This seminar includes- 1. Characteristics of Antigens and Antibodies 2. Characteristics of Antigen-Antibody Reactions 3. Types of Antigen-Antibody Reactions 4. Factors affecting Antigen-Antibody reactions 5. Grading of reactions 6. Antiglobulin tests 7. Classification to immunodeficiency diseases

1. SEMINAR ON
ANTIGEN-ANTIBODY REACTION
AND
IMMUNODEFICIENCY DISEASES
PRESENTED BY- DR. SHINY
MODERATOR- DR. NIDHI BANSAL

2. TOPICS
1. Characteristics of Antigens and Antibodies
2. Characteristics of Antigen-Antibody Reactions
3. Types of Antigen-Antibody Reactions
4. Factors affecting Antigen-Antibody reactions
5. Grading of reactions
6. Antiglobulin tests
7. Classification to immunodeficiency diseases

3. CHARACTERISTICS OF ANTIGENS AFFECTING IMMUNE RESPONSE
• Size- Antigens too small in size (Molecular weight <10000 D)
cannot produce an immune response
Eg- Haptens produce immune response only when
coupled with a heavy carrier protein
• Conformation- Specific for a specific antibody
• Charge- net charge of the molecule
• Accessibility- of epitopes
• Solubility- antigenic substances which are less soluble
elicit lesser immune response
• Chemical composition- of the stimulus

4. ANTIGENS ARE EITHER RBC ANTIGENS OR LEUKOCYTE ANTIGENS
• RBC antigens are very diverse in structure and composition.
They may be proteins (such as the Rh, M, and N blood group
substances) or glycolipids (such as the ABH, Lewis,
and P blood group substances).
• Human leukocyte antigens are glycoproteins.
• Because of these differences in structure, conformation and
molecular nature, NOT all antigens are equally immunogenic in vivo

5. Characteristics of Blood Group Antibodies
• POLYCLONAL ANTIBODIES-
 An antigen consists of numerous epitopes
 Polyclonal antibodies are produced in response to a single
antigen with more than one epitopes
 They give immunity against the entire antigen, such as a
pathogen
 Example- antisera

6. • Monoclonal Antibodies-
 A homogenous antibody population that targets a single epitope
of an antigen.
 Preferred in testing, to detect molecules of interest.

7. ANTIGEN ANTIBODY REACTIONS
• The antigen and antibody combine with each other
specifically in an observable manner
• to provide immunity in body and to diagnose
diseases in vitro.
• If an antigen is present over the red cells of an
individual, the individual will not form the
corresponding antibody.
• If an antibody is detected in the plasma of an
individual, he/she should lack that corresponding
antigen over red cells.

8. TYPES ON ANTIGEN-ANTIBODY REACTIONS-
• Agglutination- clumping of red cells
• Sensitization- coating of antigen on surface of red cell antigen
• Haemolysis- destruction of red cells with release of hemoglobin
• Neutralization (inhibition)-solubilization of antibodies by soluble
antigens
• Precipitation- insoluble Ag-Ab complex
• Complement fixation
• Radio-immunoassay
• ELISA (Enzyme linked immunosorbent assay)

9. STAGES OF ANTIGEN ANTIBODY REACTIONS
1. Primary stage or the stage of sensitization-
• attachment of antibody to an antigen on red cell membrane
• or binding of antigenic determinants of red cells to a
antigen binding site (Fab region) of the antibody molecule
• Held together by non-covalent bonds
• No visible agglutination seen
2. Secondary stage or stage of lattice formation-
• Cross linking between sensitized red cells
• Visible agglutinates seen

10. FACTORS AFFECTING PRIMARY STAGE
1. Temperature- IgG antibodies- 37 degrees C (warm reactive antibodies)
IgM (ABO antibodies)- 4-24 degrees C (cold reactive antibodies)
2. Incubation time- IgG- 30-45 minutes
IgM- just an immediate spin
3. pH- most optimum 6.5-7.5
Some IgM- lower than 6.5
Change in pH changes structure of antigen and antibody
4. Ionic strength- Low ionic strength solution media (LISS) containing 0.2 % sodium chloride is
considered optimum. It increases rate of antibody uptake by reducing ionic barrier and
decreases incubation time

11. FACTORS AFFECTING SECONDARY STAGE-
1. ANTIGEN-ANTIBODY RATIO-
Prozone effect - antibody excess
-nullified by diluting the solution
Zone of equivalence- optimum proportion of antigen and antibody
Postzone- antigen excess
-nullified by increasing the amount of antibody
Dosage effect- weak expression of antigen on RBCs alters the amount of
antigen

12. 2. Centrifugation- speed and time of centrifuging the solution
3. Distance between red cells- called zeta potential, which is reduced
by using potentiators or enhancement
reagents like LISS, albumin, polybrene etc.
4. Media used- can be enhancement media, protein/enzyme media or
media with low ionic strength further decrease
zeta potential
5. Reagents used- antihuman globin reagents which have specificity
for Fc portion of IgG

13. WHAT IS ZETA POTENTIAL?
• Red cells have a negative charge due to neuraminic
acid on the red cell membrane which makes them to
repel each other.
• This repulsive force that holds the cells apart is called
Zeta Potential
• This distance is very small but sufficient to prevent
small IgG molecules to bridge the gap and agglutinate
the red cells. Hence, they cause coating of red cells
• IgM molecules are large, so they bridge the gap , bring
cells together and cause agglutination.

