Serology and

Immunology Laboratory
Immunology : is the study of the body's immune system and its functions and disorders.

Serology: is the study of blood serum (the clear fluid that separates when blood clots).

Immunology and serology laboratories focus on the following:

• Identifying antibodies. These are proteins made by a type of white blood cell in response to a foreign
substance (antigen) in the body.
• Investigating problems with the immune system. These include when the body's immune system
attacks its own tissues (autoimmune diseases) and when a body's immune system is underactive
(immunodeficiency disorders).
• Determining organ, tissue, and fluid compatibility for transplantation

# Sample Receiving:

• Plain Gel Tube ( Yellow Cap ) : Serum ; For Ab and Viral studies, have clot activator and gel
separator. purpose of gel is to separate serum from cells.

• EDTATube( Lavender Tube) : (Ethylene Diamine TetraAcetic acide ); For NAT and PCR
Studies.

# Sample Rejection Slip:

• Specimen Hemolyzed
• Specimen Clotted
• Quantity not sufficient (QNS)
‣ Under filled
‣ Over filled
• Incomplete request form
‣ No Doctor sign or stamp
‣ No Diagnosis
‣ Test not recommended or indicated
• Mismatched file name / number on request and tube
‣ Specimen Unlabeled
‣ Specimen Mislabeled
‣ Specimen Inadequately labeled
• Wrong collection tube
• Contaminated requests
• Inconsistent with previous results
# Agglutination immunoassay:
( Agglutination occurs due to the cross-linking of antibodies with particulate antigens to form
clumping of insoluble particles. when antigen an erythrocyte the term hemagglutinin is often used)

# ZONE PHENOMENON: #
The amount of precipitate formed is greatly influenced by the relative
proportions of Ags & Abs
If increasing quantities of Ags are added to the same amount of antiserum in different
tubes, precipitation is found to occur most rapidly & abundantly in the middle tubes

> Preceding tubes – Ab excess ( Prozone )

> Middle tubes – Ag & Ab in equivalent proportions ( Zone of equivalence )
> Later tubes – Ag excess ( Post zone )

A. LATEX:
Antigen— Antibody reactioncan’tbevisualizedunlessweuseLATEX "Polystyrene
latex beads", result will be in the form; agglutination, clump, aggregation.

test includes under LATEX are;

 1. CRP ( C — REACTIVE PROTEIN ) : "used as follow—up test"

CRP is non-specific protein that appears in serum as a response to inflammatory conditions.

* Principle; The assay is done using a suspension of latex particles coated with
anti- human CRP antibodies against unknown serum. The presence of agglutination
indicates an increase of CRP levels in serum (clinically significant).

Procedure:
1- 50 µl of samples, 50 µl of positive control, 50 µl of negative
control. 2- Add 1 Drop of CRP reagent to each separate circles on
the slide.
3- Mix well using disposable stirrer spreading the mixture over
the whole test area, 4 minutes on the shaker.
4. Look for the results.
 2. RF ( RHEUMATOID ARTHRITIS FACTOR ) :

is an autoantibodies that react with individuals own immunoglobulin. These antibodies are
usually directed against the Fc portion of the human IgG. RF have been associated with three
major immunoglobulin classes: IgM, IgG, and IgA. Of these IgM and IgG are the most common. The
formation of immune complex in the joint space leads to the activation of complement and
destructive inflammation, causing rheumatoid arthritis (RA).

It is used for the diagnosis of Rheumatoid arthritis, But it is not the conformational
test because it is observed in other related and unrelated diseases.

3. ASO ( Anti — Sterptolysin O ) :

screening test for determination the presence of antibodies generated by the body against
infections by group A streptococcus pyogen mostly for Tonsillitis and Rheumatic fever, this
m/o produce the toxin is called streptolysin O, which cause destruction of RBCs cell walls. The
O stands for oxygen-labile; the other toxin that secreted is an oxygen-stable “streptolysin S”.
* Principle of the test :
A stabilized buffered suspension of polystyrene latex particles have
been coated with streptolysin O when is mixed with a serum
containing antibodies to streptolysin O ,agglutination occur.
Procedure:

1- Bring all reagents and specimens to room temperature.

2- Place one drop (50 µl) of the positive control and 50 µl of the patient
serum into separate circles on the glass slide.
3- Shake the ASO latex reagent gently and add one drop (45 µl) on each
circle next to the sample to be tested and control.
4- Mix well using disposable stirrer spreading the mixture over the whole
test area and tilt the slide gently. Agitate for about 2 minutes with rotator or
by hand and observe for the presence or abscence of agglutination.

Negative result:
No agglutination of the latex particles suspension within two minutes.
Positive result:
An agglutination of the latex particles suspension will occur within two minutes,
indicating an ASO level of more than 200 IU/ml.

4. Paul Bunnel
* Principle of the test :
It is non specific test is used to screen for infectious mononucleosis (IM), which is seen
most commonly in adolescents and young adults. IM is caused by the Epstein-Barr virus.
This infects B-lymphocytes which subsequently produce a heterophile antibody ( in
patients serum) which is identified through its reaction with the Paul Bunnell antigen (
sheep RBCs Ag) and is detected by a latex agglutination method.
 Procedure:

1- Bring all reagents and specimens to room temperature.

2- Place one drop (50 µl) of the positive control and 50 µl of the patient
serum into separate circles on the slide.
3- Shake the latex reagent gently and add one drop (45 µl) on each
circle next to the sample to be tested and control.
4- Mix well using disposable stirrer spreading the mixture over the whole
test area and tilt the card gently.

5. Brucella Abortus & Melitensis

* Principle of the test : To detect antibodies in sample against Brucella Ag, the bacteria
that causes the disease brucellosis.

Antigen A and M are common to the three main brucella species

- In the Br. Abortus, there is more A antigen than M antigen
- In the Br. Melitensis there is more M antigen than A antigen

— Brucellosis; is a zoonotic infection transmitted to humans contact with fluidsfrom

infectedanimals(sheep,cattle,goats,pigs)derivedfoodproductssuch as unpasteurized milk and
cheese .

Serologic tests are based on rising and falling of of IgM and IgG. In relapses, IgG
may be the only antibody which rises.
- IgM: rise during 1st week of acute illness, peak at 3 months, and may
persist during chronic disease

- IgG & IgA: rise during 3 weeks after onset of acute illness, peaks at 6-8 weeks, and
remains high during chronic disease.

Procedure:

1- Bring all reagents and specimens to room temperature.

2- Place one 2 drop (50 µl) of the positive control and 2 drops of negative.
3- Add 40 µl of sample on first well, 20 µl of sample on the second well, 10 µl of the
sample on the third well. ( Do the same in another row.
4- in first samples row add 1 drop form reagent A, and for the second Row add M
reagent.
5- Mix 4 min. in shaker, read the results.
Negative: if there is no Agglutination suggest less than titer 1:80
Positive: if Agglutination appear
> after 15 seconds = (1:640)
> after 30 seconds = (1:320)
> after 1 minutes = (1:160)
> after 1.30 minutes = (1:80)

• Acute brucellosis is most likely to be associated with a titer above 1:160

• A great majority of patients will have titers of 1:160 to 1:320

6. Widal Test ( Salmonella):

* Principle of the test : The main principle of widal test is that if homologous
antibody is present in patients serum, it will react with respective antigen in the
reagent and gives visible clumping on the test card and agglutination in the tube.

The antigens used in the test are “H” flagellar antigens and “O” somatic antigens of
Salmonella Typhi and S. Paratyphi.

