serological techniques

• Used to detect, identify and quantitate antigen in clinical samples, evaluate the antibody response to infection & a person’s history of exposure to infectious agents

→ use antibody to look for an antigen (presented by microorganisms) in a patient sample

→ use antigen to look for the presence of an antibody in a patient sample

• specificity of the antibody-antigen interaction and the sensitivity of many of the immunologic techniques make them powerful laboratory tools

• in many cases, the same technique used can be adapted to evaluate BOTH antigen and antibody

• many assays are designed to give a positive or negative result (i.e. the presence or not of a specific antigen/antibody)

• quantitation of the antibody response strength is determined = given as a titer

• titer of an antibody ⇒ the greatest dilution of the sample that retains a detectable activity

Seroconversion:

• serology is used to determine the time course of an infection

• seroconversion → occurs when antibody is produced in response to a primary infection

• specific lgM (immunoglobulins M) antibody found during the first 2-3 weeks of a primary infection is a good indicator of a recent primary infection

• reinfection of recurrence later in life causes a anamnestic (secondary or booster) response → more likely to see lgG antibodies

• antibody titers may remain high in patients whose disease recurs frequently (ongoing infection → ongoing stimulus of immune system → continuous antibody response)

How do we detect seroconversion?

• seroconversion or reinfection is indicated by the finding of at least a fourfold increase in the antibody titer between serum obtained during the acute phase of disease (patient actively symptomatic) and that obtained at least 2-3 weeks later during convalescent phase

• therefore must recieve 2 samples from the patient to demonstrate such a response (sometimes tricky to get patients involved in giving 2 samples)

• the result is therefore ⇒ retrospective

Antibodies:

• antibodies can be used as sensitive and specific tools to detect, identify and quantitate the antigens from a virus, bacterium, fungus or parasite

• antigen/antibody detection can be carried out directly on patient sample, no need to grow/culture the microorganism

• specific antibodies may be obtained from (1) convalescent patients (e.g. antiviral antibodies) or (2) prepared in animals

• two categories… often a combination of both is used in microbiology

→ polyclonal: heterogenous antibody preparations that can recognise many epitopes on a single antigen (e.g. used when looking for salmonella bacterium, not looking for specific strain)

→ monoclonal: antibodies that recognise individual epitopes on an antigen (e.g. used when looking for specific strains or specific variants)

How we would raise these monoclonal antibodies within animals:

Monoclonal antibodies:

Diagnostic methods: Latex Agglutination

• particles of latex are coated with (usually) antigen

• patient serum is mixed with the latex and suspension is rocked very gently for 2-3 minutes

• latex indicates reaction between antigen on latex and antibody in patient’s serum

• agglutination (particulates forming) is positive reaction

• cheap and easy to (<£1 per test)

• requires little skill to perform the test

• reading can be subjective on borderline results (autoagglutination/ poor sample collection)

Diagnostic methods: Immunochromatography

• reaction carried out on chromatographic paper by capillary action

• 2 kinds of specific antibody are used:

1. immobilised on the chromatographic paper

2. labelled and infiltrated onto the sample pad

• sample is added, if antigen is present, a complex is formed with the labelled antibody

• complex moves along strip (via capillary action)with liquid sample - binds to immobilised antibody resulting in a visible colour change

• monoclonal antibody technology is being revisited with new detection technology to improve sensitivity

e.g.

→ add 100μl of sample

→ wait 15 mins

→ 1 line = negative

→ 2 lines = positive

→ any line in the sample window is positive as long as the control has also worked

• if control line is present = accept the sample result

• if no control line present = cannot accept sample result as you cannot guarantee that the sample has actually moved through the chromatographic paper using capillary action

ELISA

• Enzyme-Linked ImmunoSorbent Assay uses antigen immobilised on a plastic surface, bead or filter to capture and separate the specific antibody from other antibodies in a patient’s sample

• can also be used to quantitate the soluble antigen in a patient’s sample

• several types of ELISA are used in a diagnostic laboratory

ELISA: general principles:

There are 4 basic elements;

1. coating/capture

2. plate blocking (to prevent any other antigens or antibodies being detected)

