Comparación Banco Compartamos con Nacional Financiera S.N.C

ANTIGEN PREPARATION

M.vaccae was subcultured from LJ slopes onto non-antigenic Sauton’s medium and incubated at 32°C for two weeks to allow them to reach their logarithmic phase of growth. Next, sterile plastic loops were used to remove the mycobacteria and to suspend them in M/15 borate buffered saline (pH 8.0) in a water-jacketed sonicator tube (about 10 loopfuls in 10 ml borate buffer). The ultra-sonicator was switched on at the maximum amplification for twenty minutes. The sonicated sample was dispensed in to tubes and centrifuged in a Beckman centrifuge at

15,000 rpm for 1 hour at 4°C. The supernatant was sterilised by membrane filtration using 0.45 jxm and 0.2 pm filters sequentially.

ADJUSTING PROTEIN CONCENTRATION

The supernatant was diluted 1:100 in borate buffer. Borate buffer was used as the reference sample, and values for extinctions at 260 mm (E260) and 280nun (E280) were taken using a spectrophotometer (Ultrospec II, LKB Biochrom, Cambridge Science Park). Protein concentration was calculated using W arburg and Christian’s method (extinction at 280mm x factor (E280/E260)). The protein concentration was adjusted to 2 mg/ml in M/15 borate buffer. Sonicated antigens of

M.tuberculosis was previously prepared in a similar manner by Professor John Stanford.

SEPARATION OF LYMPHOCYTES

The blood was centrifuged at 3000 rpm for 10 minutes. The supernatant or plasma was removed using a sterile pasteur pipette. Remaining blood cells were divided equally in two uni versais. The culture medium (RPMI-1640 supplemented with 2mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin) was added to both universals so their total volume was about 18 ml in each. Blood diluted in RPMI was then gently layered over 9ml Ficoll-Hypaque solution in universal tubes. The PBMCs were separated by centrifuging on Ficoll-Hypaque at 1800 rpm for 28 minutes. The peripheral blood mononuclear cells (PBMC) layer between the RPMI and red blood cell layers was removed using a sterile pasteur pipette. PBMC were washed by centrifuging in 20 ml of RPMI at 1500 rpm for 10 minutes. The supernatant was

poured off and the pellet was flicked to spread the cells. The cells were diluted in 20 ml of RPMI; 100 pi of this cell suspension was mixed with an equal volume of trypan blue, and the cells were counted using a haemocytometer. One drop of autologous serum was added to the cell suspension and then spun at 1000 rpm for 10 minutes. The supernatant was poured off; the pellet was flicked and the cells were suspended in suitable volumes of RPMI with 20% autologous serum (separated earlier) to give lO^cells per ml.

W HOLE BLOOD CULTURE METHOD

This method was adapted from Dr. Hazell Dockerell, London School and Hygiene and Tropical Medicine. Venous blood from the subjects was diluted 1 in 10 with RPMI-1640 medium supplemented with 2mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin. Each well had 10 p i of whole blood cells was diluted in 90 pi of RPMI-1640 culture medium (1 in 10 dilution) before the addition of 100 pi of double strength antigen in 100 pi RPMI culture medium. The eventual 1 in 20 dilution gave the best results for lymphocyte proliferation counts.

PREPARING CULTURES OF CELLS AND ANTIGENS

Ten two-fold serial dilutions of mycobacterial Ags in RPMI starting from 20 mg/ml were prepared in triplicates of 100 pi volumes in sterile 96-well cell culture plates. 100 pi of whole blood diluted 1 in 10 was put in each well. Control wells contained cells with RPMI-1640 culture medium only. The PBMC-antigen mixture, in a total volume o f 200 pi, was incubated in a humidified 5% C 02 atmosphere at 37 °C for 6 days. (Initial time-course study of healthy individuals showed maximal proliferation response on day 6). On the 6th day 100 pi of supernatant

was added to the culture before measurement of DNA incorporation of thymidine.