Procedure:

1- Place one drop (50 µl) of positive control and Negative control on reaction
circles of the slide.
2- Pipette 50 µl of the patient undiluted serum to be tested onto the
remaining reaction circles.
3- Add one drop of Widal TEST antigen 'H’, 'AH’, 'BH’, ‘CH’ to the
first row ( four reaction circles) and (PC & NC).
4- Add one drop of Widal TEST antigen 'O’, 'AO’, 'BO’, ‘CO’ to the
second row ( four reaction circles) and (PC & NC).
5- Mix contents of each circle uniformly over the entire circle
with separate mixing sticks.
6- Rock the slide, gently back and forth and observe for agglutination
macroscopically within one minute.
 Negative: if there is no Agglutination suggest less than titer 1:80
Positive: if Agglutination is visible within 1 minute, proceed for quantitative slide test for
the quantitative estimation of the titre of the antibody.
Salmonella typhi O +ve > mean recent (acute) infection.
Salmonella typhi H +ve > mean old (chronic) infection.
Salmonella paratyphi bO,bH +ve > mean carrier can infect other).

# Quantitative slide test:

1- Clean the test card and make it dry
2- Put 5 µl, 10 µl, 20 µl, 40 µl and 80 µl of undiluted serum in 1st, 2nd, 3rd, 4th and
5th circles respectively in the test card.
3- Add a drop of appropriate antigen suspension which showed
agglutination in rapid slide test, to each of the above circles.
4- Mix the contents of each circle with separate mixingsticks.
5- Rock the slide slowly for 1 minute and observe for agglutination.
6- The titre of the antibody is the highest dilution of serum up to which there is
clear agglutination.
7- Repeats steps 1 to 6 with all the antigens, which showed agglutination in rapid slide
test.
Result interpretation of Widal test:

▪ Antibody titre greater than 1:80 is considered significant and usually suggests positive test
for Salmonella infection.
▪ Low titres are often in normal individuals
▪ A single positive is less significant than the rising antibody titre, since rising titre is
considered to be a definite evidence of infection.

7. Toxoplasma:
Is an agglutination test to detect specific antibodies in serum of toxoplasmic patients usually in a
pregnant woman, unborn baby, or in a person with a weakened immune system
(immunocompromised) who has flu-like symptoms; sometimes to determine if a person has been
previously infected or to help determine if complications are due to an active Toxoplasma infection.

Procedure:

1- Bring all reagents and specimens to room temperature.

2- Place one drop (40 µl) of undiluted serum and one drop of positive control and Negative
control on reaction circles of the slide.
3- add one drop (20 µl) of Toxo latex reagent (Latex particles coated with soluble [Link]
antigen,) to the sample and control.
4- Mix the contents of each circle with separate mixing sticks and rotate the slide slowly.
5- After 3 minutes check for agglutination, at the same time compare with reaction of the control.
# If specific antibodies are present in the sample a clear agglutination will
appear
.

B. CARBON:
1. RPR (VDRL) : "Rapid Plasma Reagin" , "Venereal Disease Research Laboratory"
RPR is just the VDRL antigen, but RPR tests can be done without the use of a microscope. In
contrast, a VDLR test requires a microscope.

The Rapid Plasma Reagin (RPR) test is a macroscopic, non-treponemal, flocculation card test. It
detects the IgM and IgG antibodies to lipoidal material released from the damaged host cells, as well
as to lipoprotein-like material and possibly cardiolipin released from the treponemes.

* Principle of the test:

The antigen used in RPR is a modified VDRL (Cardiolipin) antigen, in which micro particulate
charcoal particles are used to enhance the visual difference between positive and negative results. A
cardiolopin lecithin-cholesterol antigen coated with carbon particle is mixed with patient’s serum. If
the specimen contains reagin, flocculation occurs with a coagglutination of carbon particles contained
in the antigen suspension, which appears as black clumps. Non- reactive specimens appear as an even
light gray homogeneous suspension.
Procedure:

1- Bring the RPR carbon antigen suspension, controls and samples to room temperature.
2- Add 50 µl of sample and positive and negative controls
3- Add one drop of well-mixed RPR reagent next to the test specimen, positive control and
negative control.
4- Using a mixing stick mix the test specimen and the RPR reagent thoroughly
spreading uniformly over the entire reaction circle.
5- Rotate the slide gently for 8 minutes.
6- Observe for agglutination.

Limitation:
Rapid Plasma Reagin test (RPR) and Venereal Disease Laboratory test (VDRL), are not
definitive tests for syphilis and may have a false positive result due to other medical conditions
such as viral infections (measles, hepatitis), pregnancy, and some autoimmune diseases.

C. IHA ( Indirect Haemagglutination Assay )

1. Bilharziasis:

Bilharziosis or schistosomiasis represent a group of parasitic diseases caused by non-segmented flat

worms of the genus Schistosoma. The diagnosis of bilharziosis can be made by eggs
identification. The problem is that eggs cannot be detected before the “disease state” phase.
Immunological diagnosis, via the detection of antibodies, is thus essential in the initial stages of
this disease.
schistosoma kit enables the quantitative determination, by indirect hemagglutination, of serum
antibodies from patients with bilharziosis caused by Schistosoma mansoni (intestinal form),
Schistosoma hæmatobium (urinary form) and Schistosoma intercalatum (rectal form).
* Principle of the test: Based on indirect heaemaggluination testing.

Sensitized sheep red blood cells are coated with Schistosoma antigens and added to the test serum.
> If the specific antibodies are present in the serum, they will agglutinate with the red blood cell and a
red/brown deposit will appear on the bottom of the well.
> With a non-reactive serum, no agglutination occurs with the sensitized cells and they drop to the
bottom of the well, forming a compact button.

* Kit Content:
- R1: containing 2.4mL of Sensitized red blood cells.
- R2: containing 1 mL of non-Sensitized red blood cells.
- BUF: containing 55mL phosphate buffer.
- Microplate: U-bottom.
- Control + : containing 0.2mL of tired positive control.
- Control - : containing 0.2mL of negative control.

* Procedure:

1- Deliver 50 µl of buffer reagent in 8 wells of microplate. 2- Mix 50 µl

of patients serum with buffer in first well.

3- Transfer 50 µl of diluted serum from 1st well to 2nd well, and from 2nd to 3rd. and so on until the
6th well then discard 50 µl from 6th well.
4- Add 50 µlof serum dilution to 7th well, mix it with buffer and discard 50 µl. 5- Carefully shake
R1 and R2 reagents:
……….distribute 1 drop of R1 in first 6 wells.
……….distribute 1 drop of R2 in the 7th well ( Serum control ).
……….distribute 1 drop of R1 in the 8th well ( Reagent Control ).
6- Very carefully homogenize wells manually.
7- Read results 2 hours later.
2. Leishmaniasis:
Leishmania leads to the disease leishmaniasis, commonly infecting hyraxes, canids, rodents and
humans. Leishmania are transmitted by the bite of a Phlebotomine sandfly.

* Principle of the test: Based on indirect heaemaggluination testing.

Sensitized sheep red blood cells are coated with Leishmania antigens and added to the test serum.
> If the specific antibodies are present in the serum, they will agglutinate with the red blood cell and a
red/brown deposit will appear on the bottom of the well.
> With a non-reactive serum, no agglutination occurs with the sensitized cells and they drop to the
bottom of the well, forming a compact button.

