5/8/2020

Immunoelectrophoresis

Ankur Gautam, PhD.,

Merck High End Skill Development Centre
Chandigarh, India
20th April, 2020

CSIR-IMTech Merck High-Skill Development Center

CSIR-IMTech Merck High-Skill Development Center

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Contents
❑Antibody and antigen
❑Immune complex formation
❑Precipitation reaction
❑Precipitation curve
❑Application of precipitation reaction
❑Immunodiffusion
▪ Radial immunodiffusion
▪ Double immunodiffusion
▪ Immunoelectrophoresis
▪ Rocket immuneelectrophoresis
▪ Counter immunoelectrophoresis

Antibody, Antigen and Epitopes

Virus

Antigen

Antigen

Bacteria

Antibody Types

➢ Group of different antibodies (heterogenous) ➢ Group of identical antibodies (homogeneity)

➢ All binds to different respective epitopes on ➢ All binds to identical epitope on antigen
same antigen ➢ Do not cross react
➢ Can cross react

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Antibody-Antigen Reaction

Antigens

Antibody Antigen
Antibody

➢ Antibody antigen reaction is highly specific.

➢ Known antibody, can detect unknown antigen and vice versa

Immune complex formation (Precipitation reaction)

Antigen & Insoluble complex

Antigen antibody Soluble complex (Precipitation)

➢ Crosslinking of antibodies leads to the formation of immune complex/lattice

➢ As the lattice grows, loss solubility, come out of solution, visible precipitate

Effect of Ab and Ag concentration on Precipitation

Tube 1 Tube 2 Tube 3

▪ Abs are in excess ▪ Ag and Abs are in ▪ Ags are in excess

▪ No cross linking equal proportions ▪ No cross linking
▪ No lattice formation ▪ Cross linking ▪ No lattice formation
▪ No precipitation ▪ Lattice formation ▪ No precipitation
▪ Visible precipitation

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Ag increases
Precipitation Curve Ab constant

Zone of Ab excess Zone of Ag excess

Amount of precipitate formed

Zone of
equivalence

Antigen concentration

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Applications of Precipitation reaction Corona suspects

1. Qualitative detection
(Diagnosis)

3. Identification and characterization

of Ag and Ab in samples
Yes No

2. Quantitation
Amount = ? ng/ml

Both Covid-19 and HCV positive

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Q1. Which statement is correct?

(A) Antigen is a part of an epitope

(B) Epitope is a part of an antigen
(C) One antibody can binds to different antigens
(D) One antibody can binds to different epitopes

Correct Answer: B

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Q2. Precipitation reactions occurs only at

(A) zone of equivalence

(B)Zone of Antibody excess
(C)Zone of Antigen excess

Correct Answer: A

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Agarose/agar gel preparation

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Immunodiffusion

Agarose/ agar
Ag gel

Line of Precipitation
(at the zone of equivalence)

Types
➢ Single immunodiffusion (1D)
➢ Double immunodiffusion (1D)
Ab ➢ Single immunodiffusion (2D)
➢ Double immunodiffusion (2D)

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Single and double immunodiffusion (1D)

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Single Immunodiffusion (2D)

Agar gel containing Ab ➢ Single Radial Immunodiffusion

➢ Only Ag moves

➢ Relatively slow
Ag
➢ Uses: Determination of concentration

of antigen or antibody in a sample

Precipitin ring

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Procedure Ab Solution

Requirements
Ab solution in molten
agar
Ags of known concentration Molten agar

Concentration: 4>3>2>1
1 2 3 4

Ab Solution

Glass slide

Molten agar
Solidified agar with Ab molecules

Ag of unknown conc.
1 2 3 4 Test

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Single Radial Immunodiffusion (2D)

Standard Curve

Precipitin rings 24-72 hrs

Measure the diameter

Diameter α Concentration of Ag

Concentration of test antigen can

be determined by standard curve

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Double Immunodiffusion (1D Vs 2D)

One dimension Two dimension

Ag Ab

Precipitin band

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Molten agar

Double Immunodiffusion

Identification and comparison of

Antigens/antibodies
Glass slide

a a a b ax bx

A A+B AX

Can use anti-serum

a a a b ax bx

A A+B AX

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Interpretation of the results a a

Pattern of identity
➢ Ab reacts to both Ags Ab
➢ Ab cannot distinguish between the two Ags
➢ Both Ags are immunologically identical (share all epitopes)

a b
Pattern of Non-identity
➢ None of the Abs react to the epitopes
that may be present in both Ags A and B
➢ Both Ags are immunologically unrelated
➢ No epitopes are common between two antigens.

Pattern of Partial identity Ax Bx

➢ Cross reaction occurs
➢ Ab reacts more with one Ag than the other
➢ Spur is due to epitopes present in one Ag Ax
and lacking in the other Ag
➢ Both Ags are partially similar
(can share at least one epitope)

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Serological test for the diagnosis of fungal disease

Example: Aspergilosis, Candidiasis

PC: Positive control (known antibodies of directed against different fungal antigens)
PT: Patient serum
Ag: Known Fungal antigen (specific fungi)

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Q4. What is the relation of antigens in this pattern?

