Immunological techniques in Diagnosis

M. Dankyau

June 2023

• Based on WHO Laboratory training for Field Epidemiologists

Outline

• Introduction
• Antigen-Antibody detection
• Advanced techniques
• Application implications
• Discussion
• Summary
• Conclusion

Introduction

The Diagnostic-Therapeutic Cycle: A Simplified View

Patient

Data collection:

-History

-Physical examinations

-Laboratory and other tests

Decision

making

Planning

Information

Diagnosis/assessment

Therapyplan

Data

Brief history of antibodies

• Study of antibodies began in 1890 when Emil von Behring and Shibasaburo Kitasato described antibody activity against diphtheria and tetanus toxins.
• Behring and Kitasato put forward the theory of humoral immunity, proposing that a mediator in serum could react with a foreign antigen.

Brief history of antibodies 2

• 1891: Term “Antikörper” coined by Paul Ehrlich
• Substance in the blood that confers immunity
• "if two substances give rise to two different antikörper, then they themselves must be different”
• “Lock-and-Key” theory
• 1920’s: Heidelberger and Avery identified antibodies as proteins
• 1940’s: Linus Pauling confirms lock-and-key theory

Brief history of antibodies 23

• 1948:Immunoprecipitation: use of antibodies for detection
• 1956: Glick and Chang- Bursa of Fabricius: Antibodies come from B cells
• 1962: Rodney Porter elucidated the structure of antibody gammaglobulin (IgG)
• 1976: Hozumi and Tonegawa, antibody gene rearrangement

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Useful characteristics of antibodies

• Epitope: the portion of an antigen that makes contact with a particular antibody or T cell receptor
• Specificity: Ability to recognize individual epitopes
• Cross-reactivity:
• Ability to bind to more than one epitope
• Shared epitopes between different antigens

Epitope

Useful characteristics of antibodies 2

• Strength of binding:
• Affinity: Strength of binding between F_ab and epitope
• Avidity: Overall strength of binding of serum and antigen

Antigen-Antibody reactions

Antibody specificity

Good: recognize and identify related antigens

Bad: Confuse one antigen with a related antigen

Antibody types

• Polyclonal antibodies:
• Isolated from immune serum
• Many different idiotypes
• Recognize many different epitopes
• Strong avidity
• Highly sensitive
• Cross reactive: Less specific
• Monoclonal antibodies:
• Single idiotype
• Single epitope
• Highly specific
• Lower avidity

Immunological assays

• Diagnostic:
• Assay for antigen
• Assay for antibody

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• Research:
• Identify cells
• Identify cell products
• Isolate cells or macromolecules
• Assay cell function

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Examples

Diagnostic assays

• Hemagglutination
• Hemagglutination inhibition
• Enzyme-linked Immunosorbent Assay (ELISA)
• Radioimmunoassay (RIA)
• Immunofluorescent assay (IFA)

Research techniques

• ELISA
• Immunohistochemistry
• Flow cytometry
• Cell sorting
• ELIspot
• Immunoblot
• Immunoprecipitation
• Mixed lymphocyte response
• Chromium release assay

� �Antigen and antibody detection�

Laboratory Training for Field Epidemiologists

Laboratory Training for Field Epidemiologists

Detection

• Detection of antigen-antibody complex
• Antigen-antibody complex requires specific conditions
• temperature
• pH
• Complex may be directly visible or invisible

Laboratory Training for Field Epidemiologists

Detection

Directly visible – agglutination

Invisible

• requires specific probes (enzyme-labelled anti-immunoglobulin, isotope-labelled anti-immunoglobulin, etc.)
• binds Ag-Ab complex and amplifies signals
• signals can be measured by naked eyes or specific equipment e.g. ELISA, RIA, IFA

Laboratory Training for Field Epidemiologists

Methods for Ag-Ab detection

• Precipitation
• Agglutination
• Hemagglutination and hemagglutination inhibition
• Viral neutralization test

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• Radio-immunoassays
• ELISA
• Immunofluorescence
• Immunoblotting
• Immunochromatography

Precipitation

Principle

• soluble antigen combines with its specific antibody
• antigen-antibody complex is too large to stay in solution and precipitates

Examples

• flocculation test
• immuno-diffusion test
• counter-immuno-electrophoresis (CIEP)

Laboratory Training for Field Epidemiologists

Flocculation test �(precipitation reaction)

Principle

• precipitate, a concentrate of fine particles, is usually visible (macroscopically or microscopically) because the precipitated product is forced to remain suspended