3. Echinococcosis:
Hydatidosis or echinococcosis is a parasitic disease caused by the larvae (hydatids)
of a cestode of the Echinococcus genus. The life-cycle of Echinococcus granulosus requires both
definitive and intermediate hosts. In general, the dog is the definitive host, with sheep and in rare
cases humans being the intermediate host.

* Principle of the test: Based on indirect heaemaggluination testing.

Sensitized sheep red blood cells are coated with Echinococcus granulosus antigens and added to the
test serum.
> If the specific antibodies are present in the serum, they will agglutinate with the red blood cell and a
red/brown deposit will appear on the bottom of the well.
> With a non-reactive serum, no agglutination occurs with the sensitized cells and they drop to the
bottom of the well, forming a compact button.
* Kit Content:
- R1: containing 2.4mL of Sensitized red blood cells.
- R2: containing 1 mL of non-Sensitized red blood cells.
- BUF: containing 55mL phosphate buffer.
- Microplate: U-bottom.
- Control + : containing 0.2mL of tired positive control.
- Control - : containing 0.2mL of negative control.

* Procedure:
4. TPHA " Treponema pallidum Hemagglutination Assay"
Syphilis is a complex disease which is normally sexually transmitted. The
causative organism, Treponema pallidum.
Infection is normally diagnosed by detecting antibodies specific for T. pallidum in the
patient’s serum or CSF.
# Two groups of antibodies are formed:

- one reacting with the non- treponemal antigens used in the VDRL/Carbon Antigen and RPR
tests ( found normally in active disease and the levels subside after successful treatment).
- and the other reacting with the specific antigens of [Link] ( Specific antibody persists
long after the infection has been successfully treated).

* Principle of the test: Based on Indirect Haemagglutination Assay.

is a specific, sensitive indirect haemagglutination test for the detection of antibodies to Treponema
pallidum in serum or CSF.
For professional use only.

Kit Content:
- Test Cells: T. Pallidum antigen coated preserved fowl erythrocytes in
buffer.
- Control Cells: Preserved fowl erythrocytes in buffer.
- DIL: Diluent.
- Control + : Positive Control. Serum prediluted (1/20) in buffer
containing antibodies to T. pallidum.
- Control - : Negative Control. Serum prediluted (1/20) in buffer
free of antibodies to T. pallidum.

* Procedure:
Each test requires 4 wells of a microtitre plate.

1- Dispense Diluent into the microtitration plate as follows 25 µl in

rows1,3&4 and 100 µl in row 2.
2- Dispense 25 µl of each sample into a well in row 1. 3- Mix
well and transfer 25 µl from row 1 to row 2.
4- Mix well and transfer 25 µl from row 2 to row 3. 5- Mix
well and discard 25 µl from row 3.
6- Transfer 25 µl from row 2 to row 4.
7- Mix well and discard 25 µl from row 4
8- Add 75 µl of well mixed Control Cells to row 4. 9- Add
75 µl of well mixed Test Cells to row 3.
10- Tap plate gently to mix. The final dilutions in row 3 and 4 are 1/80.
11- Cover and let stand at room temperature for 45 to 60 minutes (alternatively the plates can be
left overnight).
12- Examine for agglutination patterns.
Note: Kit controls are prediluted and should be added directly into individual wells in row 3 and 4 (
no diluent required ).

# Results interpretation:
> Agglutinated cells form an even layer over the bottom of the well.
Agglutination of the Test Cells but not the Control Cells indicates the presence of specific
antibody to [Link].
> Weakly agglutinated cells form a characteristic ring pattern.

> Non-agglutinated cells form a compact button in the centre of the well.
Absence of agglutination indicates that antibody is below the limit of detection of the system.
D. Nephelometry:
Nephelometry, in analytical method for determining the amount of cloudiness, or turbidity, in a
solution based upon measurement of the effect of this turbidity upon the scattering of light at an
angle about 13 - 24 degree.

# ( Turbidity in a liquid is caused by the presence of finely divided suspended particles [Ag-Ab
complexes] )
If a beam of light is passed through a turbid sample, its intensity is reduced by scattering, and the
quantity of light scattered is dependent upon the concentration and size distribution of the particles.
# The amount of light scattered is proportional to the concentration of antigen or antibody(protein)
in the solution
# Types of Nephelometry:
In the Lab, two types of tests can be run:

1. End point nephelometry:

tests are run by allowing the antibody/antigen reaction to run through to completion (until all of the
present reagent antibodies and the present patient sample antigens that can aggregate have done so
and no more complexes can form).
However, the large particles will fall out of the solution and cause a false scatter reading (falsely
decreased results) , thus kinetic nephelometry was devised.

2. Kinetic (rate) nephelometry

the rate of scatter is measured right after the reagent is added. As long as the reagent is constant the
rate of change can be seen as directly related to the amount of antigen present.
Kinetic is better than End point!!
# Clinical applications:
These techniques used in the laboratory to determine the levels of several blood
plasma/ serum proteins.
This technique is widely used in clinical laboratories because it is relatively easily automated

(1.) Quantitative Immunoglobulins: IgA, IgG, IgM

Immunoglobulins function to neutralize toxic substances, support
phagocytosis, and destroy microorganism
Functions:

1. IgA takes two forms: serum and secretory. Serum IgA is present in blood
serum; secretory IgA is found in saliva, tears, colostrum, and bronchial,
gastrointestinal, and genitourinary secretions, where it can protect against
microorganism invasion.

2. IgE is involved in allergic reactions.

3. IgD is involved in humoral immunity.
4. IgG, the only immunoglobulin that can cross the placenta, is
responsible for protection of the newborn during the first months of life.

5. IgM possesses antibody activity against gram- negative organisms

and rheumatoid factors and forms natural antibodies such as the ABO
blood group.

Reference Values: Adults

1. IgG: 700–1,500 mg/dL or 7.0–15.0 g/L
2. IgA: 60–400 mg/dL or 600–4,000 mg/L
3. IgM: 60–300 mg/dL or 600–3,000 mg/L
4. IgE: 3–423 IU/mL or 3–423 kIU/L
5. IgD: 0–14 mg/dL or 0–140 mg/L
 (2.) C3 Complement Component:
C3 constitutes 70% of the total protein in the complement system and is essential to
the activation of both classic and alternative pathways. C3 is synthesized in the liver,
macrophages, fibroblasts, lymphoid cells, and skin. This test is done when it is suspected
that individual complement component concentrations are abnormally reduced. This
test and the C1q and C4 tests are the most frequently ordered complement
measurements. There is a correlation between most forms of nephritis, the degree of
nephritis severity, and C3 levels. C3 is useful for assessing disease activity in SLE.

Reference Values: Normal 75–175 mg/dL or 0.75–1.75 g/L

Clinical Implications
1. Decreased C3 levels are associated with Severe recurrent
bacterial infections due to C3 homozygous deficiency,
Absence of C3 b inactivator factor, Acute post streptococcal
glomerulonephritis, Immune complex disease, Active SLE,
Membranoproliferative g. Nephritis or End-stage liver
disease.
2. Increased levels are found in numerous inflammatory states.
C4 Complement Component.
(3.) C4 Complement Component:

C4 is another of the components of the complement system and is synthesized in bone

and lung tissue. This is a follow-up test done when total complement levels are
abnormally decreased.

Reference Values; Normal 14–40 mg/dL or 140–400 mg/L

Clinical Implications

1. Decreased C4 levels are associated with the following conditions:

• Acute SLE • Early glomerulonephritis • Immune complex disease

• Cryoglobulinemia • Inborn C4 deficiency

[Link] C4 levels are associated with malignancies.

