Serology New

Serological Tests:An introduction

Chalee Sienes-Reyes, RMT, MS-MT

Jasmen S. Pasia, RMT, MS-MT

Aileen Grace Ang, RMT, MAST-Bio

Francis Ian L. Salaver, RMT

Antigen-Antibody Interactions 3 categories:

1.Primary or Initial Reactions2.Secondary interactions3.Tertiary interactions

Primary or Initial Interactions

Initial Ag-Ab interaction occurs almost instantaneously and is rarely directly visible

Visualization is accomplished by labelling Ab or Ag withRadioactive labelsFluorescent dyesEnzymatic markers

Primary or Initial Interactions Techniques

Are known as primary binding tests

Considered as the most sensitiveInclude the following

Radioimmunoassay (RIA)Enzyme immunoassay (EIA) or Enzyme-

linked immunosorbent assay (ELISA)Immunofluorescent (IF) test

Secondary Interactions

Ag-Ab interactions which involve building a lattice to form visible aggregates of precipitates or agglutinates

Less sensitive but do not require sophisticated instruments

Secondary Interactions Techniques

Reactions are known as secondary binding tests

IncludePrecipitationAgglutinationNeutralizationComplement-fixation

Tertiary Interactions

Biologic expressions of Ag-Ab interaction that may either be beneficial or harmful to the body

These consequences of immune response in vivo are measured by tertiary binding tests

SECONDARY BINDING TESTS

Francis Ian L. Salaver, RMT

1. Affinity and Avidity

2. Ag:Ab ratio

3. Location and concentration of antigenic determinants on the particle

4. pH 6.8-7.0

5. Physical forms of the antigen

1. Soluble

2. Cellular/Particulate

6. Temperature

7. Length of incubation

8. Centrifugation

Factors Affecting Measurement of Ag/Ab Reactions

Effect of temperature

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Equivalence point: (suitable proportion between the virus particles and

RBCs)

Precipitation Curve

• Prozone – antibody excess, many antibodies coat all antigen sites- results in false negative

• Postzone – antigen excess, antibody coats antigen but cannot get lattice formation, results in false negative

• Zone of Equivalence – antigen and antibody present in optimal proportions to bind and give visible reaction

Affinity and Avidity

Agglutination

• When the antigen is particulate, the reaction of an antibody with the antigen can be detected by agglutination (clumping) of the antigen.

• The general term agglutinin is used to describe antibodies that agglutinate particulate antigens.

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All antibodies can theoretically agglutinate particulate antigens but IgM, due to its high valence, is particularly good agglutinin and one sometimes infers that an antibody may be of the IgM class if it is a good agglutinating antibody.

Agglutination• Qualitative and quantitative• Quantitation of agglutination tests to detect

specific Abs is done by titration• The reciprocal of the highest dilution giving

a positive reaction is known as titer and provides a measure of the amount of antibody in the serum

• A fourfold or more increase in the antibody titer between a paired sera is significant

Agglutination

Tube 1

Tube 2

Tube 3

Tube 4

Tube 5 Tube 6 Tube 7

SALINE 1 1 1 1 1 1 1

SERUM 1 1 of1:2 1 of1:4 1 of1:8 1 of1:16 1 of1:32 1 of1:64

FINAL DILUTION

1:2 1:4 1:8 1:16 1:32 1:64 1:128

Agglutination• Agglutinins

– Antibodies that produce such reactions

• Involves two-step process:– Sensitization or initial

binding– Lattice formation or

formation of large aggregates

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SENSITIZATIONSENSITIZATION

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Secondary Phenomenon • Lattice Formation• The Fab portion of the Ig molecule attaches to

antigens on 2 adjacent cells-visible results in agglutination

• If both antigen and antibody are SOLUBLE reaction will become visible over time, ie, precipitation

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Agglutination

• Direct Agglutination– Slide Agglutination Test– Plate Agglutination Test– Tube Agglutination Test

• Passive Agglutination Test– Slide Agglutination Test– Plate Agglutination Test– Tube Agglutination Test

• Haemagglutination test (HAT)

Direct Immune Agglutination

• Ag is an intrinsic component of the particle (bacteria, rbc)

