Immunity – lines of reaction

1. 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

2. 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

3. 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

4. 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.

5. Demonstration –bactericidal activity • Inactivated 1:32 • 1:16 undiluted o o o o o o o o o o O o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o oo o o o o o o o o o o o o o o o o o O o o o o o o o o o o o o o o oo o o o o oo o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o

6. 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

7. 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.

8. 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.

9. 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