14. GRADING OF AGGLUTINATION REACTION
 ++++ Complete agglutination of all cells, single large agglutinate.
No free cells
 +++ Numbers of large agglutinates. No free cells
 ++ Small to medium clumps with few free cells
+ Fine granular appearance visually but definite small clumps
per low field
-ve No agglutination
When different grades are seen together, it is called mixed field
agglutination

15. ANTIGLOBULIN TESTS
Antiglobulin test is based on the following principle:
• Antibody molecules (IgG) and complement components are
globulins.
• Antihuman globulin (AHG) is an antibody against human
globulin molecules that reacts with them either bound to the
RBCs or present free in the serum.
• Washed red cells coated with human globulins (IgG and
complement) are thus agglutinated by AHG, resulting in visible
agglutination.

16. NEED FOR AHG TESTING- TO DETECT AUTO/ALLO ANTIBODIES ON
SENSITIZED RED CELLS
Autoantibodies- The immune system recognizes and
tolerates self-antigens. The failure to tolerate self-
antigens may result in the formation of antibodies to
self antigens, and such antibodies are called
autoantibodies.
Alloantibodies- The immune response to the
transfused antigens is mainly humoral, mediated by B
cells and resulting in the formation of antibodies
towards foreign antigens of same species called
alloantibodies.

17. TYPES OF AHG TESTS-
1. Direct antiglobulin test / direct Coombs test (DAT/DCT)-
DAT is used to detect in-vivo sensitization of red cells where
the addition of AHG directly into a suspension of washed red
cells of the patient will result in visible agglutination
2. Indirect antiglobulin test / Indirect Coombs test (IAT/ICT)-
An indirect antiglobulin test is used to detect the presence of
red cell antibodies in patient’s plasma by sensitizing the red
cells in-vitro and then detecting the in-vitro sensitized red cells
using antiglobulin reagent

20. CLASSIFICATION OF IMMUNODEFICIENCY DISEASES
• B- cell disorders
• T- cells disorders
• Combined B and T cell disorders
• Phagocytic dysfunction disorders

21. ANTIBODY (B CELL) IMMUNODEFICIENCY DISORDERS
• X-linked hypogammaglobulinemia (congenital hypogammaglobulinemia)
• Transient hypogammaglobulinemia of infancy
• Common, variable, unclassifiable immunodeficiency (acquired by
hypogammaglobulinemia)
• Immunodeficiency with hyper-IgM
• Selective IgA deficiency
• Selective IgM deficiency
• Selective deficiency of IgG subclasses
• Secondary B-cell immunodeficiency associated with drugs
• X-linked lymphoproliferative disease

22. CELLULAR (T CELL) IMMUNODEFICIENCY DISORDERS
• Congenital thymic aplasia (DiGeorge syndrome)
• Chronic mucocutaneous candidiasis (with or without
endocrinopathy)
• T-cell deficiency associated with purine nucleoside phosphorylase
deficiency
• T-cell deficiency associated with absent membrane glycoprotein
• T-cell deficiency associated with absent class I or II MHC antigens or
both (base lymphocyte syndrome)

23. COMBINED ANTIBODY-MEDIATED (B CELL) AND CELL
MEDIATED (T CELL) IMMUNODEFICIENCY DISORDERS
• Severe combined immunodeficiency disease (autosomal recessive, X-linked, sporadic)
• Cellular immunodeficiency with abnormal immunoglobulin synthesis (Nezelof syndrome)
• Immunodeficiency with ataxia-telangiectasia
• Immunodeficiency with eczema and thrombocytopenia (Wiskott-Aldrich syndrome)
• Immunodeficiency with thymoma
• Immunodeficiency with short-limbed dwarfism
• Immunodeficiency with adenosine deaminase deficiency
• Immunodeficiency with nucleoside phosphorylase deficiency
• Biotin-dependent multiple carboxylase deficiency
• Graft-versus-host disease
• AIDS

24. PHAGOCYTIC DYSFUNCTION
• Chronic granulomatous disease
• Glucose-6-phosphate dehydrogenase deficiency
• Myeloperoxidase deficiency
• Chediak-Higashi syndrome
• Job’s syndrome
• Tuftsin deficiency
• Lazy leukocyte syndrome
• Elevated IgE, defective chemotaxis, and recurrent infections

25. TRANSFUSION ASSOCIATED GRAFT VERSUS HOST
DISEASE (TA-GVHD)
• It is a type of Combined Antibody-Mediated (B Cell) and Cell
Mediated (T Cell) Immunodeficiency Disorder
• Mortality rate of TA-GVHD- 90%
• TREATMENT-
• Immunosuppresive drugs post transfusion
• Gamma irradiation for all blood components pre-transfusion

26. HEMOLYTIC DISEASE OF NEWBORN
• Hemolytic disease of the fetus and newborn (HDFN) can result when the
maternal immune system produces an antibody directed at an antigen
present on fetal cells but absent from maternal cells.
• The mother is exposed to fetal RBCs as a result of feto maternal transfer of
cells during pregnancy or childbirth.
• Maternal memory cells can cause a stronger response during a second
pregnancy if the fetus is positive for the sensitizing antigens.
• IgG1, IgG3, and IgG4 are capable of crossing the placenta and attaching to
fetal RBCs, whereas IgG2 and IgM are not.

27. REFERENCES
• Harmening DM. Modern Blood Banking & Transfusion Practices 7th edition
• DGHS. Tranfusion Medicine Technical Manual 3rd edition
• Makroo RN. Principles & Practice of Transfusion Medicine 2nd edition