Immunodiagnosis SCBM 343 CLINICAL PATHOLOGY. Lect. Dr. Witchuda Payuhakrit

Transcription

1 Immunodiagnosis SCBM 343 CLINICAL PATHOLOGY Lect. Dr. Witchuda Payuhakrit

2 Objectives 2 Understand the antigen and antibody interaction Understand the principle of immunoassay Give the example of applications from each immunoassay method

3 Immunoassay u The immunochemical techniques that Ab combine with an Ag or analyte of interest and react with an indicator u Rapid, sensitive and easily automated method for routine analyses u Antibody-antigen interactions u Hormones, pharmaceuticals and disease biomarkers u Used in conjunction with histochemistry to visualize specific cells Immunohistochemistry

4 Antigen and Antibody Antigens (allergen, Infectious agents, autologous antigen) Small molecules (hormone) Macromolecules (protein, lipids, carbohydrates or polysaccharides) Antibodies are immunoglobulin (Ig) à IgG, IgA, IgM, IgD and IgE Polyclonal antibodies Monoclonal Antibodies

5 Antibodies 5 Polyclonal Antibodies Ab that produced by different B-cell lineages against a specific antigen Ab attach to different epitope sites on the same Ag

6 Antibodies 6 Monoclonal antibodies Ab from single B-cell clone Specific to one epitope of Ag

7 Immunoassay 7

8 Principle of immunochemical method 1. Heterogenous Immunoassays 2. Homogenous Immunoassays 3. Competitive Immunoassays 4. Noncompetitive Immunoassays

9 Heterogenous & Homogenous Heterogenous require separation of the free labeled Ag from the bound labeled Ag in the solution Precipitation, Liquid-phase adsorption, Solid-phase adsorption Homogenous do not require separation of bound and free forms of labeled Ab or Ag Quantitate drugs of abuse and therapeutic drugs Free labeled Ag Ag-Ab*

10 Competitive Immunoassays Based on competition between the unlabeled analyte in the sample and the labeled Ag in the assay Analyte concentration is inversely proportional to the absorbance of the solution

11 Noncompetitive Immunoassays11 Have the highest level of sensitivity and specificity Cardiac markers, hepatitis markers One and two-step formats

12 12 Type of immunochemical method 1. Label methods 2. Particle methods 3. Light scattering methods

13 13 Label Methods 1. Enzyme-Linked Immunosorbent Assay 2. Enzyme-Multiplied Immunoassay Technique 3. Fluorescence Polarization Immunoassay 4. Microparticle Enzyme Immunoassay

14 Enzyme-Linked Immunosorbent Assay 14 ELISA Ab are absorbed to the surface of solid phase Enzymes coated-ab are added à wash à substrate à measure absorbance Cytokine, hormones, antibodies, drugs, tumor markers, cardiac markers and toxic substances

15 Microparticle Enzyme Immunoassay 15 MEIA Uses the surface of small beads called microparticles to isolate Ab-Ag complexes The solid phase consists of latex particles coated with Ab The enzyme substrate is fluorescence 4-methylumbelliferone phosphate (4-MUP) Measure large molecules e.g. cardiac markers, fertility hormone, tumor markers, hepatitis antigens, thyroid hormones and metabolic makers in an automated instrument

16 Microparticle Enzyme Immunoassay 16 Microparticles coated with anti-analyte and the sample are incubated An aliquot of the reaction mixture is transfer to the glass fiber matrix Alkaline phosphatase-labeled anti-analyte are allowed to bind to microparticle complex The substrate MUP is added to the matrix and the methylumbelliferone (MU) is measured

17 17 Particle methods Characterized by the precipitation of Ag-Ab-complex, no label and both qualitative and quantitative applications 1. Immunodiffusion 2. Immunoelectrophoresis 3. Counterimmunoelectrophoresis 4. Western Blot

18 Particle methods 18 Ag-Ab complex is formed in the zone of equivalence

19 Immunodiffusion 19 Laboratory technique that allows Ag and Ab to diffuse through agar or agarose Determine relative concentrations, compare Ag or determine the purity of a preparation More sensitive than liquid assays Radial immunodiffusion Ouchterlony Double immunodiffusion Laurell Technique

