31 Difference Between Immunoblotting and Immunodiffusion in Serology

Serology, the study of blood serum and other body fluids for diagnostic and research reasons related to immune responses, uses both immunoblotting and immunodiffusion as laboratory techniques. These methods are employed to find and recognise particular proteins, especially antibodies, in a sample.

In serology and molecular biology, immunoblotting, commonly referred to as Western blotting, is a common laboratory procedure. It’s employed to identify particular proteins in a complicated blend of proteins that have been isolated from cells or tissues. Immunoblotting is frequently used in serology to identify antibodies against particular antigens in patient serum samples, assisting in the diagnosis of a variety of illnesses or infections.

The ability to specifically detect antibodies against specific antigens using immunoblotting is a strong serological approach that helps with the diagnosis and understanding of a variety of illnesses, including autoimmune diseases and infectious diseases.

In serology, which is the study of blood serum, immunodiffusion is a laboratory technique used to identify and measure the presence of particular antibodies or antigens in a patient’s blood sample. It is based on the idea that when antibodies and antigens interact, it can result in the formation of observable precipitate lines.

Historically, immunodiffusion has been employed in a variety of diagnostic procedures, particularly for the identification of antibodies to certain infections or antigens linked to disease. The enzyme-linked immunosorbent assay (ELISA) and numerous forms of immunoassays using fluorescence or chemiluminescence detection, however, have largely superseded it as more sensitive and precise methods. These more recent methods provide more accurate measurement, speedier outcomes, and the capacity to identify a greater variety of chemicals.

Even while immunodiffusion may not be used as frequently these days, it nevertheless has its place in laboratory research as one of the original techniques that served as the basis for the creation of more sophisticated immunological testing techniques.

S.No.	Aspects	Immunoblotting	Immunodiffusion
1	Technique	Western blotting technique	Immunodiffusion technique
2	Purpose	Detection of specific proteins	Detection of antigen-antibody
3	Result format	Band patterns	Precipitation lines
4	Electrophoresis	Involved	Not involved
5	Separation of components	By molecular weight	By diffusion distance
6	Sensitivity	High	Lower
7	Specificity	High	Moderate
8	Number of antibodies used	Multiple	Typically one
9	Antigen-antibody interaction	On a solid membrane	In a gel medium
10	Detectable antigens	Proteins	Mostly large molecules
11	Detection range	Wide	Limited to specific antigens
12	Quantitative analysis	Possible with calibration	Limited or not applicable
13	Required equipment	Electrophoresis setup, membrane	Glass slide or agar plate
14	Time-consuming	Yes	Typically faster
15	Sample size	Smaller	Larger
16	Resolution	High	Lower
17	Antibody labeling	Often labeled antibodies used	Unlabeled antibodies used
18	Cross-reactivity	Less common	More common
19	Antibody specificity	Must be well-characterized	Less critical
20	Antigen confirmation	Often denatured	Native conformation preserved
21	Affected by antigens’ charge	Yes	No
22	Quantitative data	Yes, through densitometry	No
23	Cost	Higher	Lower
24	Ease of interpretation	Requires expertise	Easier for simple reactions
25	Utility in clinical diagnosis	Common	Limited
26	Detection of antibodies	Possible	Not suitable
27	Antibody screening	Useful for screening antibodies	Not suitable
28	Diagnostic applications	Used in disease diagnosis	Limited diagnostic potential
29	Sample preparation	More complex	Simpler
30	Handling of multiple antigens	Suitable	Not suitable
31	Typical use cases	Protein analysis, disease research	Initial antigen detection

Frequently Asked Questions (FAQ’S)

Q1. What is the immunoblotting procedure?

Proteins are separated by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), transferred to a membrane (typically nitrocellulose or PVDF), blocked to prevent non-specific binding, incubated with primary antibodies specific for the target protein, followed by secondary antibodies conjugated to enzymes or fluorophores for detection.

Q2. What does immunoblotting's chemiluminescence mean?

In the immunoblotting technique known as chemiluminescence, a secondary antibody that has been enzyme-conjugated produces light when it reacts with a substrate. For visualization, this light emission is recorded on X-ray film or via a digital imaging system.

Q3. What varieties of immunodiffusion are there?

Radial immunodiffusion (RID) and double immunodiffusion (Ouchterlony technique) are the two primary varieties. The Ouchterlony technique enables the visualization of antigen-antibody interaction by producing a grid pattern, whereas RID counts antigens depending on the diameter of the precipitation ring.

Q4. What is the immunodiffusion principle?

The idea behind it is that when antigens and antibodies are allowed to drift towards one another in a gel media, the points of contact and reaction will generate clear precipitation lines.

Q5. What distinguishes immunoelectrophoresis from immunodiffusion?

In contrast to immunoelectrophoresis, which combines electrophoresis with immunodiffusion to separate proteins based on their charge and size before antibody contact in a gel, immunodiffusion uses diffusion in a gel. The resolution and information about complicated protein mixtures are improved by immunoelectrophoresis.