(4.) Ceruloplasmin

Measurement of ceruloplasmin aids in the diagnosis of copper metabolism disorders,

that is, Wilson’s disease. Copper bound to ceruloplasmin constitutes the largest
amount of Cu2+ in the circulation. In Wilson’s disease, Cu2+ mobilization from the
liver is drastically reduced because of the low production of ceruloplasmin.

The test gives a quantitative measurement of the amount of ceruloplasmin in the

patient’s serum. Values can vary considerably from patient to patient and may be 50%
of normal (pointing to some other primary defect). Patients with Wilson’s disease are
not always extremely low in ceruloplasmin.

Reference Values: Normal 25–63 mg/dL or 250–630 mg/L

Clinical Implications

Values<14mg/dL are expected in Wilson’s disease. Deficient ceruloplasmin, however,

is not the primary defect in Wilson’s disease and therefore can vary considerably
from patient to patient.

(5.) α1-Antitrypsin

α1-Antitrypsin (AAT) is the most abundant serum protease inhibitor and inhibits
trypsin and elastin as well as several other proteases. The release of proteolytic
enzymes from plasma onto surface organs and into tissue spaces results in tissue
damage unless inhibitors are present. AAT deficiency has been associated with
neonatal respiratory distress syndrome, severe protein- losing disorders, and pulmonary
emphysema. The test is useful for individuals in whom familial chronic obstructive lung
disease is suspected.

Reference Values: Normal

Adults: 100–200 mg/dL or 1.0–2.0 g/L

Newborns: 145–270 mg/dL or 1.45–2.70 g/L

 Enzyme-linked immunosorbent assays

Enzyme-linked immunosorbent assays (ELISAs) are the most common immunoassay type and used to detect the
presence and measure the amount/concentration of antigens or antibodies by producing an enzyme-triggered color
change.

In diagnostics, ELISAs are frequently used to determine whether and how many antibodies have been produced in
response to pathogen exposure or vaccination.

The EIA method uses the catalytic properties of enzymes to detect and quantitate immunologic reactions. An
enzyme-labeled antibody or enzyme-labeled antigen conjugate is used in immunologic assays. The enzyme, with
its substrate, detects the presence and quantity of antigen or antibody in a patient specimen.

In a representative EIA test, a plastic plate is coated with antigen. The antigen reacts with antibody in the patient’s
serum. The plate is then incubated with an enzyme-labeled antibody conjugate. If antibody is present, the conjugate
reacts with the antigen-antibody complex on the plate.

The enzyme activity is measured spectrophotometrically after the addition of the specific chromogenic substrate.
For example, peroxidase cleaves its substrate, o-dianisidine, causing a color change.

Types of ELISA
A number of variations of ELISA have been developed, allowing qualitative detection or quantitative measurement
of either antigen or antibody.

1. Indirect ELISA

The indirect ELISA detects the presence of antibody in a sample. The antigen for which the sample must be
analyzed is adhered to the wells of the micro titer plate. The primary antibody presents in the sample bind
specifically to the antigen after addition of sample. The solution is washed to remove unbound antibodies and then
enzyme conjugated secondary antibodies are added. The substrate for enzyme is added to quantify the primary
antibody through a color change. The concentration of primary antibody present in the serum directly correlates
with the intensity of the color.
 2. Sandwich ELISA

The sandwich ELISA is used to identify a specific sample antigen. The wells of micro titer plate are coated with
the antibodies. Non-specific binding sites are blocked using bovine serum albumin. The antigen containing sample
is applied to the wells. A specific primary antibody is then added after washing. This sandwiches the antigen.
Enzyme linked secondary antibody is added that binds primary antibody. Unbound antibody-enzyme conjugates
are washed off. The substrate for enzyme is introduced to quantify the antigens.

3. Competitive ELISA

This type of ELISA depends on the competitive reaction between the sample antigen and antigen bound to the
wells of microtiter plate with the primary antibody.
First, the primary antibody is incubated with the sample. This results in the formation of Ag-Ab complex which are
then added to the wells that have been coated with the same antigens. After an incubation, unbound antibodies are
washed off. The more antigen in the sample, more primary antibody will bind to the sample antigen. Therefore
there will be smaller amount of primary antibody available to bind to the antigen coated on well. Secondary
antibody conjugated to an enzyme is added, followed by a substrate to elicit a chromogenic signal. Concentration
of color is inversely proportional to the amount of antigen present in the sample.

Figure: Microplate ELISA: colored wells indicate Figure: ELISA Kit; Reagents, Conjugate, Diluent, Buffer and the
reactivity. The darker the color, the higher the Micro plate.
reactivity.
AUTOMATED SYSTEMS FOR ELISA TESTING
When using the automated system, only dispense the required amount of sample into the test plate well; the instrument
performs all the subsequent processing steps fully automatically. (Refer to the manual for each specific test).

Figure: Bio Rad Evolis Microplate Processor

The EVOLIS™ System is a self-contained microplate processor for automated EIA testing. The System
has a capacity of up to 180 primary tubes with racks accommodating tubes 8–16 mm in diameter. Other
features include four independent temperature controlled incubators, four ambient light protected
incubators and three different wash buffers.
ELISA basics/ELISA principle
ELISA’s rely on specific antibodies to bind the target antigen, and a detection system to indicate the
presence and quantity of antigen binding.

Figure: DSX-4-plate ELISA Processing system from Dynex

The instrumentation available for signal-detection is spectrophotometer which is designed to read results
of the ELISA test, this instrument works on the principle of "Photoelectric Calorimetry"

ELISA Data Interpretation

The ELISA assay yields different types of data output:

Quantitative: ELISA data can be interpreted in comparison to a standard curve (a serial dilution of a
known, purified antigen) in order to precisely calculate the concentrations of antigen in various samples.
Qualitative: ELISAs can also be used to achieve a yes or no answer indicating whether a particular
antigen is present in a sample, as compared to a blank well containing no antigen or an unrelated control
antigen.
 Viruses Tests
Hepatitis A (HAV)
Hepatitis A virus (HAV), which is acquired through enteric transmission, infects the gastro- intestinal
tract and is eliminated through the feces. Serologically, the presence of the IgM antibody to HAV (IgM
anti-HAV) and the total antibody to HAV (total anti-HAV) identifies the disease and determines previous
exposure to or recovery from HAV.

Hepatitis B (HBV)
Hepatitis B virus (HBV) demonstrates a central core containing the core antigen and a surrounding
envelope containing the surface antigen: less than 0.01 pg/mL for viral load. Detection of core antigen
(HBcAg), envelope antigen (HBeAg), and surface antigen (HBsAg) or their corresponding antibodies
constitutes hepatitis B serologic or plasma assessment. Viral transmission occurs through exposure to
contaminated blood or blood products through an open wound (eg, needle sticks, lacerations). Hepatitis
monitoring panel for serial testing includes four B markers: HBsAg ,HBeAg, anti-HBe, and anti-HBs.
Interpretation depends on clinical setting.

• HBsAg (hepatitis B surface antigen)

A positive test result means you are infected with hepatitis B.

• HBsAb (hepatitis B surface antibody)

A positive test result means you have antibodies to hepatitis B. If you also test positive for HBcAb, then
you were infected or exposed to hepatitis B virus in the past, cleared the virus, and are now "immune"
(protected) against another infection with hepatitis B. If you test positive for HBsAb but negative for
HBcAb, then you were vaccinated against hepatitis B and are protected by the vaccination.

