Serological Tests PDF

‫ميحرلا نمحرلا هللا مسب‬ Serological tests Presented by Abeer Mohamed Assistant lecturer at Microbiology department  Antigen ▪ Means any substance, which when introduced into body stimulate the immune system to produce immune response specific to the injected...

‫ميحرلا نمحرلا هللا مسب‬ Serological tests Presented by Abeer Mohamed Assistant lecturer at Microbiology department  Antigen ▪ Means any substance, which when introduced into body stimulate the immune system to produce immune response specific to the injected substance and no to un-related materials.  Antibody Specific to Fab Microbe Specific to FC species & (in same Ab) Labelling Antibody Enzyme or Fluoresence isothiocynate  Antigen- antibody reactions ❖ Are serological reactions and are used as serological diagnostic tests for the identification of infectious disease.  Serological tests Used to detect and measure antigen –antibody reaction Primary binding Secondary Tertiary binding test binding test test Immunofluorescence Neutralization ELISA and Precipitation Agglutination test Immunoperoxidase test Radioimmunoassay Complement Fixation test  Primary binding test Tests that directly measure the binding of antigen and antibody  Enzyme-linked immunosorbent assay (ELISA) Principle: 1.ELISA is a serological test in which the antigen-antibody reaction is detected by labeling the antibody by an enzyme (horseradish peroxidase and alkaline phosphatase). 2.The enzymatic activity is detected by addition of substrate which destructed into certain a color detected by ELISA reader (Spectrophotometer device) 3.The intensity of the developed color is proportional to the amount of the antibody or antigen. 4. Performed in wells of polystyrene plates which used to absorb Ag or Ab (Microtiter plate) 5.Types of ELISA according to the solid surface used: - Cell ELISA: performed in wells of microtiter plate. - Dot ELISA: performed on high protein binding paper (nitrocellulose membrane). Dot ELISA Cell ELISA  Types of ELISA assays: Direct ELISA: Indirect ELISA: Sandwich antigen antibody ELISA: antigen detection detection. detection  Direct ELISA: antigen detection Substrate Any free Conjugated 1ry Ab Ab washed away Fetal 5.Washing PBS Ag 2.Washing PBS 3-5 times bovine 3-5 times serum 1.Incase of uncoated 3.Add fetal bovine 4.Add 6.Add substrate and plate load Ag serum or casein conjugated 1ry incubated in dark place (Sample) to well of (blocking substance) Ab with enzyme 15 min at room temp microtiter plate and To allow Ab attached to and incubated which converted by incubate overnight at Ag not to wall of wells 37 °C for 1hr. enzyme into detectable 4°C for complete substance then add absorption to wells stop solution H2SO4 wall then measure colour by ELISA reader Advantages of direct ELISA: Disadvantages of direct ELISA: Fewer steps. (Ag, labeled Ab) The direct labeling of primary Quick results. antibodies for every Ag (expensive)  Indirect ELISA: antibody detection& Secondary quantitively determined. Ab Any free 1ry Ab washed away Fetal Washing PBS 3-5 2.Washing PBS times Ag 3-5 times bovine serum 1. Ag (known) coated 3.Add fetal bovine 4.Add serum or 5.Add to well of microtiter serum or casein sample contain conjugated 2ry plate and incubate (blocking substance) primary Ab Ab that binds to overnight at 4°C for To allow Ab attached to (unknown) the primary Ab complete absorption to Ag not to wall of wells unconjugated wells wall and incubated 37 °C for 1hr.  Substrate Unbounded 2ry Ab washed away Washing PBS 3-5 times 6.Add substrate so enzyme hydrolyzed substrate to form colored product then measure by ELIZA reader (the amount of colored end product is directly proportional to Ab Advantages of indirect ELISA: Disadvantages of indirect ELISA: -Cross-reactivity might occur with the Highly sensitive and more flexible secondary antibody, resulting in than direct ELISA due to use 2ry Ab nonspecific signal. -An extra incubation step is required in the procedure.  Used for antigen detection but Sandwich ELISA: the target antigen must contain at antigen detection least two antigenic sites (epitopes).  