Class 3 Ab + L AbL

Reviewd/dt [AbL] = kon [Ab][L] – koff [AbL] koff [1/s], kon [1/Ms], koff / kon = KD [M] why the lower the KD the tighter the binding?typical values for KD, kon, koff for Ab binding L

fx X binding Y @ [YT]/KD /(1 + [YT]/KD) when Y in excessD [m2/s], t~distance2/6D (in 3-d),Dg=kBT (4*10-21J at room temp), g=6phrflux j [#/m2s] = D Dc Fick’s law

New Single molecule ELISA – why miniaturization helps step-by-step binding equilibria and kinetics specificity and backgroundImmuno-chromatography and intro to pcr

kon

koff

Single-molecule ELISA assay Nat Biotech 28, 598 (2010)

Why does miniaturization help?

Enzyme catalytic rate kcat ~100 substrate molecules/sper enzyme molecule

Need threshold conc of dye ~100nM to see it the smaller the vol, the higher the conc how many molecules/50fl for 100nM? how many can one enz. molecule make in 30s? what are dimensions of 50fl volume

fluorescence = advantage =

emit longer wavelength than absorbedcan block bright excitation lightthat would otherwise -> signal

6*1023-7-15*50=3*103

[50x10-15*10-3m3 ] @ (4mm)3

Why measure PSA?

Why might it beuseful to measure[PSA] after prost-x?

Why might it notbe useful?

Sensitivity of SiMoA

LOD std assay

Nat Biotech 28, 598 (2010)

Fig. 2A J Immunol Methods 378:102 (2012)

[AbL]2 - [AbL] {[LT] + [AbT] + KD} + [LT] [AbT] = 0

or, since Ab in excess, [AbL]/[LT] @ [AbT]/KD / (1 + [AbT]/KD)

given [AbT] @ 2nM

Fig.3 J Immunol Methods 378:102 (2012)

Is Ab or L in excess? Then t @ koff-1 / (1 + [AbT]/KD)

Simplify [AbT]/KD Do results agree with figure?Are reactions B, C near equilibrium at 1000s? *

What is equil [AbL]/[LT]? Does this agree with * ?

The one in excess

Fig. 4 J Immunol Methods 378:102 (2012)

What step of rxn is this modeling? Why so flat?

Then fx AbL in complex @ [Det AbT]/KD / (1 + [Det AbT]/KD) independent of [AbL]

assumes KD of interaction = 1nM

For which symbols is detection Ab in excess?

Fig. 5 J Immunol Methods 378:102 (2012)

What step is this modeling?What is the KD of this interaction?What is in excess? What is formula? Does this

explain why almost all the points are 100%?

Specificity and background

Background can come from non-tgt protein in sampleor signal-generating species (e.g. enzyme)

How many specific binding steps are there in label-freeassay, like SPR?

Suppose capture Ab binds tgt protein 107x more tightly(in terms of KD) than it binds high conc proteinlike albumin in biological sample [alb]plasma @ mM

What fx of capture Ab binds albumin? KD @ 10-9+7

, so [alb]/KD / (1 + [alb]/KD) @ 0.1

What would this do in SPR assay?

What would happen in sandwich assay?

What would be [alb] after it is captured(wrongly) by capture Ab?

If detection Ab also binds alb with mM affinity,what fraction of capture Ab-alb complexesbind detection Ab?

If capture Ab is nM in device , [cap Ab-alb] ~0.1nM

If [det Ab] = nM, it is in excess over bound albumin, so f @ [det Ab]/KD /(1 + [det Ab]/KD) = 10-7

so 1011 capture Ab to start would -> 104 det Ab from alb

This is likely << threshold for signal in standard ELISA, but would -> signal in SiMoA

Background in SiMoA was ~1% of beads

Pcr is similar to sandwich immunoassays in that2 specific binding events are requiredfor signal (exponential amplification of tgt DNA)

Biology often uses binding of multiple proteins to increase specificity of reactions

Lateral flow immunochromatography assays

Common format for home tests (e.g. HCG - pregnancy)and now many medical lab tests

immobil. capt. Ab Neg control

Det Ab on membrane but not attached;gets picked up and carried along by sample

Questions – how many gold particles at what density for easily visible line? why are they visible – ? just concentration or something special about plasmon resonance, which gives them color

Flipped left-right from schematic; don’t know why C and T are diff. colors

Polymerase chain reaction = method to replicate pieceof DNA “exponentially”

1. DNA polymeraseN

strands are“anti-parallel”

adds bases to 3’-end

pol requires primer to start synthesis

primertemplate

Short pieces of DNA (“oligo”nucleotides ~1-100 bases) can be chemically synthesizedN , commercially available for <$1/base for 100nmol = 6x1016 molecules, serve as “primers” to start DNA synthesis at particular place on DNA template

Pcr amplification of DNA using thermostable DNA polymerase (e.g. Taq pol)

forward primer

templatestrand (A)

copy strand (B)

Taq pol

Melt DNA (94oC), cool (60oC) to anneal primers, extend (72oC)

reverse primernew strand A

Old and new templates are not destroyed by melting, so repeated cycles of melting and polymerization ->

1 -> 2 -> 4 -> 8 -> … -> 2n copies of DNA region lyingbetween 2 primer-annealing sites on initial template

Polymerase copies ~1000b/min => ~1 hour for 230 =1010-fold amp. of a kb piece of DNA

http://www.youtube.com/watch?v=_YgXcJ4n-kQ

http://www.dnalc.org/ddnalc/resources/animations.html

Note target DNA seq., to ends of which oligos bind in right orientation, is amplified exponentially 2N, (N=#cycles)

Non-tgt DNA, to which oligos may bind weakly, may becopied but only increases arithmetically ~N

Different from signal amplification, e.g. use of enzymesthat generate multiple fluors or dyes from eachbound ligand in ELISA; here you make multiple copies of DNA ligand if there is 1 present to start with

Next week – first homework due

Will begin paper that considerseffects of flow and diffusion in sensors that use microfluidics – seeBlackboard

Considers “simple” revisions to model of bindingkinetics when analyte concentrationscan’t be considered spatially uniform