6: Color Tests, Spectrophotometry, Immunoassays Flashcards by Mariela Torres

Trinder’s Reagent for Salicylate

Tridner’s reagent: mercuric chloride, water, 1 M HCl, ferric nitrate

Purple = salicylate

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FPN Test for Phenothiazines

Ferric Chloride, Perchloric Acid, Nitric Acid

Color results depend on specific phenothiazine (pink red, orange, violet, blue)

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20 % NaOH + pyridine + heat

Red in pyridine layer = trichloro +

Used for chloral hydrate, chloroform

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Conway Diffusion for Ethanol

Outer ring = sample
Inner ring = potassium chromate

Green = ethanol (or other reducing agent)

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Paraquat/ Diquat

0.1% sodium dithionite in 1 M NaOH

Blue = paraquat
Green = diquat

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Reinsch test for Heavy Metals

Copper wire + acidified and heated sample matrix result in black or silvery deposits/staining

Dull black = arsenic
purple black = antimony
shiny black = bismuth
silver =mercury

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Marsh Test for Arsenic

Covert arsenic into arsine gas and deposit it into a black film

Black = arsenic

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technique that incorporates the binding reaction of a target substance (antigen) w/ an antibody

Immunoassay

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In immunoassays, the drug of interest

Antigen

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In immunoassays, the part of the antigen that is recognized by the immune system

Epitope

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In immunoassays, part of antibody that binds with antigen

Paratope

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In immunoassays, antibodies are the same as

immunoglobulions (Ig)

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These are produced by animals in antiserum.

Polyclonal antibodies

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the concentration of an antibody as determined by finding the highest dilution at which it is still able to cause agglutination of the antigen

Titer

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What is produced when an animal is given antigen (hooked with protein)

Monoclonal antibodies

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Type on immunoassay that allows measurement of labeled antigen w/o separating bound and free antigen

Homogeneous

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When two substances (antigen and labeled antigen) compete for the same antibody binding sites

Competitive Binding

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Type on immunoassay that requires separation of bound and free antigen before labeled antigen is measured

heterogeneous

*more common; think ELISA

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Homogenous Immunoassay technique

-When labeled drug is bound to antibody, enzyme is inactive
-When enzyme is active (unbound bc specimen had antigens that bound) NAD is converted to NADH (measured at 340 nm)

Enzyme Multiplied Immunoassay Technique (EMIT)

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Homogenous Immunoassay technique

When Fluorescein-linked drug is bound to antibody, the fluorescein label does not rotate freely and the absorbed polarized light is emitted; when the label is free, it rotates in solution and the amount of emitted light is reduced

Fluorescence Polarization Immunoassay (FPIA)

*Think Fluorescein, polarized light = FP

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Homogenous Immunoassay Technique

-Genetically engineered fragments of e-coli as enzyme label; activity of the enzyme requires an enzyme acceptor and an enzyme donor

-Reassociated enzyme hydrolyzes CPRG to CPR and galactose; CPRG does not absorb at 570 nm, but CPR does

-If a drug is found in the donor’s sample, the unbound Enzyme Donor part reassembles and reacts with the substrate and causes a change in the color absorbance.

Cloned Enzyme Donor Immunoassay (CEDIA)

*Think e-coli = C E

Homogenous Immunoassay Technique

-Label = microparticle w/ several linked drugs
-No drugs present: microparticle binds multiple antibodies to form large aggregates (increase absorbance w/ time)

Kinetic Interaction of Microparticle in Solution (KIMS)

*Think: microparticle = aggregate; microparticle = M

Heterogeneous Immunoassay Technique

Antibody coated wells; add analyte and labeled analyte, incubate, wash material, add substrate, develop color, add stop acid, measure color

Enzyme Linked Immunosorbent Assay (ELISA)

Quants antigens between two layers of antibodies (capture and detection); antigen must have at least two epitopes capable of binding

Sandwich ELISA

excessive antigen interferes with ability of detection antibodies to bind (resulting in reduced signal)

Hook Effect

the degree of response in an immunoassay to a substance other than the analyte of interest

Cross-reactivity % cross reactivity = (concentration reading of assay analyte)/(concentration of cross-reactivity analyte) x 100

measures ability of test to correctly ID the positive cases

Sensitivity *think opposite; positive = seNsitivity Sensitivity (%)=(true positive)/((true positive+false negative)) x 100

measures ability of test to correctly ID negative cases

Specificity *think opposite; negative = sPecificity Specificity (%)=(true negative)/((true negative+false positive)) x 100

the study of absorption and emission of light by matter as a function of wavelength

Spectroscopy

method to measure how much a chemical substance absorb light; done so by measuring the intensity of light as a beam passes thru a sample

Spectrophotometry *think reading ELISA plates

distance between two successive peaks

wavelength (λ)

the number of peaks passing through a point in a second

frequency (v)

range of light wavelengths

Shorter wavelength, higher frequency UV 10-380 Visible 380-780 IR 780 - 300,000 Microwave 300,000 - 1,000,000,000 longer wavelength, lower frequency

change in spectral band position in the absorption spectrum of a molecule to a longer wavelength (lower frequency); red shift

Bathachromic shift *think batha = bad = red long = bad = red

change in spectral band position in the absorption spectrum of a molecule to a shorter wavelength (higher frequency); blue shift

Hypsochromic shift

for a defined path length (b) the transmitted intensity (I) decreases exponentially with the increase in concentration of the solution (c)

Beer's Law

at a given concentration (c) the transmitted intensity of light (I) decreases exponentially with increased path length (b)

Lambert's Law *think transmission decreases with length = L = lambert

kcb= log⁡ of Io/I k = molar absorptivity c = concentration b = path length l = transmitted intensity

Beer-Lambert Law (measuring absorbance)

In spectrophotometry, a device based on separating capability of refraction (prism) or diffraction (diffraction grating)

Monochromater

Type of detector used in spectrophotometry consists of photo emissive cathode, several dynodes, and an anode involves multiplication of electrons that are ejected from photons that are then measured as an amplified current

Photomultiplier tube

Type of detector used in spectrophotometry can obtain info over wide range of wavelengths at one time

Photodiode Array (DAD):

A low molecular weight substance that can induce an immune response when coupled to high molecular weight immunogenic molecules.

Hapten

True/False: A hapten will not stimulate an immune response

True (must be coupled)

The portion of the antibody that contains the peptide sequences that form the antigen binding site.

the FAB region

Hapten + Immunogenic Molecule =

Immunogen

A substance that stimulates an animal lymphocyte to produce an antibody that specifically binds to it

Antigen

True or False: The Hook Effect can produce a false positive or an inaccurately high result.

True

True or False: In an EMIT immunoassay, more drug = increased NADH production.

True *Think homogenous so result is proportional to amount of drug in sample

This can produce false positive FPIA results. Blood proteins Iron bound to hemoglobin Bile salts Lipids Amines

Bile Salts

True or False: High concentrations of diphenhydramine cause false-positive results in some urine PCP assays.

True

90% of Ig in the serum is: IgM IgG IgD IgE IgA

IgG In normal serum, about 80% is IgG, 15% is IgA, 5% is IgM, 0.2% is IgD and a trace is IgE.

The methamphetamine antibody binds methamphetamine in a sample more strongly than the labeled amphetamine. This is an example of: Competitive binding Non-cooperative binding Cooperative binding Non-competitive binding

Cooperative Binding