• HBcAb (hepatitis B core antibody)

A positive test result means you once were infected with hepatitis B.
 • HBeAg and anti-Hbe

If HBeAg is detectable in a blood sample, this means that the virus is still active in the liver (and can be
transmitted to others). If HBeAg is negative and anti-HBe is positive, this generally means that the virus
is inactive. However, this is not always the case. Some people with chronic hepatitis—especially those
who have been infected with HBV for many years—may have what is known as a precore or core variant
mutated form of HBV. This can cause HBeAg to be negative and anti-HBe to be positive, even though
the virus is still active in the liver.
A single reactive ELISA test by itself cannot be used to diagnose HBV. The test should always be repeated in
duplicate using the same blood sample. If repeatedly reactive do the test again in new second sample if still
reactive follow-up tests using neutralization test should be done. A positive neutralization test is considered
confirmatory for HBV.

Hepatitis C (HCV)
Hepatitis C virus (HCV), formerly known as non-A, non-B hepatitis, is also transmitted parenterally.
HCV infection is characterized by presence of antibodies to hepatitis C (anti-HCV) and levels of alanine
aminotransferase (ALT) that fluctuate between normal and markedly elevated. Levels of anti-HCV
remain positive for many years; therefore, a reactive test indicates infection with HCV or a carrier state
but not infectivity or immunity. PCR or reverse transcriptase PCR (RT-PCR) (viral load), which detects
HCV RNA, should be used to confirm infection when acute hepatitis C is suspected. A negative hepatitis
C antibody (recombinant immunoblot assay [RIBA]) does not exclude the possibility of HCV infection
because seroconversion may not occur for up to 6 months after exposure.

A single reactive ELISA test by itself cannot be used to diagnose HCV. The test should always be repeated in
duplicate using the same blood sample. If repeatedly reactive do the test again in new second sample if still
reactive follow-up tests using Western blot should be done. A positive Western blot is considered
confirmatory for HCV.
Human Immunodeficiency Virus (HIV-1/2) Antibody Tests

This test detects human immunodeficiency viruses’ types 1 and 2 (HIV-1/2), which cause AIDS. The
diagnosis of AIDS must be clinically established. Combination HIV antibody and HIV antigen test—this is
the recommended screening test for HIV. It is available only as a blood test. It detects the HIV antigen
called p24 plus antibodies to HIV-1 and HIV-2. The level of p24 antigen and the amount of virus (viral
load) increase significantly soon after initial infection. Testing for p24 allows for detection of early
infections, before HIV antibody is produced. A few weeks after exposure, antibodies to HIV are
produced in response to the infection and remain detectable in the blood thereafter, making the antibody
test useful for detecting infections weeks after exposure. By detecting both antibody and antigen, the
combination test increases the likelihood that an infection is detected soon after exposure. These tests can
detect HIV infections in most people by 2-6 weeks after exposure.
A single reactive ELISA test by itself cannot be used to diagnose AIDS. The test should always be repeated in
duplicate using the same blood sample. If repeatedly reactive do the test again in new second sample if still
reactive follow-up tests using Western blot should be done. A positive Western blot is considered
confirmatory for HIV.
Rubella Antibody
Rubella, a mild, contagious illness characterized by an erythematous maculopapular rash, is observed
primarily in children 5 to 14 years of age and in young adults. The disease, commonly called German or 3-
day measles, may be asymptomatic or may involve a 1- to 5-day prodromal period of malaise, headache,
cold symptoms, low-grade fever, and suboccipital lymphadenopathy.
Although the illness is mild in children, it may cause the congenital rubella syndrome in the fetus of a
mother infected early in pregnancy. The classic abnormalities associated with the rubella syndrome include
congenital heart disease, cataracts, and neurosensory deafness. After 20 to 24 weeks of gestation, congenital
abnormalities are rare.
The quantitative measurement of IgG antibodies to rubella virus aids in the determination of immune status.
Assay results of 10 IU/mL of antibody are negative or not immune. Assay results >10 IU/mL are considered
positive or immune. A positive result of IgM antibody indicates a congenital or recent infection.
Result interpretation:
Negative for rubella IgG antibodies (<7 IU/mL: no immunity) Negative for IgM antibodies (<0.9 IU/mL: no
infection)
Positive for rubella IgG antibodies (>10 IU/mL; immunity, indicates a current or previous exposure or
immunization to rubella) Positive for rubella IgM antibodies (with or without positive IgG) (>1.1 IU/mL;
indicates a current or recent infection with rubella virus)
Cytomegalovirus (CMV) Antibody

Cytomegalovirus (CMV) is a ubiquitous human viral pathogen that belongs to the herpesvirus family.
Infection with CMV is usually asymptomatic (up to 60 days) and can persist in the host as a chronic or latent
infection. Cytomegalovirus has been linked with sexually transmitted infections. This test determines the
presence of CMV antibodies in the serum.
Reference Values: Normal
Negative for CMV-specific IgG and IgM

Herpes Simplex Virus (HSV) Antibodies (HSV-1 and HSV-2 Tests)

Two types of herpes simplex virus exist. Herpes simplex virus type 1 (HSV-1) causes orofacial herpes; type
2 (HSV-2) causes genital and neonatal herpes. Serologic differentiation is difficult; therefore, type-specific
antibody tests are required.
These tests identify the herpes simplex infections. Human herpes simplex virus (HSV) is found worldwide
and is transmitted by close personal contact. The clinical course is variable, and symptoms may be mild
enough to go unrecognized. Major signs and symptoms include oral and skin eruptions, genital tract
infections and lesions, and neonatal herpes. Herpes simplex is also common in individuals with immune
system deficiencies (eg, cancers, HIV/AIDS, chemotherapy treatment). HSV antibody testing is also widely
used for bone marrow recipients and donors.
Reference Values: Normal
Negative for HSV-1 and HSV-2 specific IgG and IgM.

Epstein-Barr Virus (EBV) Antibody Tests

Epstein-Barr virus (EBV) is a herpesvirus found throughout the world. The most common symptomatic
manifestation of EBV infection is a disease known as infectious mononucleosis (IM). This disease induces
formation of increased numbers of abnormal lymphocytes in the lymph nodes and stimulates increased
heterophile antibody formation. IM occurs most often in young adults who have not been previously
infected, through contact with infectious oropharyngeal secretions. Symptoms include fever, pharyngitis,
and lymphadenopathy.
Reference Values: Normal - Negative for EBV antibodies.
 Bacteria Tests
Chlamydia Antibody IgG Test
Chlamydia is caused by a genus of bacteria (Chlamydia spp.) that require living cells for growth and are
classified as obligate cell parasites. Recognized species include Chlamydia psittaci, Chlamydia
pneumoniae, and Chlamydia trachomatis. C. psittaci causes psittacosis in birds and humans. C.
pneumoniae is responsible for approximately 10% of cases of community-acquired pneumonia. C.
trachomatis is grouped into three serotypes. One group causes lymphogranuloma venereum (LGV), a
venereal disease. Another group causes trachoma, an eye disease. The third group causes genital tract
infections different from LGV.

Reference Values: Normal

Negative for chlamydia antibody

Parasite Tests

Toxoplasmosis (TPM) Antibody Tests

Toxoplasmosis is caused by the sporozoan parasite Toxoplasma gondii and is a severe, generalized,
granulomatous CNS disease. It may be either congenital or acquired and is found in humans, domestic
animals (eg, cats), and wild animals. Humans may acquire the infection through ingestion of inadequately
cooked meat or other contaminated material. Congenital toxoplasmosis may cause fetal death. Symptoms of
subacute infection may appear shortly after birth or much later. Complications of congenital toxoplasmosis
include hydrocephaly, microcephaly, convulsions, and chronic retinitis. The CDC recommends serologic
testing during pregnancy.
Reference Values: Normal ,Negative
 ❖ Confirmatorytest for HBsAg"Neutralization"
used to confirm the presence of hepatitis B surface Ag (HBs Ag) in human serum or plasma by
means of specific antibody neutralization.

