Comprehensive Guide to Immunology and Serology Techniques

Immunology

The study of the body's immune system and its functions and disorders.

Serology

The in-vitro study of blood serum and its components to detect antigens and antibodies.

Antigen-antibody reactions in-vitro

Reactions that occur outside a living organism, typically in a laboratory setting, involving the interaction between antigens and antibodies.

Monoclonal antibodies

Antibodies that are made by identical immune cells that are all clones of a unique parent cell.

Polyclonal antibodies

Antibodies that are produced by different B cell lineages within the body.

Immunoglobulins production

The process by which antibodies are produced commercially, often involving hybridoma technology.

Precipitation

The process of combining soluble antigen with soluble antibody to produce insoluble complexes that are visible.

Agglutination

The process by which particulate antigens, such as cells, aggregate to form larger complexes when a specific antibody is present.

Serological tests

An in-vitro study of blood serum and its components to detect antigens and antibodies.

Quantitation of antigen and antibodies

The measurement of the amount of antigens and antibodies present in a patient sample.

Specific serological tests

Tests designed to diagnose specific viral and bacterial infections.

Non-specific serological tests

Tests that provide evidence for some diseases but are not definitive.

Qualitative tests

Tests that provide a yes or no answer regarding the presence of a substance.

Quantitative tests

Tests that measure how much of a substance is present in a sample.

Semi-quantitative tests

Tests that provide an estimate of the amount of a substance present, but not an exact measurement.

Detection of antibody

The process of identifying the presence of antibodies in a sample.

Detection of autoantibody

The identification of antibodies that mistakenly target and react with a person's own tissues.

Detection of offending drugs

The identification of drugs that may cause adverse reactions in patients.

Immunoassay of hormones

A test that can measure minute amounts of hormones in a sample.

Label-free immunoassays

Broadly classified as label-free immunoassays.

AG AB interaction

Based on physical change, it usually involves AG AB interaction.

Antibody

A glycoprotein produced by B lymphocytes which is capable of specifically binding antigen via an antigen binding site.

Antigen

A molecule which is capable of interacting with an antibody molecule (or T cell receptor) via the antibody molecule's antigen binding site.

Specific binding capacity

The ability of an antibody to bind specifically to its corresponding antigen.

Affinity

The strength of interaction between an antigen and an antibody.

Association

Ag + Ab forms an Ag-Ab complex.

Dissociation

The process where the antigen-antibody complex breaks apart.

Epitope binding site

The specific site on an antigen that is recognized and bound by an antibody.

Avidity

The collective affinity of multiple binding sites of an immunoglobulin.

Avidity formula

Avidity = Affinity + Valence.

Valence

The number of epitope binding sites in an antibody.

Heterogeneous mixture of antibodies

A mixture produced by different B cell clones.

Polyclonal antibodies production

Antibody responses differ from one individual to another.

Hybridoma technology

A method used to produce monoclonal antibodies using hybridoma cells.

Cell Fusion

The process of fusing B cells with myeloma cells to produce hybridomas.

HAT Medium Selection

A selection medium containing Hypoxanthine, Aminopterin, and Thymidine used to select hybridomas.

HGPRT enzyme

An enzyme inherited from B cells that allows hybridomas to survive in HAT medium.

Specificity

The ability of an antibody to bind to a specific epitope, with monoclonal antibodies having high specificity and polyclonal antibodies having lower specificity.

Sensitivity

The ability of an antibody to detect its target, with polyclonal antibodies generally having higher sensitivity.

Reproducibility

The consistency of antibody production across batches, with monoclonal antibodies being highly reproducible and polyclonal antibodies showing low reproducibility.

Cost

Monoclonal antibodies are expensive, while polyclonal antibodies are more cost-effective.

Production Time

Monoclonal antibodies take longer to produce using the hybridoma method, whereas polyclonal antibodies can be produced in a shorter time through animal immunization.

IgM

An antibody that appears first after infection, detectable around 1-2 weeks, peaks quickly, and is an early marker of recent/acute infection.

IgG

An antibody that rises more slowly than IgM, detectable around 2 weeks, reaches a higher sustained level, and provides longer-term immunity.

IgA

An antibody that appears around the same time as IgM/IgG, important for mucosal immunity, and increases in levels but not as strongly sustained as IgG.

Neutralising Antibodies

Antibodies that provide functional protection by blocking the pathogen and follow a similar pattern to IgG.

Uncertainty in Antibody Response

Reflects variability between individuals in terms of antibody levels, timing, or persistence.

Polyclonal Response

The body's natural production of a variety of antibodies (IgM, IgG, IgA) from different B-cell clones after infection.

Monoclonal Antibody Use

Lab-made antibodies that mimic one specific antibody and are often used for therapy or diagnostics.

Serum

Clotted blood collected using a Serum Separator Tube, commonly used for serological testing.

Plasma

Anticoagulated blood used for various serological tests.

