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BioMedChem M2 > 16/17 - Immunoassays > Flashcards

16/17 - Immunoassays Flashcards

1
Q

For a DIRECT assay

Interference causes a ________ in Signal

A

Direct = linear response to signal with analyte

DECREASE

Less analyte would be bound, so less signal would be shown

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2
Q

Adv & Disadvantages of

Immunometric Elisa

A

VERY PRECISE / ROBUST / SENSITIVE

neg:

Analyte / Antigen** has to be **>6000da MW

to have 2 antigenic sites (haptens)

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3
Q

Cross Reactivity

A

Response by antibody to substances

OTHER THAN THE ANYLYTE

similar in structure / family member

larger antigens = folding patterns / AA sequences

POLYCLONAL ANTIBODIES

main culprit

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4
Q

Causes for INTERFERENCE

A

not from cross reactivity

  • Detection System
    • wrong substances giving the signal
  • Seperation Issues
    • ​incomplete linkage / poor linkages of AB-enzyme/antigen
  • Variation over batches = Heterogenesity
  • Alteration
    • ​of AB / Enzyme / Ag
  • Displacement
    • of ​analyte by other biochemical substances (hormones / FAs)
  • Blockage
    • ​of analyte by binding proteins (albumin etc)
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5
Q

Most Important Antibody Properties

A

STERIC ALIGNMENT

NON-Covalent Interactions

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6
Q

What type of Assay?

A

INDIRECT Assay

Competitive Immunoassay

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7
Q

Immunometric Assay Steps

Heterogeneous Assay

#ABs & What is Labeled?

A

DIRECT ASSAY

  • 2 ANTIBODIES
    • Unlabeled AB bound to SOLID phase
      • ​recognizes 1 of the anylyte binding sites
    • LABELED AB is free
      • recognizes OTHER binding site
  • 1 Antigen = Analyte
    • with 2 different binding sites
  • ​we measure the labeled AB which in turn measures the
    • Analyte Concentration
      • ​POSITIVE expenential curve,
        • more analyte = greater signal
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8
Q

EMIT

Enzyme Multiplied Immunoassay Technique

A

HOMOgeneous assay that

Uses ENZYME

that has covalently attached to drug of interest

measure the amount of Free Drug in sample

does not use radioisotopes, uses colored product

Used for:

Assaying THERAPEUTIC DRUGS

URINE TESTS

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9
Q

Clinical uses of Western Blots

A

Detection of VIRAL PROTEINS

Hep B / C

HIV

lyme disease / prion disesase

athletic doping w / erythropoietin

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10
Q

How Pregnancy Tests work

A

We detect for HCG in URINE(human chorionic gonadoropin)

uses Monoclonal Antibodies

  • 2 Antibodies
    • 1* AB = NOT immobilized
      • ​binds to HCG, and has DYE
    • 2* AB = immobilized
      • ​downstream of 1*, also binds HCG
        • binds the HCG + 1* dye complex
  • 2* AB retains the complex of DYED 1*AB + HCG
    • –> forms 1st blue line = pregnant
      • 2nd blue line = control
        • ​simply binds to the 1* AB no matter what
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11
Q

MONOclonal Antibody

A

MonoSPECIFIC AB that is

produced by a single plasma cell

or single CLONE of plasma cells

  • ADV:
    • Identical protein / same IG Class (usually IgG)
    • highly specific –> recognize same epitope
    • reduces: cross-reactivty
    • UNENDING Supply
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12
Q

Low MW molecule/compound

that is not immunogenic itself

It still can bind to the Antibody, but it does not induce a response

Becomes immunogenic & induces an antibody response

after conjugation to a larger carrier/protein

A

Hapten

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13
Q

Immunometric ELISA

What Antibodies / Antigens?

What is Measured?

A

“Sandwich” / DIRECT

  • 2 AB’s
    • 1 is bound to solid phase
    • 2* AB is enzyme-labeled
      • enzyme reacts with the substrate
        • –> product/color development is MEASURED
  • 1 substrate
    • ​that reacts with the enzyme
  • 1 Hapten
    • with 2 binding sites for each AB
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14
Q

Any FOREIGN material specificallly bound by:

_antibody or lymphocyte_s;

does not have to induce an immune response

A

Antigen

Ag

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15
Q

IRMA

A

ImmunoRadioMetric Assay

uses a Radioisotope Label on the

SECOND Antibody (not Ag like RIA)

easier to label AB vs Ag

Direct & Indirect Varients

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16
Q

Precipitation/Agglutination Assays

Types / Uses

A

2 Ag binding sites = double ended AB

+

Soluble Antigen (ag) or Epitope

  • Various Types:
    • Precipitin Assay
    • Blood Typing / Coombs Test
    • Hemagglutination assay for HIV
    • Nephelometry/turbidimetry
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17
Q

Flexible Hinge

A

Hinge + 2 Ag-binding sites (Fv)

can ROTATE along various axis

Allow for CROSS-LINKING

of 2 different bacterium or virus

Allow for the most important antibody property:

STERIC AIGNMENT

& Non-covalent interactions

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18
Q

Which ImmunoAssays use ENZYMES?

