Immune system of the body is capable of distinguish the molecules

1. IMMUNOGLOBULINS &ELISA

2. IMMUNE SYSTEM
• Immune system of the body is capable of distinguishing the molecular
'self' particles from'nonself' (foreign) particles and then destroying
the nonself particles.

3. Basic terminology
Antigen
Antibody
Immunity

4. Antibody
• Substance produced in response to antigens is called antibody.

5. Antigen
• Any substance that is capable of stimulating the synthesis of specific
antibody is antigen

6. Immunity
• Immunity is the capacity of body to resist against pathogenic foreign
particles.

7. Types of Immunity
a) Innate immunity :It is the immunity that an individual has from birth.
It can be species, racial or individual specific immunity.
b) Adaptive immunity: It is the immunity that an individual gets during
life.

10. Humoral (or antibody mediated) immunity: B lymphocytes are
responsible for humoral immunity. During course of immune response,
B-cells mature into plasma cells & secrete immunoglobulins to fight
against antigens.

11. Cell mediated immunity: T lymphocytes (T cells) are responsible for cell
mediated immunity They can directly attack the antigens.

12. There are 4 types of T cells:
1) Killer or cytotoxic T cells (CD8 cells): Destroys infected cells.
2) Helper T cells (CD4 cells): Modulate activities of other immune cells.
3) Suppressor T cells: Turn off an immune response.
4) Memory T cells: Key components of acquired immune response

13. Immune response
• It is the capacity of body to produce antibodies in response to
antigen.
There are two types
a) Primary immune response &
b) Secondary immune response

14. a) Primary immune response:
• When an antigen is injected for the first time, the immune response is
called primary immune response. Antibodies in blood appear in about
10 days, peaks in 20 days & declines by about a month. IgM
molecules are predominant in primary immune response.

15. Secondary immune response:
• When the same antigen is re-injected into same animal after some
duration, the immune response is called secondary immune
response.
• IgG molecules will be predominant in secondary immune response.
Antibody production is faster (within 3 days) stronger (100 to 1000
times more quantity), and longer (lasts for months).
• This is due to the memory cells produced during primary immune
response. This forms the basis of vaccination.

16. Immunoglobulins (Ig)
• The immunoglobulins are γ -globulins, called antibodies.
• Constitute about 20% of all the plasma proteins
• produced by plasma cells and by lymphocytes in
response to a variety of antigen.

17. Schematic structure of immunoglobulin G.

18. ▪ The basic immunoglobulin is Y shaped consist of four
polypeptide chains:
– two H and
– two L chains
▪ The four chains are linked by disulfide bond
▪ L chain may be either of two types, Kappa (қ) or Lambda
(λ) but not both

19. ▪ The heavy chains may be of five types and are designated by
Greek letter:
– alpha (α)
–gamma (γ),
– delta (δ),
– mu (μ) and
– epsilon (ε)
▪ Immunoglobulins are named as per their heavy chain type as
IgA, IgG, IgD, IgM and IgE

21. ▪ The L and H chains are subdivided into variable ( towards
the carboxyl terminal end) and constant ( towards the
amino terminal end) regions.
▪ L chain consists of one variable (VL) and one constant
(CL) domain or region.
▪ Most H-chains consist of one variable (VH) and three
constant (CH-1, CH-2, and CH-3) domains.

22. ▪ The hinge region between the CH-1 and CH-2 domains
confers flexibility and allows both Fab arms to move
independently , thus helping them to bind to antigenic site

23. Light chain type
• Light chain may be either of two types, Kappa (k) or
Lambda (l) but not both.
• In a given immunoglobulin either 2k or 2 l but not the
mixture of kappa and lambda
• Most abundant light chain in human is k

24. ▪ Enzyme (papain) digestion splits the immunoglobulin
molecule into two fragments
1. Fab: Fragment for antigen binding. Located in variable
region.
2. Fc: Crystallisable fragment or fragment for
complement binding

30. Monomeric, dimeric, and pentameric forms of
immunoglobulins.

33. ▪ Most common Ig in serum: IgG
▪ Least common Ig in serum: IgE
▪ Largest Ig: IgM
▪ Pentamer Ig: IgM
▪ Dimmer: Secretary IgA
▪ Ig fixes the complement: IgG and IgM
▪ Ig present in secretion: IgA

34. ▪ Ig can cross placenta: IgG
▪ IgG can not cross placenta: IgG2Ig involved in primary
immune response: IgM
▪ Ig secondary immune response: IgG
▪ Ig with J chain: IgM and IgA
▪ Ig with secretary piece: IgA

35. Disorders due to changes in immunoglobulins
Quantitative changes in the amount of immunoglobulins in the
plasma and urine are known in several pathological conditions in
human.
• Multiple Myeloma
• Amyloidosis
• Bence Jones Proteins
• Cryoglobulinaemia
• Waldenstrom’s Macroglobulinaemia

36. ELISA
• ELISA, or Enzyme-Linked Immunosorbent Assay, is a laboratory
technique and diagnostic test commonly used to detect and measure
the presence of specific proteins, antibodies, antigens, or other
molecules in a sample of biological fluid (such as blood, serum,saliva
or urine).
• It is widely employed in medical and scientific research, clinical
diagnostics, and various fields of biology.

37. A Direct Enzyme-Linked Immunosorbent Assay (ELISA) is a laboratory
technique used to detect the presence of a specific antigen (a molecule or
substance of interest, like a protein) in a sample.
Materials Needed:
• A microplate with wells.
• The sample containing the antigen of interest.
• A known primary antibody specific to the antigen.
• An enzyme-linked secondary antibody that specifically binds to the primary
antibody.
• A substrate solution that the enzyme can act on.
• Equipment for washing and measuring absorbance (a microplate reader).

38. PROCEDURE
Coating the Microplate:
➢Take a microplate with wells.
➢Add the sample containing the antigen directly to each well.
➢The antigen sticks to the bottom of the wells through physical adsorption.
Blocking:
➢To prevent non-specific binding of other molecules, add a blocking solution
(e.g., BSA or milk) to fill any remaining empty spaces in the wells.
➢Incubate to ensure thorough blocking.

39. Primary Antibody Binding:
➢Add a known primary antibody that specifically recognizes and binds
to the antigen you want to detect.
➢Allow the primary antibody to bind to the antigen in the wells.
Washing:
➢Wash the wells multiple times with a buffer solution to remove
unbound primary antibodies or any other molecules that may be
present.
Secondary Antibody Binding:
➢Introduce an enzyme-linked secondary antibody that specifically
binds to the primary antibody.
➢This secondary antibody is usually labeled with an enzyme like
horseradish peroxidase or alkaline phosphatase.

40. Washing:
➢Wash the wells again to remove any unbound secondary antibodies.
Enzyme Substrate Addition:
➢Add a substrate solution that the enzyme can act on. The enzyme
converts the substrate into a detectable signal, which may be a color
change or fluorescence.
Signal Measurement:
➢Use a microplate reader to measure the intensity of the signal (e.g.,
absorbance at a specific wavelength or fluorescence intensity).
➢The intensity of the signal is directly related to the amount of antigen
present in the sample.

41. Data Analysis:
➢Compare the signal from your sample wells to a standard curve or
control wells to determine the concentration of the antigen in your
sample.