Principles of immunological testing

Views:
 
     
 

Presentation Description

No description available.

Comments

Presentation Transcript

Principles of immunological testing:

Principles of immunological testing

Introduction :

Introduction Ab- Ag specificity for Dx of diseases Detected / measured to aid in Dx & Mx (serum) Serology: use of serum Abs to detect & measure Ags & vice versa Immunoassay: assays, or tests using immunological rgts (Ags & Abs) Monoclonal Abs are often used in immunoassays

PowerPoint Presentation:

1 o 2 o (Anamnestic) Structure 1 st Exposure to Ag 2 nd Exposure to Ag Lag Phase Days to months Hours Type of Antibody IgM switch to IgG 2-3 wks after (isotype switching) IgG Titer Rises slow, Peaks then declines Rises after & higher. Stays high longer

PowerPoint Presentation:

IgG (7S) IgM (19S) Monomer Pentamer Crosses Placenta Cannot cross placenta 160,000 MW 900,000 MW Reacts best at 37 o C Reacts best at RT Binds complement Binds complement Reacts best in albumin or LISS Reacts best saline Incomplete/ Blocking / Immune Abs Complete/ Agglutinating/ Naturally occuring Abs Always clinically significant for BB (HTR or HDN) Occasionally clinically significant for BB

PowerPoint Presentation:

Factor Comments Sensitization Stage 1. Temperature Attachment of antibody to antigen Clinically significant antibodies React best at 37ºC 2. pH Most antibodies react at pH 5.5- 8.5 3. Ionic strength Reducing the ionic strength of the Medium facilitates interaction of Ab w/ Ag (LISS) 4. Antigen: antibody ratio Too much antibody can cause prozone (false-negative). Optimum serum to cell ratio 80:1. Usually 2 drops serum + 1 drop Ag. Follow manufacturer’s directions

PowerPoint Presentation:

PRECIPITANT CURVE : Constant antigen concentration: increasing addition of antibody Amount of Precipitin Increasing amount of antibody added Ag > Ab Ab ~= Ag Ab > Ag Ag Ab Equivalence point (physical band) The goal of humoral responses is to achieve Ab excess to remove the pathogen

PowerPoint Presentation:

Agglutination Stage Type of antibody molecule Formation of antigen-antibody bridges between RBCs IgM is larger and can span the distance between RBCs more easily. Density of antigens and location on RBC surface Affects ease of attachment of antibodies Zeta potential Difference in charge between negatively charged RBC surface & cloud of positive ions surrounding the RBCs. Reduce the zeta potential allows RBCs to move closer together (enzyme treatment of the test cells)

Quantifying antigen-antibody reactions:

Quantifying antigen-antibody reactions Detectable specific Abs in serum until 7-10 days after infection Seroconversion: change from negative serum w/out specific Abs to serum positive for specific Abs. Progression of infection, amount of Abs in the blood called ‘titer’ increases Rise in titer of Abs is characteristic of an active infection

Quantifying antigen-antibody reactions:

Quantifying antigen-antibody reactions Blood serum, plasma, urine, CSF, sputum & other body constituents may contain Ags or Abs (detected in solid tissues also) Amount of Abs or titer of a serum sample is usually determined in serial dilutions

Quantifying antigen-antibody reactions:

Quantifying antigen-antibody reactions Reciprocal of the last dilution showing a detectable Ag-Ab rxn is taken as the titer Thus if positive reaction is observed in the dilution 1:256 but not in 1:512, the titer is 256.

Quantifying antigen-antibody reactions:

Quantifying antigen-antibody reactions Both pos & neg controls should be included Uses plastic microtiter plates so that tests can be done on very small samples

Immune Phenomena:

Immune Phenomena

Antigen-antibody based immunological tests:

Antigen-antibody based immunological tests 1. Precipitation reactions: Abs + soluble Ags = precipitate Ring test Immunodiffusion tests Immunoelectrophoresis

Antigen-antibody based immunological tests:

Antigen-antibody based immunological tests 2. Agglutination reactions: Abs + particulate Ags = agglutination Larger aggregates easier to visualize Direct agglutination tests Indirect agglutination tests Hemagglutination Inhibition

Antigen-antibody based immunological tests:

Antigen-antibody based immunological tests 3. Immunofluorescence test Fluorescent dyes (fluorescein or rhodamine) attached to known specific Abs, used to detect presence of Abs in serum or Ag (microorganisms) in a sample Direct fluorescent Ab test: used to detect Ag or microorganism Indirect fluorescent Ab test: used to find a specific Ab in the serum

Antigen-antibody based immunological tests:

Antigen-antibody based immunological tests 4. Radio immunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) Both RIA & ELISA widely used assays Extremely sensitive tests Analyze large number of samples quickly, using very little samples & reagents

Antigen-antibody based immunological tests:

Antigen-antibody based immunological tests 5. Western Blot or immunoblot Combines electrophoresis + ELISA to separate & then detect specific proteins in a given sample using Abs

Antigen-antibody based immunological tests:

Antigen-antibody based immunological tests 6. Complement fixation test Bacteria, RBCs, or other cells in the presence of Abs specific for their surface Ags sometimes get lysed as the result of complement activity. Tests for specific Abs in a patient's serum Complicated & lengthy Replaced by newer & easier techniques (ELISA)

Antigen-antibody based immunological tests:

Antigen-antibody based immunological tests 7. Neutralization tests ( virus neutralization test) Serum + known viral suspension If Abs to the virus are present, Abs bind to the virus, preventing its attachment to & subsequent infection of cells When virus is then added to an appropriate cell culture, it is unable to replicate & cause cell death Also to used to test for toxins

Tests used in cellular immunology:

Tests used in cellular immunology Serologic tests are cheaper & easier vs. tests performed using lymphocytes or other cells Serological tests are more widely used in clinical settings Cellular tests are also useful clinically but more for research purposes

Laboratory Identification of Lymphocytes :

Laboratory Identification of Lymphocytes

Common Lymphocyte Markers :

Common Lymphocyte Markers

Tests used in cellular immunology:

Tests used in cellular immunology 1. Identification of subsets of lymphocytes: Fluorescent linked Abs used to ID subsets of lymphos (CD4 & CD8) Fluorescent microscope or a fluorescence-activated cell sorter (FACS) technique is more efficient as it counts & separates cell quickly

Tests used in cellular immunology:

Tests used in cellular immunology 2. Lymphocyte response to mitogens: Useful to determine immunodeficiency Failure of human lymphocytes to proliferate in response to phytohemagglutinin (PHA) indicates a deficiency in Tcell responses

Tests used in cellular immunology:

Tests used in cellular immunology 3. Cytotoxic T-cell (Tc) function: Ability of Tc cells to kill target cells measured by labeling target cells w/ radioactive chromium Count radioactivity in the released chromate gives a measure of cytotoxicity

Tests used in cellular immunology:

Tests used in cellular immunology 4. Cell mediated immunity to infectious agents: Measures uptake of radioactive thymidine into DNA in lymphocyte cultures stimulated by a specific Ag Skin testing also helpful in assessing CMI responsiveness to certain pathogens. TB, leprosy, various fungi

authorStream Live Help