Describes the how heterophile antibodies interfere with Ag-Ab reaction

2. Interfering antibodies affecting the results
of immunoassays
Dr. Arwa Mohammed Othman
Associate professor of immunology and microbiology

3. Outlines
Antibody structure
Antigenic epitope
Antibody affinity and avidity
Antibody cross reactivity
Heterophile antibodies
Hetrophile blocking reagents

4. Antibody structure

5. Antibody structure
Antigen binding site
Antigen binding site
Fc portion

6. Antibody structure
Idiotype
Variation in the variable region
Isotypes
Variation at constant region
IgG, IgM, IgA, IgE

7. Antibody structure
Allotypes
Variation between individuals

8. Epitope

9. Multivalent
Epitope

10. Antibody specificity

11. Antibody specificity
Variable region determines the antibody specificity

12. Antibody affinity and avidity
Antibody affinity
is the strength of the reaction between a single antigenic determinant
and a single combining site

13. Antibody affinity and avidity
Avidity
is a measure of the overall strength of binding of an antigen
with many epitopes and multivalent antibodies.

14. Epitope

15. Types of bonds
Antibodies bind reversibly to epitopes through non-covalent
interactions which include
Hydrogen bonds
Ionic bonds
Hydrophobic bonds

16. Factors Affecting Antigen-Antibody Interaction
1- Temperature:
It depends on the chemical nature of epitopes, and bonds involved.
Example
hydrogen bonds are stable at low temperatures
Hydrophobic bonds are stable at high temperatures.

17. Factors Affecting Antigen-Antibody Interaction
2- pH:
Optimal pH range is 6.5 to 8.5.
Extreme pH values change the conformation of antibodies and
inhibit the reaction.

18. Factors Affecting Antigen-Antibody Interaction
3- Concentrations of Ag and Ab:
Increase in the concentration of antigen and antibody enhances the
reaction.
Antigen + Antibody ⇄ Ag-Ab complex

19. Cross reactivity
Refers to the ability of one antibody to react with more than
one epitope

20. Cross reactivity
Common source of interferences in diagnostic
immunoassays.

21. When cross reaction occurs?
Cross reactivity

22. Cross reactivity
How to overcome cross reactivity?

23. 2- Site immunometric (sandwich) assays
Contains two antibodies:

24. Immunochromatographic assays

25. ELISA vs immunochromatography
Area reaction in Immunochromatographic is small, so low amount of
Ag/Ab can give false negative results (lower sensitivity)

26. ELISA vs immunochromatography
No washing in Immunochromatographic, so Abs that bind weakly
can give false positive results (lower specificity)

27. Enzyme-labelled antibodies
EDTA and sodium fluoride is not recommended since they inhibits
horseradish peroxidase.

28. Heterophile Antibodies
Heterophile Abs are endogenous Abs which can cross-react
with several antigenic epitopes.

29. Heterophile Antibodies
Heterophile Abs can be of the IgG, IgM, or IgA isotype.

30. Heterophile Antibodies
They bind to the
Fc region (mostly) of the assay Abs
idiotope
hinge region

31. Heterophile Antibodies
Heterophilic antibodies have normally low binding affinity

32. Heterophile Antibodies
They can bind to Igs of other species, including the species
used to generate the antibodies used as reagents for
immunoassays
interfere with test results

33. Heterophile Antibodies
They reduce assay sensitivity and specificity of the test

34. Common heterophilic interfering antibodies
1- Rheumatoid factor
•autoantibodies (usually IgM isotype)
•Found in patients with RA

35. Common heterophile interfering antibodies
1- Rheumatoid factor
•Found in 1% to 4% of the general population
•can react against different species of Fc-IgG, including
human and rabbit.

36. Common interfering antibodies
2- Human anti-animal antibodies (HAAAs)
Human anti-mouse antibodies (HAMA)*
They are human antibodies which can bind specifically mouse
antibodies.
They could be both HAMA-IgG or HAMA-IgM subtypes.

37. Common interfering antibodies
3- Other heterophilic antibodies
•goat (HAGA)
•sheep (HASA)
•rabbit (HARA)

38. Heterophile Antibodies
Increase the use of “sandwich” assay has led to growing
concern over the heterophilic antibodies.

39. Types of heterophiles interference
Bridging Blocking

40. Heterophile blocking reagents
They are preparations that prevent non-specific binding of
heterophilic antibody
They are antibodies

41. Heterophile blocking reagents
They must be the same species of capture and detection
assays Abs
so typically more than
one type must be used

42. Heterophile blocking reagents
Characteristic of ideal HBR
1- It should have the ability to correct interference from all
samples
2- It should be effective at low concentrations
3- It should not interfere with linearity of dilution of a true
positive patient sample.

43. Heterophile blocking reagents
Characteristic of HBR
4- It should have long term stability
5- Effective cost