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Immunodiagnostics Cheat Sheet (DRAFT) by

diagnostic assays run to determine Ab-Ag interaction

This is a draft cheat sheet. It is a work in progress and is not finished yet.

Immuno­flu­ore­scence Assays

Fluore­scent Antibody Test (FA)

Mechanism
Fluore­scently labeled mAb to bind and illuminate a target Ag/Ab
Designed to Detect
A sample's Antigen OR Antibody
Reagent
mAb-FITC conjugate ~ Ab tagged with a Fluore­scein
Compatible Sample Types
Serum or tissue section
 
Types
1. Direct (DFA)
Detection of sample's antigen
Sample
Unknown Antigen in blood
2. Indirect (IFA)
Detection of sample's antibody
Sample
Unknown Antibody in blood (Ag = known)
Reagent
Secondary mAb-FITC conjugate

Direct Fluore­scent Antibody

Uses
Bovine Viral Diarrhea Virus (BVDV)
Detection of live BVDV in bovine blood
Rabies in Brain Necropsy
Detection of the Rabies virus in the brain tissue
 
Sample
Ag from cultur­e/slide
Known
mAb-FITC conjugate against antigen of interest
Detects (unknown reactant)
Antigen from sample
Reagent
mAb-FITC conjugate
 
Results
Positive Test
Fluore­scence = Ag present
Negative Test
No fluore­scence = No Ag
*Only ONE 'known' Ab is used in this test ~ so the known test component IS the Reagent

DFA Mechanism of Action

DFA Example ~ BVDV

1. Incubate patient serum (conta­ining the virus) with a cultured cell line
~ Cell-line must be permissive to BVDV infection
2. Probe with mAb-FITC conj. that targets the viral Ag of BVDV

BVDV DFA Results

DFA ~ Rabies Brain Necropsy Dx

DFA is required for an official Rabies Dx
- An impression or tissue section of the euthanized animal's **Cere­bellum, Hippoc­ampus, and/or
Brains­tem** is collected
- mAb-FITC targeting the Rabies virus' antigen

Positive DFA of Rabies in the Brain

Indirect Fluore­scent Antibody Test (IFA)

Uses
Porcine Reprod­uctive and Respir­atory Virus (PRRSV)
Detection of PRRSV Antibody in Porcine serum
Titers
Highest serial dilution of serum with Ab - that fluoresces
Dengue Fever/­Chi­kV/Zika Virus
 
Sample
Serum
Known
Antigen
Detects (unknown reactant)
Antibody from serum (1ºAb)
Reagent
Anti-spp. Ab-FITC conjugate (2ºAb)
 
Results
Positive Test
Fluore­scence = Ab present
Negative Test
No fluore­scence = No Ab
*This test uses TWO antibodies ~ a 1º and 2º antibody

IFA Mechanism

IFA Example ~ PRRSV

Detection of Antibody against PRRSV in Swine serum
> Known: PRRSV infected cell line
> Sample: Porcine serum incubated with cell line
> 2ºAb Probe: Anti-pig IgG conjugated with FITC
> Unknown: Antibody against PRRSV

Results of PRRSV IFA Test

IFAs Results for DENV 1-4/CH­IKV­/ZIKV

  

Enzyme­-Linked Immuno­sorbent Assay (ELISA)

ELISA

High sensit­ivity / Low specif­icity
 
Test Types that Detect Antigen
>
Direct ELISA
or
Sandwich ELISA
or
Antigen Capture ELISA
or
Antigen ELISA
USES
Heartworm Test (HWTM)
Using the Anti-H­TWM­-Ab-HRP
 
Test Types that Detect Antibody
>
Indirect ELISA
or
Antibody ELISA
USES
Titrations (titers)
Quantifies the amount of Ab present

DIRECT (Ag Capture) ELISA

Sample
Serum from patient
Known
1º Capture Ab (coats wells in tray)
Detection of (unknown)
Antigen
Reagent + Substrate*
2º Detection Ab ~ specific to disease conjugated to enzyme
Positive Test
Color change = Ag present
Negative Test
No color change = No Ag
* substrate = activates enzyme
** The capture Ab and the detection Ab may be the same Ab > BUT ONLY the detection Ab will be tagged with the enzyme

Ag-Capture ELISA ~ MOA

Ag-Capture ELISA ~ MOA

Ag-Capture ELISA ~ MOA

Ag Capture ELISA ~ HTWM Ag Detection

> 1º Capture Ab: Ab that targets Ab
> Sample with unknown: Serum with HTWM Ag
> Wash slide
> 2º Detection Ab: Anti-H­TWM­-Ab-HRP
> Add substrate to activate enzyme to show color change if bound

HTWM Ag-Capture ELISA Results

Ag-Capture ELISA ~ MOA

INDIRECT (Antibody) ELISA

Sample
Serum from patient
Known
Antigen (coating wells)
Detection of (unknown)
1º Ab in serum (spec. for Ag)
Reagent + Substrate*
Anti-Ab 2º Detection Antibo­dy-HRP conj. ~ *Targets hosts own Ab (1º Ab from serum)
Positive Test
Color change = Ab present
Negative Test
No color change = No Ab

Ab ELISA ~ MOA

Ab ELISA ~ MOA

Ab ELISA Titration

 
> Coat ELISA wells with Ag (can get commercial Ag of interest)
> Add serial dilutions of patient's serum into wells
> wash off unbound Ab
> Add 2º Ab like Rabbit­-An­ti-­Hor­se-Ig thats conjugated w enzyme, to the wells
> wash off unbound Ab
> add substrate

