Immune system; Antigens and antibodies; Epitopes, haptens and adjuvants; Complement system; Raising of anti-sera; Antigen-antibody reactions; Cellular antigens; ABO blood group system: A, B and H antigens; Bombay blood group; Rh blood group system; Blood transfusion; Transfusion reaction; Maternal reaction; Other blood groups: MN, Lewis, Lutheran, Kidd, Duffy and P systems; Secretor phenomenon; Origin testing.

1. FORENSIC SEROLOGY

3. Antigens
• The term antigen is derived from antibody generation, referring to any substance
that is capable of eliciting an immune response (e.g., the production of specific
antibody molecules). By definition, an antigen (Ag) is capable of combining with
the specific antibodies formed by its presence.
• Generally, antigens are foreign proteins or their fragments that enter host body
via an infection.
• However, in some cases, the body’s own proteins (Autoantigens) may act as
antigens and induce an autoimmune response.
• Bacteria and viruses contain antigens, either on their surface (Exogenous
antigen) , or inside (endogenous antigen). These antigens can be isolated and
used to develop vaccines.

4. Antigens

5. Antigens

6. Epitopes • The small site on an antigen to which a
complementary antibody may specifically bind is called
an epitope or antigenic determinant.
• This is usually one to six monosaccharides or five to
eight amino acid residues on the surface of the
antigen.
• Because antigen molecules exist in space, the epitope
recognized by an antibody may be dependent upon the
presence of a specific three dimensional antigenic
conformation (e.g., a unique site formed by the
interaction of two native protein loops or subunits).
This is known as a conformational epitope.
• The epitope may also correspond to a simple linear
sequence of amino acids and such epitopes are known
as linear epitopes.

7. Epitopes

8. Epitopes

9. Antigens
• However, this specific immune response is highly variable and
depends much in part on the size, structure, and composition of
antigens.
• Proteins or glycoproteins are considered as the most suitable
antigens due to their ability to generate a strong immune response; in
other words, they are strongly immunogenic.
• Antigens are recognized by the host body by two distinct processes:
• by the T cell receptor on T cells.
• by B cells and their surface antibodies (IgM) and

10. Immune response

11. HAPTENS
• Antigens are generally of high molecular weight, and commonly are proteins or
polysaccharides. Polypeptides, lipids, nucleic acids, and many other materials
can also function as antigens.
• Immune responses may also be generated against smaller substances, called
haptens, if these are chemically coupled to a larger carrier protein, such as
bovine serum albumin, keyhole limpet hemocyanin (KLH), or other synthetic
matrices.
• A variety of molecules such as drugs, simple sugars, amino acids, small peptides,
phospholipids, or triglycerides may function as haptens.
• .

12. Antigens

13. Antigens

15. ADJUVANTS VERSUS HAPTENS

16. ANTIBODIES
• An antibody is defined as “an immunoglobulin capable of specific
combination with the antigen that caused its production in a
susceptible animal.”
• Antibodies are produced in response to the invasion of foreign
molecules in the body. An antibody, abbreviated as Ab, is commonly
referred to as an immunoglobulin or Ig.
• Human immunoglobulins are a group of structurally and functionally
similar glycoproteins (82-96% protein and 4-18% carbohydrate) that
confer humoral immunity.

17. ANTIBODIES
• Antibodies exist as one or more copies of a Y-
shaped unit, composed of four polypeptide
chains.

18. ANTIBODIES
• Each Y contains two identical copies of a heavy
chain and two identical copies of a light chain,
named as such by their relative molecular weights.

19. ANTIBODIES
This Y-shaped unit is composed of the
two variable, antigen-specific F(ab) arms,
which are critical for actual antigen
binding, and the constant Fc “tail” that
binds immune cell Fc receptors and also
serves as a useful “handle” for
manipulating the antibody during most
immunochemical procedures.