A

EMIT

Enzyme Multiplied Immunoassay Technique

typically uses G6P-DH

ELISA

Enzyme-Linked ImmunoSorbent Assay

4 formats

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19
Q

Iodine Radioisotopes

A

125I

131I

attach to Tyr on proteins/peptides

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20
Q

HETEROgeneous mixture of ABs

with diverse affinities/regions towards an ANTIGEN

produced by a LARGE # of plasma cell clones

polyclonal response -> CROSS-REACTIVITY

Ex. Whale Myoglobin

from various Pharma Farms: goats/sheeps/cows

A

POLYclonal Antibody

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21
Q

Hemagglutination Assay for HIV Antibodies

A

2 Antibodies –> Sandwich

  • 1* HIV AB
    • AB for HIV (produced by the BODY if you had HIV)
  • 2* AB
    • Ytip Fab Region = anti-RBC = binds to RBC
    • Fc region has HIV-ANTIGEN
  • 2*AB will bind all over the ​RBC’s
  • ​​If 1* HIV Antibody is present
    • –> will bind to HIV Antigen on 2* AB
      • –> Cause AGGLUTINATION!
        • ​= POSITIVE
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22
Q

How are BLOOD TYPES determined by immunoassays?

COOMBS Test

A

More Effective than Agglutination Assay

Uses 2 Antibodies = Coombs reagent

Avoids issue of repulsion due to ZETA potential

  • 1* AB
    • normal, binds to surface of RBC
  • 2* AB
    • recognizes the TAIL = Fc region of 1*AB
      • crosslinkage of 2 RBC’s via the tails
        • –> Agglutination
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23
Q

ImmunoRadioMetric Assay

uses a Radioisotope Label on the

SECOND Antibody (not Ag like RIA)

easier to label AB vs Ag

Direct & Indirect Varients

A

IRMA

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24
Q

5 Antibody Types

A

G A M E D

Differentiated by the type of Heavy Chain

  • IgG
    • ​main type produced in immune response, kind most used in most immunoassays
  • IgA - mucus membranes
  • IgM - ME FIRST - first responder in immune response
  • IgE - AlEEErgic reactions
  • IgD - autoimmunity protection, D-Unknown
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25
Q

EMIT

Homogeneous or HETEROgeneous?

What is Immobilized?

A

Homogeneous

nothing is immobilized

faster & more convenient

only other homogeneous is FPIA

we are measuring the enzyme + substrate =

PRODUCT (typically colored)

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26
Q

Hapten

A

Low MW molecule/compound

that is not immunogenic itself

It still can bind to the Antibody, but it does not induce a response

Becomes immunogenic & induces an antibody response

after conjugation to a larger carrier/protein

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27
Q

Uses for ELISA?

Immunocapture / Indirect

A

PRIMARY ASSAY for detection of:

AB –> Pathogen

SPECIFIC ANTIBODIES

Antiviral IgG

IgM against HEP A

Measels / mumps etc.

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28
Q

Competitive Immunoassay

Heterogeneous Format

#ABs & What is Labeled?

A

Indirect Assay

  • 1 Antibody
    • ​ATTACHED (by Fc region) to Solid Phase
  • ​​2 Analytes
    • 1 is LABELED, other unlabeled
  • After Binding, we wash away the unbound materials
    • measure signal from the bound-labeled analytes
  • As analyte concentration increases,
    • signal strength decreases
      • ​regative exponential slope
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29
Q

Other Radioisotopes

Proteins

A

Fluorescein

fluorophore

GFP

green fluorecent protein (whole protein)

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30
Q

Which immunoassays are COMPETITIVE?

