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
**_EMIT_** ## Footnote **Homogeneous or HETEROgeneous?** **What is Immobilized?**
***_Homogeneous_*** *nothing is immobilized* faster & more convenient _only other homogeneous is **FPIA**_ *we are measuring the enzyme + substrate =* **PRODUCT** (typically colored)
26
**Hapten**
***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**
27
**Uses for ELISA?** **_Immunocapture / Indirect_**
PRIMARY ASSAY for detection of: **AB --\> Pathogen** **_SPECIFIC ANTIBODIES_** _Antiviral IgG_ _IgM_ against HEP A Measels / mumps etc.
28
**Competitive Immunoassay** Heterogeneous Format **#ABs & What is Labeled?**
***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*
29
**Other Radioisotopes** **Proteins**
**Fluorescein** ## Footnote fluorophore **GFP** green fluorecent protein (whole protein)
30
**Which immunoassays are COMPETITIVE?**
Competitive = ***_indirect_*** **RIA** **Competitive ELISA** ***Indirect IRMA*** FPIA measures the ***Unbound*** ***inversely proportional*** to amount of Ag
31
**Monoclonal Antibodies** **ADVANTAGES**
Highly **SPECIFIC** = recognize **exactly the same EPITOPE** **Identical** protein molecules Same **Ig Class** (usually **IgG**) **Unending supply** *reduce background binding* ***less cross reactive binding***
32
**RIA** **R**adio **I**mmuno **Assay**
**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*
33
**RIA Uses & Their Issues**
**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
34
**Common ENZYMES for immunoassays** that use enzymes
_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_**
35
**ELISA**
**_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**
36
**Antigen** **Ag**
Any **FOREIGN** material specificallly bound by: _antibody or lymphocyte_s; *does not have to **induce an immune response***
37
**Medical Uses for** **EMIT****?**
Enzyme that covalently attaches to drug of interest **_HbA1C Levels_** **_URINE TESTS /**_ _**Free Drug Ratio_** **_Pregnancy_** (plasma / serum tests) **Hydrocodone** Other **Therapeutic Drugs**
38
What type of Assay?
**DIRECT** **ASSAY** sandwich / immunoMETRIC
39
**Radiolabel** **=** **Radioactive Label**
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
40
**Name the SPECIFIC Immunoassay**
**_DIRECT_** **_IRMA_** ImmunoRadioMetric Assay Positive correlation We count the **BOUND 2nd antibody**, which is proportional to the **Ag**
41
For a ***INDIRECT*** assay ## Footnote **Interference causes a ________ in Signal**
**_INCREASE_** Indirect = *inverse signal response to more analyte* Since less analyte would detected, we would see a GREATER signal
42
**_IRMA_** **Homogeneous or HETEROgeneous?** **What is Immobilized?**
**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*
43
**_RIA_** ## Footnote **Homogeneous or HETEROgeneous?** **What is Immobilized?**
**_HETEROgeneous_** *Nothing is bound*
44
**Labels for Antibodies Vs Antigens?**
* ***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
**Haptens / Homopolymers / Polysaccharides** **as ANTIGENS**
* 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
**Heavy Chain** Antibody Structure Components
**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
**Variable Region** Contain the **Tips of the "Y"** where **_binding/recognition of the antigen occurs_** **Large variety of AA sequences** for **AB Specificity & Affinity!**
**F-v Region**
48
**What are often the BEST ANTIGENS?** **Why?**
**_PROTEINS_** Lots of **Variability** in **AA Sequences** More **Differences** = **More Antigens recognized** MORE **CHANCES to ELICIT a RESPONSE**
49
**F-c** **Region**
Antibody Structure Component **Base of the chunky "Y"** consist of the end of the: **2 dark chains** c=first part crystallized, abc
50
**Name this Assay**
**_IMMUNOMETRIC ASSAY_** **Sandwich Assay** **_Direct Assay_** more Analyte --\> Greater Signal Signal is on the 2\* ANTIBODY
51
**Main Enzyme RadioIsotopes**
**HRP** ## Footnote Horseradish Peroxidase **G6P-DH** glucose 6-phosphate dehydrogenase **ALP** alkaline phosphatase
52
**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**
**Heavy Chain** Antibody Structure Components
53
**HOMOgeneous Assay**
Analyte is labeled, *Does **_NOT REQUIRE SEPERATION_*** of **bound & free antigen** Faster & Convenient We simply collect the **product = colored** **_Ex._** **_EMIT_**
54
**F**-**ac** **Region**
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
**Source of POLYclonal ABs**
* 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
_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_
**Label**
57
Response by antibody to substances ***OTHER THAN THE ANYLYTE*** similar in _structure / family member_ _larger antigens_ = folding patterns / AA sequences **_POLYCLONAL ANTIBODIES_** main culprit
**Cross Reactivity**
58
**Epitope**
The **Specific Portion** of an **_antigen_** that **_Binds to an ANTIBODY or T-cell receptor_** *antigenic determinant*
59
**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**
**MONOclonal Antibody**
60
**EMIT** **2 Ways the Antibody can *INHIBIT*** **the enzyme**
**_STERIC INTERFERENCE_** Antibody PHYSICALLY gets in the way of the binding **_INDUCED CONFORMATIONAL CHANGE_** *keeps the substrate from binding*
61
**FPIA**
**Fluoresence** **Polarization** **Imunno-** **Assay**
62
**Isotope**
**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
**Antibody Structure**
64
**Interference**
**_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
Analyte is labeled, *Does **_NOT REQUIRE SEPERATION_*** of **bound & free antigen** Faster & Convenient We simply collect the **product = colored** **_Ex._** **_EMIT_**
**HOMOgeneous Assay**
66
