INNOTEST^® β-AMYLOID(1-40)

If the species has the same amino acid sequence as humans or a highly conserved amino acid sequence, particularly in the areas that are recognized by the antibodies (epitopes) in the assays, it is possible to use the CSF or plasma of other species on these assays.*

The human amino acid sequence for β-amyloid(1-42) and Tau are listed below, together with the epitopes of our antibodies used in the Neuro assays. You can compare these with the sequence of the other species.

A recommended site to look up amino acid sequences of other species is www.uniprot.org.

• 81576 (CE) / 81583 (RUO): INNOTEST β-AMYLOID(1-42): antibodies used are 3D6 and 21F12.

• 80462 (CE) / 81585 (RUO): INNOTEST β-AMYLOID(1-40): antibodies used are 3D6 and 2G3.

• 81572 (CE) / 81579 (RUO): INNOTEST hTau Ag: antibodies used are AT120, HT7 and BT2
• 81574 (CE) / 81582 (RUO): INNOTEST PHOSPHO-TAU_(181P): antibodies used are AT270 and HT7

• 80933: INNO-BIA plasma Aβ forms: antibodies used are 3D6, 2G3 and 21F12 (cf. INNOTEST assays)

* Use on other samples than CSF is not validated by Fujirebio Europe N.V.

Yes, below you can find a protocol and references to publications where it has been used in combination with our Neuro INNOTEST® assays.*

Protocol for brain Aβ42 and Aβ40:

• Homogenize mouse brain in 6.5 volumes of 20 mM Tris-HCl, pH 8.5 with
  5 mM EDTA,
  2 mM phenylmethylsulfonyl fluoride
  0.5 mg/ml leupeptin
  0.7 mg/ml pepstatin
  0.1 mg/ml phenanthroline
  0.1 mg/ml benzamidine
• Clear the homogenate as follows: centrifuge at 135,000 x g for 1h at 4°C and at 220,000 x g for 2h at 4°C.
• Concentrate on Sepak C18 cartridges (Waters, Milford, MA).

This protocol has been used in the following publications:

• Moechars et al., 1999 J Biol Chem
• Lee et al., 2003 J Biol Chem

* These protocols were not validated by Fujirebio Europe N.V.

Although there is a difference in absolute concentrations between both assays, INNOTEST and INNO-BIA AlzBio3* results are highly correlated (cf. publication list).  However, for the conversion of INNOTEST concentrations into INNO-BIA concentrations or vice versa (e.g. pooling of two different data sets), we highly recommend to define your own conversion factor by analyzing at least a subset of your study population with both technologies, given the fact that:

• different publications mention different correlation factors and conversion factors
• the conversion factor might not be the same for the entire measurement range
• there is no certified reference material available to which the concentrations obtained with these assays can be linked.

Publication list:

• Olsson et al. Clin Chem 2005;51(2):336-345
• Reijn et al. Clin Chem 2007;53:859-65
• Lewczuk et al. Neurobiol Aging 2008 Jun;29(6):812-8
• Wang et al. J Alzheimers Dis 2012;31(2):439-45
• Irwin et al. Arch Neurol 2012 Aug ;69(8) :1018-25
• Le Bastard et al. J Alzheimers Dis 2013;33(1):117-31
• Vanderstichele et al. Clin Chem 2013 Apr;59(4):710-2.

* The INNO-BIA AlzBio3 assay is no longer commercially available.

Cut-off values are usually calculated using samples from clinically diagnosed AD patients and healthy controls or non-AD dementia patients that have been followed up for some years.  This follow-up is important since it possibly impacts the obtained optimal cut-off value and/or its associated sensitivity and specificity, because:

1. a significant number of patients with an AD clinical diagnosis do not have an underlying AD pathology and;
2. AD pathology may be present decades before the onset of the clinical symptoms. 

The samples used for setting the cut-off values should preferably have undergone the same pre-analytical track as the samples that will be analyzed and interpreted in routine, as many of these pre-analytical factors influence the final biomarker results (Vanderstichele et al. Alzheimers Dement 2012 Jan;8(1):65-73 - del Campo et al. Biomark Med 2012 Aug;6(4):419-30).

Different statistical approaches to choosing the right cut-off are described in Bartlett et al. Biomark Med 2012. Sample sizes should be chosen so that cut-off values can be estimated with adequate precision and this will depend on the statistical approach.    

According to the consensus report, the (combined) assessment of biomarkers for Alzheimer’s disease should achieve at least 80% sensitivity and specificity (Working Group on Molecular and Biochemical Markers of Alzheimer’s disease, Neurobiol Aging 1998). Cut-off values determined in one population should preferably be cross-validated in an independent cohort to avoid overestimation of the diagnostic performance of the chosen cut-off values (Bartlett et al. Biomark Med 2012 Aug;6(4):391-400).