A

Competitive = indirect

RIA

Competitive ELISA

Indirect IRMA

FPIA

measures the Unbound

inversely proportional to amount of Ag

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31
Q

Monoclonal Antibodies

ADVANTAGES

A

Highly SPECIFIC = recognize exactly the same EPITOPE

Identical protein molecules

Same Ig Class (usually IgG)

Unending supply

reduce background binding

less cross reactive binding

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32
Q

RIA

Radio Immuno Assay

A

heterogeneous competition immunoassay that uses

radiolabeled antigens or antibodies;

in the original liquid-phase format, the assay

would require a precipitation step to

separate antibody-antigen complexes;

Used for Small molecule analytes (steroids / cytokines / peptides)

Not well suited for LARGE analytes (whole proteins

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33
Q

RIA Uses & Their Issues

A

RadioImmunoAssay

Currently used for Small Molecule Anayltes

(Steroids / Cytokines / Peptides)

not well suited for large analytes = whole proteins

Radioactivity is a MAJOR drawback:

Hazard / waste disposal / decay

Licensing / unable for automation

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34
Q

Common ENZYMES for immunoassays

that use enzymes

A

EMIT

Commonly uses Glucose 6-Phosphate Dehydrogenase

adding NAD + GlC6P –> we monitor NADPH formation by fluoresence

ELISA also uses enzymes

Other enzymes:

Horseradish Peroxidase

Alkaline Phosphatase

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35
Q

ELISA

A

Enzyme-Linked ImmunoSorbent Assay

Uses ENZYMES to detect/quantify HAPTENS

has 4 different formats

also is HETEROgeneous;

uses microtiter plates w/ bound AB or bound Ag

to form sandwich

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36
Q

Antigen

Ag

A

Any FOREIGN material specificallly bound by:

_antibody or lymphocyte_s;

does not have to induce an immune response

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37
Q

Medical Uses for

EMIT?

A

Enzyme that covalently attaches to drug of interest

HbA1C Levels

URINE TESTS /** **Free Drug Ratio

Pregnancy (plasma / serum tests)

Hydrocodone

Other Therapeutic Drugs

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38
Q

What type of Assay?

A

DIRECT ASSAY

sandwich / immunoMETRIC

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39
Q

Radiolabel

=

Radioactive Label

A

A Label that uses

Unstable Isotopes that spon. transform

into a more stable state

–> emitting ENERGY in the form of Particles / EM pulses

Ex. RIA = RadioImmunoAssay

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40
Q

Name the SPECIFIC Immunoassay

A

DIRECT

IRMA

ImmunoRadioMetric Assay

Positive correlation

We count the BOUND 2nd antibody, which is proportional to the Ag

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41
Q

For a INDIRECT assay

Interference causes a ________ in Signal

A

INCREASE

Indirect = inverse signal response to more analyte

Since less analyte would detected,

we would see a GREATER signal

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42
Q

IRMA

Homogeneous or HETEROgeneous?

What is Immobilized?

A

HETEROgeneous assay

Both Direct & Indirect :

1* Antibody is immobilized

Direct measures the labeled 2* AB, which is proportional to Ag

Indirect measures the unbound labeled 2* AB, inverseley proprotional

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43
Q

RIA

Homogeneous or HETEROgeneous?

What is Immobilized?

A

HETEROgeneous

Nothing is bound

44
Q

Labels for Antibodies Vs Antigens?

A
  • ANTIGENS: quite small (MW <600)
    • difficult to find a site to attach a LABEL
      • ​​without compromising AB recognition
  • ​​Antibodies: LARGE proteins (MW ~150,000)
    • MANY possible sites to attahch labels
    • Ex. Radioisotopes (IRMA)
    • Enzymes (ELISA)
    • GFP = green fluorescent protein
    • EMIT - use enzymes!
45
Q

Haptens / Homopolymers / Polysaccharides

as ANTIGENS

A
  • Hapten:
    • Does NOT elicit a immune response on its OWN
    • can bind, but not likely if only ONE
  • ​Polysaccharide/Homopolymer:
    • Consist of many epitopes
      • all have the SAME specificity
      • NOT VERY IMMUNOGENIC
46
Q

Heavy Chain

Antibody Structure Components

A

Longer darker Chain

2 of them, identical to one another

linked together by DiSulfide bonds (cys-residues)

contain the Fc Region

Linked to light chain by Di-sulfide bonds

47
Q

Variable Region

Contain the Tips of the “Y”

where binding/recognition of the antigen occurs

Large variety of AA sequences

for AB Specificity & Affinity!

A

F-v Region

48
Q

What are often the BEST ANTIGENS?

Why?