3. probing/detection

4. signal measurement

Non-competitive ELISA:

• used for detecting presence of antibodies to infections

• between each stage we are washing away any of the unbound material

• so amount of colour that develops is proportional to the amount of antibody in the patient sample

Sandwich ELISA:

• used to detect viral antigens; active infection

→ capture antibody coated along the bottom of the solid surface

→ add patient sample

→ if target antigen is present, it will bind to the antibody

→ use detection antibody to detect antigen-antibody complex

→ HRP-conjugated (horse-radish peroxidase conjugated) will bind to that complex (2 antibodies + antigen)

→ add TMB which will combine with the horseradish peroxidase to show colour change

Class Capture ELISA:

• designed to detect a specific type of antibody e.g. lgG or lgM

• amount of colour that develops is proportional to the amount of antibody in the patient sample

Competitive ELISA:

• presence of antibody in patients serum stops reaction

• the chromogenic substrate/ colour change will only be detectable where we see the enzyme labelled antibody has bound to the antigen

• high amount of antigen labelled antibody if the patient sample has not already bound to the antigen

• no colour = positive reaction

• amount of colour is inversely proportional to the amount of antibody in the patient specimen

• e.g. Hepatitis B core antibodies

ELISA comparison:

• type used is dependent upon the target (e.g. antibody or antigen, type of antibody, using a secondary or tertiary antibody, expecting a colour change or not etc)

Advantages of ELISA:

✅ cheap simple tests

✅ easy to perform

✅ easy to automate (on a large scale)

✅ robust and reliable technology

✅ not easily contaminated

✅ new kits have sensitivity, specificity, approaching 100% and PPV over 98%

✅ can diagnose active infections or immunity (class capture IgM recent infection or IgG past infection)

✅ can be performed on various platforms

Automated EIA: BioMerieux VIDAS

• this is a single shot EIA

• similar technology to other ELISA methods

→ add patient sample to first well

→ load it into the analyser

→ analyser will take some of the patient’s sample and add it to second well (second well has bound antigen)

→ if theres antibody present in the patients sample, it will form a complex with the antigen

→ the sequential step is either a wash step OR the addition of an enzyme labelled antibody OR the detection/chromogenic step

Focus on: Epstein-Barr virus

• EBV is a member of the subfamily Gammaherpesvirinae

• known as human herpesvirus 4

• limited host range and a tissue tropism defined by the limited cellular expression of its receptor

• the primary receptor for EBV is also the receptor for the C3d component of the complement system expressed on B cells of humans

baltimore classification?

define tissue tropism?

what receptors are required for EBV to gain entry into host cells?

Baltimore Classification System:

• a way for us to identify what type of genome a virus has

• consider what process would have to happen in order to generate that mRNA transcript

Epstein-Barr symtoms:

the triad of classic symptoms for infectious mononucleosis is:

• lympadenopathy (swollen glands)

• splenomegaly (large spleen)

• exudative pharyngitis (excess mucous production)

• high fever

• malaise (muscle aches)

• hepatosplenomegaly (large liver)

→ primary infection followed by persistent infection

→ we are able to distinguish between a primary infection and a persistent infection based upon the types of antibody that can be detected in the patient’s sample

• viral proteins produced during a productive infection are serologically defined and grouped as early antigen (EA), viral capsid antigen (VCA) and the glycoproteins of the Nuclear antigen (NA)(EBNA)

→ we can determine whether a patient is having a primary infection or reactivation of previous infection by looking at the levels of those individual antigens/antibodies

• serological tests for antibody to viral antigens are a dependable method

• EBV infection is indicated by the finding of any of the following:

→ LgM antibody to the VCA

→ the presence of the VCA antibody and the absence of the NA antibody

→ elevation of antibodies to VCA and early antigen

• the finding of both VCA and EBNA antibodies in the serum indicates that the person had a previous infection

• generation of antibody to EBNA (epstein-barr virus nuclear antigen) requires lysis of the infected cell and usually indicates T-cell control of active disease (requires patient to be immunocompetent and able to have T-cell response)