• Used to determine whether Ab is present in biological fluids such as serum, urine, CSF

• Used primarily for diagnosis of infectious disease

• In case in which particle is RBC, reaction is referred to as hemagglutination

Direct Immune Agglutination

• ABO Blood Grouping

• Hemeagglutination• Cold Hemagglutinatinin Test

• Heterophile Agglutinin Test

• Bacterial Agglutination• Widal test

• VDRL, RPR

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Slide Agglutination TestQualitative Agglutination

• Antigen: isolated Salmonella in suspension• Antibody: specific antisera against Salmonella• Place test Salmonella in a drop of saline on a

slide • Add a drop of antiserum, mix and rock slide for

approx 1 minute• Examine for agglutination

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Slide Agglutination Test

Agglutination TechniquesSlide Test

• primarily for screening large number of sera (undiluted sera)

• rapid and sensitive

• use glass slides or disposable test cards

Agglutination TechniquesSlide Test

• If (+) reactivity is obtained using undiluted serum, serial dilution of serum is done to determine Ab titer

• False (-) can occur when Ab titer in undiluted serum is too high

Reading/Grading Agglutination Reading/Grading Agglutination ReactionsReactions

• Pseudo agglutination or the Rouleaux Formation also occurs– Red blood cells appear as stacks of coins.

• Addition of physiologic Nacl will disperse pseudo agglutination

• Saline Replacement is done after pseudo agglutination is observed so that it may not give false negative result due to the dilution effect of the saline

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Agglutination TechniquesMicrotiter Plate Tests

• serial dilution of serum sample to determine the Ab titer

• Constant amount of Ag is added to each serum containing well, rotated and titer is read

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Tube Agglutination Test

• Also known as the standard agglutination test or serum agglutination test (SAT)

• Test serum is diluted in a series of tubes (doubling dilutions)

• Constant defined amount of antigen is then added to each tube and tubes incubated for the required time.

• Particular antigen clumps at the bottom of the test tube

No agglutinationAgglutination

1/10 1/20 1/40 1/80 1/160 1/320 Neg. ctrl

In this case, the titre is 1/40

Tube Agglutination Test

GRADING AGGLUTINATION REACTIONS

GRADE DESCRIPTION Appearance

Negative (-) No aggregates

Weak (+/-) Tiny aggregates that are barely visible macroscopically; turbid and reddish supernatant

1+ A few small aggregates just visible macroscopically; turbid and reddish supernatant

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2+ Medium-sized aggregates; clear supernatant

3+ Several large aggregates; clear supernatant

4+ One solid aggregate; clear supernatant

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Identify what type of Agglutination reaction

Indirect (Passive)Agglutination

• Refers to agglutination of–Antigen-coated RBC

–Inert particles (latex, charcoal, bentonite or colloidal gold) which are passive carriers of otherwise soluble Ags

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Passive Agglutination Test

• Converting a precipitating test to an agglutinating test???

• Chemically link soluble antigen to inert particles such as LATEX or RBC

• Addition of specific antibody will cause the particles to agglutinate

• Reverse PAT: antibody linked to LATEX

e.g. Lancefield grouping in Streptococci.

Passive hemagglutination: The agglutination test only works with particulate antigens. However, it is

possible to coat erythrocytes with a soluble antigen and use the coated red blood cells in an agglutination test for antibody to the soluble antigen. This is called passive hemagglutination.

The test is performed just like the agglutination test. Applications include detection of antibodies to soluble antigens and detection of antibodies to viral antigens.

Passive (indirect) agglutination

Examples of types

– latex agglutination– passive hemagglutination (treated red blood

cells made resistant)Examples of tests - agglutination for leptospirosis

Widal test (typhoid fever)

Reverse Passive Agglutination

Antibody is bound to the carrier molecule, which is then mixed with patient’s sample to detect antigen.