Ab ELISA ~ MOA

• Can run serum from multiple patients at once
• Can determine titer by running serial dilutions of the serum

Indirect ELISA Ab Titer

  

Western Blot (WB)

WB

Higher specif­icity than ELISA
 
Designed to ID/Detect PROTEINS:
1. Detection of Antibody
if patients serum contains Ab against a specific protein in a complex protein mixture
2. ID specific protein antigen in mix
Use of a known reagent Ab to the protein of interest
 
MOA
This is a three-­stage primary binding test
Stage I
Electr­oph­oresis of a protein mixture on gels so that each component is resolved into a single band
Stage II
Blotting of these protein bands to an immobi­lizing nitroc­ell­ulose membrane
Stage III
Visual­ization of transf­erred Ag by either directly or indirectly probing the membrane with Ab's
 
WB Probing Methods
Direct
Detection of the Protein Antigen
Indirect
Detection of the Antibody

WB MOA

DIRECT WB

1. Separate out proteins by size and charge in the unknown antigen using gel
electr­oph­oresis
2.Transfer molecules to secondary matrix
3.Probe with enzyme­-la­beled known antibody to the antigen of interest
4.Wash
5. Add substrate and observe change (color or light)

DIRECT WB ~ Bovine Spongiform Enceph­alo­pathy (BSE)

Sample
Serum w Ag from pt. (separated by electr­oph.)
______­___­___­___­___­_____
Brain tissue
Known
BSE specific Ab-tagged with an enzyme (reagent)
Detects
BSE Prion protein Antigen (Unknown)
 
RESULTS
Positive Test
Banding that match the positive band pattern = Antigen present
Negative Test
Banding that match the negative band pattern = No Antigen
Direct WB Required for Dx of BSE! ~ Because:
- The BSE Ag that causes a disease is a normal brain protein in Bovine that is malfun­cti­oning because it is folded incorr­ectly
- Since this is a normal protein in the Cow brain ~ There is NO IMMUNE RESPONSE that will generate

>>> THUS: We have to test for the Ag since the Ab will never be produced

Direct WB BSE Results

- 3 brain preps from 3 suspect cow with brain proteins separated
- Abnormal BSE-sp­ecific prion protein molecules can be detected using antibodies linked to an enzyme that results in a chemical reaction
- For this test a monoclonal antibody was made that recognizes BSE-sp­ecific abnormal prion protein >>> This antibody is a reagent antibody (tagged with an enzyme).

RESULTS
Cow #3 has BSE-sp­ecific prion proteins in its brain.

INDIRECT WB

USES
ELISA Dx Confir­mation
Feline Immuno­def­iciency Virus (FIV)
 
Human Immuno­def­iciency Virus (HIV)
 
Sample
Patient serum w Ab (separated by
Known
HIV Ag (from known HIV-in­fected cells)
Detects (unknown reactant)
Ab spec. to HIV Ag
Reagent
Anti-spp Ab conj. to enzyme
 
INTERP­RET­ATION
Positive Test
Banding Pattern matches that of known positive = Ab present
Negative Test
Banding Pattern matches that of known negative = No Ab

INDIRECT WB MOA

1. Separate out proteins by size and charge in the known antigen using gel electr­oph­oresis
2.Transfer molecules to secondary matrix
3.Probe with patient's serum antibody
4.Add enzyme­-la­beled antibody to patient's antibody
5.Wash
6. Add substrate and observe change (color or light)

INDIRECT WB ~ FIV Confir­mation

Source for Ag
Proteins from FIV-in­fected cells (separated out by electr­oph­oresis)
Sample with unknown Ab
Cat's serum w/ Ab
Detection Reagent
Anti-cat Ab conju. w enzyme

Immuno­his­toc­hem­istry (IHC)

IHC Test

- Always detects antigen
- Horser­adish peroxidase (brown color)
- (-) Controls = irrelevant Ab OR normal tissue section
 
Sample
Thin tissue section
Known ~ 1º reagent
1º Reagent Antibody ~ probes Ag
Detects (unknown)
Antigen in the Tissue sect.
2º Detection reagent
2º Detection Ab conj. ~ spec. for 1º Ab
 
CONTROLS
Positive Control
1º Reagent
1º Reagent Ab ~ spec. for tissue Ag
Positive Result
Brown in color = Ag is present
 
Negative Control
1º Reagent
1º Reagent Ab ~ spec. for tissue Ag NOT IN SAMPLE
Negative Result
No color change = No Ag

IHC MOA

IHC Microscopy Result

IHC Negative Control

USES
Detection of mammary tumor Ag*
Use of an irrelevant rabbit mAb spec. to tumor Ag
Detection of Brucella Melitensis

IHC ~ Brucella Melitensis

Sample
Histo section of a Goat's tissue from the Prepuce of the Penis and the Seminal Vesicular Gland
1º Reagent Ab
mAb spec. to B. melitensis Ag
2º Detection Ab
2º mAb spec. to 1º Ab
 
RESULTS
Positive Test
Observed brown color when compared to controls = Ag present
Negative Test
No color change from controls = No Ag

B. Melite­nsis IHC Results

Top: Mucosal epithelium of the Prepuce of the Penis
Bottom: Seminal Vesicular Gland epithelia