20. ANTIBODIES

21. ANTIBODIES

22. COMPLEMENT SYSTEM

24. Polyclonal Antibodies Monoclonal Antibodies
Short production time (2-3)
and low cost.
Developing a monoclonal takes
time (>6 months) and requires
high technical skills.
Mixed population of antibodies Single population of antibodies
High affinity Highly specific recognition of
only one epitope of an
antigen
Cross-reactivity possible Cross reactivity low
Polyclonal antibodies are
less sensitive to antigen
changes (slight
denaturation, polymorphism,
heterogeneity of
glycosylation)
Monoclonal antibodies are
sensitive to antigen changes
(slight denaturation,
polymorphism,
heterogeneity of
glycosylation)

25. Polyclonal Monoclonal

26. BLOOD
Erythrocytes

27. BLOOD
Erythrocytes

32. ABO GROUP• The ABO blood group is determined by the presence or
absence of Antigens on the RBC's named A and B.
• At present, the human transfusion regulating body has
recognised 43 significant blood group systems, of
which the ABO system is most important.
• A mismatch in the ABO system during transfusion can
cause potentially fatal adverse reactions.
• ABO type group is present in other primates such as
apes and even in the old world monkeys.

33. ABO GROUP
 ABH Antigens
 ABH antigens synthesis (glycosyltransferase
enzymes that sequentially add specific
monosaccharides to glycoproteins and
glycolipids.
 H antigen is a precursor for A and B antigens.
 H antigen concentration by ABO blood group
varies (O>A2>B>A2B>A1>A1B).
 ABH antigen (secretor status) found on other
tissues, including endothelial and epithelial
cells of lung, gut, and urinary and
reproductive tracts.
 ABO antigens are not fully developed at birth.
But appears on the RBCs by 2–4 years.
 Approximately, 80% of group A individuals are A1, while
∼20% are A2; subgroups A3, Ael, Ax, etc.
 A1 and A2 is both quantitative (∼five times fewer A antigens
on A2 than A1 RBCs) and qualitative (structural differences)
 Dolichos biflorus lectin is used to distinguish A1 from A2, as
the lectin is diluted to agglutinate A1 but not A2 or weaker
subgroup RBCs.

34. ABO GROUP
 These antibodies are
termed “naturally
occurring,”
 Antibody production
begins after birth and is
usually detectable by 4–
6 months of age,
reaches peak at age 5–
10 years, and then
declines with increasing
age.
 Immunodeficient
patients
 These antibodies are
primarily IgM with some
IgG

36. ABO GROUP

37. ABO GROUP
• The ABO blood type was discovered by Karl Landsteiner in
1901 for which he received the Nobel prize in Physiology or
Medicine in 1930.
• He was working in the Pathological-Anatomical institute of
the University of Vienna.
• In 1900, using a microscope and a workbook Karl Landsteiner
for the first time mixed blood of two individuals in a test tube
and described "clumped blood cells" which later got to be
known as "agglutination".
• In 1901, Karl Landsteiner again made a refined discovery
stating that "blood serum of an individual would agglutinate
with only those of certain individuals".
• In 1910, Ludwik Hirszfeld and Emil Freiherr von Dungern
were also the first to explain the genetic inheritance of the
blood groups.

38. ABO GROUP• The first practical use of blood typing in transfusion was by an
American physician Reuben Ottenberg in 1907. And the
large-scale application started during the First World War.
• Felix Bernstein demonstrated the correct blood group
inheritance pattern of multiple alleles at one locus in 1924.
• Watkins and Morgan, in England, discovered that the ABO
epitopes were conferred by sugars, to be specific, N-
acetylgalactosamine for the A-type and galactose for the B-
type.
• 1978, Yamamoto's group showed the precise glycosyl
transferase set that confers the A, B and O epitopes.

40. 0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50%
Caucasian
African-American
Asian
45%
49%
43%
40%
27%
27%
11%
20%
25%
4%
4%
5%
AB B A O

41. BLOOD
Erythrocytes

42. Bombay and Para-Bombay Phenotypes:
• Bombay individuals lack ABH antigens on RBCs, tissues, and secretions
and make anti-A, anti-B, and potent anti-H. These individuals must be
transfused with RBCs from other Bombay individuals, which are very
rare.
• Para-Bombay individuals lack H antigen on their RBCs but have H
antigen in their secretions. /Para-Bombay also describes individuals who
have minimal H on their RBCs, regardless of secretor status.