Principle– antigen binds to soluble antibody coated on

carrier particles and results in agglutination – detects antigens

Example– detecting cholera toxin

Reverse Passive agglutination

Reverse Agglutination

• Ab is attached to the particles and the biological sample is tested for the presence of Ag

• Used to detect the presence of CRP

• A protein secreted into the blood during a inflammatory response

Reverse Agglutination: CRP

• Anti-CRP antibodies are affixed to to the latex particles

• Mixed with the serum of patients to detect the presence of CRP

Reverse Passive Latex Agglutination Test for RF

• RF is found in 75-80% of patients with RA

• RF is usually IgM Ab that reacts with new antigenic determinants on the Fc portion of IgG

• RF reagent• Latex particles coated with anti-RF

antibodies

Co-agglutinationCo-agglutination

• Co agglutination is similar to the latex agglutination technique for detecting antigen.

• Protein A, a uniformly distributed cell wall component of Staphylococcus aureus, is able to bind to the Fc region of most IgG isotype antibodies leaving the Fab region free to interact with antigens present in the applied specimens.

• The visible agglutination of the S. Aureus particles indicates the antigen-antibody reactions

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Co agglutination TestCo agglutination Test

Agglutination test in which inert particles (latex beads or heat-killed S aureus Cowan 1 strain with protein A) are coated with antibody to any of a variety of antigens and then used to detect the antigen in specimens or in isolated bacteria.

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Haemagglutination

Direct Non-Immune Hemagglutination

Principle– many human viruses have the ability to bind to

the surface structures on red blood cells from different species thereby causing agglutination

Example– influenza virus binds to fowl’s red blood cells

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Haemagglutination

RBC

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Viral Haemagglutination• Some viruses and microbes contain proteins

which bind to erythrocytes (red blood cells) causing them to clump together

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Viral Hemagglutination

• the attachment of viral particles by their receptor sites to more than 1 cell.

• As more and more cells become attached in this manner agglutination becomes visible

Negative control well (only RBCs+ buffer) (no haemagglutinin)

Positive control well (contains haemagglutinin)

Titer = 32 HA units/ml

Hemagglutination test: method

1:8

1:2 1:21:21:21:2

8 16 32 64 128 256

virus

serial dilution

mix with red blood cells

side view

top view

One HA unit :minimum amount of virus that causes complete agglutination of RBCs

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HEMAGGLUTINATION INHIBITION TEST (HI)

VIRUSE SERUM

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In the absence of anti-virus antibodies

Erythrocytes

Virus

Virus agglutination of erythrocytes

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In the presence of anti-virus antibodies

Erythrocytes

Virus Anti-virus antibodies

Viruses unable to bind to

the erythrocytes

Coombs Test : Agglutination Test

• The Coombs test is actually two separate tests: the "direct" and "indirect" Coombs tests.

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Coombs (Antiglobulin)TestsCoombs (Antiglobulin)Tests

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• Incomplete Ab• Direct Coombs Test

– Detects antibodies on erythrocytes

+ ↔

Patient’s RBCs Coombs Reagent(Antiglobulin)

Coombs Test Direct ant globulin test (also called the

Coombs’ test,

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Coombs (Antiglobulin)TestsCoombs (Antiglobulin)Tests • Indirect Coombs Test

– Detects anti-erythrocyte antibodies in serum

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Patient’s Serum

TargetRBCs

+ ↔Step 1

+ ↔

Coombs Reagent(Ant globulin)

Step 2

Application of Coombs Application of Coombs (Antiglobulin)Tests(Antiglobulin)Tests

• Applications–Detection of

anti-Rh Ab

–Autoimmune hemolytic anemia

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WASHING RBCs

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Why do we have to wash RBCs?

• To obtain pure RBCs and to get rid from any other blood components such as WBCs, immune complexes, and Abs

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Washing process

• Take place 4-5 time .

• Until get clear solution above the RBCs after centrifugation .

• Using PBS or normal saline .

Note :(avoid using water to wash RBCs because it will definitely lead to RBCs lyses)

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Procedure

• Obtain blood samples in tubes, spin at 1500 RPM for 5 minutes.

• Draw off the supernatant using Pasteur pipette.

• Add 2ml PBS to each tube and move to a clean test tube.

• Centrifuge again.

• Each time draw off the washing solution and add 10 ml PBS until the solution above the RBCs layer becomes clear.

Neutralization Test

• Ag-Ab reaction in which the harmful effects of a bacterial exotoxin or a virus are blocked by specific antibodies (antitoxins).

