Caso Loayza Tamayo

MoAb iodinations with 1^1I and 125I were performed with either the stationary phase chloramide, Iodogen™ [210] or using a modification of the Chloramine-T technique [211].

(a ) The io d o g en tech n iq u e.

Iodogen was dissolved in chloroform to a concentration of 100 pg/ml. 2 0 0 pi aliquots of the solution ( 2 0 pg iodogen) was then dispensed into

borosilicate glass vials (12 x 37 mm) (Wheaton) and evaporated under a gentle stream of nitrogen gas. A fine deposit of iodogen remained at the base of the vials which were sealed and stored at 4°C. Iodogen-coated vials were used within seven days of preparation. For the reaction procedure, MoAb in PBS (maximum volume 1 2 5 pi) was dispensed into an iodogen tube with a

gently agitated for 1 0 minutes before the reaction was terminated by removal of products.

(b ) The m od ified C hloram ine-T techn iqu e.

MoAb in 100 pi PBS was dispensed into conical polypropylene tubes and radiolabelled by the addition of iodine and 1 0 pi of a 0 . 8 mg/ml solution of Chloramine-T. The reaction was terminated after two minutes by the addition of 2 0 pi of a 1 mg/ml solution of sodium metabisulphite and an excess ( 6 0 0 pg) of potassium iodide.

Following iodination by either technique, the radiolabelled protein was separated from unreacted iodine by Sephadex G-25™ gel Filtration. A 15 ml column (Analytichem) was packed with 12 ml of swollen Sephadex G-25™ and equilibrated in PBS containing 1% HPPF as carrier protein. The radiolabelled antibody was loaded on the column and washed through with PBS/1% HPPF. The effluent was collected in sequential 0.6 ml fractions and assayed for radioactivity using an Isocal™ radionuclide assay calibrator (Vinten). Two or three fractions corresponding to the chromatographic peak were collected and the rest discarded.

(c ) D eterm in ation o f Radioactivity.

Quantitative measurement of gamma radioactivity was performed using a 1282 Compugamma™ automated gamma counter (LKB Wallac), providing automatic deadtime correction, interfaced with an IBM computer using the Ultroterm™ 2 software package. Results are given in counts per minute (cpm). The activity in MBq is given by:

cpm

F

MBq = z-

60 x 1 0 °

where,

F is the conversion factor for counts to disintegrations. For 125I, F = 1.25 and for *311, F * 4, given gamma counter efficiencies of 80 and 2 5% respectively (see below)

and 60 x 106 is the number of disintegrations per minute in 1 MBq.

The gamma counter was calibrated for 125I by means of a standard capsule of 12^I (LKB) and for 131I by repeated measurement of a standard source of 131I. This gave counting efficiencies of 80% for 125I and 25% for 13ii> which remained unchanged throughout the period of this project. A dual channel facility was also available for determining individual isotopic activities in a mixture of *3il and 125I. In this situation, a computer calculated correction was applied to account for spillover of radiation into the 125I counting cham ber.

(d ) D eterm in ation o f free-iod in e contam ination b y trich loracetic acid (TCA) p recip itation .

Radiolabelled antibody was diluted 1:100 in PBS and 100 pi mixed by vortexing with 400 pi of cold 10% TCA and 500 pi of PBS containing 1% HPPF. The mixture was incubated on ice for 15 minutes and then centrifuged at 10,000 x g for a further 15 minutes. The supernatant was separated from the precipitate and both assayed for radioactivity. The protein-bound iodine was estimated as the proportion of total activity present in the protein precipitate.

(e ) A ssessm en t o f structural integrity o f rad iocon ju gates by FPLC g el filtration .

Radiolabelled protein was diluted in PBS to give an activity of 105 - 107 counts per minute in a volume of 200 pi. This was loaded onto a Superose-12™ gel filtration column incorporated into the FPLC apparatus, and run under conditions described in section 3h. Ninety sequential 0.5 ml fractions were

individually assayed for radioactivity, and an elution profile plotted of counts per minute against fraction number.

