Assay Technologies

How do you calculate percent inhibition?

((1-(experimental-min)/(max-min))x100

How do you calculate % CV?

((SD)/(mean))x 100

monoclonal antibodies recognizes how many epitopes? why?

1 because it is very specific and selective

what is a general protocol for AlphaLISA?

1. Add sample 2. Add biotinylated antibody and alphaLISA acceptor bead 3.add donor beads 4. Read plate

What are the general steps to DELFIA(Dissociation-enhanced lanthanide fluorescence immunoassay)

1. Coat well with capture Ab 2. Incubate w/analyte and Eu labeled Ab* 3. Wash away non-bound Eu/Ab 4.Add enhancer solution (chelator) 5.Measure fluorescence

How does a general PCR occur?

1. Denaturation 2. Annealing 3. Extension

3 requirements for FRET

1. Donor and acceptor molecules must be in close proximity 2. The absorption spectrum of the acceptor must overlap the fluorescence emission spectrum of the donor 3. Donor and acceptor must have appropriate orientation with respect to each other

when measuring fluorescence, what are the different state the molecule goes through in order?

1. Ground state 2. Excited state-high energy 3. Excited state-low energy 4. Ground state

Why are lanthanides ideal for TRF?

1. Large stokes shift 2. Narrow emission peaks(allows for multiplexing using other lanthanides)

What are two things that can limit sensitivity in an fluorescence assay?

1. Reagent background(unbound or nonspecific bound probes) 2. Endogenous(autofluorescence)

What are the general steps for indirect sandwich ELISA?

1.Coat plate with capture ab 2.Wash plate 3.Add blocking 4.Wash 5.Add test solution 6.Wash 7.Add detection antibody 8.Wash 9.Add enzyme labeled anti-antibody or streptavidin 10.Wash 11.Add substrate - stop or read within a time range 12.Detect in microplate reader

2 types of FRET

1.Donor-Acceptor: causes fluorescence of acceptor 2. Donor-Quencher: only quenches donor

How many fluorescence intensity readings are needed for a FP value calculation?

2

How do you calculate Fold difference?

2^(-∆∆CT)

In Alphascreen what is the wavelength for the excitation light?

680nm

How do you calculate ∆CT ?

=Gene of interest ct- Control gene ct

Define Analyte

A substance that is determined in an analytical procedure

In a protein, what cause it to peak at 280nm when measuring for absorbance?

Amino acids with aromatic rings

In FP what kind of light is exciting the fluorophore? A. Regular light B. Polarized light C. non polarized light

B. Polarized light(By using a polarization filter)

what does Streptavidin have an high affinity for?

Biotin

What are the two types of epitopes?

Conformational and Linear

Relative QPCR compares the _______ value of one target gene to another

Ct value

What are some advantages to FP? A.Homogeneous B.Relatively simple to develop C.Competitive FPIAs are very sensitive D.Independent of fluorophore concentration E. all of the above

E. all of the above

how do you measure stoke shifts?

Emission max - Excitation max

For FRET to work the ____ of the donor must overlap with ___ acceptor

Emission wavelength Excitation wavelength

What does ELISA stand for?

Enzyme-Linked Immunosorbent Assay

What phase does the product double in Q-PCR?

Exponential phase

What is a type of instrument used to measure fluorescence? A. spectrofluorometers B. Microplate readers C. fluorescence microscopes D. Flow cytometers E. a and c F. all of the above

F. all of the above

True or False: PCR can not detect a single molecule of DNA

False, PCR can detect a single molecule of DNA, it is highly sensitive

True or false: In flow cytometry more than one cell is dropped at a time.

False, only one cell is dropped at a time

True or False: Donor fluorescence increases while acceptor decreases, unless the acceptor is a non-fluorescent quencher

False. Donor fluorescence decreases while acceptor increases, unless acceptor is a non-fluorescent quencher

True or false: Decreasing the concentration of the fluorophore in assay will reduce autofluorescence.

False. Increasing the concentration of the fluorophore will reduce autofluorescence not not always a desirable option

True or false: most antigens have one epitope that only recognizes one antibody

False:most antigens have many different epitopes that can be recognized by different antibodies

If a molecule has a low molecular weight, how does it rotate in solution when excited? and does it give high or low polarization?

Fast and low polarization

What does FRET stand for?

Fluorescence Resonance Energy Transfer

How are hybridomas made?

Fused immortal myeloma cells with antibody-producingB-lymphocytes

What protein is used as fluorophore?

GFP-Green Fluorescence Protein

Tags are added the ____ level.

Gene

Side scatter (SSC) measures

Granularity and structure complexity

What are the common enzyme label used in ELISAs?

HRP:Horseradish peroxidase AP: Alkaline Phosphatase

If a fluorophore that is linked to a protein becomes bound to its receptor and it excited, what we the FP reading be?

High polarization or High mp

what is the max or positive control?

Highest signal or a signal the indicated that the assay worked

What method is used help the cells to flow one at a time?

Hydrodynamic focusing-flowing stream of fluid

FP value is ___________ of fluor concentration Dependent or Independent

Independent because FP value is a ratio

Which common type of fluorophore is used for TRF and list some examples?

