Basic Serological Concepts and Principles Flashcards by Julia Kristine Cocal

scientific study of serum and other bodily fluids
measures the amount of Ag or Ab in serum

useful in:
- diagnosing a disease
- efficacy of vaccine
- monitoring therapy

SEROLOGY

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The Antigen & Antibody bind specifically with each
other.

Antigen – Antibody Reaction

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3 Stages of Ag-Ab Reactions

Formation of Ag-Ab complex
Formation of visible events
Destruction of Antigen

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Formation of Ag-Ab complex

Binding/Recognition

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Destruction of Antigen

Immune Response Activation and Neutralization

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Formation of visible events

Agglutination and Precipitation

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The combining site of an antibody is located in the Fab portion of the molecule and antigen nestles in a cleft.

Lock and Key Concept

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Bonds that hold the antigen to the antibody

Non-covalent Bonds

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Since Ag-Ab reactions occur via non-covalent
bonds, they are by their nature reversible.

Reversibility

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formed between hydrogen of one molecule to usually, a nitrogen or oxygen in another molecule

Hydrogen bonds

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attraction between oppositely charged ions (positive and negative charges)

Electrostatic bonds

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Non-polar regions of the antigen and antibody tend to cluster together to avoid water, resulting in a stabilizing interaction.

Hydrophobic bonds

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nonspecific attractive forces generated by the interaction between electron clouds and hydrophobic bonds

Van der Waals

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refers to the strength of a single antibody-antigen
interaction. Each IgG antigen binding site has high
affinity for its target.

Affinity

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refers to the strength of all interactions combined. IgM typically has low affinity antigen binding sites, but there are ten of them, so avidity is high.

Avidity

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measures the acidity or alkalinity of a
solution

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neutral pH between

6.8-7.0

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incubation period allows Ag-Ab to
optimally bind to each other

Time

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shortens distance between Ag and Ab, allowing interaction to occur

Centrifugation

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influences antibody action

Temperature

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Best at 37oC
Can cross the placenta

Warm-reacting Ab

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Physical Form of the Antigen that is more visible reactions

Cellular antigens

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Best at 4-22oC
Can activate complement

Cold-reacting Ab

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Physical Form of the Antigen that is less
visible reactions

Soluble antigens

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detects presence or absence of Ab/Ag

Qualitative test

determine concentration of Ab/Ag

Quantitative test

– concentration of an antibody expressed as the highest dilution of a serum that produces a positive result.

Titer

time where antibodies start to appear the development of detectable specific antibodies in serum

Seroconversion

time where antibodies start to disappear

Seroreversion

The ability of a population of Ab molecules to react with more than one antigen.

Cross reactivity

ability of a test to detect a very small amounts of a substance

analytical sensitivity

ability of a test to give positive result if px has the disease

clinical sensitivity

represent cases in which individuals with the particular disease test negative by a certain test

False-negatives

ability of test to detect substance without interference from cross-reacting substances

analytical specificity

ability of test to give negative result if patient does not have disease

clinical specificity

with visible effects

SECONDARY

represent cases in which individuals without the particular disease test positive by certain tests

False-positives

– rapid initial reaction without visible effects

PRIMARY

– In vivo testing

TERTIARY

* Detects initial interaction between antigen and antibody * Not visible to the naked eye

Primary / Initial Reactions

any substance that will complex to another substance

Ligand/Analyte

pros of Primary / Initial Reactions

– Highly sensitive – Can detect picogram or nanogram amounts of Ag or Ab concentration

one reactant is labeled so that the amount of binding can be measured

Ligand assay

Cons of Primary / Initial Reactions

– Expensive – Hazardous to health – Requires special training

* Patient antigen in the serum competes with labelled antigen to limited binding sites in immobilized antibodies on the test medium * Detects antigens (+) * Inverse relationship

Competitive Reaction

* Patient antigen in the serum competes with labelled antigen to limited binding sites in immobilized antibodies on the test medium * Detects antibodies (+)

Indirect Noncompetitive

Immobilized antibodies specific for patient Ag Detects antigens (+)

Sandwich / Capture Noncompetitive

* Uses radioisotopes as labels (Carbon14, 135I, 131I, tritium) * Measured by scintillation counter or gamma/beta rays counter * More sensitive than ELISA

Radioimmunoassays

Detects total IgE (+)

Radioimmunosorbent test (RIST)

Detects allergen specific IgE (+)

Radioadsorbent test (RAST)

* Use enzyme as ligand + chromogenic substrate → colorimetric rxn * Measured by spectrophotometer * Less sensitive than RIA

Enzyme-Linked Immunosorbent Assay (ELISA)

* Detected by flow cytometer * Uses fluorescent dyes

Immunofluorescence assay (IFA)

color of Fluorescein isothiocyanate

green

color of Tetramethyl rhodamine

red

color of 4’,6-diamidino-2-phenylindole

blue

color of Alexa flour dyes

multiple colors

Detects consequence (outcome) of antigen-antibody binding * Pros: – Directly visible to the naked eye – No label needed * Cons: – Less sensitive

Secondary Reactions

* Involves the actions of antibody (agglutinin) and cellular/particulate antigen (agglutinogen)

Agglutination

Upfront reaction of antibodies to antigens which are part of the cell surface ; detects antibodies (+)

Direct Agglutination

* Antigen binds to soluble antibody coated on carrier particles resulting to agglutination * Detects antigens (+)

Reverse Passive Agglutination

An agglutination reaction that employs particles that are coated with antigen not normally found in the cell surface Detects antibodies (+)

Passive Agglutination

* Autoantibody binds to Fc portion of labprepared antibodies in which Fab sites have coated carrier particles causing agglutination * Detects antibodies (+)

Modified Reverse Passive Agglutination

* Using protein A of inert bacteria S. aureus, in which Fc portion of IgG binds to, leaving the Fab sites free to interact with antigen of patient sample * Detects antigens (+)

Coagglutination

* Makes use of some viruses that have receptors for red blood cell (RBC) antigens * Detects antigens (+)

Viral Hemagglutination

Also known as Coombs’ Test or Antiglobulin Test (AGT)

Coombs Test

the degree of negative charge on the surface of a red blood cell

Zeta potential

* Detects antibodies (+) ; positive result is absence of agglutination since RBCs not clumped

Agglutination Inhibition Reaction

Autoantibodies (IgG) + own red cells

Autoimmune Hemolytic Anemia

Recipient antibodies + donor red cells

Hemolytic Transfusion Reactions

Detect in vivo binding of IgG to RBCs

Direct Coombs’ Test

used to detect very low concentrations of antibodies present in a patient's plasma/serum prior to a blood transfusion

Indirect Coombs Test

Maternal antibodies + fetus red cells

Hemolytic Disease of the Newborn)

used to detect antibodies or complement proteins bounded to the surface of RBCs

direct Coombs test

* Detects in vitro binding of IgG to RBCs * Also known as indirect antiglobulin test (IAT)

Indirect Coombs’ Test

* Ability of specific antibodies to block the site(s) on bacteria or viruses that they use to enter cell * Positive result is no cell lysis

Neutralization

* Detects fixed amount of antibodies (+) * Positive result is no hemolysis

Complement Fixation Test

* Uses soluble antigen to detect IgG (+) * Positive result is a precipitate (+)

Precipitation

* In vivo serologic reactions * Determination of the protective value of antiserum in the test

Tertiary Reactions

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Basic Serological Concepts and Principles Flashcards

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