you are running the samples for research purposes, there is the possibility to have a break after the extraction step. For more details please contact support@ldn.de.  

On a regular base we participate at the Ringversuch of INSTAND (www.instand-ev.de) for plasma and urine samples. INSTAND offers this proficiency testing every quarter.

The patient should ask his medical doctor who will give him guidelines for the collection of the sample.

No, currently we do not offer such ranges.

This was not tested yet. If you are interested in this kind of measurement, we would recommend using the Research ELISA (serial BA E-5xxxR) and running a proof-of-principle. Our specialists would assist you in establishing a testing protocol (support@ldn.de).

Yes, there are automats which are capable for the automated runs of Catecholamines. For more details please contact our specialists at support@ldn.de.

If you need to dilute the samples, you can use 0.01 N HCl. The samples have to be diluted before the extraction procedure.

Our assays detect the free Adrenaline, Noradrenaline or Dopamine.

To prevent catecholamine degradation, add EDTA (final concentration 1 mM) and sodium metabisulfite (final concentration 4 mM) to the sample as early as possible.

The pH plays a critical role during the extraction procedure: an excess of acid should be avoided to preserve the buffer capacity of the extraction buffer.
A pH > 7.0 during the extraction is mandatory.

SEROTONIN

The most common sample matrix used for IVD is serum. The Serotonin ELISA Fast Track (BA E-8900) was validated for serum. In general, plasma is not recommended for testing of Serotonin, as the thrombocytes might undergo lysis and release their high content of Serotonin. As the degree of lysis is very different and varying from sample to sample, it will interfere with the measurement in an unpredictable way.

Yes.
For platelet-rich plasma the Serotonin ELISA Fast Track (BA E-8900) can be used as an IVD-application. For the RUO-determination of Serotonin in platelet-poor plasma and CSF the research ELISA BA E-5900R has to be used.

This was not tested yet. If you are interested in this kind of measurement, we would recommend using the Serotonin Research ELISA (BA E-5900R) and running a proof-of-principle. Our specialists would assist you in establishing a testing protocol (support@ldn.de).

Yes, on a regular base INSTAND (www.instand-ev.de) offers Ringversuch for urine and plasma samples. INSTAND offers this proficiency testing every quarter.

Yes, there are automats which are capable for the automated determination of Serotonin. For more details, please contact our specialists at support@ldn.de.

For homogenates and cell culture samples the Serotonin Research ELISA (BA E-5900R) is recommended. Special attention has to be paid to the pH as Serotonin is not stable in acidic milieu (the use of a stabilizer is mandatory). The use of cold buffers is mandatory and the samples should not be stored too long under acidic conditions.
Our general advise for the preparation of tissues is homogenisation in 0.01 N HCl + 1 mM EDTA + 4 mM sodium metabisulfite. Homogenise, spin down and use the clear supernatant for the assay. The ratio tissue to HCl depends on the expected concentrations and also the sample volume that should be extracted depends on expected concentrations. If this is unknown then it is best to use relatively low HCl volumes and extract different sample volumes.
For different animal serum samples – such as mouse or rat samples – the Serotonin Fast Track ELISA (BA E-8900) might be an alternative. Our specialists do have a lot of experience with different kinds of Serotonin studies, and they will assist you in establishing your specific testing protocol (support@ldn.de).  

HISTAMINE and HISTAMINE RELEASE

No, currently we do not offer such ranges.

Yes, there are automats which are capable for the automated determination of Histamine. For more details please contact our specialists at support@ldn.de

For the Release, heparin is recommended as less basophile cells are destroyed.

No, because too many cells are destroyed which will give a high background.

For the measurement of Histamine in different animal plasma samples, the Histamine Research ELISA (BA E-5800R) is recommended. The following chart shows some examples of Histamine concentrations measured in different animals:

Animal species

Concentration (ng/ml)

Mouse

22.9

Rat

20

Cat

1.1

Dog

0.3

Horse

0.6

GABA (Gamma-aminobutyric acid) and GLUTAMATE

In general, there will be many different buffers which can be used for preparing the cell lysates and which will not interfere with our assay. To be sure that the buffer is compatible with the assay, we always advise to perform a proof of principle. You could spike your buffer with GABA and measure the recovery. You could use the highest standard provided in the kit for spiking.

An excess of acid should be avoided: For GABA, a pH of 3.0 during the extraction procedure of the assay is mandatory, for Glutamate a pH of 5.0.

Our general advise for the preparation of tissues is homogenisation in 0.01 N HCl + 1 mM EDTA + 4 mM sodium metabisulfite. Homogenise, spin down and use the clear supernatant for the assay. The ratio tissue to HCl depends on the expected concentrations and also the sample volume that should be extracted depends on expected concentrations. If this is unknown, then it is best to use relatively low HCl volumes and extract different sample volumes.

TRYPTOPHAN

The precipitation procedure releases the bound fraction in the sample so that the assay detects the total Tryptophan.

Yes, the kit can be used for various biological samples. For more details, please contact our specialists at support@ldn.de.

