- Assessing expression of neuronal targets is an important part in Alzheimer’s disease research
- SimpleStep ELISA® Kits quantify BDNF, Tau and TREM2 in a one-wash 90 min protocol
- SPECTROstar®Nano detects assays and results are analyzed automatically with MARS analysis software
Table of contents
Alzheimer’s disease (AD) is a multifactorial neurodegenerative condition. While it is accepted that AD results in progressive dementia, the causes and mechanisms leading to this condition are still poorly understood1,2. Numerous candidates for potential intervention have been proposed, but further research on their roles in AD and normal neural function is required to develop safe and effective therapies. Here, we describe the detection of 3 targets; Human BDNF (Brain derived neurotrophic factor), Tau and TREM2 (Triggering receptor expressed in myeloid cells 2) for which changes in expression have been implicated in AD progression. Each of the analytes was tested using the innovative SimpleStep ELISA® kits and detected with the SPECTROstar Nano microplate reader. SimpleStep ELISA® technology streamlines ELISA experiments to a semi-homogeneous format that results in a simple 90-minute, single-wash protocol (Figure 1). Combining these technologies with MARS data analysis allows you to get great results in the fastest, easiest way possible.
Materials & Methods
- SPECTROstar® Nano
- SimpleStep ELISA® kits
- Human BDNF ELISA Kit (ab212166)
- Human Tau ELISA Kit (ab210972)
- Human TREM2 ELISA Kit (ab224881)
|Product Name||Human BDNF ELISA Kit||Human Tau ELISA Kit||Human TREM2 ELISA Kit|
|Dynamic Range (pg/ml)||15.6-1000||31.25-2000||78.1-5000|
|Species Reactivity*||Hu, Ms, Rt||Hu, Rt, Bv||Hu|
|Sample type#||CS, S, HP, EP, CP||CS, S, CE, HP, EP, CP, CSF||S, CE, TE, CS, HP, EP, CP, CSF|
|Assay Time||1h 30 Min||1h 30 Min||1h 30 Min|
* CS: Cell Culture Supernatant; S: Serum; HP: Heparin Plasma; EP: EDTA Plasma; CP: Citrate Plasma; CE: Cell Culture Extracts; TE: Tissue Extracts; CSF: Cerebral Spinal Fluid. # Hu: Human; Ms: Mouse; Rt: Rat; Bv: Bovine
For all kits, samples were handled in accordance with instructions3. For all tests, standard curves were prepared based on an 8-point , 2-fold dilution series using duplicate samples. Starting concentrations were 5000, 2000 and 1000 pg/mL for TREM2, Tau and BDNF, respectively. Two different dilutions of biological replicates and blank replicates were used to determine assay reproducibility. The biological replicates were: human citrate plasma for BDNF, human brain extract for Tau and human serum for TREM2. Plates were read on a SPECTROstar Nano with the following settings:
|Optic settings||Absorbance spectrum, Endpoint test|
|Wavelength range (step width)||400-700 (2) nm|
|General settings||Number of flashes||45|
|Settling time||0.2 s|
A MARS data analysis template was used to select appropriate wavelengths from spectral data set, generate a 4-parameter ﬁt curve based on reference corrected data (OD450 – OD630), assess %CV of blanks and recalculat biological replicates, and determine MDD (minimum detectable dose) based on the formula: MDD = recalculated concentration of AvgBlank + 2*SDBlank
Results & Discussion
BDNF is a growth factor in the neurotrophin family. It is best characterized for promoting survival and maintenance of neurons, thus decreased BDNF might participate in the disrupted function of neurons associated with AD2.
|MDD ||%CV of blanks (n=15) ||%CV of bio-replicates|
|10% Plasma Citrate (n=8)||5% Plasma Citrate (n=8)|
Fig. 2: Fit curve, sensitivity and reproducibility from Human BDNF ELISA Kit (ab212166). The standard curve exhibits an R2 of 0.997 for the 4-parameter ﬁt.
Tau proteins are associated with the microtubule network in neurons and as such are implicated in maintaining the unique structure of these cells which is vital for neuronal function. Tau is the protein component of neuroﬁbrillary tangles, the most recognizable alterations associated with AD, which expression decreases with age1.
|MDD ||%CV of blanks (n=15) ||%CV of bio-replicates|
|2 ug/ml brain |
|1 ug/ |
Fig. 3: Fit curve, sensitivity and reproducibility from Human Tau SimpleStep ELISA® kit (ab210972). Standard curve exhibits great correlation to the 4-parameter ﬁt (R2 =0.997).
TREM2 is a receptor expressed exclusively by brain immune cells. Elevated levels of TREM2 is a biomarker for increased AD risk1. (Fig. 4).
|MDD||%CV of blanks (n=15)||%CV of bio-replicates|
Fig. 4: Fit curve, sensitivity and reproducibility from Human TREM2 ELISA Kit (ab224881). Standard curve exhibits excellent correlation to the 4-parameter ﬁt (R2 =0.9994)
SimpleStep ELISA® technology provides fast sample analysis with little hands-on time required. The SimpleStep ELISA® protocol only requires one washing step, providing results in just 90 minutes while delivering high sensitivity, speciﬁcity and reproducibility. Each single kit has been validated using biological samples to ensure it works the ﬁrst time with high performance standards. The combination of SimpleStep ELISA® kits, SPECTROstar Nano microplate reader and MARS data analysis templates provide further beneﬁts in performance, saving signiﬁcant time and effort. These tools offer immediate and accurate results to support current and future neuroscience research needs.
1. Bolós, M., Perea, J.R., Avila, J. Alzheimer’s disease as an inﬂ ammatory disease Biomol. Concepts (2017) 8: 37- 43
2. Fumagalli, F., Racagni, G., Riva, M.A. The expanding role of BDNF: a therapeutic target for Alzheimer’s disease? Pharmacogenomics J. (2006) 6: 8-15
3. SimpleStep ELISA® kits www.abcam.com/kits/simplestep-elisa-kits