Principles:
uses the principle of specific antibody neutralization to confirm the presence of HBsAg in
samples found to be repeatedly reactive.
Antibody to Hepatitis B Surface Antigen (Human) (anti- HBs) is incubated with a sample. If
HBsAg is present in the sample, it will be neutralized by the antibody.

The neutralized HBsAg is blocked from binding to the anti-HBs coated microparticles in a test
for HBsAg. A reduction of signal occurs when compared to the signal of a paired sample that
has not been treated with the antibody reagent. A sample is considered to be positive if its
reactivity in the kit HBsAg Confirmatory test procedure is neutralized by the addition of
antibody reagent and the reduction in signal is 50% or greater.

* Kit Content:
- RA: Neutralization reagents (Antibody to Hepatitis B surface Antigen (Human)
nonreactive for HBsAg).
- RB: Negative control diluent.
- RC: Sample Diluent.

* Procedure:
7. Quickly dispense 50 μl of conjugate solution (R6 + R7) into all wells, the conjugate solution
must be shaken before use. Homogenize the reaction mixture. NB : The sample distribution can also
be visually controlled at this step of the manipulation, as well as the conjugate distribution : The
conjugate solution (R6+R7), which is coloured red, can be visually controlled at this step of the
manipulation. (refer to section 14 for automatic verification).

[Link] possible, cover the plate with new adhesive film and incubate for 1hour and 30 ± 5
minutes at 37±1ÆC.

9. Remove the adhesive film, empty all wells by aspiration and wash a minimum of 5 times. The
residual volume must be lower than 10 μl (if necessary, dry the strips by turning them upside down
on absorbent paper).

10. Quickly dispense into each well 100 μl of prepared development solution (R8+R9), freshly
prepared before use. Allow the reaction to develop in the dark for 30 ± 5 minutes at room
temperature (18 - 30ÆC). Do not use adhesive film during this incubation.
N.B.: The distribution of the development solution, which is coloured pink, can be visually
controlled at this step of the manipulation : There is a clear difference of colouration between empty
well and well containing the pink substrate solution. (refer to section 14 for automatic verification :
SPECTROPHOTOMETRIC VERIFICATION OF SAMPLE AND REAGENT PIPETING)

11. Add 100 μl stopping solution (R10) by using the same sequence and rate of distribution as for
the substrate solution. Homogenize the reaction mixture. N.B.: The distribution of the stopping
solution, which is not coloured, can be visually controlled at this step of the manipulation. After
the addition of the stopping solution the pink colouration of the substrate disappears (for the
negative samples) or turns from blue to yellow (for the positive samples).

12. Carefully wipe the plate bottom. Wait at least 4 minutes after stopping solution addition before
reading and within 30 minutes of stopping the reaction, read the optical density at 450/620-700 nm
using a plate reader.
13. Check for agreement between the spectrophotometric and visual readings and against the plate
and sample distribution and identification plans.
 #PolymeraseChainReaction(PCR):
Methods based on the detection of viral genome are also commonly known as molecular
methods

PCR, polymerase chain reaction: is an in-vitro technique for amplification ( cloning) of a region of
DNA whose sequence is known or which lies between two regions of known sequence.

* Purpose: To duplicate DNA molecule.

* Principle: The method relies on thermal cycling, consisting of cycles of repeated heating and
cooling of the reaction for DNA melting and enzymatic replication of the DNA.

# Reaction components:
1. DNA template
• DNA must be extracted from the sample, containing region to be sequenced.
2. Primers ( 2 sets of primers ; Forward & Reverse )
• Short stretches of DNA that initiate the PCR reaction, designed to bind to either side of the
section of DNA you want to copy.
• The selectivity of PCR results from the use of primers that are complementary to the DNA region
targeted for amplification under specific thermal cycling conditions.

3. Enzyme -Thermostable DNA Polymerase -

• Usually Taq Polymerase or anyone of the natural or Recombinant thermostable polymerases,
an enzyme originally isolated from the bacterium Thermus aquaticus stable at T0 up to 950 C

• Taq Pol has 5’-3’ exo only, no proofreading.

4. dNTPs (deoxyribonucleoside triphosphates)

(sometimes called "deoxynucleotide triphosphates" ), the building-blocks from which the DNA
polymerase synthesizes a new DNA strand.

5. Mg2+ ions - cofactor of the enzyme

6. Buffer solution

Buffer solution, providing a suitable chemical environment for optimum activity and stability of
the DNA polymerase.
 # DNA extraction:
DNA extraction is a routine procedure used to isolate DNA from the nucleus of cells..

What does DNA extraction involve?

Step 1. Breaking cells open to release the DNA

The cells in a sample are separated from each other, often by a physical means such as
grinding or vortexing , and put into a solution containing salt.
The positively charged sodium ions in the salt help protect the negatively charged
phosphate groups that run along the backbone of the DNA.
A detergent is then added. The detergent breaks down the lipids in the
cell membrane and nuclei. DNA is released as these membranes are disrupted.

Step 2. Separating DNA from proteins and other cellular debris

To get a clean sample of DNA, it’s necessary to remove as much of the cellular debris as
possible. This can be done by a variety of methods.
Often a protease ( protein enzyme) is added to degrade DNA-associated proteins and other
cellular proteins. Alternatively, some of the cellular debris can be removed by filtering the
sample.

Step 3. Precipitating the DNA with an alcohol

Finally, ice-cold alcohol (either ethanol or isopropanol) is carefully added to the DNA
sample. DNA is soluble in water but insoluble in the presence of salt and alcohol.
By gently stirring the alcohol layer with a sterile pipette, a precipitate becomes visible and
can be spooled out. If there is lots of DNA, you may see a stringy, white precipitate.
Step 4. Cleaning the DNA
The DNA sample can now be further purified (cleaned). It is then resuspended in a slightly
alkaline buffer and ready to use.

Step 5. Confirming the presence and quality of the DNA

For further lab work, it is important to know the concentration and quality of the DNA

When an ice-cold alcohol is added to a solution of

DNA, the DNA precipitates out of solution.

If there is enough DNA in the solution, you will see

a stringy white mass.

# The PCR Cycle:

cycles of heating and cooling called thermal cycling which is carried out by machine
known as a " thermal cycler" , is a DNA amplifier that regulates temperature and
amplifies segments of DNA via the polymerase chain reaction
 ✤ There are three main stages:

1. Denaturing –During this stage the cocktail containing the template DNA and all the other
core ingredients is heated to 94-95⁰C.

The high temperature causes the hydrogen bonds between the bases in two strands of template
DNA to break and the two strands to separate.

This results in two single strands of DNA, which will act as templates for the production
of the new strands of DNA.

It is important that the temperature is maintained at this stage for long enough to ensure that
the DNA strands have separated completely.

This usually takes between 15-30 seconds.

2. Annealing – During this stage the reaction is cooled to 50-65⁰C. This enables the primers
to attach to a specific location on the single- stranded template DNA by way of hydrogen
bonding (the exact
temperature depends on the melting temperature of the primers you are using).