( f ) D eterm in ation o f th e im m unoreactive fraction o f ra d io la b elled an tib od ies.

Direct radiobinding assays were established to determine the immunoreactive fraction of radioimmunoconjugates. For antibodies

recognising leukaemia associated cell surface antigens, a modification of the m ethod described by Lindmo was used. For antibodies raised against neural antigens, a conventional direct binding assay was employed, estimating the immunoreactive fraction under conditions of antigen excess.

(i) M odification o f th e m ethod o f Lindm o for d eterm in in g im m unoreactive fraction [212] [213].

Lindmo described a method, based on a modified Lineweaver-Burk plot, by which the fraction of immunoreactive radiolabelled antibody is accurately determined by linear extrapolation to binding at infinite antigen excess. The theoretical background to this approach has been previously described [212]. This method was employed in a liquid phase direct

radioimmunoassay for the investigation of iodinated antibodies recognising leukaemia associated antigens. An appropriate human leukaemic cell line (T- ALL or B-ALL) was cultured and harvested in exponential growth phase. Cells were washed in PBS and centrifuged at 400 x g for five minutes. The cell pellet was resuspended in PBS and the proportion of live cells determined by

Trypan Blue exclusion. The live cells were concentrated to 25 x 10^/ml in a volume of 4 ml which was divided into two aliquots. From each aliquot a series of six doubling dilutions were made in LP3 tubes pre-coated for 6 0 minutes at 37°C with 1% HPPF in PBS. Final concentrations of 25.0, 12.5, 6.25, 3.13, 1.56 and 0.78 x lOfyml in 1 ml PBS were prepared. In order to saturate cell binding sites for subsequent measurement of non-specific binding, 50 - 1 0 0 |ig of

unlabelled antibody was added to each tube of one series of 1 ml dilutions. These were incubated at 4°C for one hour. Radiolabelled antibody was added to both series of cell suspensions at a concentration of 0 . 5 - 1 . 0 ng/ml

(maximum volume 50 pi). The tubes were incubated for four hours at 4°C on a Roto-Torque™ rotator (Cole Palmer Instruments) at 10 rpm. Following

incubation, the total activity in each tube was measured in a gamma counter. The cells were washed four times by centrifugation at 400 x g for five minutes and resuspended in 2 ml of PBS. Cell bound activity was then measured. For each cell concentration, specific cell-bound activity was determined as the difference between bound activities of unblocked and pre-blocked cells. A plot of T/B as a function of I/C was then constructed where, T is the total incubated activity, B is the specific cell-bound activity and C is the cell concentration (x lOfyml). By extrapolation, the origin of the abscissa

represents infinite antigen excess, and the intercept with the ordinate defines the value Y0 - 1/r, the inverse of the immunoreactive fraction (r).

( il) D eterm ination o f the im m unoreactive fraction at antigen e x c e s s .

For antibodies recognising neural antigens, immunoreactivity was

estimated at antigen excess by a conventional binding assay. A homogenate of fresh whole adult brain was prepared in PBS and stored in 1.5 ml aliquots at -70°C. To assay for immunoreactivity, aliquots of brain were thawed,

centrifuged at 10,000 x g and washed in PBS. 1 ml samples were placed in LP3 tubes, pre-blocked at 37°C for 1 hour with PBS/1% HPPF. Approximately 1 ng 13il-labelled test antibody was diluted in 100 pi PBS, together with an equivalent quantity of a 1 2 5I-labelled irrelevant antibody. The antibody solution was then added in triplicate to the brain homogenate and incubated for four hours at 4°C on a tube rotator. Following incubation, the total activity of each isotope was measured using the dual channel facility of the gamma counter and

the homogenate washed four times in PBS by centrifugation at 10,000 x g. The supernatant was discarded and the activity of each isotope in the brain pellet measured. The immunoreactive fraction was determined by subtracting the m ean percentage binding of the irrelevant antibody from that of the specific antibody.

CHAPTER 3.

SELECTION AND CHARACTERISATION OF