Complement Fixation Test (CFT)

• IgM and IgG can activate complement system forming immune complexes

• Hemolytic CFT is performed in 2- parts– Test system: Ag + Serum (Ab?) +

guinea pig serum which provide the complement

– Indicator system: sensitized sheep RBC + anti-SRBC

Precipitation Reactions

- is the reaction between a soluble antigen and its specific antibodies-soluble antigens are smaller and in solution; complexing with antibodies make these bigger and they fall out of solution as a precipitate –visible to the eye.

• IgG is a much better precipitating Ab than IgM (the reverse is true of agglutination)

• 2 types:• Precipitation in liquid medium• Precipitation in gel medium

Measurement of Precipitation by Light

• Antigen-antibody complexes, when formed at a high rate, will precipitate out of a solution resulting in a turbid or cloudy appearance.

• Turbidimetry measures the turbidity or cloudiness of a solution by measuring amount of light directly passing through a solution.

• Nephelometry indirect measurement, measures amount of light scattered by the antigen-antibody complexes.

Turbidimetry

• Measures turbidity or cloudiness of a solution by measuring the amount of light PASSING THROUGH the solution.

• Soluble antigen and antibody join and once they join in sufficient amounts precipitate, results in cloudiness.

• The more cloudy the solution, the less light can pass through.

Nephelometry

• Measures SCATTERED light bouncing off antigen-antibody complexes.

Nephelometry

• Antibody reagent is combined with patient

sample. • If antigen is present in

the patient’s sample, Ag/Ab complexes will

form and precipitate out of solution.

Y

Y Y

Nephelometry

• When light is passed through the solution, the precipitates cause the light to scatter at various angles.

• The light that is scattered at a particular angle is measured. This corresponds to the level of antigen in the sample.

Light source

Detector

Passive Immunodiffusion

• Reactions in gels/liquids

• Migrate towards each other and where they meet in optimal proportions form a precipitate.

Four Methodologies

• Single diffusion, single dimension

• Single diffusion, double dimension

• Double diffusion, single dimension

• Double diffusion, double dimension

Immunodiffusion Tests

Single diffusion test: if only one reactant (usually Ag) is moving

Double diffusion test: if both Ag and Ab are moving through the gel medium

Single dimension: if there’s only one effective dimension for Ag or Ab migration, like up and down

Double dimension: if circular holes or wells are cut in a gel on a flat surface like petri dish,Ag or Ab diffuses from the wells radially

Ouidin Single Diffusion, Single Dimension

Oudin Precipitation

• Solution of antibody is carefully layered on top of a solution of antigen, such that there is no mixing between the two.

• With time at the interface where the two layers meet, antigen-antibody complexes form a visible precipitate.

Single Diffusion, Double Dimension

Radial Immunodiffusion (Mancini/Fahey) –

• In radial immunodiffusion Antigen (IgG antibody) is incorporated into the agar gel as it is poured.

• Different dilutions of the antibody are placed in holes punched into the agar.

• As the antibody diffuses into the gel it reacts with the antigen and when the equivalence point is reached a ring of precipitation is formed.

• The diameter of the ring is proportional to the concentration of antibody since the amount of antigen is constant.

Radial Immunodiffusion

Immunodiffusion TestRadial Immunodiffusion or Mancini

TestAb is fixed to the agar in a petri

dishCircular wells are cut in the gel

and are loaded with AgA ring of precipitate is formed as

the Ag diffuses radially towards its equilibrium concentration

Radial Immunodiffusion

• 1. Fahey Method– Also known as Kinetic Method or Incomplete Immunodiffusion– Diameter of precipitin ring measured at 24 hours of diffusion– Diameter proportional to log of antigen concentration

• 2. Mancini Method– Also known as End Point Method or Complete Immunodiffusion– Area of precipitin ring formed at 48 hours of diffusion measured– Area proportional to antigen concentration

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Double Diffusion, Single Dimension

• -Two wells cut in agar plate (1 for antigen; one for antibody)

• -Antigen and antibody diffuse towards one another• -At optimal concentrations, a precipitate line is formed

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Double Diffusion, Double Dimension

• Ouchterlony Gel Diffusion– Holes punched in agar.– Known antibody or antigen added to center

well.– Known sample added to outer well.– Unknown sample added to outer well next to

unknown sample.– Wait for bands to form.