Primers serve as the starting point for DNA synthesis. The polymerase enzyme can only add
DNA bases to a double strand of DNA. Only once the primer has bound can the polymerase
enzyme attach and start making the new complementary strand of DNA from the loose DNA
bases.

The two separated strands of DNA are complementary and run in opposite directions (from one
end - the 5’ end – to the other - the 3’ end); as a result, there are two primers – a forward primer
and a reverse primer.

This step usually takes about 10-30 seconds.

3. Extending (DNA synthesis) – During this final step, the heat is increased to 72⁰C to enable
the new DNA to be made by a special Taq DNA polymerase enzyme which adds DNA
bases.

Taq DNA polymerase is an enzyme taken from the heat-loving bacteria?

Thermus aquaticus.

This bacteria normally lives in hot springs so can tolerate temperatures above 80⁰C.

The bacteria's DNA polymerase is very stable at high temperatures, which means it can
withstand the temperatures needed to break the strands of DNA apart in the denaturing stage
of PCR.

DNA polymerase from most other organisms would not be able to withstand these high
temperatures, for example, human polymerase works ideally at 37˚C (body temperature).

72⁰C is the optimum temperature for the Taq polymerase to build the complementary strand.
It attaches to the primer and then adds DNA bases to the single strand one-by-one in the 5’ to
3’ direction.

The result is a brand new strand of DNA and a double-stranded molecule of DNA.

The duration of this step depends on the length of DNA sequence being amplified but usually
takes around one minute to copy 1,000 DNA bases (1Kb).
These three processes of thermal cycling are repeated 20-40 times to produce lots of copies of
the DNA sequence of interest.

The new fragments of DNA that are made during PCR also serve as templates to which the DNA
polymerase enzyme can attach and start making DNA.

The result is a huge number of copies of the specific DNA segment produced in a relatively
short period of time.
Detection of amplification products:

* Agarose Gel Electrophoresis

Gel electrophoresis :

is a process of separating bio molecules of different sizes by running them

through a sievelike matrix using electricity. The larger molecules move more
slowly, while smaller molecules slip through the matrix and move faster and
farther, thus separating the different fragments based on size and charge.

Materials Required:

An electrophoresis chamber and power supply.

Gel casting trays, which are available in a variety of sizes and composed of UV- transparent
plastic.

Sample combs, around which molten agarose is poured to form sample wells in the gel.

Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE).

Loading buffer, which contains something dense (e.g. glycerol) to allow the sample to
"fall" into the sample wells, and one or two tracking dyes, which migrate in the gel and
allow visual monitoring or how far the electrophoresis has proceeded.

Ethidium bromide, a fluorescent dye used for staining nucleic acids.

Transilluminator (an ultraviolet light box), which is used to visualize ethidium bromide-
stained DNA in gels.
Principle:

The first step to gel electrophoresis is to set the gel matrix. Agarose is used to separate
DNA molecules, and acrylamide is used to separate proteins. The gel starts off as a liquid,
which is poured into a molding tray. A comb is placed in the liquid matrix so that when the
matrix solidifies, wells are formed to load samples in them. Once the gel has solidified, it
is removed from the mold and placed in a special apparatus where current can be applied. A
buffer that can act as a conductor of electricity is poured around the matrix.

The samples of bio molecules are usually mixed with a substance of high density (a viscous
dye) so that they sink to the bottom of the well instead of floating away in the buffer. The dye
also helps track the progress of the experiment. Each sample is loaded in a separate well.
One of the wells is usually assigned for loading a marker, which has a set of fragments whose
sizes are already known in order to allow for comparison with the samples being loaded.

When the current is switched on, the samples tend to move towards the positively charged
side of the apparatus since the phosphate backbones of the molecules confer a negative
charge on them. After the samples have run a sufficient distance, the matrix is studied to
view the bands that are formed by the separation of the molecules.

# UV Transilluminator

An ultra-violet (UV) transilluminator is a standard piece of equipment used in life science

laboratories for visualization of target DNAs and proteins.

The UV transilluminator works by emitting high levels of UV radiation through the viewing
surface. The key application for a UV transilluminator is for visualization of DNA and
protein agarose and polyacrylamide gels after electrophoresis. Gels can be directly placed
onto the UV transilluminator; wavelength will vary on your particular application.

The size(s) of PCR products is determined by comparison

with a DNA ladder (a molecular weight marker), which
contains DNA fragments of known size, run on the gel
alongside the PCR products
After the gel has been run, the ethidium bromide
that was added to stained PCR products and
causes the gel to glow under ultraviolet light.
While it is glowing, a picture can be taken of it
to record the results
 #NucleicAcidTesting(NAT):
Nucleic Acid testing (NAT) for HIV, HCV and HBV became available in the latter 1990’s and is
now routinely used in many countries.

* Nucleic Acid Technology (Nucleic Acid Amplification Testing) ; is a molecular technique

advance in blood screening that enables to detect a virus or a bacterium These tests
were developed to shorten the window period a time between when a patient has been
infected and when they show up as positive by antibody tests.

* It reduces the window period by detecting low levels of viral genomic materials that are present
soon after infection but before the body starts producing antibodies in response to a
virus.

* Serology tests will fail to detect Window Period cases, use of NAT is an additional layer of
safety to the supply of blood and blood products.

> Detects very low levels of viral RNA or DNA that may be present in donated blood
> Highly sensitive & specific — targets specific viral nucleic acid sequences

All involve extraction or capture of nucleic acid, amplification, and detection.

Have same principle as PCR.

HBV, HCV and HIV commercially

manufactured primers are used. Differences
between PCR and NAT are:

NAT PCR
Runs 3 primers at the same time Runs 1 primer only
Qualitative Quantitative
Screens both donor and others Screens patient (follow up every 3
months)
More sensitive (any qualitative method is more
sensitive than the quantitative)

# Types of NAT machines:

Qualitative, Quantitative.

> When NAT gives “Non-reactive” it’s for the 3 viruses, but when it gives “Reactive” it
may be for one or more viruses, some machines specify it for you. If not, serology
testing helps in specification of the results.

> PCR negative results are written as TND (target not detected). Maximum cycles
number is 40 cycle.

> Nucleic acid is replicated in till the machine can detected (cycles). Depending on the
cycles number the infection is categorized.

0 - 20 cycles 20 - 30 30 - control value > control value

Highly Reactive Reactive Weak Reactive False Positive
The Recombinant ImmunoBlot Assay (RIBA) Test

This test is confirmatory for positive HCV ELISA.

Qualitative immunoblot assay - RIBA. Utilizes recombinant HCV encoded antigens and synthetic HCV
encoded peptides that are immobilized as individual bands onto test strips.
The INNO-LIA™ HCV Score is a line immunoassay which incorporates HCV antigens derived from the
core region, the E2 hypervariable region (HVR), the NS3 helicase region, the NS4A, NS4B, and NS5A
regions. The antigens were coated as 6 discrete lines on a nylon strip with plastic backing. In addition,
four control lines are coated on each strip: streptavidin control line, 3+ positive control (anti-human Ig)
which is also used as sample addition control line, 1+ positive control (human IgG), and the ± cut-off line
(human IgG). The INNO-LIA™ HCV Score is based on the principle of an enzyme immunoassay. A test
sample is incubated in a trough together with the test strip. If present in the sample, HCV antibodies will
bind to the HCV antigen lines on the strip. Subsequently, an affinity-purified alkaline phosphatase-
labelled goat anti-human IgG (H+L) conjugate is added and reacts with specific HCV antigen/antibody
complexes, if previously formed. Incubation with the enzyme substrate produces a chesnut-like color, the
intensity of which is proportionate to the amount of HCV-specific antibody captured from the sample on
any given line. Color development is stopped with sulfuric acid.
Test Procedures: 16 hours sample incubation
Sample Diluent 1 ml
Specimen 10 µl
Controls 10 µl
LIA® test strips 16 hours ± 2 hours
Washing 1 ml/3 x 5 min
RTU* Conjugate 1 ml/30 min
Washing 1 ml/3 x 5 min
RTU* Substrate 1 ml/30 min
Stop Solution 1 ml/10 - 30 min

The possible serological profiles defined by this assay include the following: Negative, Positive, Indeterminate.