(A) Identical
(B)Non-identical
(C)Partial identical

Correct Answer: B

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Immunoelectrophoresis
➢ Combine immunodiffusion with electrophoresis
➢ Fast, Qualitative
➢ Can identify and characterize mixture of Ags

Step 1 Step 2
Separate different Identification of each
antigens antigen

Electrophoresis Double Immunodiffusion

(One dimensional single electroimmunodiffusion)

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Requirements
Antibody Antigen solution
Molten Agarose Solution Or serum
➢ Gel casting tray Or anti-serum

➢ Molten agarose

➢ Antibody solution

➢ Antigen solution

➢ Flask

➢ Electrophoresis apparatus

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Steps involved in Immunoelectrophoresis

Preparation of gel coated glass slide

Electrophoresis of antigen mixture

Loading of Antibody in trough

Double diffusion of antigen & antibody

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1. Preparation of gel coated glass slide

Trough
Agarose slab

Ag mixture or
serum

Why agarose?
➢ Neutral
➢ No reaction with antigen or antibody

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Electrophoresis
Movement of macromolecules in electric field

Agarose Gel
Buffer
Migration depends on

➢ Charge
Electrophoresis
➢ Size Tank

Large molecule:
Move slowly
Cover short distance

-
Small Molecule:
Move faster
Cover large distance
+
Power
supply

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Ab / anti-serum

- +

Agarose gel with precipitation lines

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Interpretation of the results

➢ Presence of precipitin line indicates the presence of antibody specific to the antigen

➢ Absence of precipitin line indicates the absence of antibody specific to the antigen

➢ Single precipitin line: Homogeneity of the antiserum to the antigen

➢ More than one line: Heterogeneity of the antiserum to the antigen

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Applications of immunoelectrophoresis

➢ Detection and identification of proteins present in serum

➢ Normal and abnormal proteins in serum

➢ Analysis of complex protein mixture

➢ Antigen-antibody purity.

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Q6.Which technique is used to identify mixture of antigens?

(A) Single radial immunodiffusion

(B)Double immunodiffusion
(C)Immunoelectrophoresis

Correct Answer: C

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Q3. Which of the following technique is quantitative?

(A) Immunoelectrophoresis
(B) Double immunodiffusion
(C) Radial immunodiffusion

Correct Answer: C

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Q8. Immunoelectrophoresis techniques are designed to separate the

mixture components from each other by using electrophoresis

(A) After reaction with antibody

(B)Prior to reaction with antibody
(C)Prior to reaction with antigen
(D)After reaction with antibody

Correct Answer: B

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Rocket Immunoelectrophoresis
➢ Adaptation of radial immunodiffusion.
➢ Used for determining concentration of antigen in unknown sample

Agarose gel containing Abs

Purified antigens

24-72 hrs 30 min

Radial immunodiffusion With Current

+

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Requirements
Sample
Standard Antigen (unknown
concentration)

➢ Gel casting tray/ slide

➢ Molten agarose

➢ Antibody solution

➢ Standard antigen (known concentration) Antibody

Solution

➢ Sample (unknown concentration of antigen) Molten Agarose

➢ Electrophoresis apparatus

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Steps involved in Rocket Immunoelectrophoresis

Preparation of agarose gel (with antibody)

Loading standard and test antigen solution

Electrophoresis of antigen in agarose gel

Visualization and measurement of height of

rockets

Preparation of standard curve and calculation

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Preparation of agarose gel Loading standard and test antigen

(with antibody) solution on gel

Ab Solution

Ab solution in molten Molten agarose

agarose

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Electrophoresis of antigen & Standard curve preparation &

measurement of heights + calculation

Height of rocket
Concentration of
unknown Ag

Concentration of Ag

Height α Concentration of Ag

- Concentration of test antigen can

be determined by standard curve
(One dimensional single electroimmunodiffusion)

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Counter Immunoelectrophoresis

Ag and Ab should have

opposite charges
Ag Ab
(Two dimensional) (One dimensional)
Double immunodiffusion Electric current

- +

24-72 hrs 30 min

Precipitin band

- +

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Interpretation of Results

➢ Qualitative- Rapid (major advantage).

➢ Presence of precipitin line indicates the presence of specific Ab

➢ Absence of precipitin line indicates absence of specific Ab

➢ More than one precipitin line, heterogeneity of the antibody

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COVID-19 testing

Covid-19
Threee COVID-19 suspected persons

Which one is positive?

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Q9. What is correct for Countercurrent immunoelectrophoresis?

(A) One dimensional

(B) Two dimensional
(C) Adaptation of single immunodiffusion

Correct Answer: A

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Q10.In Rocket Immunodiffusion the length of the rocket is

(A)proportional to the amount of antibody placed in each well

(B)Inversely proportional to the amount of antibody placed in each well
(C)Inversely proportional to the amount of antigen placed in each well
(D)Proportional to the amount of antigen placed in each well

Correct Answer: D

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Q5.More than one lines of precipitin in double immune

diffusion suggests

(A) Heterogeneity of the antibody in the test sera

(B)Homogeneity of the antibody in the test sera
(C)Heterogeneity of the antigens in the test sera
(D)Homogeneity of the antigens in the test sera

Correct Answer: A

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Thank you

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