Examples

• VDRL slide flocculation test
• RPR card test
• Kahn’s test for syphilis

Laboratory Training for Field Epidemiologists

Flocculation test �(A precipitation reaction)

RPR card test

(1) Non Reactive (2) Weakly Reactive (3,4) Reactive

Laboratory Training for Field Epidemiologists

Precipitation: Performance, applications

• Advantages
• sensitive for antigen detection
• Limited applications
• Time taken - 10 minutes

Laboratory Training for Field Epidemiologists

Direct agglutination

Principle

• combination of an insoluble particulate antigen with its soluble antibody
• forms antigen-antibody complex
• particles clump/agglutinate
• used for antigen detection

Examples

• bacterial agglutination tests for sero-typing and sero-grouping e.g., Vibrio cholerae, Salmonella spp

Laboratory Training for Field Epidemiologists

Passive (indirect) agglutination

Principle

• precipitation reaction converted into agglutination - coating antigen onto the surface of carrier particles like red blood cells, latex, gelatin, bentonite
• background clears

Examples of types

• latex agglutination
• co-agglutination
• passive hemagglutination (treated red blood cells made resistant)

Examples of tests - agglutination for leptospirosis; Widal test (typhoid fever)

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Laboratory Training for Field Epidemiologists

Reverse passive agglutination

Principle

• antigen binds to soluble antibody coated on carrier particles and results in agglutination
• detects antigens

Example

• detecting cholera toxin

Laboratory Training for Field Epidemiologists

Reverse passive agglutination

Positive

Negative

Laboratory Training for Field Epidemiologists

Agglutination:�Performance, applications

Advantages

• sensitive for antibody detection

Limitations

• Prozone phenomenon:
• requires the right combination of quantities of antigen and antibody
• handled through dilution to improve the match

Time taken

• 10-30 minutes

Laboratory Training for Field Epidemiologists

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 red blood cells

Laboratory Training for Field Epidemiologists

Hemagglutination inhibition

Principle

Antibodies to the virus in the patient serum bind to the virus; blocks binding sites on the viral surfaces

• prevents the virus from agglutinating the red cells

Example

• detecting antibodies to influenza and dengue viruses

Positive

Negative

Hemagglutination inhibition for detection of Dengue antibodies

Laboratory Training for Field Epidemiologists

Hemagglutination:�Performance, applications

Advantages

• highly specific
• can be used as gold standard

Limitations

• technically demanding
• time consuming
• cannot distinguish IgG from IgM

Time taken

• 1 day

Laboratory Training for Field Epidemiologists

Neutralization assays

Principle

• antibodies in serum neutralize antigens on the surface of viruses �(neutralizing antibodies)
• inhibited viruses cannot infect cell lines

Example

• plaque neutralization assay for dengue virus, Japanese encephalitis virus
• antibodies to bacterial toxins and other extra-cellular products that display measurable activities (e.g., ASLO, diphtheria toxin, clostridium toxin)

Laboratory Training for Field Epidemiologists

Neutralization:�Performance, applications

• Advantages
• Highly specific
• Often used as gold standard
• Limitations
• Technically demanding
• Time consuming
• Can only be used for viruses that can be grown
• Complexity limits the use beyond gold standard
• Time taken
• 1 week

Laboratory Training for Field Epidemiologists

Neutralization Assay

Laboratory Training for Field Epidemiologists

Radio-immunoassays

• Principle
• Radioactively labelled-antibody (or antigen) competes with the patient’s unlabelled antibody (or antigen) for binding sites on a known amount of antigen (or antibody)
• Reduction in radioactivity of the antigen-patient antibody complex compared with control test is used to quantify the amount of patient antibody/antibody bound
• Limited use due to the problems with handling radioisotope
• Example
• HBsAg
• Thyroid function test

Laboratory Training for Field Epidemiologists

Radio-immunoassays:�Performance, applications

Adantages

• highly sensitive
• can be used for detection of small quantities
• quantification possible

Limitations

• expensive
• requires isotopes

Time taken

• 1 day

Laboratory Training for Field Epidemiologists

Enzyme-linked immunosorbant assay (ELISA)

Principle

• use of enzyme-labelled immunoglobulin to �detect antigens or antibodies
• signals are developed by the action of �hydrolyzing enzyme on chromogenic substrate
• optical density measured by micro-plate reader

Examples

• Hepatitis A (Anti-HAV-IgM, anti-HAV IgG)

Labeling technique

Laboratory Training for Field Epidemiologists

ELISA

Laboratory Training for Field Epidemiologists

Types of ELISA (Ag Ab tests)