A

PROTEINS

Lots of Variability in AA Sequences

More Differences = More Antigens recognized

MORE CHANCES to ELICIT a RESPONSE

49
Q

F-c Region

A

Antibody Structure Component

Base of the chunky “Y”

consist of the end of the:

2 dark chains

c=first part crystallized, abc

50
Q

Name this Assay

A

IMMUNOMETRIC ASSAY

Sandwich Assay

Direct Assay

more Analyte –> Greater Signal

Signal is on the 2* ANTIBODY

51
Q

Main Enzyme RadioIsotopes

A

HRP

Horseradish Peroxidase

G6P-DH

glucose 6-phosphate dehydrogenase

ALP

alkaline phosphatase

52
Q

Longer darker Chain

2 of them, identical to one another

linked together by DiSulfide bonds (cys-residues)

contain the Fc Region

Linked to light chain by Di-sulfide bonds

A

Heavy Chain

Antibody Structure Components

53
Q

HOMOgeneous Assay

A

Analyte is labeled,

Does _NOT REQUIRE SEPERATION_

of bound & free antigen

Faster & Convenient

We simply collect the product = colored

Ex. EMIT

54
Q

F-ac Region

A

Antibody Structure Component

“V” part of the chunky Y

Contains the Variable Region

both the light and dark chain

does not include the Fc region

55
Q

Source of POLYclonal ABs

A
  • Pharma Farms:
    • Goats / Sheep / Cows / Horses
  • ​Ag INJECTED –> into an animal
    • animal’s B-Cells produce Antibodies to the Ag
      • Various Antibodies with slightly different AA’s are formed**​
        • => POLYCLONAL RESPONSE
56
Q

Any substance with a

measurable property that can be attached to an

Ag’s / AB’s

any other binding substance

(biotin / protein A / Avidin)

includes:

Chromophores / Fluorophores

Radioactive Isotopes / Enzymes

A

Label

57
Q

Response by antibody to substances

OTHER THAN THE ANYLYTE

similar in structure / family member

larger antigens = folding patterns / AA sequences

POLYCLONAL ANTIBODIES

main culprit

A

Cross Reactivity

58
Q

Epitope

A

The Specific Portion of an antigen that

Binds to an ANTIBODY or T-cell receptor

antigenic determinant

59
Q

MonoSPECIFIC AB that is

produced by a single plasma cell

or single CLONE of plasma cells

  • ADV:
    • Identical protein / same IG Class (usually IgG)
    • highly specific –> recognize same epitope
    • reduces: cross-reactivty
    • UNENDING Supply
A

MONOclonal Antibody

60
Q

EMIT

2 Ways the Antibody can INHIBIT the enzyme

A

STERIC INTERFERENCE

Antibody PHYSICALLY gets in the way of the binding

INDUCED CONFORMATIONAL CHANGE

keeps the substrate from binding

61
Q

FPIA

A

Fluoresence

Polarization

Imunno-

Assay

62
Q

Isotope

A

Atomic species that

in the nucleus

have the SAME # of PROTONS

but a different # of neutrons

radioisotopes:

used to radioactively label Ag’s or AB’s

63
Q

Antibody Structure

A
64
Q

Interference

A

ANY FACTOR causing BIAS in an assay result

that is NOT in the presence of a

Cross-Reacting Substance

  • Numerous Causes:
    • Detection system / Seperation problems
    • Batch Variation / Enzyme Alteration
    • Analyte Displacement / Blocakage
65
Q

Analyte is labeled,

Does _NOT REQUIRE SEPERATION_

of bound & free antigen

Faster & Convenient

We simply collect the product = colored

Ex. EMIT

A

HOMOgeneous Assay

66
Q

Western Blot

Assay

A

Used AFTER Protein Gel Electrophoresis

cut channel/column and lay on polymer sheet (support)

1* AB -> specific to 1 protein

2* AB w/ DYE or enzyme -> recognized fc region of 1*ab

Signal detects protein bands that bound to primary AB

illuminate / fluorecence

Used for:

HIV INFECTION / Viral Proteins (hep B/C) / Doping

67
Q

Agglutination (aggregation) vs Precipitation

Using Antibodies

A

Aggregation = Agglutination Using ABs

when the Antigen = LARGE / INSOLUBLE

ex. Whole Cell

68
Q

Name This Assay

A

COMPETITIVE ASSAY

Indirect Assay

Adding more unlabeled Analyte (x) –> lower signal (y)

more analyte is competing with LABELED analyte (with signal)

69
Q

Light Chain

Antibody Structure Components

A

2 identical shorter chains

not linked together

linked to the heavy chain by DiSulfide Bonds (cys-residue)

70
Q

Uses for Immunometric=Sandwich ELISA?