LABELLING. HARVESTING. COUNTING

Tritiated thymidine at 1 mCi of %-thymidine was prepared by adding 200 !|1I of thymidine (Amersham) to 25 ml of RPMI. WO jdL of 1.0 mCi of ^H-thymidine was added to the cultures using a plastic Pasteur pipette, and incubated for the last 8 hours. The cells were harvested using glass-fibre filter paper. These were put in vials and 3 ml of scintillation fluid was added. The radioactivity incorporated was counted using a liquid scintillation counter.

DATA ANALYSIS

The results were expressed as mean counts per minute (CPM) of triplicate cultures and the stimulation indices (SI) were calculated from the ratio between counts given by antigen-stimulated and antigen-free cultures.

SKIN-TESTING

Skin-testing was done with 0.1 ml of sonicated mycobacterial antigens (vaccin, chelonin and tuberculin) of 1 Tig/ml concentration injected intradermally on the forearm. An individual was described as skin-test positive if he/she developed an induration diameter of 10 mm or above, 72 hours after the injection. Twenty three healthy subjects were skin- tested. Peripheral venous blood was collected from each individual p rio r to skin-testing.

2.4 DETECTION OF CYTOKINES (IFN-y & IL-41 USING ENZYME-LINKED IMMUNOASSAY TELISA)

ELISA FOR IFN-y

The protocol provided by Amersham Sciences (AMS) was adapted as described below:

A 96-well ELISA plate was coated with 50 |ll of 1 Ab at 1 p-g/ml in phosphate-buffer saline* (PBS) per well, and left at 4°C overnight. The wells were washed twice with 200 p.1 PBS/Tween (PBS/T). For each wash, PBS/T was left in the wells for 3 minutes before aspirating the PBS/T and patting the plate dry on paper towels. Wells were blocked with 200 |ll of PBS/1%BSA at 37°C for two hours, then washed three times with PBS/T. Duplicate supernatant samples (50 p,l) were added to the wells, and incubated at 37°C for two hours. The plate was washed three times with PBS/T. Biotinylated 2 Ab at 1 |ig/m l (50 jxl) was added to each well, and incubated at 37°C for one hour. The plate was washed three times. Streptavidin-alkaline phosphatase solution (supplied by AMS) was diluted 1:1000 in PBS, added to each well (50 |il), and incubated at 37°C for one hour. The plate was washed three times. Next, p-nitrophenyl phosphate (p-NPP, Sigma) was added to each well (50 |Lil),

and incubated at 37°C for 30 minutes. The plate was read using dual filters at 405 nm / 450 nm using a spectrophotometer (Dynatech MR5000).

* SOLUTIONS

PBS (1 litre): 8g NaCl, 0.2g KH^PO^, 1.135g Na2HP0 4.2H0 z, 0.2g KCl and 0.5ml Tween 20.

ELISA FOR IL-4

The protocol provided by Pharmingen was adapted so the plate could be run together with ELISA for IFN-y as described above:

The wells were coated with 50 |il of 2 |ag/nil 1 Ab in PBS and incubated overnight at 4°C. The wells were washed twice with 200 pi PBS/Tween (PBS/T). For each wash, PBS/T was left in the wells for 3 minutes before aspirating the PBS/T and patting the plate dry on paper towels. Wells were blocked with 200 pi of PBS/1%BSA at 37°C for two hours, then washed three times with PBS/T. Duplicate supernatant samples (100 pi) were added to the wells, and incubated at 37°C fo r four hours. The plate was washed three times with PBS/T. Next, the wells were coated with 100 pi of 1 mg/ml biotinylated 2 Ab, and incubated for 1 hour at room temperature in the dark. Streptavidin- peroxidase (Sigma) was diluted 1:1000 in PBS/1%BSA and incubated at room temperature for 30 mins. The plate was washed three times with PBS/T. 100 p i of substrate buffer* with ABTS (2,2-azino-bis(3- ehtylbenz-thiazoline-6-sulphonic acid. Sigma) was added to each well and incubated for 30 minutes. The plate was read at 405 nm using a spectrophotometer (Dynatech MR5000).

* SOLUTIONS

Substrate buffer: 150 mg of ABTS in 500 ml of O.IM citric acid (pH4.35). Aliquot 10 mis per vial and store at -20’C. Add 10 ml of 30% H2O2 per 10 ml buffer just before use.