43. BLOOD
Erythrocytes

44. BLOOD
Erythrocytes

46. Secretors and Non-
Secretors
*mutation in FUT2 gene causes non secretor
status
FUT gene is located on chromosome 19

47.  This method involves the addition of tittered
antiserum (1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256)
to the stain (bodily secretions).
 The titer of the antiserum is adjusted so that most the
antibodies will be bound by the quantity of the stain
employed in the test if homologous antigen is present.
 Dry cavity tile mark A, B, H and place one drop of
suitable dilution of Anti A serum, Anti B serum, Anti H
serum.
 Add 1 drop of extract in cavity and incubate at 4
degree for 2 hours. If the corresponding antigen is
present in the stain, it will react with antibody and
decrease the titer of antiserum, so that it is no longer
available for agglutination of known test cells.
 Add one drop of 0.2% indicator cells and keep at 4
degree for 30 minutes.
 Thus the absence of agglutination of known test cells is
indicative of the presence of the antigen in the stain.
Absorption – inhibition test employed for ABO grouping of body
fluid stain.

49. Erythroblastosis Fetalis

52. OTHER
BLOOD
GROUP
SYSTEMS
• Other than the ABO and RH systems, there are many
other blood group systems.
• Usually the pattern follows ABO system - the RBCs have
the antigens coated on the cell surface & the antibodies
are found in the plasma.

53. OTHER BLOOD GROUP SYSTEMS
• These antigens also have a physiological function in our body.
• They are membrane transporters or channels - e.g. Rh, Kidd
• Membrane-bound enzymes - e.g. kell
• Structural proteins - e.g. MNS
• Chemokine receptor - e.g. Duffy
• Cell adhesion molecule - e.g. Lutheran
• Complementary regulatory proteins - e.g. Cromer

54. OTHER BLOOD GROUP SYSTEMS
Classification of the different blood groups can be Antigen or
Antibody-based.
Antigen based – Carbohydrate / Protein antigens
• Carbohydrate antigens are ABO, Lewis, P1PK, I, etc.
• Protein-based antigens are MNS, Rh, Lutheran, Kell, Duffy etc.
Antibody based - Warm or cold antibodies.
• Cold Antibodies are Lewis.
• Warm antibodies are Rh, Kell, Duffy, Kidd

55. Blood group Gene name (locus) Transfusion reaction Antigen Antibodies
Rh RHD,RHCE (1p36.11) delayed hemolytic
transfusion reactions
(HTRs) and hemolytic
disease of the fetus
and newborn (HDFN).
 56 (C, c, E, e, D, and
more other antigens)
 12-transmembrane
span model
 Presence or absence of
the D antigen confers
the Rh-positive or Rh-
negative status
Rh antibodies are IgG
(some have IgM
component) and are
clinically significant
MNS GYPA,GYPB,GYPE
(4q31.21)
Anti-M reactivity at
37°C has occasionally
been associated with
(HTR) and (HDFN)
 50 (major antigens are
M, N, S, s, and U.
 single-pass membrane
sialoglycoproteins
Anti-M is primarily
IgM but may have an
IgG component .

56. Blood group Gene name
(locus)
Transfusion reaction Antigen Antibodies
Lewis FUT3 (19p13.3) Antibodies are naturally
occurring and are not usually
clinically significant
 6
 (Lea and Leb) adsorbed onto
RBCs, platelets, and
lymphocytes from plasma
glycolipids.
 also widely distributed on
human tissues, present in
soluble form in body fluids
 serve as receptors for some
pathogenic bacteria.
 predominantly found in
persons with Le(a−b−)
RBCs
 primarily IgM and
reactive at temperatures
below 37°C; however,
mixtures of IgM/IgG or
pure IgG, which are
reactive at 37°C, do exist.
Lutheran BCAM (19q13.2)  Anti-Lua has not been
associated with HTRs and
has rarely been
associated with mild
HDFN.
 Anti-Lu b reported
causing mild HTRs and
mild HDFN.
 Anti-Lu3 is found in
immunized individuals (Lu
null phenotype) may
cause delayed HTRs or
HDFN
 27
 major antigens are Lua (low
prevalence) and Lu b (high
prevalence).
 The rare Lu null phenotype

57. Blood group Gene name (locus) Transfusion reaction Antigen Antibodies
Kidd SLC14A1(18q11-
q12)
 Kidd antibodies
are responsible
for about one-
third of all
delayed HTRs
(severe).
 rarely cause
HDFN (but if
present it is not
severe).
 3
 Jka , Jkb, and Jk3;
however, Jka and
Jkb are the most
important
antigens in
routine practice.
 rare null
phenotype,
Jk(a−b−) lack the
high-prevalence
Jk3 antigen.
 The Kidd protein
functions as a
urea transporter
 mainly IgG but
can be partially
IgM.