Protein A one of cell wall of Staphlyococoous consider as receptor for FC Unbounded Substrate 1ry Ab washed Ag away Washing Washing PBS 3-5 PBS 3-5 Washing PBS 3-5 times times Ab times 1. Primary Ab (known) 2. Sample contain 4. Add detection 6.Add 8.substrate added to coat well of suspected Ag is antibody (specific to a enzyme- so enzyme microtiter plate and added to well, different epitope on conjugated hydrolyzed incubate overnight at then incubation the antigen) and secondary substrate to 4°C for complete ex.37 °C for 1 hr allowed to react antibody is form colored absorption to wells added and product wall or already coated allowed to then plate react. measure by ELIZA reader Reading ELISA results: Qualitative result (especially for antibody detection) is determined by naked eye as a simple colour change. Quantitative result (antibody detection) is determined by measuring the intensity of colour in a spectrometer (ELISA reader) or by testing dilutions of the test serum and determining the highest dilution that shows a colour change.  Dot ELISA Dotted Ag allowed to dry at room temp Nitrocellulose membrane floated with reference serum containing specific Ab then incubated at room temp for 2hr Washing 3 times by buffer Immersed in pool contain antispecies serum (2ry Ab) conjugated with peroxidase enzyme incubate 37C/2hr Blue dot Immersed in substrate working for 15 min at room temp in dark positive place ,stop reaction by washing  Immunofluorescence (IF) fluorescent antibody test (FAT) Principle: ❑The intracellular location of antigen could be determined in the infected cell cultures, impression smears or thin frozen sections of infected tissues by using specific antibodies prelabeled with fluorescent dye (fluorescene isothiocyanate). Two types of fluorescent antibody test Direct Indirect  Direct IF Detection of Ag within cell Detection of Identify Ag location In nucleus In cytoplasm Procedure of the direct method: 1. Acetone-fixed tissue or smear containing (Ag) (unknown or suspected) is fixed on a slide, then incubated with the fluorescence antibody. 2.Wash (3 times by PBS) to remove unbounded antibody 3. Examined by dark field illumination under a microscope with UV light source Result The antigen particles that bounded with labeled antibody gives yellow-green fluorescence  Indirect IF Detection of Ab Needed Primary Secondary unlabeled labeled antibody antibody Procedure of the indirect method: 1. Acetone-fixed tissue or smear is treated with (unlabeled primary Ab) then incubated for 30 min at room temp 2.Wash (3 times by PBS) to remove unbounded antibody 3. A fluorescent labeled antibody (Secondary Ab) then incubated for 30 min at room temp 4. Wash 5. Examined by fluorescence microscope  Points of Direct IF Indirect IF difference For Detection Ag Detection Ab Labeled Ab Primary Secondary Steps 2-steps 3-steps Time Rapid Slow Advantage Rapid Conjugated Ab for each species Disadvantage Conjugated Ab for each Ag Slow Specificity Less specific More specific Disadvantages of immunofluorescence staining: ▪ High cost of fluorescent microscope and reagents. ▪ The need of well-trained person.  Immunoperoxidase test (IP) Principle: The intracellular location of antigen could be determined in the infected cell cultures, impression smears or thin frozen sections of infected tissues by using specific antibodies prelabeled with enzymes (peroxidases). Immunoperoxidase (IP) staining It is an alternative method for IF test. Its procedures and principles are similar to those of IF test, but the antibody is conjugated to peroxidase enzyme. In the presence of appropriate substrate, the enzyme forms a colored insoluble precipitate appear as yellowish-brown dots in the cytoplasm or nucleus of the infected cell. Advantages of IP staining in comparison with IF staining: Requires an ordinary light microscope. Produces morphologically clearer, non-fading and permanent preparation. Disadvantages of IP staining: ▪ The presence of endogenous peroxidase enzyme in the cells of many tissues, particularly leukocytes, may induce false positive result with IP staining. ▪ This problem can be overcomed by destruction of the endogenous peroxidase before staining by treatment of the tissue sections with hydrogen peroxide. Endogenous peroxidase Points of difference IP ELISA Physical nature of Ag Ag in paraffine embedded Ag is free in fluid as saliva, tissue section, fixed on In tissue homogenate, slide, monolayer of filtrate infected cell culture, impression smear of lymph node Labeling Peroxidase enzyme Alkaline phosphatase enzyme Result of hydrolyzed Insoluble granules so Soluble color substrate detect location of Ag either in nucleus or cytoplasm Read Light microscope ELISA reader  Same idea as ELISA but different label: Radioactive isotope (instead of enzyme) Measurement of Degree of radioactivity (instead of degree of colour change) As sensitive as ELISA C. Radioimmunoassay Disadvantage: Hazards of radioactivity (RIA) Applications: Measurement of biological substances (Ags) (e.g. drugs, hormones, tumour markers) Measurement of antibodies Thanks for your attention