Serology my introduction

serology Introduction 

SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES 
•The scientific study of blood sera and their effects 
•Subdivision of immunology concerned with in-vitro Ag-Ab reaction 
•Concerned with the laboratory study of the activities of the component of serum that contribute to immunity. 

METHODS OF DETECTION OF ANTIBODIES Immuno-precipitation Assays = detect antibodies in solution = qualitative indication of the presence of antibodies = end-point is visual flocculation of the antigen and antibody in suspension

METHODS OF DETECTION OF ANTIBODIES Immunofluorescence = requires use of microscope equipped to provide ultraviolet illumination or an instrument capable of irradiating the assay with UV light and detecting resulting fluorescence with a fluorometer

IMMUNOFLUORESCENCE 
These are Ag-Ab reactions in which Ab or anti-human immunoglobulin is labelled with fluorescein. 
Fluorescein is a dye which emits greenish fluorescence under UV light. 
There are two ways for this test; 
Direct immunofluorescence (detect antigen with labelled antibody),
Indirect immunofluorescence (detect antibody with labelled anti-human immunoglobulin).

METHODS OF DETECTION OF ANTIBODIES Enzyme Immunoassay = the most sensitive = usually indirect assay that depends on the use of an antihuman IgG or IgM antibody conjugate = the antibody conjugate (if present) is made to attach to enzyme which catalyzes conversion of the substrate to a colored product which will then be read with the use of a spectrophotometer

ENZYME-LINKED IMMUNO SORBENT ASSAY (ELISA) 
This technique is ; 
Very sensitive 
 Does not require specialized equipment
  Avoid the hazards of radioactivity. 
The method depends on conjugation of an enzyme to either Ag or Ab, then substrate is added as a quantitative measure of enzyme activity.

ELISA  Indirect ELISA: 
 In this test an enzyme- labelled anti-human Ig is used to detect the presence of specific Abs in patients’ sera. 
 Known Ag is fixed by adsorption onto a plastic surface. 
 The serum sample is added (if specific Ab is present, it will bind the fixed Ag).
 Wash 
 Add the enzyme-labelled antihuman Ig 
 wash the excess 
 add the substrate, then quantitatively measure for the degree of colour change.

TO IMPROVE SPECIFICITY – WESTERN OR IMMUNOBLOTTING
 Separation of antigens by gel electrophoresis 
 Antigens blotted onto nitrocellulose
 Antigens printed directly onto nitrocellulose 
 Strips of separated antigens on nitrocellulose reacted with serum
 Washed
 React with anti-human conjugate 
 Washed
 Colour detected with substrate

SEROLOGICAL DIAGNOSIS OF INFECTIOUS DISEASES = HIV • Laboratory diagnosis of HIV infection Western Blot Testing = interpretation of result 
 no bands, negative 
 in order to be interpreted as positive a minimum of 3 bands directed against the following antigens must be present : p24, p31, gp41 or gp120/160 
 CDC criteria require 2 bands of the following : p24, gp41 or gp120/160

ROLE OF SEROLOGY IN MICROBIAL DIAGNOSTICS 
 Generally considered as the back-up when cultivation is not appropriate
 Organisms too slow to grow 
 Organism cannot be grown  Organism to hazardous to grow
 Imprecise compared to isolation
 Antigens may cross-react with those of other organisms
 Antigens may be shared between organisms (lateral gene transfer)


GOOD THINGS ABOUT SEROLOGY 
 Cheap 
 Easy to automate 
 Good for population screening 
 Can be used for several specimen types 
 Serum 
 CSF
 Synovial fluid 
 Milk 
 Can be used for retrospective diagnosis 
 Sensitive if used 10-14 days post-infection

BAD THINGS ABOUT SEROLOGY
 Poor for acute infection 
 Often require repeat samples 
 Poor if the infection is endemic 
 Poor for determination of reinfection or relapse 
 Specificity variable (depending upon antigen) 
 Vaccination can complicate results 
 Early treatment can interfere with development of positive response 
 Risk of dealing with blood samples (bloodborne pathogens)

**!!REMEMBER TO STAY POSITIVE LIKE A PROTON!!**