ELISA / AlphaLISA / PCR / SPR / DNA Synthesis / Cell Signaling Assays

The majority of assays are sandwich-based and consist of a Multi-Spot microplate, where each spot is coated with unique capture antibodies. After incubation with the sample, binding of analytes are detected via SULFO-TAG conjugated antibodies. When electricity is applied to the plate, light is emitted from the SULFO-TAG, and intensity measured for quantification of analytes in the sample.

V-PLEX (Validated assays)	U-PLEX (Custom multiplex of assays)	R-PLEX (Custom assay design)	S-PLEX (Extra sensitivity assays)
V-PLEX assays are designed to maximize reproducible performance, consistency and reliability in results and confidence in data. The assays and panels are fully validated in multiple matrices according to fit-for-purpose principles. The validated assays come in both separate assays or multiplex variants and are designed to ensure optimal and consistent performance. Lot-to-lot consistency is guaranteed to support long-term studies.	The U-PLEX assays offer full flexibility to custom design your own singleplex or multiplex assays. In the U-PLEX assays biotinylated capture antibodies are coupled to U-PLEX linkers, which bind to specific spots on the plate. Besides antibodies, the linkers can be used with other biotinylated capture-reagents such as proteins, peptides, or nucleic acids, allowing you to study the desired proteins unique for your project.	The R-PLEX antibody sets can also be used for development of multiplex or single analyte immunoassays. Each R-PLEX antibody set contains a biotinylated capture antibody, electrochemiluminescence labelled detection antibody and a recombinant protein for standard.	S-PLEX assays can be used for detection of very low concentrations of protein. An optimized detection antibody increases the sensitivity of the assays, with a detection limit down to the low fg/m,

ELISA vs WB

		WB	ELISA
Common	In principle, WB (immunoblot) and ELISA are identical systems
difference	Solid phase	nitrocellulose membrane	polystyrene [PS] in ELISA
	Readout	optical density	Visual interpretation
	sensitivity		↑
	precise		↑
	easy		Better for large number of samples
		Semiquantitative, There is a quantitative WB: {Taylor, 2014 #2066} Marianthi: Paul will be generating a standard curve (like with ELISA) and we will get absolute numbers. , but Kathy: this is relative-absolute	quantitative
	linearity	Narrow

AlphaLISA

• a biotinylated antibody + an antibody-conjugated AlphaLISA Acceptor bead (to capture) + Donor bead
• Upon excitation, a photosensitizer inside the Donor bead converts ambient oxygen to an excited singlet state. Singlet oxygen diffuses up to 200 nm to produce a chemiluminescent reaction in the Acceptor bead, leading to light emission

Figure labels (AlphaLISA mechanism diagram, evidence-only):

Excitation 680 nm
Emission 615 nm
Streptavidin-coated Alpha Donor Bead
Biotinylated Anti-analyte
Analyte
Anti-Analyte Conjugated AlphaLISA Acceptor Bead

PCR

• Method
• Terminology (Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines)

	RT-PCR	qPCR	RT-qPCR'
Meaning	reverse transcription PCR	quantitative real-time PCR	reverse transcription quantitative real-time PCR.
But commonly used to			to describe RT-qPCR.
Steps	RNA is isolated → Using the reverse transcriptase enzyme, a single-stranded copy of cDNA is generated. → amplified by a DNA polymerase, → generating ds- cDNA, feeding into a standard PCR-based amplification process	DNA is isolated → PCR amplification of DNA in real time, measured by a fluorescent probe, most commonly an intercalating dye or a hydrolysis-based probe, enabling quantitation of the PCR product	RNA is isolated → cDNA generated → qPCR procedure
Applications	- molecular cloning of genes of interest (GOIs), - but most commonly, it serves as first step in RT-qPCR	- to detect the presence of pathogens and - to determine the copy number of DNA sequences of interest	- to measure RNA levels (RNA-Seq)
Sample preparation	-	- extraction is carried out using commercially available kits,	most common extraction method used is with total RNA extraction kits (RNeasy). This isolates mRNA, rRNA, ribosomal RNA, But often no smaller RNAs 9eg. non-coding RNA, miRNA) → quality control to check degradation: RIN (RNA integrity number, 10 maximum, >7.0필요) → cDNA generation: using oligo (dT) primers
Detection	-	- Basically fluorescence method	i) fluorescence method: using a fluorescent dye that binds to dsDNA ii) HYDROLYSIS PROBES (eg TaqMan): DNA polymerase extends the primer, the probe is cleaved

SPR (surface Plasmon Resonance)

DNA Synthesis assays

	bromodeoxyuridine (BrdU),	EdU (5-ethynyl-2'-deoxyuridine)	[3H] thymidine
	thymidine analog	thymidine nucleoside analog
	BrdU is incorporated into newlysynthesized DNA strands of actively proliferating cells.
detection method	Antibody-based, incorporated BrdU is detected immunochemically by anti-BrdU antibody.	Non-Antibody-based, Fluorophore-labeled azide reacts with the incorporated EdU to allow detection by microscopy or flow cytometry.
disadvantage	The majordisadvantage of the BrdU method is that BrdU antibodies will only react with single stranded DNAsince double-stranded DNA blocks the access of anti-BrdU antibody to incorporated BrdU molecules		slow, labor intensive andhas several limitations including the handling and disposal of radioisotopes, as well as the need forexpensive equipment.
advantage		therefore do not require DNA denaturation for detection of the incorporated nucleosideis compatible with flow cytometry &microscopy.

Cell Signaling Assay

assay	company			Applications
FLIPR (Fluorescene Imaging Plate Reader)	Molecular Devices'
FDSS (Functional Drug Screening System),	Hamamatsu	its imaging-based plate reader, high-throughput, cell-bases assay, a CCD camera-based plate reader that uses xenon lamp excitation and a variety of excitation and emission filters for imaging multiple fluorophores, and, in some cases, for performing FRET-based assays. It can accommodate 96- or 384- well plates	• voltage-sensitive fluorescent dyes or fluorescent indicaors for different ions • BRET (bioluminescence resonance energy transfer), FRET	GPCR, calcium, cAMP, ion channels, where it is limited to calcium assays."

Qualitative / Quantitative / Semi-quantitative

	qualitative	quantitative	Semi-quantitative
definition	the detection or identification of the constituent elements in the sample	accurate determination of their concentrations	estimation of their approximate concentrations,

Uncertain Spans

location	text/status	reason
ELISA vs WB header	only WB and ELISA headers visible; intermediate columns are blank	The grid has empty narrow columns between the two product columns; treated as layout spacing rather than content.
Cell Signaling Assay header	two unlabelled middle columns between company and Applications	Header cells are blank in the visible source; columns are kept as empty <th></th> in the table.
FDSS Applications cell	trailing punctuation assays."	A close-quote is visible without a matching open-quote; preserved as written.
Sample preparation row (RT-qPCR column)	non-coding RNA, miRNA) 9eg.	The opening parenthesis appears to be misrendered in the source as 9eg. instead of (eg.; the 9 may be a display artefact but is preserved as printed.