Biomarkers
A
B
10,000,000 1,000,000 100,000 10,000 1,000 100
LDL = 29.4pg/mL
ation system, allows for sensitivity. ELISA assays can detect protein in complex matrices like serum, urine, saliva or plasma often down to the picogram per mil- lilitre range, and numerous commercial kits are avail- able with various degrees of validation. The detec- tion method can involve colorimetric or fluorescence measurement, with fluorescent readouts generally exhibiting increased assay sensitivity. In addition, a horseradish peroxidase activated tyramine signal amplification system, TSA ELAST® (PerkinElmer, Inc) has been shown to increase sensitivity further in traditional ELISA format. The utility of various lan- thanide chelates as fluorophores (commercially avail- able in DELFIA® products from PerkinElmer, Inc) offers the ability to run ELISA assays in time- resolved mode, which further reduces potential inter- ference due to the prompt fluorescence of many bio- logical matrix components.
-∞ -12 -11 -10 -9 -8 -7 Log (human Adiponectin) (g/mL) -6 -5 Figure 2
A) Generic assay configuration of an AlphaLISA assay for easy quantification of biomarkers. B) Standard curve for quantification of adiponectin obtained using AlphaLISA, a non-wash assay. The assay can detect as little as 29pg/mL of total adiponectin in serum
methodologies combined with adequate data han- dling and analysis can lead to the discovery of inter- esting unbiased biomarkers in either biological flu- ids10 or in cell-based models11. Whether biomark- ers are identified using unbiased strategies or select- ed based on a priori insights of signalling pathways, it is subsequently critical to develop rapid and accu- rate assay protocols that will allow their measure- ment on multiple samples in a broad range of matrices using proven, easy and reliable techniques.
ELISA and other antibody-based approaches
Sandwich ELISA is the gold standard in the quantifi- cation of protein biomarkers for research applica- tion. It is an easy and sensitive method relying on the use of two specific antibodies to the protein of inter- est. A microplate-bound capture antibody confers specificity and allows for washing to remove inter- fering matrix components, while a detection anti- body coupled directly or indirectly to a signal gener-
18
Although considered as a gold-standard in research and analytical laboratories, ELISA suffers from many disadvantages. It can require a large amount of input sample, depending on detection technology used. Moreover, the numerous wash steps make it difficult to automate and labour intensive. These large numbers of manipulations make ELISA prone to variability due to minor dif- ference in procedure and skills between operators. A method similar to ELISA, but using electro- chemiluminescence detection methodology is avail- able from MesoScale Discovery. This technology requires a unique ruthenium-chelate label on the detection antibody which is read via integrated electrodes contained in proprietary microplates. By spotting discrete areas of a well, up to 9-plex ana- lytes can be simultaneously measured, reducing the sample requirement. Application of the Mesoscale platform was recently described in a multicentre study to monitor Alzheimer’s biomarkers in cere- brospinal fluid12.
An alternative to microtiter plate ELISA-type assays is bead-based immunodetection technology using flow cytometry methods. Examples of this approach are the BD™ Cytometric Bead Array from BD Biosciences and Luminex Corp. xMAP® platform, reagents for which are available from various suppliers. The xMAP® system uses 5.6µm polystyrene internally dyed beads coated by specif- ic antibodies to a given analyte. Labelling with two distinct dyes at various ratios allows for the cre- ation of 100 uniquely identified beads that can be individually monitored by standard flow cytome- try principles in a dedicated detection instrument. While this allows for potential theoretical multi- plexing of up to 100 analytes, rarely more than 15 analytes are validated in single panels due to the
Drug Discovery World Summer 2010
AlphaLISA signal (counts)
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84 |
Page 85 |
Page 86 |
Page 87 |
Page 88 |
Page 89 |
Page 90 |
Page 91 |
Page 92