20 Immunodiffusion 20 Radial immunodiffusion Used to determine concentration of Ag Ag is suspended in agar or agarose, a small well is punched Ag diffuses into the medium à Ab-Ag complex precipitates, forming circles that mark the zone of equivalence

21 Western Blot 21 Quantitation of small amount of Ag Separated by SDS-PAGE, which is the anionic detergent Proteins are overall negative charge and separated on their size

22 Western Blot 22 Electroblotting = separated protein are transferred to nitrocellulose membrane Radioactive isotope or enzyme probes are used to detect protein

23 23 Light-Scattering Methods When Ab-Ag complex are created in a liquid solution à cloudy 1. Turbidimetry 2. Nephelometry

24 Light-Scattering Methods Turbidimetry by using spectrophotometer measure the amount of light transmitted and the amount of absorbed by suspended particles in a solution More turbidity = less light transmitted Protein determinations in urine or cerebrospinal fluid

25 Light-Scattering Methods Nephelometry by using spectrophotometer with specific angle (90, 70 or 37 degrees) in relation to the solution More sensitive than turbidimetry Quantitating serum proteins (e.g., haptoglobin, transferrin, C-reactive protein, α 1 -antitrypsin) and Immunoglobulin

26 Conclusion 26 The Antibodies are ideally suited for immunochemical techniques because they are specific and sensitive, allowing small amounts of antigen to be detected Immunochemical techniques (label, particle, light scattering) selection depend on nature of interested analyte

27 LAB6- RPR test 27

28 28 RPR test u Used to diagnose syphilis u Treponema pallidum u Rapid: Its rapid test that can be done within few minutes. u Plasma: The sample is plasma (or serum) u Reagin: is autoantibody directed against cardiolipin Ag Cardiolipin = lipoprotein-like material released from damaged host cells Treponema pallidum Chancre Maculopapular rash

29 RPR test 29 RPR test is one of the nonspecific tests (Nontreponemal tests) Measures IgM and IgG antibodies to cardiolipin Agglutination assay RPR reagent contains cardiolipin, lecithin, cholesterol, 10% choline chloride, EDTA, charcoal etc. in Buffer

30 Method Label a RPR card with patient and control information being careful (not to interfere with the test areas of the card) 2. Adding one free-falling drop (50 µ) of Positive control and Negative control using a new dispenser for each sample 3. Using disposable serum dispensers 50 µl of serum sample onto a circle on the test card

31 Method Spread the sample smoothly across the circle area using the paddle side of the dispenser as shown by instructor. Take care not to scratch the test area 5. After mixing the antigen solution by swirling, add one drop of the antigen suspension to each sample / control testing area

32 Method Place the card on an automatic rotator. Rotate at 100 ± 5 rpm for 8 minutes. Following rotation, a brief hand rotation and tilting of the card (3 4 times) should be performed to aid in differentiating nonreactive from minimally reactive results 7. Immediately read results macroscopically in the wet state under a high intensity light source

33 Interpretation of RPR Test 33 Non-reactive (NR) smooth suspension, no clumping or slight roughness Reactive (R) any degree of clumping Reactive with titer 1:64

34 False positive results 34 Patients with following infectious diseases may produce reaginic antibodies Chancroid Chickenpox Hepatitis Infectious mononucelosis Leprosy Leptospirosis Non-infectious conditions Autoimmune disorders (rheumatoid disease) Drug addiction Lymphogranuloma venereum (LGV) Malaria Measles Ricekttsial disease Trypanosomiasis and Tuberculosis Other factors (old age, pregnancy, recent immunization)

35 Confirm results 35 Treponemal test to detect antibody to T.pallidum Fluorescent treponemal antibody-absorption (FTA-ABS) Treponema pallidum Hemagglutination Assay (TPHA)

36 References 36 Donna L. Larson. Clinical Chemistry Fundamentals and Laboratory Techniques Elsevier Frances Fischbach Marshall B. Dunning III. A manual of laboratory and diagnostic test. Ninth edition Wolters Kluwer health.