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Methods of Enumeration and Activity of
Microorganisms (Part 2)
Immunological Methods
Immunoassays are analytical methods used for detection and quantification of the
antigen-antibody interaction
Antigen: any compound or substance which induces the production of antibodies in
warm-blooded animals
Antibody: Y-shaped complex protein produced in response to the antigen by the immune
system by b-cells

Immunoglobulins (Antibodies)/IGG
Antibody molecule has 4-polypeptide chains:
a
...
Two identical light chains (L-chains)
These chains are joined together by disulfide linkages to form “Y” shaped structure
Heavy chain consists of 400 amino acids, while light chain has 200 amino acids

Structure of Immunoglobulins (IGG)
Within each polypeptide chain there are two regions:
a
...
Variable region
The arms of the antibody are identical, and a single molecule of antibody may combine with two
antigens to form Antibody-Antigen Complex
The antibody molecule can be broken down into two parts with an enzyme “Papain”:
- Fab-fragment: binds to Ag
...
IgM
2
...
IgA
4
...
IgD
5 Types of Immunoglobulins
Ab Type

% in serum

Mol
...
of subunits

Crosses
placenta

IgM

5-10

900,000

5

No

IgG

80

150,000

1

Yes

IgA

10

400,000

2

NO

IgE

1

200,000

1

No

IgD

0
...
IgM
-

The first antibody to appear in the circulation by B-cells
The largest one

2
...
IgA

4
...
IgD

Commonly Used Immunoassays

1
...

3
...

5
...
Fluorescent Immunolabeling
-

Fluorescent signal molecules are conjugated to antibodies
The signal when reacting with antigen produces fluorescent light
The sample antigen is attracted microscopic slide and a fluorescent-antibody conjugated
specific antigen is added
This is then viewed under the microscope equipped with fluorescent light
The labeled antibody appears light-green against dark background
The assay is commonly used to detect protozoa parasites in water

2
...
7 cm in diameter

ELISA detects the presence of an antibody or an antigen in a sample
Antigen: Any substance or compound which stimulates the immune system to produce
antibodies
Antigens may be:
- Proteins
- Polysaccharides - sugars such as mannose
- Lipoproteins - conjugates of lipids (fats) with proteins
Epitope: a specific configuration on the protein surface
- One or more epitopes may be present on the antigen
Applications of ELISA
-

Serum antibody concentrations
Detecting potential food allergens (milk, peanuts, walnuts, almonds and eggs)
Disease outbreaks - tracking the spread of disease e
...
HIV, bird flu, common colds,
cholera
Detection of antigens e
...
pregnancy hormones, drug allergen, GMO, mad cow disease

-

Detection of antibodies in blood sample for past exposure to disease e
...
Lyme disease,
trichinosis, HIV, bird flu

Methods for AG-AB Detection
- Precipitation
- Aggulation
- Hemagglutination and hemagglutination inhibition
- Viral neutralization test
- Radio-immunoassays
- ELISA
- Immunofluorescence
- Immunoblotting
Variants of Elisa
1
...
Direct ELISA
3
...
Competitive ELISA
5
...
Antibody capture ELISA
7
...
Two-step ELISA
9
...
Indirect ELISA (DAC-ELISA)
2
...
Competitive ELISA (COM-ELISA)
Indirect Elisa Procedure (DAC-ELISA)
1
...

3
...

5
...

7
...


Coating of plate with antigen (serum samples)
Incubation (37 C for 30 min) and washing
Add virus specific antibody (e
...
Ab of HIV)
Incubation (37C for 30 min) and washing
Add secondary antibody conjugated with enzyme (ALP)
Incubation (37C for 30 min) and washing
Add substrate, wait for color development
Take reading in ELISA reader

Double Antibody Sandwich ELISA (DAS-ELISA)
1
...

3
...

5
...

7
...


Coating of plate with virus specific antibody
Incubation (37 C for 30 min) and washing
Add samples (patient’s serum) (antigen)
Incubation (37C for 30 min) and washing
Add virus specific antibody (e
...
HIV-AB) with enzyme (HRP)
Incubation (37C for 30 min) and washing
Add substrate, wait for color development
Take reading in ELISA reader

Competitive ELISA
A measured amount of known enzyme-labeled component (being measured) is added to the
reaction at the same time patient sample is added
The labeled component (known antigen) therefore competes against the unlabeled component
in the patient sample for binding sites
Results
- Negative reaction has color change
- Positive reaction no color change

Competitive - labeled known and patient unknown are added to reaction and “compete” for the
target
- For example, looking for an antibody
- Add labeled reagent antibody of known specificity, patient sample and known antigen
- Patient antibody competes with reagent antibody for the target antigen
- Concentration is inversely proportional to results
Competitive ELISA Procedure
1
...
Wash to remove unbound reactants
3
...
Soil Environments
- Microbial DNA is obtained by two methods
1
...
First separate the cells from the soil then lyse the cells
- DNA is purified and molecular analysis is carried out
B
...
2 um size filters
- Viruses are concentrated by electronegative or electropositive filters
Nucleic Acid Based Methods
1
...

3
...

5
...

7
...
Southern Blotting (for DNA)
2
...
Denaturation at around 94C

-

During the denaturation (unwinding), the double strand melts open to single
stranded DNA, all enzymatic reactions stop (for example, the extension from a
previous cycle)
2
...
Extension at around 72C
- The bases (complementary to the template) are coupled to the primer on the 3’
side (the polymerase adds dNTP’s from 5’ to 3’, reading the template from 3’ to 5’
side, bases are added complementary to the template)

Restriction Fragment Length Polymorphism (RFLP)
-

Total DNA is extracted
The DNA is cut by endonucleases
Horizontal gel electrophoresis
Dot blot hybridization

Reporter Genes
Reporter gene: a gene or set of genes that is inserted into a target gene of interest and
subsequently reports the activity of that gene
Example:
- Lac Z gene (coding for Beta Galactosidase)
- Gus A gene from E

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