METANEPHRINES IN URINE

FREE METANEPHRINES IN PLASMA

Yes, for the interpretation of the results, a grey area has to be considered. This grey area does not depend on the methodology used and is reflected in a slight to mediate increase in Metanephrine and Normetanephrine up to 4 times the upper cut-off (Eisenhofer et al. 2003). Approx. 20 % of the tumors are found in this grey area, especially in the case of the Hereditary Syndrome, incidental tumors and in sporadic cases of Pheochromocytomas with a diameter less than 1 cm.
In case of a result in the grey area, it is recommended to collect a new sample together with an anamnesis concerning especially influences like the medication and age of the patient.

Using our kit, we recommend collecting the blood sample with the patient in an upright/sitting position.

No, serum samples are not recommended – one must use EDTA- or Heparin plasma.

Yes, there are automats which are capable for the automated determination of these Metanephrines. For more details, please contact our specialists at support@ldn.de.

In principle, it is possible to measure the Metanephrines in different animal species. For more details, please contact our specialists at support@ldn.de.

Medications like Serotonin-noradrenaline reuptake inhibitors, tryicyclic antidepressants, MAO inhibitors, antihypertensive drugs and L-DOPA can influence Metanephrine and Normetanephrine level. People who are taking such medication should consult with their doctor before specimen collection.
Sympathomimetic agents, sport and smoking can also influence Metanephrine and Normetanephrine level. Alcohol and caffeinated drinks should be avoided the day before and including the day of sample collection.

5-HIAA (5-Hydroxy-3-Indole Acetic Acid)

There is the possibility to run an extraction procedure with such samples for research applications. For more details, please contact our specialists at support@ldn.de.

CHROMOGRANIN A

Yes, certain proton pump inhibitors and type-2 receptor antagonists can influence CgA levels in serum. People who are taking such medication should consult with their doctor before specimen collection.
Sport and ingestion of meal can also influence the CgA levels.

On a regular base we participate at the Ringversuch of RfB (https://www.rfb.bio ). RfB offers this proficiency testing every quarter.

The assay was validated for serum samples and serum must be used when running samples for IVD-purposes. But for research applications, plasma might also be a suitable sample matrix.

FOOD SAFETY

Yes, LDN participates on a regular base at the external quality assessment scheme of FAPAS. The participating assay is the HistaSure ELISA Fast Track (FC E-3600).
Results are available upon request (support@ldn.de) .

Yes, the HistaSure ELISA Fast Track (FC E-3600) is participating at the Performance Tested Method (PTM) program of the AOAC Research Institute and is AOAC-certified since 2014.

For these applications, the HistaSure ELISA Fast Track (FC E-3600) had been tested and is recommended. For more details, please contact our specialists at support@ldn.de .

If not tested immediately, the extracted samples can be stored frozen at -20 ⁰C.

No, we do not offer such a program. But there are good and reliable online calculation programs for free which can be used, such as Myassays.com.

Yes, for running an assay on ELISA processors, we recommend the HistaSure ELISA Fast Track (FC E-3600). For example, this assay can easily be adapted on the Gemini and other automats identical in construction.

HISTAMINE FOOD ELISA

The histamine concentration read from the standard curve is not the final concentration – but has to be multiplied with the corresponding dilution factor used for the sample preparation. In the IFU, the dilution factors for the most frequently kinds of samples preparations (fish, sausage, cheese, milk, wine and champagne are listed).

HistaSure ELISA Fast Track

All fish samples tested so far are suitable for the HistaSure ™ ELISA Fast Track. The lists below depict some major applications in different matrices.

Species validated through AOAC Certification

Fish species

Presentation

Tuna

canned chunk light

fresh/frozen yellow fin

Mahi Mahi

fresh/frozen

Sardines

canned in oil

Fishmeal

 

Species validated through in-house testing

Fish species

Presentation

Mackerel

smoked

Anchovy

fresh

brined

in sauce

Shad

dry salted

fermented

Herring

smoked

Salmon

smoked

Bonito

lakerda

Swordfish

fresh

Marlin

fresh

Fish products validated through in-house testing

Maldive fish

Fish sauce (sardines/anchovy)

Oyster sauce (oyster/sardines)

Please contact us (support@ldn.de) if you cannot find your sample of interest in this list – we will check your application.

No, if you have prepared an uniform homogenate you can just take a small aliquot of a few ml for the filtration/centrifugation.

No, the use of a shaker is mandatory.

Yes, the following controls are available upon request:

  • 0 ppm (FC C-0051)
  • 50 ppm (FC C-0052)
  • 100 ppm (FC C-0053)

HistaSure Fish Rapid Test

The cut-off can easily be adjusted during the sample extraction step by varying the amount of distilled water in which the fish sample is homogenized:

Designated cut-off *)

Procedure

100 ppm

Homogenize 10 grams fish sample in 490 ml dist. water

50 ppm

Homogenize 10 grams fish sample in 240 ml dist. water

20 ppm

Homogenize 10 grams fish sample in 90 ml dist. water

10 ppm

Homogenize 10 grams fish sample in 40 ml dist. water

*) Any other cut-off can be derived from the examples given above.

No, if you have prepared an uniform homogenate you can just take a small aliquot of a few ml for the filtration/centrifugation.

Please contact us (support@ldn.de) if you cannot find your sample of interest in this list – we will check your application.

Yes, the run is valid although there is only a weak signal. A weak signal arises, if the Histamine content of the samples is very low.