Introduction to Serology Lecture 1 & 2 PDF

Prepared by: Dr. Mohamed S. Abdel-Latif Assoc. Prof. of Immunology  Learning Objectives At the end of the course, the students should be able to:  List different types of serological tests  State the principle of serological tests  Describe the application of different serologi...

Prepared by: Dr. Mohamed S. Abdel-Latif Assoc. Prof. of Immunology  Learning Objectives At the end of the course, the students should be able to:  List different types of serological tests  State the principle of serological tests  Describe the application of different serological techniques  Identify the advantages and drawbacks of different serological techniques  Identify factors, which affect antigen-antibody reaction.  Out line 1. Introduction to Serology 2. Immunological technique primary binding sites Secondary binding sites Tertiary binding tests 3. Factors affecting antigen antibody reactions Introduction to Serology Safety rules Serological reactions Terms/Items  Safety rules  Wear lab coat  It is a must to wear gloves  Never mouth pipette  Cover any cuts or burns  Do not eat or drink in lab  In case of accident report to instructor  Avoid hand to face operations  Wash hands before you leave  What is serology  Is a branch of immunology dealing with study of Ag – Ab interactions in Vitro by different serological tests.  In other term; is the scientific study of blood serum  Importance of Lab diagnosis: 1- Save patient’s life 2- Prevent spread of disease 3- Treatment therapy 4- Confirm clinical diagnosis  Introduction  Immune system: the structures, cells, and soluble constituents that allow the host to recognize and respond to foreign stimulus.  Secondary immune response: the cellular and humoral events that occur when an antigen is encountered for a second or subsequent time.  Introduction Application of serologic tests  Serological tests may be performed for diagnostic purposes when an infection is suspected, in rheumatic illnesses, and in many other situations, such as checking an individual's blood type.  Serology blood tests help to diagnose patients with certain immune deficiencies associated with the lack of antibodies. In such cases, tests for antibodies will be consistently negative.  Introduction Antibody molecules combine reversibly with antigens to form immune complexes. Ag+Ab Ag.Ab complex The detection and measurements of these reactions form the basis of serology a sub discipline of immunology. Therefore Serology - is the science of measuring antibody or antigen in body fluids. Lab diagnosis of infectious diseases 1. Isolation and identification of causative agent by: a. Morphological tests (microscopy) b. Biochemical reactions c. Cultural identification d. Serological reactions e. Biotechnology: PCR-DNA probe- DNA finger printing 2. Detection of specific Ab in sera of infected patients using serological techniques  Serological Reactions Primary binding tests: It measures the direct interaction between Ag and Ab in Vitro (test tube). Secondary binding tests: It measures the consequences of interaction between Ag and Ab in Vitro. Tertiary binding tests: It measures Ag and Ab interactions in Vivo (in body). Primary binding tests E.g.  Enzyme linked Immunosorbent assay (ELISA) tests and  Radioimmunoassay (RIA)  Western blotting  Northern blotting  Southern blotting  Fluorescence tests Primary binding tests  Widely used in the serological diagnosis of  bacterial,  viral,  fungal, and  parasitic diseases.  They are usually sensitive and give reproducible results.  Secondary binding tests E.g. Agglutination tests Precipitation tests Complement fixation tests (CFT)  In district laboratories, agglutination tests are frequently used because compared with other serological tests, they are:  Simpler to perform  Require no special equipment  Usually less expensive.  Secondary binding tests  The fundamental and most commonly used reaction in medical serology laboratory  Mostly used in clinical lab to demonstrate the interaction of antigen and antibody  By broad definition, agglutination is simply the clumping of cells into aggregates, often as a result of the combination of an antibody's binding sites with antigen binding sites of the cells  Tertiary binding tests  Tertiary binding tests measure the consequences of immune responses in vivo.  These tests are much more complex than primary and secondary tests but their results reflect the practical significance of the immune response.  E.g. measurement of the protective effects of antibody. Factors affecting antigen antibody reaction  specificity  cross reactivity  temperature  pH  ionic strength  concentration  intermolecular specificity  Terms  Serum: the fluid portion of the blood after the blood clots.  Specificity: ability of a test to identify correctly those who do not have the disease.(have least cross reactivity).  Sensitivity: Ability of a test to identify correctly those who have the disease(can detect v. small amounts).  Validity: A serological test should provide an indication of which individuals actually have the disease and which do not.  Susceptibility: having little resistance.  Example Sensitivity: True positive rate of the test (no false –ve) Specificity: True negative rate of the test (no false +ve) Test result Test result Total No positive negative of people Really have AIDS 99 1 100 Do not have AIDS 199 9701 9900 Totals 298 9802 7 10,000 Sensitivity = 99/100 x100 = 99% Specificity= 9701/9900x100= 98%  Terms  Quantitative test:  It measures the amount of Ag or Ab.  Qualitative test :  It detects the presence or absence of Ag or Ab.  Terms  Seroconversion: is development of detectable specific Ab to microorganisms in serum as a result of infection or immunization  Sero reversion: is the opposite of seroconversion. This is when the test can no longer detect Ab or Ag in patient's serum  Criteria for Diagnosing Primary infection Re-infection  Seroconversion  Absence to slight  Presence of IgM increase of IgM  4 fold rise or more  4 fold rise increase in IgG titer in IgG  Serum Separation What is serum? Serum: Blood- cells and clotting factors Plasma: blood – cells Separation: Use plain tube (no anticoagulant) Leave blood for 1 hour at room temp. Serum Separate the clot cells Centrifuge at 3000rpm for 10 min.  Serum preservation  Aliquoting  Must aliquot the serum into different tubes to avoid freezing and thawing(Why)  Keep serum in fridge at 4⁰C for 1 day  Keep in freezer at -20⁰C for more the 1 day.  Use frozen serum only once, discard after use. Disposal of serum and contaminated lab ware  Dispose used serum tubes, microtiter plates, universal tubes, bijou bottles in autoclave bags.  Dispose used serological pipettes, microtiter tips, slides in disinfectant jars.  Do not throw tissue, gloves, paper in disinfectant jars.  Items  Micropipettes Fixed volume / adjustable volume Ejectable / non ejectable Multichannel micropipette  Microtiter plates U -bottom V- bottom Flat bottom  Dilution  It is important to dilute patient samples for serological tests  Dilution= Serum volume Total volume Total dilution = serum volume + diluent volume Serum volume=Total volume-diluent volume  Dilution= dilution of preceding tube x Serum volume Total volume Doubling serial dilution Monoclonal antibodies  Assignment: As a part of the semester activity, one student is selected every week to prepare a short seminar about his/her point of interest in one of the lecture topics. That to be discussed and evaluated during the next lecture.