Whole Blood

Blood collected with anticoagulants like EDTA or Heparin, used in flow cytometry and HLA typing.

Cerebrospinal Fluid (CSF)

Fluid collected via lumbar puncture, used for testing neurosyphilis and viral encephalitis.

Bronchoalveolar Lavage (BAL)

Fluid collected via bronchoscopy, used for testing Pneumocystis jirovecii.

Synovial Fluid

Joint fluid collected via arthrocentesis, used for testing rheumatoid factors and autoimmune markers.

Urine

Collected midstream, used for testing Bence Jones Protein and Legionella antigen.

Stool

Sample collected for testing Clostridium difficile and Rotavirus.

6 main uses of serological test

1. identification of microorganisms

2. detection of antibodies

3. detection of autoantibodies

4.detection of offending drugs

5.immunoassay of hormones (measure minute amount of hormones)

6.detection of immunologic abnormalities

Nasopharyngeal Swab

Swab collected from the nose/throat, used for testing Influenza and COVID-19 Antigen.

Dried Blood Spot (DBS)

Blood collected via heel or finger prick, used for newborn screening and HIV/Hepatitis testing.

Collection Tube

Container used to collect blood samples for testing.

EDTA (Purple)

Anticoagulant used in blood collection that prevents clotting.

Heparin (Green)

Anticoagulant used in blood collection that prevents clotting.

Citrate (Blue)

Anticoagulant used in blood collection that prevents clotting.

Type of blood collection tube used for serum collection.

Red-top, serum separator tube - SST (Gold/Tiger-top)

Clotting Factors

Proteins in blood that help control bleeding.

Processing Time

Duration required to prepare blood samples for testing.

Antibody Testing

Testing method to detect the presence of antibodies in the blood.

Complement System Testing

Testing method to evaluate the complement system's function.

Acute Serum

Serum collected from a patient with a current infection.

Convalescent Specimen

Blood sample collected during the recovery phase of an infection.

Paired Sera

Two serum samples tested together, one from acute and one from convalescent phase.

Seroconversion

The change of an antibody test from negative to positive over time.

Confirming a Recent or Active Infection

Using paired serology to detect rising IgG titers to confirm an infection.

Retrospective Diagnosis of Viral or Bacterial Infections

Using paired testing to detect past infections when recovery occurs before diagnosis.

Diagnosing Diseases That Require Seroconversion Detection

Identifying diseases by observing the change in antibody presence over time.

Confirming Immunity or Vaccine Response

Measuring antibody development after vaccination or post-infection.

what is the acute sample, convalescent sample and diagnosis for dengue Fever

acute sample: high IgM/ low IgG

convalescent sample: low IgM/ high IgG (4x increase)

diagnosis: recent infection

COVID-19 IgG Serology

Example of retrospective diagnosis using paired testing.

Hepatitis A, B, C Serology

Examples of diseases that may require seroconversion detection.

Hepatitis B (Anti-HBs IgG response)

Example of confirming immunity post-vaccination.

Measles, Mumps, Rubella (MMR vaccine response)

Example of confirming vaccine response.

Varicella-Zoster Virus (Chickenpox Immunity Testing)

Example of testing for immunity against chickenpox.

The Early Phase of infection will use what sample of patients serum?

acute serum

when should convalescent specimens be followed up?

2-4 Weeks Later

Criteria for diagnosis of Primary Infection

4-fold or more increase in titre of IgG or total antibody between acute and convalescent

IgM present

Recent infection

Presence of IgM

Typhoid (Widal Test)

4× rise in titers

what is the acute sample, convalescent sample and diagnosis for Typhoid Fever

acute: Low O & H agglutinin titers

convalescent: 4x rise in tires

diagnosis: typhoid fever

name 2 Criteria's for reinfection

4-fold or more increase in IgG antibody or total

IgM absecent/ slight increase

what is the acute sample, convalescent sample and diagnosis for Syphilis (RPR/VDRL)

acute sample: Low titer

convalescent sample: 4x increase/ decrease (post-treatment)

diagnosis: active or resolving infection

Presence of IgM

Similar to reinfection

7 examples of UNLABELLED IMMUNOASSAYS

Examples include Precipitation, Agglutination, Complement fixation, Neutralisation, Antibody dependent cell mediated cytotoxicity (ADCC), Opsonisation

Formation of an antigen-antibody lattice

Depends on the valence of both the antibody and antigen.

Bivalent antibody

A precipitate will not form with monovalent Fab fragments.

Zone of Equivalence

The optimal ratio of antigen and antibody affects the amount of precipitation.

Zone of antibody excess

Initially, there is not enough antigen to produce visible lattice formation.

Equivalence zone

Where both the optimal antigen-antibody interaction and maximal precipitation occur.

Zone of antigen excess

If even more antigen were added, the amount of precipitation would decline.

Prozone Effect (Antibody Excess Zone)

A phenomenon in which visible agglutination or precipitation does not occur in mixtures of specific antigen and antibody because of antibody excess.