Download Lysosyme

Immunity – lines of reaction
Nonspecific - cellular- quantitative - no. of lymfocytes
(rosettes), Blood count, - leu, lymfo, mono, eosino………
•
- functional - phagocytosis,
Activity, Index, test of activation of lymfocyts
•
- humoral - quantitative - IgG,IgA,IgM,
C3,C4,CH50,CIK, proteins of acute phase of inflamation CRP,
•
- functional – clinically,
bactericidal activity of serum, activity of lysosym
Demonstration
• Lysosyme – function of macrophage sy
• Bactericidal activity of serum - humoral immunity –
functional test
• CH50/ C3, C4 - humoral immunity,
functional/quantitative
• CRP – protein of acute phase inflamation
Determination of lysosyme
• Lysosyme – basic low molecular protein present in saliva, tears.....
• Micrococcus lysodeiticus – bacterium soluble by lysosyme (G+)
• Sample in which we are testing presence and level of lysosyme saliva, blood, urine, CSF
• Agar with suspension of Micrococcus lysodeiticus.on glass dish
• Wells in agar with standards with known concentration of lysosyme
and unknown sera
• We read the clarification next to the well with serum with lysosyme
that causes the lysis of bacteria in suspension in agar
• Diameter of the zone of clarification is directly influenced by the
concentration of lysosyme and we calculate it by comparison using a
formula
Standards of lysosyme
Unknown samples
Agar + bacterial suspension
Bactericidal activity of blood
• Escherichia coli - Suspension of bacteria – nephelometry,
photometry – measurement of cloud with different
concentration of bacteria ex. 10, 100, 103 104 105 106
• + serum (activity of which we measure) – diluted
• On agar plates – cultivation
• Reading the no of bacteria that grew in the presence of
serum.
• Bactericidal activity is calculated using the formula in
comparison of unknown sera result and result of known
standard
• Base – in the presence of healthy serum a certain no of
bacteria are killed.
Demonstration –bactericidal activity
• Inactivated
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• 1:16
undiluted
CRP - C reactive protein,
protein reacting with C substance present in
the wall of Streptococcus pneumoniae
• Antibody agains CRP is poured to the capillary
and carefully the serum is added to the top
• If CRP is present in the serum, it will react with
anti CRP present in the bottom of the capillary and
a white precipitate will appear
• Qualitative test in capillary
• Quatitative test is done in the agar – difusion
(compare with lysosyme, IgG, protein of acute
phase of inflamation, C3, C4). Where zone of
precipitation is compared with the zone of the
standard
• CRP positive in bacterial infections
Immune-diffusion in agar
• Test for determination of concentration of proteins – ex.:
IgG, IgA, IgM, C3, C4, proteins of acute phase of
infalmation - ceruloplasmin, macroglobulin………….
• Base – the antibody against the substance we want to
measure is added to the agar. In preformed wells the
standards (4-5) with known concentrations and unknown
samples are added. The diffusion occurs and a ring of
precipitation is formed based on the reaction of AgAb. The
diameter of the ring is directly dependent of the
concentration of measured sample and is compared with
standard.
CH50
• Complement system - cascade of protein components,
each of which is activated by the previous. The system is
activated in nonspecific or/and specific way.
The aim is to form the membrane attac complex – the
structure that is incorporated to the bacterial cell wall
and this causes the lysis of the microbial cell.
• Determination of the amount of serum that is able to lyse
50% of standard concentration of erytrocytes.
CH50 – Kaber Mayer
• Erytrocytes + ab + C´= hemolysis
• Hemolytical system – erytrocytes + ab
• Control serum – amount of C´ lysing 50% of ery = 1
unit CH50
• Patient serum – unknown amount of C´
Extinction of control serum
Extinction of patient serum
= 38 j