LAB 1 - Direct agglutination. Serology-the study of the in vitro reactions between antibody and antigen

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1 LAB 1 - Direct agglutination Serology-the study of the in vitro reactions between antibody and antigen Serological reaction: - quantitative (weight/volume) - qualitative Agglutination - the aggregation of particulate antigen by antibodies. Direct agglutination -the antigen is a natural constituent of a particle. The titer of agglutination is the highest dilution of serum that still causes agglutination, but beyond which no agglutination occurs. 1. Hemagglutination test Place on the slide 1 drop of serum Nr 1 and add 1 drop of red blood cells (rbc). Mix and read agglutination after 5 min. Make appropriate control. 2. Bacterial agglutination Place on the slide 1 drop of serum Nr 2 and add 1 drop of bacterial suspension. Mix and read agglutination after 5 min. Make appropriate control. 3. Titer of serum Make dilution of serum Nr 1 from titer 1 : 2 in the 0.25 ml 0.9% NaCl. 10 th tube use as control. Add 1 drop of rbc to each dilution. Read the titer after 1 hour. 4. Presentation of Widal test (diagnosis of Salmonella infections) 5. Presentation of Wright test (diagnosis of brucellosis) Prozone-A region of diminished agglutination or precipitation of antigen-antibody complexes in titration curve due to excess of antibody.

2 LAB 2- Passive agglutination Passive (indirect) agglutination- reaction between antibodies and soluble antigen that had been attached to an insoluble particle (rbc, latex). The passive agglutination is divided into: 1. Passive agglutination "per se", when insoluble particle is coated by antigen. In this case antibody is looked for. A/ TPHA- detection antibodies against T. pallidum (passive hemagglutination) B/ test for detection of rheumatiod factor- RF (usually IgM that reacts with individual's own IgG) Arthri- Slidex - presentation The latex particles are sensitized with human gamma globulin. In the presence of RF agglutination is observed. RF is detected in 80 % of sera from patients with clinically suspected rheumatoid arthritis (RA). RF may be also detected in other diseases or cases than RA; - 4 % normal subjects (percentage increases with age) - in certain patients with systemic lupus erythematosus, hepatitis, cirrhosis of liver, syphilis. In these cases the RF titer is generally lower than in RA. If the latex slide test is positive, confirmation of the test is Waaler - Rose test. In Waaler- Rose test (semiquantitative test) sheep rbc are coated by gamma globulin. The coated rbc are added to the dilution of tested serum from dilution 1:20 to 1:2560. Test is positive when the titer is 1:160 or higher. 2. Reversed passive agglutination - insoluble particle is coated by antibodies. In this case antigen is looked for. A/ detection of HCG in urine of pregnant women B/ CRP detection (CRP slidex-presentation) CRP is an acute phase protein. The concentration of CRP is increased in case of inflammation: infection, autoimmunity, trauma, malignant tumors. CRP is risk factor of coronary heart disease and stroke C/ detection of bacterial antigen: Strepto-slidex-test, Meningitis-slidex -test, and other D/ detection of viral antigens: Adenovirus, Rotavirus in the feces in the cases of viral diarrhea The passive agglutination is more sensitive than direct agglutination and precipitation.

3 1. Direct immunofluorescence LAB 3 - Immunofluorescence - detection of antigen: Chlamydia trachomatis, rabbies virus, influenza virus, HSV (Herpes Simplex Virus) - deposition of immunoglobulin in kidney or skin in SLE (Systemic Lupus Erythematosus) 2. Indirect immunofluorescence A/ detection of antigen (Chamydophila pneumoniae) B/ detection of antibodies Autoimmune diseases : - detection of antinuclear antibodies (anti DNA, ssdna, dsdna, anti RNA, anti histones) in SLE - anti mitochondrial antibodies (AMA): primary biliary cirrhosis (PBC), chronic active hepatitis (CAH) - anti smooth muscle antibodies (ASMA): autoimmune hepatitis (AIH), PBC anti-neutrophil cytoplasm antibody (ANCA): canca (cytoplasmatic) Wegener s granulomatosis, panca (perinuclear) SLE, RA (rheumatoid arthritis), Sjögren syndrome, ulcerative colitis, primary sclerosing cholangitis - Infectious diseases: - syphilis FTA (Fluorescent Treponemal Antibody Test) - Respiratory tract infections (influenza, parainfluenza, Chlamydophila pneumoniae, Mycoplasma pneumoniae, Haemophilus influenzae, Legionella pneumophila, Bordetella pertusis)

4 LAB 4 - ABO Blood Groups Determine the ABO blood groups according to the scheme: standard anti sera standard rbc group anti A anti B anti AB O A 1 B

5 LAB 5 - Antiglobulin (Coombs) test 1. Antiglobulin (Coombs) test a) indirect - cross-matching between donor and recipient for detection of alloantibodies before blood transfusion - detection of antibodies anti-rh (and other) in the blood of a pregnant women b) direct - detection of antibodies in hemolitic disease of the newborns (detection mother's antibodies bound to newborns' rbc - hemolytic anemia (after drugs or autoimmune anemia) 2. Indirect antiglobulin (Coombs) test: - add 3 drops of tested serum to 3 drops of rbc - incubate 20 min.at 37 C - wash by centifugation 4 times - add 2 drops of antiglobulin serum - centrifuge 1 min.