Download LAB 1 - Direct agglutination Serology

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.
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.
LAB 3 - Immunofluorescence
1. Direct 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)
LAB 4 - ABO Blood Groups
Determine the ABO blood groups according to the scheme:
standard anti sera
anti A
anti B
anti AB
O
standard rbc
A1
group
B
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.