I have an urgent question for which I have been given a deadline. So please help!
In the acute stage of Brucellosis there is an initial production of IgM antibodies, followed closely by production of IgG antibodies(in the chronic stage). IgG-class antibodies may decline after treatment( so the active phase of the disease is literally over); however, high levels of circulating IgM-class antibodies may be found without any active disease( and gradually decrease over a period of time after the end of the disease ).
Having said all that, I've been told that for other infections ( maybe not all of them ) the story is the other way round. I mean it's the IgM that drops by the end of the active phase of the disease (treatment). And IgG stays for quite some time after the disease.
Now this is the question I've been asked:what's the reason for this difference?
BTW I tried to draw the charts the prof. showed us in the attachment. Sorry if it's not so good.
Please... Help.
THANKS
-ethanjvn-
Brucella IgG/IgM immunoassay was performed using a home-made kit according Sippel J.E., El-Marry N.A., Farid Z. (1982) Diagnosis of human brucellosis with ELISA. Lancet, 3, 19, modified by myself. In brief, dispensed 200 µl of suspension Brucella antigen (Sclavo) diluted 1:10 in PBS (dissolve in dH2O, 8.0 g/L NaCl, 0.2 g/L KCl, 1.15 g/L Na2HPO4, 0.2 g/L KH2PO4 obtaining a solution having pH 7.4) in wells of HB-MTP (BioHit) and incubate at 4 °C for 48 hours. After one wash with washing solution (PBS+0.05% Tween-20), dispense 200 µl of blocking solution (2.5% BSA in washing solution or blocked for 90 min with PBS containing 0.1% casein, 0.5 M NaCI and 0.1% Tween 20) and incubate for 24 hours at 4 °C. After twice wash as above, the plate is dried ad store at 4 °C in sealer plastic bag. At the time of the assay, take the number strips useful for the duplicate samples/controls assay and blank and curry up to room temperature. Dispense 100 µl serum samples and controls (positive and negative sera), diluted 1:100 in diluent assay (1% BSA in washing solution) in respectively wells, sealer the wells whit adhesive plastic to avoid evaporation, and incubate for 1 hour at room temperature. Wash thrice the IgG wells with physiologic saline, do not to use washing solution, and for IgM wells, once with RF neutralization buffer (citrate buffer 0.1 M at pH 4.8) and twice with physiologic saline. Dispense 100 µl of stable solution of goat anti-human IgG or IgM phosphatase alkaline conjugate (dilute 1:100 goat anti-human IgG or IgM alkaline phosphatase conjugate in 1% BSA in PBS containing 1% NaN3. Store in transparent glass bottle at 4 °C, resulting stable 8 months) diluted, just before use, 1:5 in 1% BSA in PBS and incubate for 1 hour at room temperature or for 30 minutes at 37 °C in a moist chamber. Repeat step wash with only physiologic saline and add 100 µl of pNPP substrate 10 mg in 10 ml DEA buffer (dilute in 300 ml dH2O, 2.9 ml <30 mM> of DEA, adjust at pH 9.8 with 1 M HCl at 25 °C. Dissolve 0.514 g <5.4 mM> MgCl2 anhydrous and 17 mg (0.124 mM) ZnCl2 and mix for 15 min. Check again pH, and add 0.22 ml of Tween-20, continue to mix avoid foam, not less than 30 mins. And add 15.2 ml (0.053%) Proclin 300 and fill to 1 liter with dH2O, mixing for further 30 mins. Aliquote in opaque plastic bottle and store at 4 °C resulting stable for several months) and incubate for 30 min. at room temperature protect from light. Stop reaction dispensing 50 µl of 3 N NaOH. Mix and read the Abs at 405 nm. Under the described conditions, is considered positive values when Abs is > to 0.500 for IgG and > 0.200 for IgM
-Zagami Francesco-