**Western Blot** **Assay**
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
**Agglutination (aggregation**) vs Precipitation Using Antibodies
**_Aggregation = Agglutination_** Using ABs when the **Antigen = LARGE / INSOLUBLE** ex. Whole Cell
68
**Name This Assay**
**_COMPETITIVE ASSAY_** ***Indirect Assay*** Adding more *unlabeled Analyte (x)* --\> *lower signal (y)* more analyte is competing with LABELED analyte (with signal)
69
***Light Chain*** Antibody Structure Components
**2 identical *shorter chains*** *not linked together* linked to the **heavy chain by DiSulfide Bonds** (cys-residue)
70
**Uses for Immunometric=Sandwich ELISA?**
Good for **large antigens/proteins \>6000da** **INSULIN** **TSH / Thyroxine** **Cortisol / ACTH**
71
**Immunoassay**
a _procedure_ for **_detecting or measuring_** specific **proteins/substances** through their **_properties as Ag's or AB's_**
72
**HETEROgeneous Assay**
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
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**
**Western Blot** **Assay**
74
**_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**
**Interference**
75
**Monoclonal Antibody Production**
* 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
Antibody Structure Component **Base of the chunky "Y"** consist of the end of the: **2 dark chains** c=first part crystallized, abc
**F-c** **Region**
77
**AntiBody** **AB**
**Proteins** made by the **_B-Cells_** of the immune system They serve to identify **Antigens** in the body by binding them
78
Agglutination (aggregation) vs **Precipitation** Using Antibodies
***Precipitation Using ABS*** When the **Ag is** ***SMALL &*** **_SOLUBLE_** ex. PROTEIN
79
**_G A M E D_** Differentiated by the type of **Heavy Chain** * Ig**G** * **​**main type produced in _immune response_, kind most used in **most immunoassays** * Ig**A -** mucus membranes * Ig**M** - **M**E FIRST - first responder in immune response * Ig**E -** Al**EEE**rgic reactions * Ig**D** - autoimmunity protection, **D-Unknown**
**5 Antibody Types**
80
Format that uses **_TWO PHASES_** typically **liquid + solid** to seperate **bound/reacted** from ***unbound/unreacted*** Ex. **_ELISA**_ & _**IRMA_** both **_direct & *indirect*_** (Immunometric / sandwich) & *(competitive)*
**HETEROgeneous Assay**
81
**List several different types of LABELS for antigens**
**Radioisotopes** *dangerous and difficult* **Fluorophores** Fluorecein = LARGE dye, for **UV** light **Whole Proteins** GREEN fluorescent protein **Whole Enzyme** G6PDH - color the ENZYME
82
**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_
**Isotope**
83
**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_**
**_EMIT_** **E**nzyme **M**ultiplied **I**mmunoassay **T**echnique
84
**Proteins** made by the **_B-Cells_** of the immune system They serve to identify **Antigens** in the body by binding them
**AntiBody** **AB**
85
**Substances succeptable to CROSS-REACTIVITY**
**_PolyClonal Antibodies_** Compounds of Similar **Family / Structure** = **_Steroids_** Closely related **folding patterns / AA sequences** **_Large Antigens = Proteins_**
86
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
**Radiolabel** **=** **Radioactive Label**
87
**HCG Test for Pregnancy Photo**
88
**Name this SPECIFIC immunoassay**
***INDIRECT*** **_IRMA_** ImmunoRadioMetric Assay ***Negative correlation*** We REMOVE and count the ***unbound 1st antibody*** which is ***inversely proportional to _Ag_***
89
**Immunogen Vs Antigen**
**_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
How are **BLOOD TYPES** determined by **immunoassays?** **_Blood Type Agglutination Assay_**
**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
**POLYclonal Antibody**
**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
**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)*
**RIA** **RadioImmunoAssay**
93
**Common Isotopes used in Radiolabels**
**_RIA = RadioIsotope Labeled Antigens_** **Iodine** = 125I or 131I to **Tyr** on **Peptides/Proteins** **Tritium** = 1H exchange to 3H **Phosphorylate** = 32P-Containing-**ATP**
94
**F-v Region**
**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
a _procedure_ for **_detecting or measuring_** specific **proteins/substances** through their **_properties as Ag's or AB's_**
**Immunoassay**
96
**Competitive ELISA** **Adv / Disadvantages**
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
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*
**F**-**ac** **Region**
98
**Competitive ELISA** What **ABs / Ags?** What is **Measured?**
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
**2 identical *shorter chains*** *not linked together* linked to the **heavy chain by DiSulfide Bonds** (cys-residue)
***Light Chain*** Antibody Structure Components
100
**Which Immunoassays are** **IMMUNOMETRIC?**
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
**Western Blot Definition**
**Membrane-based assay** in which **proteins are separated** by **electrophoresis**, followed by **transfer to a membrane** and **_probing with a labeled antibody_**
102
**Hydrogen & Phosphorous** **Radioisotopes**
**Tritium** **3H** ## Footnote exchange 1H to 3H **32P** **phosphoralated-ATP**
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The **Specific Portion** of an **_antigen_** that **_Binds to an ANTIBODY or T-cell receptor_** *antigenic determinant*
**Epitope**
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**_ELISA_** ## Footnote **Homogeneous or HETEROgeneous?** **What is Immobilized?**
**_HETEROgeneous_** **AB** is bound
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**Label**
_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_