A

Good for large antigens/proteins >6000da

INSULIN

TSH / Thyroxine

Cortisol / ACTH

71
Q

Immunoassay

A

a procedure for detecting or measuring

specific proteins/substances through their

properties as Ag’s or AB’s

72
Q

HETEROgeneous Assay

A

Format that uses TWO PHASES

typically liquid + solid to

seperate bound/reacted from unbound/unreacted

Ex.

ELISA** & **IRMA

both direct & indirect

(Immunometric / sandwich) & (competitive)

73
Q

Used AFTER Protein Gel Electrophoresis

cut channel/column and lay on polymer sheet (support)

1* AB -> specific to 1 protein

2* AB w/ DYE or enzyme -> recognized fc region of 1*ab

Signal detects protein bands that bound to primary AB

illuminate / fluorecence

Used for:

HIV INFECTION / Viral Proteins (hep B/C) / Doping

A

Western Blot

Assay

74
Q

ANY FACTOR causing BIAS in an assay result

that is NOT in the presence of a

Cross-Reacting Substance

  • Numerous Causes:
    • Detection system / Seperation problems
    • Batch Variation / Enzyme Alteration
    • Analyte Displacement / Blocakage
A

Interference

75
Q

Monoclonal Antibody Production

A
  • Inject mouse with Ag
    • collect Spleen Cells (contain B-cells that produce ABs)
      • infuse with HAT resistance
    • ADD: Immortal Myeloma Cell line + HAT medium
      • PEG –> causes the cells to FUSE
        • Unfused cells will DIE
  • ​Fused spleen cells = IMMORTAL (hat resistance + myeloma)
    • ​​​continuously producing ANTIBODIES
      • DILUTE into wells then, SCREEN for Desired Cell
        • Isolate desired Monoclonal Spleen cell
          • ​that produces MONOCLONAL ANTIBODIES
76
Q

Antibody Structure Component

Base of the chunky “Y”

consist of the end of the:

2 dark chains

c=first part crystallized, abc

A

F-c Region

77
Q

AntiBody

AB

A

Proteins made by the B-Cells of the immune system

They serve to identify Antigens in the body by binding them

78
Q

Agglutination (aggregation) vs Precipitation

Using Antibodies

A

Precipitation Using ABS

When the Ag is

SMALL & SOLUBLE

ex. PROTEIN

79
Q

G A M E D

Differentiated by the type of Heavy Chain

  • IgG
    • ​main type produced in immune response, kind most used in most immunoassays
  • IgA - mucus membranes
  • IgM - ME FIRST - first responder in immune response
  • IgE - AlEEErgic reactions
  • IgD - autoimmunity protection, D-Unknown
A

5 Antibody Types

80
Q

Format that uses TWO PHASES

typically liquid + solid to

seperate bound/reacted from unbound/unreacted

Ex.

ELISA** & **IRMA

both direct & indirect

(Immunometric / sandwich) & (competitive)

A

HETEROgeneous Assay

81
Q

List several different types of LABELS for antigens

A

Radioisotopes

dangerous and difficult

Fluorophores

Fluorecein = LARGE dye, for UV light

Whole Proteins

GREEN fluorescent protein

Whole Enzyme

G6PDH - color the ENZYME

82
Q

Atomic species that

in the nucleus

have the SAME # of PROTONS

but a different # of neutrons

radioisotopes:

used to radioactively label Ag’s or AB’s

A

Isotope

83
Q

HOMOgeneous assay that

Uses ENZYME

that has covalently attached to drug of interest

measure the amount of Free Drug in sample

does not use radioisotopes, uses colored product

Used for:

Assaying THERAPEUTIC DRUGS

URINE TESTS

A

EMIT

Enzyme Multiplied Immunoassay Technique

84
Q

Proteins made by the B-Cells of the immune system

They serve to identify Antigens in the body by binding them

A

AntiBody

AB

85
Q

Substances succeptable to CROSS-REACTIVITY

A

PolyClonal Antibodies

Compounds of Similar Family / Structure

= Steroids

Closely related folding patterns / AA sequences

Large Antigens = Proteins

86
Q

A Label that uses

Unstable Isotopes that spon. transform

into a more stable state

–> emitting ENERGY in the form of Particles / EM pulses

Ex. RIA = RadioImmunoAssay

A

Radiolabel

=

Radioactive Label

87
Q

HCG Test for Pregnancy Photo

A
88
Q

Name this SPECIFIC immunoassay

A

INDIRECT

IRMA

ImmunoRadioMetric Assay

Negative correlation

We REMOVE and count the unbound 1st antibody

which is inversely proportional to Ag

89
Q

Immunogen Vs Antigen

A

Immunogen:

is a substance that is

capable of inducing a

IMMUNE RESPONSE

  • Antigen does NOT have to induce an IMMUNE RESPONSE*
  • it just has to specifically bind*
90
Q

How are BLOOD TYPES determined by immunoassays?