Competitive

• Antigen or antibody are labelled with enzyme and allowed to compete with unlabeled ones (in patient serum) for binding to the same target
• Hydrolysis signal from Ag-Ab complex (enzyme-labelled) is measured
• Antigen or antibody in serum is then calculated
• No need to remove the excess/unbound Ag or Ab from the reaction plate or tubes)

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Labeling technique

Laboratory Training for Field Epidemiologists

Types of ELISA used in the detection of antigens and antibodies

• Non-competitive
• must remove excess/unbound Ag�or Ab before every step of reactions
• Direct ELISA
• Indirect ELISA
• Sandwich ELISA
• Ab Capture ELISA (similar to sandwich ELISA but in 1^st step,� anti-Ig (M or G) is coated �on the plate
• Then antibodies in patient serum �are allowed to capture in next step

Labeling technique

Laboratory Training for Field Epidemiologists

ELISA:�Performance, applications

• Advantages
• Automated, inexpensive
• Objective
• Small quantities required
• Class specific antibodies measurable
• Limitations
• Expensive initial investment
• Variable sensitivity / specificity of variable tests
• Cross contamination
• Time taken - 1 day

Laboratory Training for Field Epidemiologists

Immuno-fluorescence

• Principle
• Use fluorescein isothiocyanate labeled-immunoglobulin to detect antigens or antibodies according to test systems
• Requires a fluorescent microscope
• Examples
• Herpes virus IgM
• Dengue virus
• Rabies virus
• Scrub and murine typhus

Labeling technique

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Cell infected with Dengue virus

V. Cholerae

Laboratory Training for Field Epidemiologists

Immuno-fluorescence:�Performance, applications

• Advantages
• Sensitive and specific
• Can be used for discrepant analysis
• Limitations
• Expensive (Reagents and equipment)
• Subjective
• Cross reactivity
• Non-specific immuno-fluorescence
• Time taken
• 1 day

Laboratory Training for Field Epidemiologists

Advanced techniques

Laboratory Training for Field Epidemiologists

Western-blot analysis (1)

• Principle
• Antigens are separated by Poly Acrylomide Gel Electrophoresis (PAGE) and trans-blotted onto nitrocellulose/nylon membranes
• Antibodies in serum react with specific antigens
• Signals are detected according to the principles of test systems
• Antibodies against microbes with numerous cross-reacting antibodies identified more specifically
• Examples
• T. pallidum, B.burgdorferi,
• Herpes simplex virus types 1 and 2

Anti HIV-1

Laboratory Training for Field Epidemiologists

Western-blot analysis (2)

• Serum, saliva, urine can be tested
• Kits are commercially available
• Recombinant immuno-blotting assays (RIBA) uses recombinant proteins

Anti HIV-2

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Immunoblot:�Performance, applications

• Advantages
• Used for discrepant analysis
• Highly specific
• Rapid kits available
• Limitations
• Cost
• Concern validated data
• Time taken
• 1 day

Laboratory Training for Field Epidemiologists

Immuno-chromatography: �Principle (1)

• 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

Laboratory Training for Field Epidemiologists

Immuno-chromatography: �Principle (2)

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

Laboratory Training for Field Epidemiologists

Immuno-chromatography:� 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 hour

Laboratory Training for Field Epidemiologists

Application Implications

Laboratory Training for Field Epidemiologists

Interpretation of antigen detection tests

• In general, detection of the antigen denotes a presence of the pathogen
• More important in some parasitic and fungal diseases

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Interpretation of a single, acute IgM test

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Interpretation of a single IgG test

* Collected between onset and convalescence

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Interpretation of two, acute and convalescent IgG tests *

* Convalescent serum collected 2-4 weeks after onset

Laboratory Training for Field Epidemiologists

Elements influencing the sensitivity and specificity of a given test kit

• Test format
• Precipitation versus IFA, Rapid test versus ELISA
• Purity of the antigen used
• Crude versus purified antigen versus synthetic peptides
• Type of the antibody used
• Polyclonal versus monoclonal antibodies
• Interfering substances in the sample
• Presence of rheumatoid factor in the serum of the patient
• Similarity in antigenic composition of pathogens
• Cross reactivity

Laboratory Training for Field Epidemiologists

Discussion

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Summary

• Precipitation
• Agglutination
• Hemagglutination and hemagglutination inhibition
• Viral neutralization test
• Radio-immunoassays
• ELISA
• Immunoflourescence

Laboratory Training for Field Epidemiologists

Conclusion

Laboratory Training for Field Epidemiologists

Bibliography

• WHO. (2007). Antigen and antibody detection
• Eaton K. (2016). Practical immunology