Negative - if all HCV antigen lines have a negative reactivity rating

- if only one HCV antigen line has a reactivity of ±, except when the reactivity is
observed for NS3.
Positive - if at least two HCV antigen lines have a reactivity of ± minimum or higher.
Indeterminate - if one HCV antigen line has a reactivity rating of 1+ or higher - if the NS3 line reacts
with a reactivity of ± or higher and all other antigen lines are negative.
Autoantibodies Tests

.
Figure: Alegria® by ORGENTEC Diagnostika

Figure: Alegria test strips.

 TESTS FOR AUTOIMMUNITY AND SYSTEMIC RHEUMATIC DISEASE (SRD)
• Antinuclear Antibody (ANA) Test

Measurement of ANAs in serum is the most commonly performed screening test for autoantibodies in
patients suspected of having systemic rheumatic disease (SRD). SRDs are also called connective tissue
diseases or collagen diseases. Examples of SRDs include SLE, mixed connective tissue disease, Sjögren’s
syndrome, scleroderma, CREST (calcinosis, Raynaud’s phenomenon, esophageal dysfunction,
sclerodactyly, and telangiectasia) syndrome, rheumatoid arthritis, and polymyositis dermatomyositis.
The diagnosis of SLE is difficult because clinical signs and symptoms are varied and mimic other SRDs. SLE
is characterized by the production of autoantibodies to nuclear anti- gens, that is, anti-dsDNA. SLE is a
multisystem disease that can affect every organ system in the body, especially the kidneys.
Reference Values: Normal ANA screen: Negative ANA titer: <1:160

• Anti-dsDNA Antibody Test, IgG

The anti-dsDNA test is done specifically to identify or differentiate native (ie, double- stranded) DNA
antibodies, found in 40% to 60% of patients with SLE during the active phase of their disease, from other,
nonnative DNA antibodies found in other rheumatic diseases. The presence of antibodies to dsDNA
generally correlates with lupus nephritis. An anti- dsDNA test supports a diagnosis, allows monitoring of
disease activity and response to therapy, and establishes a prognosis for SLE.
Reference Values: Normal
Negative: <25 IU
Borderline: 25–30 IU
Positive: 31–200 IU Strongly positive: >200 IU

• Antibodies to Extractable Nuclear Antigens (ENAs): Anti-Ribonucleoprotein (RNP); Anti-

Smith (Sm); Anti-Sjögren’s Syndrome (SSA, SSB); Anti-Scleroderma (Scl-70); Anti-Jo-1
(Jo-1)
The extractable nuclear antigens (ENAs), another group of nuclear antigens (nonhistone proteins) to which
autoantibodies may develop. The most common ENAs are ribonucleoprotein (RNP) and Smith (Sm).

Reference Values: Normal

Negative: <20 U/mL
Borderline: 20–25 U/mL
Positive: >26 U/mL
 AUTOIMMUNE LIVER DISEASE TESTS

• Anti–Smooth Muscle Antibody (ASMA) Test

ASMA is associated with liver and bile duct autoimmune diseases. The immune response itself is
believed to be responsible for the disease process.

• Antimitochondrial Antibody (AMA) Test

AMA is non–organ specific and non–species specific and is directed against a lipoprotein in the
inner mitochondrial membrane. AMA is a marker for primary biliary cirrhosis (PBC), a chronic
inflammatory liver disease, characterized by the progressive destruction of inter- lobular bile ducts
with development of cholestasis and eventually cirrhosis.

Antineutrophil cytoplasmic antibodies (ANCA)

autoantibodies produced by a person's immune system that mistakenly target and attack proteins
within the person's neutrophils.
To help detect, aid in the diagnosis of, and sometimes monitor certain forms of the autoimmune
disorder systemic vasculitis (inflammation of blood vessels)
To help distinguish between Crohn disease (CD) and ulcerative colitis (UC), the two most
common types of inflammatory bowel disease (IBD).
Antiphospholipid Antibodies
Phospholipds are critical to platelet function in addition to various coagulation co-factors. When
antiphospholipid antibodies are produced, they interfere with the clotting process.
Antiphospholipid antibody testing is used to help determine the cause of:

• Inappropriate blood clot formation (unexplained thrombotic episode, excessive clotting)

• Recurrent miscarriage
• Low platelet count (thrombocytopenia)
• Prolonged PTT test
Depending on a person's signs and symptoms and medical history, a healthcare practitioner may order one or
more of these tests to help detect the presence of antiphospholipid antibodies and/or to help diagnose
antiphospholipid syndrome (APS):
Cardiolipin antibodies (IgG, IgM, and sometimes IgA) are frequently ordered since they are the most
common antiphospholipid antibodies.
Lupus anticoagulant assays (e.g., RVVT, LA-sensitive PTT) if a person has a prolonged PTT test.
Beta-2 glycoprotein 1 testing may be ordered along with the other antiphospholipid antibodies to detect their
presence and to provide the healthcare practitioner with additional information.

Anti Gastic Parietal Cell \ Anti Interinsic Factor

Parietal cell antibodies are autoantibodies, proteins produced by the immune system that mistakenly target a
type of specialized cells that line the stomach wall. This test detects these antibodies in the blood to help
diagnose pernicious anemia.
An intrinsic factor antibody (IF antibody) test may be used to help determine the cause of a vitamin B12
deficiency and to confirm a diagnosis of pernicious anemia.
Pernicious anemia is caused by vitamin B12 deficiency due to a lack of intrinsic factor. This condition
occurs primarily when the body's immune system targets its own tissues and develops antibodies directed
against the parietal cells and/or the intrinsic factor.
Celiac Disease Antibody Tests

• Anti-tissue transglutaminase antibody (anti-tTG), IgA: detects antibodies to tissue

transglutaminase, an enzyme that causes the crosslinking of certain proteins. Anti-tTG, IgA is the
most sensitive and specific blood test for celiac disease. The IgG class of anti-tTG may be ordered for
people who have a deficiency of IgA.

• Deamidated gliadin peptide (DGP) antibodies, IgA: detects anti-DGP IgA antibodies; like anti-
tTG, the IgG class may be performed for a person with an IgA deficiency.

• Anti-endomysial antibodies (EMA), IgA class: detects antibodies to endomysium, the thin
connective tissue layer that covers individual muscle fibers.

rheumatoid arthritis (RA) Antibody Tests

A cyclic citrullinated peptide (CCP) antibody test may be ordered along with or following a rheumatoid
factor (RF) test to help diagnose rheumatoid arthritis (RA) and to assess the severity and probable course of
the disease (prognosis). Inflammatory markers may also be measured at this time, such as ESR and C-
reactive protein (CRP).
Cyclic citrullinated peptide antibodies are autoantibodies produced by the immune system that are directed
against cyclic citrullinated peptides (CCP).
The normal level of anti-CCP is less than 20 u/ml. A level above 20 suggests the possibility of RA.