58. Blood group Gene name (locus) Transfusion
reaction
Antigen Antibodies
Duffy ACKR1(1q21-q22)  linked to acute
and delayed
hemolytic
transfusion
reactions, as well
as fetal and
newborn
hemolytic
disease.
 5
 Duffy antigens
Fya (Fy1) as well
as Fyb (Fy2)
 antigens function
as receptors for
chemokines
 also receptors
for the malaria
parasites
Plasmodium
knowlesi and
Plasmodium
vivax.
IgG subclass as well
as are clinically
significant
P1PK A4GALT(22q13.2)  Anti-P1 has not
caused HDFN
and HTR.
 3
 P1PK system is
comprised of P1,
Pk and NOR
antigens
anti-P1, which is
cold-reactive IgM
antibody that is
usually “naturally
occurring.”

59. Human Blood Animal Blood
Blood Cells Red blood cells, white blood cells, and
platelets are the cellular components of
the human blood.
Most animals comprise red blood cells,
white blood cells, and platelets. Animals
with a closed circulatory system comprise
hemocytes.
Blood Group Humans comprise ABO blood group and
Rh blood group.
Animals comprise different blood groups
Respiratory Pigments Hemoglobin is the respiratory pigment
of humans and other vertebrates
Hemoglobin, haemerythrin, haemocyanin,
and chlorocruorin are the four types of
respiratory pigments in invertebrates.
Blood Color Human blood is red in color. Red, blue, green, and pink are the colors of
the blood in animals.
Red Blood Cell Antigens A, B, and Rhesus antigen are the three
types of antigens present in the red
blood cells of humans.
Red blood cells of animals may contain
different types of antigens.
Red Blood Cell Nucleus and
Organelles
Humans and other mammals do not
have a nucleus and organelles in red
blood cells.
Animals other than mammals have a nucleus
and organelles in red blood cells.
White Blood Cells Humans have five types of white blood
cells.
Different animals have different types of
white blood cells.

60. SPECIES OF ORIGIN
Most assays for species
identification
 immunochromatographic assays
 precipitation-based assays
include ring assays, Ouchterlony
assays, and crossed-over
immunoelectrophoresis.

61. Immunochromatographic assays
OBTI and
the ABAcard
HemaTrace®
Human Hemoglobin
Protein
RSID™-
Blood
Identification of
Human Glycophorin A
Protein

62. precipitation-
based assays
include ring
assays
RING ASSAY PROCEDURE:
 Sample preparation and extraction
 Controls (positive and negative)
 Loading of antibody and samples
 Immunodiffusion reaction

63. OUCHTERLONY
ASSAY
• Cross over gel diffusion is a technique where antigens and
antibodies are left to react overnight on a gel plate.
• Agglutination / precipitation is recorded by bands that
are formed between wells.
OUCHTERLONY ASSAY PROCEDURE:
 Sample preparation
 Controls • Positive (known serum) • Negative (extraction
blank) • Substrate controls (extraction of substrate from
unstained area)
 Agarose gel preparation
 Loading antibodies and samples
 Immunodiffusion reaction

65. Immuno electrophoresis
• But cross-over gel diffusion takes a
lot of time and the little sample that
we have will get evaporate by then.
• Electrophoresis helps to speed up
the process/diffusion.
• We perform horizontal
electrophoresis using an agarose
gel.
• Antigens and Antibodies are plotted
at 2 different ends of the agarose
plate.
• Multiple samples can be tested at
the same time.

66. Immuno-electrophoresis
• The sample load (Ammonia extract)
must face the cathode (black end) and
the antisera must face the anode (red
end).
• Use filter paper/tissue paper wicks
moistened in tank buffer to connect
the gel plate with the tank buffer.
• The usual electrophoresis run is set at
150V, 15mA for 20 minutes.