Lanthanides Europium(Eu) Terbium(Tb)

Describe a linear epitope

Linear sequence of amino acids (Continuous)

If a fluorophore that is linked to a protein does not bind to its receptor and it excited, what we the FP reading be?

Low polarization because there was no weight change and the fluorphore compound is moving rapidly in solution

How do you calculate assay window?

Max-Min

If you are trying to capture antibody that has His-Tag, what would you use to coat the plate?

Nickel Chelate or anti-his tag

If you denature an antigen that has a conformational epitope, will the antibody be able to bind to it? Yes or no, and explain your answer

No because the antigen has been denature/unfolded than the conformational epitope would not be together and the antibody will not be able to recognize the spot to bind

If you decrease the fluorophore concentration from 1000 to 100, does the FP value change.?why?

No, because the concentration is independent of the FP Value

In a protein, what cause it to peak at 200nm when measuring for absorbance?

Peptide bonds

How are polyclonal antibodies made and collected?

Produced by injecting animals multiple times with antigen and collection serum

In alpha screen what color laser is used to excite the sample?

Red laser

what are the two methods of quantification using ELISA?

Sandwich(indirect and direct)and Competitive

If a molecule has a high molecular weight, how does it rotate in solution when excited? and does it give high or low polarization?

Slow rotation and high polarization

Define affinity

The strength of the interaction between a single site of an antibody and a single epitope

Why are polyclonal antibodies non-identical?

They were produced by different B-Cell

Why are monoclonal antibodies identical?

They were produced by one type of B-cell

Why is there a blocking method when running an ELISA?

To reduce background, to lower non-specific plastic binding.

In Fluorescence Polarization Immunoassay(FPIA) the fluorophore is used as a what?

Tracer

True or false: AlphaLISA can cover a range of sizes for example: Small molecules like cAMP and Big molecules like higG

True

True or false: FP is roughly proportional to apparent molecular weight

True

True or false: In QPCR fluorescence is proportional to the amount of amplified DNA

True

True or false: Polyclonal Abs epitope recognition between animals varies

True

True or false: Polyclonal Abs recognize multiple epitopes

True

True or false: SD provides the absolute variation in the units while %CV gives relative measure of variability

True

True or false: Shifting excitation/emission filter or wavelengths can reduce autofluorescence.

True

True or false: When the animal dies so does their supply of Polyclonal Abs

True

True or false: with FRET, you get Em from both donor and acceptor fluorophore

True

True or false:•%CV of ≤ 5% is generally considered excellent

True

What is absolute Quantitation used for?

Used to quantitate unknown samples by getting their quantity from a standard curve.

what state does a fluorophore emit light? When do you measure for fluorescence?

When the low energy excited state goes back to ground state is when the fluorophore emits light and you measure.

If you denature an antigen that has a linear epitope, will the antibody be able to bind to it? Yes or no, and explain your answer

Yes because the epitope is not depended on how the protein is folded.

The one main requirement for TRF is...

a fluorophore with a long fluorescence half-life

Define quencher

a non-fluorescent molecule that accepts energy from a fluorophore that is in close proximity but does not re-emit the energy as light

Define assay

a procedure where the activity, property or concentration of an analyte is measured

Define antigen

a substance the elicits and antibody response

what z-factor range is an assay considered excellent? a. less than 0 b. 0-0.5 c. 0.5-1.0

c. 0.5-1.0

Forward scatter (FSC) measures

cell size

What is a reason DNA no longer doubles in the linear and plateau?

depletion of PCR reagents

True or false: Polyclonal Abs are very expensive

false they are inexpensive

True or False: Antigen and antibody interaction are irreversible

false: Since antigen-antibody reactions occur via non-covalent bonds, they are by their nature reversible

If you are trying to capture antibody that has GST Tag, what would you use to coat the plate?

glutathione or anti-GST

what type of non covalent bonds hold antigens to the antibodies?

hydrogen electrostatic Van der Waals Hydrophobic

Define epitope

is the part of an antigen that an antibody binds too.

What is the min or negative control/background?

lowest signal in the assay

Hybridomas produce:

monoclonal antibodies

Define avidity

more than one binding site

Describe a conformational epitope

non-continuous amino acids only together when the protein is properly folded

why is protein A used to isolate Abs?

protein A has an affinity for many different Abs.

How do you calculate signal to background?

ratio of mean signals for max and min

Define multiplex

the detection of more than one analyte from a single sample

What does fluorescence polarization measure?

the rate of rotation of a fluorophore in solution

Always remember that in FP, the fluorophore should be attached too_____?

the small molecule

If droplet has more than one cell or has no cells at all where does that drop go?

the waste

True or false: TRF is one way to overcome autofluorescence

true

what is relative quantitation used for?

used to analyze changes in gene expression in a given sample relative to another reference sample(such as an untreated control sample)

If the animal dies that was used to help make the Hybridoma, are you still able to produce more of that particular monoclonal antibody?

yes, hybridomas are immortal in nature. They can be grown, frozen, thawed and re-cultured

How do you calculate ∆∆CT?

∆CT Drug (1, 2, or3)- ∆CT of untreated/control/reference