Flouremetry/ Florescence polarization

Question 1

Fluorometry principle

when certain molecules are what

Answer 1

When certain molecules are exposed to light energy,
they may absorb it & enter an excited state

Question 2

Fluorometry

The excited energy of the molecule may give off what

Answer 2

The excited energy of the molecule may be given off in
an alternate energy form as the electrons return to their
original orbits & the molecule returns to the ground state.

Question 3

Fluorometry

If the alternate energy form is a what then

Answer 3

If the alternate energy form is a photon of light, and if
this emitted light is of lesser energy (longer
wavelength) that the impinging light, the phenomenon is
known as fluorescence

Question 4

Fluorometry

example

Answer 4

a substance that absorbs at 380 nm & emits at
460 nm is fluorescing

Question 5

Fluorometry

Only certain what are capable of fluorescence

Answer 5

Only certain compounds (fluors or flurophors) are capable of
fluorescence

Question 6

Fluorometry

is the what

Answer 6

‣quantitative measurement of the intensity of light produced
by an excited fluorescent substance
‣relationship between measured fluorescent intensity &
[substance].Fluorometry

Question 7

Fluoremetry components

Answer 7

-Usually) UV light generated by xenon, deuterium, mercury
vapor arc lamp directed onto excitation filter.
* This approximately monochromatic light focused onto sample.
* If sample is a fluorophor, emits wavelength light longer than the one it
received.
* Emission filter (at 90° angle)
* Only allows emitted light to hit detector

Question 8

Fluoremetry Components

Types of lamps

Answer 8

UV light generated by xenon, deuterium, mercury
vapor arc lamp directed onto excitation filter

Question 9

Fluoremetry components

This approximately monochromatic light is

Answer 9

This approximately monochromatic light focused onto sample

Question 10

Fluoremetry Components

If sample is a fluorophor then

what filter

only allows what to hit what

Answer 10

If sample is a fluorophor, emits l of light longer than the one it
received
* Emission filter (at 90° angle)
* Only allows emitted light to hit detector

Question 11

Direct Fluorescent Immunoassay

Answer 11

Same as ELISA Sandwich, 2° antibody fluorescent
labeled

Question 12

Indirect Fluorescent Immunoassay

Answer 12

Same as above, except measure excess unbound
antibody

Question 13

Substrate Labeled Fluorescent Immunoassay

Substrate labeled fourescent Immunoassay

Answer 13

Ag(patient) + antibodies–> Bound + free measured ag (patient)

Question 14

MEIA

Answer 14

Microparticle Enzyme Immunoassay (MEIA)

anti analyte(antigen) microparticle + sample —-> antibody-antigen complex

Antibody-antigen complex + Anti-analyte specific alk phos conjugate —> anitbody- antigen - antibody complex

Question 15

MEIA

Immune complex bound to

Answer 15

Glass fiber matrix

Question 16

Flourescent Immunoassays

MEIA

Answer 16

• Substrate: 4-methylumbelliferyl Phosphate (MUP)
• Enzyme Label: Alkaline Phosphatase (ALP)
• Reaction:
  – MUP + ALP (Ab label on sandwich complex) —> MU (fluorescent)
• Rate of production of MU is measured.

Question 17

Fluorescence Polarization Immunoassay
Reaction

Answer 17

Analyte + analyte tracker —> antibody compete for antigen binding sites

Question 18

Fluorescence Polarization Immunoassay

Answer 18

Unbound antigen tracker results in decreased polarized light

Question 19

Antigen tracker bound to antibody

Answer 19

Results in Increased polarized light