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Ouchterlony Immunodiffusion

Ouchterlony - Identity

• The precipitation appears as a continuous line in the form of an angle between those two wells and the C well. There are no spurs at the angle and this type of reaction is termed a band of identity.

Ouchterlony – Partial IdentityIf a solution with antigens X and Y is placed in well 1, a solution with antigen X only is placed in well 2, and antiserum containing antibodies specific for both X and Y is placed in well 3, a reaction similar to that appearing in Fig. 2 will occur. Notice that there is a spur reaction towards the XY well. This indicates that the two antigenic materials in wells 1 and 2 are related, but that the material in well 1 possesses an antigenic specificity not possessed by the material in well 2. Such a reaction with spur formation indicates partial identity

Ouchterlony – Non-Identity

• If the material in wells 1 and 2 do not possess common antigens and the antiserum in well 3 possesses specificities for both materials, the reaction will appear as two crossed lines as in Fig. 3

Immunodiffusion Test

• Disadvantages of ordinary immunodiffusion tests

• incubation of several hours or even days are needed to ensure equilibrium of the system has occurred

• possibility that 2 or more precipitation bands may form in the same plate

Electrophoretic Techniques

Electrophoretic Techniques

• Immunodiffusion can be combined with electrical current to speed things up.

Rocket Immunoelectrophoresis

• Antigen is electrophoresed into gel containing antibody. The distance from the starting well to the front of the rocket shaped arc is related to antigen concentration.

Rocket Electrophoresis

Immunoelectrophoresis


• Two-dimensional immunoelectrophoresis. Antigens are separated on the basis of electrophoretic mobility.

• The second separation is run at right angles to the first which drives the antigens into the antiserum-containing gel to form precipitin peaks; the area under the peak is related to the concentration of antigen.

Immunofixation Electrophoresis

• Immunofixation Electrophoresis (IFE) combines zone electrophoresis with immunoprecipitation.

• This technique may be used to identify and characterise serum proteins.

• In IFE, proteins of sample are first separated by electrophoresis on a support (agarose) according to their charge and after that the medium is overlaid with monospesific antiserá reactive with specific protein - antigen.

• If the antigen is present a characteristic immunoprecipitin band will be formed.

Immunofixation Electrophoresis

Western Blot

• 1) Proteins are separated by gel electrophoresis.

• 2) The proteins are transfered to a sheet of special blotting paper called nitrocellulose, though other types of paper, or membranes, can be used. The proteins retain the same pattern of separation they had on the gel.

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Western Blot

• 3) An antibody is then added to the solution which is able to bind to its specific protein. The antibody has an enzyme (e.g. alkaline phosphatase or horseradish peroxidase) or dye attached to it which cannot be seen at this time.

• 4) The location of the antibody is revealed by incubating it with a colorless substrate that the attached enzyme converts to a colored product that can be seen and photographed.

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Immuno-chromatography: Principle

• Dye-labelled antibody, specific for target antigen, is present on the lower end of nitrocellulose strip or in a plastic well provided with the strip.

• Antibody, also specific for the target antigen, is bound to the strip in a thin (test) line

• Either antibody specific for the labelled antibody, or antigen, is bound at the control line

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Lysing agendLabled AB.

Test band(bound AB)

Control band(bound AB)

Nitrocellulose strip

BoundAB

Free labled AB

Immuno-chromatography: Principle

• If antigen is present, some labelled antibody will be trapped on the test line

• Excess-labelled antibody is trapped on the control line

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Captured Ag-labelled Ab-complex

Captured labelled Ab

Labelled AB-AG-complex

Captured by bound AB of

test band

Labelled AB-AG-complex

Captured by bound AB ofcontrol band

Immuno-chromatography:Immuno-chromatography: Performance, applications Performance, applications

• Advantages– Commercially available– Single use, rapid test– Easy to perform– Can detect antigen or antibody– Can be used in the field

• Limitations– Cost– Concern validated data

• Time taken - 1 hour04/08/23 Dr.T.V.Rao MD 116

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