6: Color Tests, Spectrophotometry, Immunoassays Flashcards
Trinder’s Reagent for Salicylate
Tridner’s reagent: mercuric chloride, water, 1 M HCl, ferric nitrate
Purple = salicylate
FPN Test for Phenothiazines
Ferric Chloride, Perchloric Acid, Nitric Acid
Color results depend on specific phenothiazine (pink red, orange, violet, blue)
20 % NaOH + pyridine + heat
Red in pyridine layer = trichloro +
Used for chloral hydrate, chloroform
Conway Diffusion for Ethanol
Outer ring = sample
Inner ring = potassium chromate
Green = ethanol (or other reducing agent)
Paraquat/ Diquat
0.1% sodium dithionite in 1 M NaOH
Blue = paraquat
Green = diquat
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
Marsh Test for Arsenic
Covert arsenic into arsine gas and deposit it into a black film
Black = arsenic
technique that incorporates the binding reaction of a target substance (antigen) w/ an antibody
Immunoassay
In immunoassays, the drug of interest
Antigen
In immunoassays, the part of the antigen that is recognized by the immune system
Epitope
In immunoassays, part of antibody that binds with antigen
Paratope
In immunoassays, antibodies are the same as
immunoglobulions (Ig)
These are produced by animals in antiserum.
Polyclonal antibodies
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
What is produced when an animal is given antigen (hooked with protein)
Monoclonal antibodies
Type on immunoassay that allows measurement of labeled antigen w/o separating bound and free antigen
Homogeneous
When two substances (antigen and labeled antigen) compete for the same antibody binding sites
Competitive Binding
Type on immunoassay that requires separation of bound and free antigen before labeled antigen is measured
heterogeneous
*more common; think ELISA
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)
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
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