Blood Type Agglutination Assay

A

Blood type = Type of ANTIGEN it HAS

  • “Eldoncard” agglutination assay:
    • Each Antibody Crosslinks RBC –> bridges Ag’s together
      • causes aggregation/agglutination if it has
        • that specific antigen
  • 4 Pads:
    • Anti-A = ​has antibody for A
    • Anti-B = has AB for B
    • Anti-D
      • ​has AB to the most common Rhesus Factor Antigen
    • CONTROL
      • ​should not aggregate at all!
91
Q

POLYclonal Antibody

A

HETEROgeneous mixture of ABs

with diverse affinities/regions towards an ANTIGEN

produced by a LARGE # of plasma cell clones

polyclonal response -> CROSS-REACTIVITY

Ex. Whale Myoglobin

from various Pharma Farms: goats/sheeps/cows

92
Q

heterogeneous competition immunoassay that uses

radiolabeled antigens or antibodies;

in the original liquid-phase format, the assay

would require a precipitation step to

separate antibody-antigen complexes;

Used for Small molecule analytes (steroids / cytokines / peptides)

Not well suited for LARGE analytes (whole proteins)

A

RIA

RadioImmunoAssay

93
Q

Common Isotopes used in Radiolabels

A

RIA = RadioIsotope Labeled Antigens

Iodine = 125I or 131I to Tyr on Peptides/Proteins

Tritium = 1H exchange to 3H

Phosphorylate = 32P-Containing-ATP

94
Q

F-v Region

A

Variable Region

Contain the Tips of the “Y”

where binding/recognition of the antigen occurs

Large variety of AA sequences

for AB Specificity & Affinity!

95
Q

a procedure for detecting or measuring

specific proteins/substances through their

properties as Ag’s or AB’s

A

Immunoassay

96
Q

Competitive ELISA

Adv / Disadvantages

A

Useful for LOW MW Analytes

does NOT have to be a hapten

w/ 2 DISTINCT immunogenic sites

Neg:

NOT as SENSITIVE as immunometric ELISA

97
Q

Antibody Structure Component

“V” part of the chunky Y

Contains the Variable Region

both the light and dark chain

does not include the Fc region

A

F-ac Region

98
Q

Competitive ELISA

What ABs / Ags?

What is Measured?

A

Enzyme-Linked ImmunoSorbent Assay

Indirect

  • 1 Antibody
    • bound to solid phase
  • 2 Analytes
    • 1 analyte is labeled w/ ENZYME
      • ​We measure the amount of bound enzyme
  • ​1 Substrate
    • reacts w/ ENZYME –> color development
99
Q

2 identical shorter chains

not linked together

linked to the heavy chain by DiSulfide Bonds (cys-residue)

A

Light Chain

Antibody Structure Components

100
Q

Which Immunoassays are IMMUNOMETRIC?

A

Immunometric = DIRECT

If 2/ 2 sites = Sandwich

Direct IRMA

Immunometric ELISA

Cortisol / ACTH / TSh / Thyroxine

Measure the bound labeled AB

which is directly proportional to the amount of Ag

101
Q

Western Blot Definition

A

Membrane-based assay in which proteins are separated

by electrophoresis,

followed by transfer to a membrane and

probing with a labeled antibody

102
Q

Hydrogen & Phosphorous

Radioisotopes

A

Tritium 3H

exchange 1H to 3H

32P

phosphoralated-ATP

103
Q

The Specific Portion of an antigen that

Binds to an ANTIBODY or T-cell receptor

antigenic determinant

A

Epitope

104
Q

ELISA

Homogeneous or HETEROgeneous?

What is Immobilized?

A

HETEROgeneous

AB is bound

105
Q

Label

A

Any substance with a

measurable property that can be attached to an

Ag’s / AB’s

any other binding substance

(biotin / protein A / Avidin)

includes:

Chromophores / Fluorophores

Radioactive Isotopes / Enzymes

BioMedChem M2 (8 decks)

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