CUADRO 1-20 INDICADORES DE LA COLABORACIÓN EN EQUIPO DE LOS EMPLEADOS

The m ethodology describing this histam ine assay was first published by Shore et al (350). The interaction between histamine and o-phthaldialdehyde (OPT), under alkaline conditions, generates a highly fluorescent product. This adduct can be stabilised and m easured by the use of a com m ercially available spectrophotom eter. The reaction between histam ine and OPT is displayed in Fig. 2.1.

In this study, test samples (2 ml) were rendered alkaline through the addition of NaOH (1 M; 267 pi) and thoroughly vortex mixed. OPT (1 % w/v in methanol;

100 pi) was added, the sample mixed once more and the mixture allowed to react for 4 min. The reaction was quenched by the addition o f HOI (3 M; 133 pi). The fluorescence generated was proportional to the amount of histamine present in the sam ple and could be measured on a spectrophotom eter (Perkin Elmer LS 5B). This machine employed an excitation wavelength of 360 nm and an emission wavelength of 440 nm. In this manner, histamine concentrations of >5 ng/ml could be measured.

If im m unoglobulin w as used as a secretag og ue in p a rtic u la rly high concentrations, protein precipitation often resulted on the addition of acid. To counteract this, sam ples were centrifuged (200 g; 20 min) before being assayed for fluorescence.

2.8.2 Automated assay of histamine

For samples other than those of RPMC (or RPMC sam ples containing an inhibitory compound which interfered with the fluorescence obtained in the manual assay), the histam ine contents w ere assayed in a com m ercial autoanalyser (Techincon A utoanalyser II). In essence, the final assay procedure w as identical to the manual m ethod. However, autom ated chemical extraction ensured purification of the histamine sample prior to its interaction with OPT.

To prepare sam ples for analysis, perchloric acid was added (0.4 M final concentration) to liberate residual histamine and precipitate proteins. This was especially important in the case of basophils, since unprecipitated blood proteins could interfere to a marked degree in the assay. The samples were vortexed thoroughly to ensure complete mixing and centrifuged (200 g; 20

min). On autoanalyser entry, samples were made alkaline and the histamine extracted into salt saturated butan-1-ol. The organic phase was separated and retained, washed once in a less alkaline medium, made less polar by the addition of 2-heptane and the histamine back extracted into dilute HCI. The amine was allowed to react with OPT under alkaline conditions and the adduct generated stabilised by acidification. The fluorescence was measured by fluorophotom eter and recorded on a chart recorder. An assay of this description perm itted basal histam ine concentrations of >1 ng/ml to be m easured.

2.8.3 Radioimmunoassay (RIA) of histamine

H is ta m in e le v e ls w e re a lso m e a su re d , fo llo w in g a c y la tio n , by im m unoanalysis using a RIA kit (Serotec). A kit of this nature was highly sensitive and specific for histamine. The principle of the assay involved the com petition between histam ine (present in sam ple) and the iodinated histamine tracer for the binding to the antibody coated tubes.

In brief, a set of histamine standards (including a zero standard Bq) and experim ental standards (100 pi) w ere added to a p p ro pria tely labelled polypropylene tubes containing the acylating agent. Acylation buffer (50 pi) was added, the tubes mixed thoroughly and left to incubate for 30 min at RT. To the m onoclonal antibody coated tubes, acylated standard or acylated sam ple (50 pi) were added, followed by iodinated tracer (500 pi). In two uncoated polypropylene tubes, iodinated tracer (500 pi) was added to determine the total radioactivity or total counts (TC). All tubes were then left for a minimum of 18 hours at 4 °C. Subsequently, all tubes (except TC) were com pletely aspirated at 2-8 °C until all liquid was removed. Aspirated (B, bound cpm) and TC tubes were then counted for 1 min in a gamma counter. A standard curve plotting B/TC (%) against histamine (nM) was constructed from which the amine levels in each sample could be determined. Samples were diluted (if necessary), to lie within the range of the standards (0.5-150

2.8.4 RIA of prostaglandin D2 (PGD2)

2.8.4.1 Experimental protocol for PGD2 release

These experim ents were performed on purified RPMC. C ells from 5-7 sensitised animals were pooled together, purified as previously described in section 2.4.1.2., and counted. An appropriate volume of FHT was then added in order to ensure that, on deposition into the reaction tubes, each sample contained a minimum of 1 x 10 ^ cells.

Samples were incubated at 37 °C and then challenged with secretagogue(s) for the pre-selected time period. On termination of the reaction, samples were centrifuged (100 g; 3 min; 4 °C) and supernatant aliquots (500 pi) swiftly pipetted into appropriately labelled Eppendorf tubes. These tubes were im m ediately ‘snap frozen’ (liquid nitrogen) to prevent both further synthesis and degradation of the prostanoid. The tubes were then stored at -70 °C until they were assayed (see below). The remaining supernatants were separated from the cell pellets and both fractions assayed for histam ine in the conventional manner.

2.5.4.2 Protocol for PGD2 assay

P G D2 levels w ere m easured using a com m ercially ava ila ble RIA kit (Amersham). The assay reflected the competition between unlabelled PGD2

and ^H-labelled PGD2 for binding to a limited quantity of an antibody raised with a high specificity to PGD2.

In sum m ary, a range of PG D2 standards (provided in the kit) and experimental samples (100 pi) were aliquoted into pre-labelled polypropylene tubes. This was followed by the addition of 3H -P G D2 tracer (100 pi), PGD2

tracer, PGD2 specific antiserum (100 pi) and assay antiserum (100 pi).

Simultaneously, a total counts (TC) tube (100 pi; 100 pi antiserum; 200 pi buffer), a zero standard (Bq) tube (100 pi tracer; 100 pi antiserum; 200 pi buffer) and a non-specific binding (NSB) tube (100 pi tracer; 300 pi buffer), were prepared. All tubes were vortex mixed and incubated overnight (4 °C).

The next day, a dextran coated charcoal suspension (500 pi) was added to each tube (with the exception of the TC tube, where 500 pi of assay buffer was added). Tubes were immediately mixed, left to stand in an ice-bath (15 min) and centrifuged (250 g; 4 °C; 15 min). All supernatants were decanted into scin tilla tio n vials, scin tilla n t added (O ptiphase; LKB; 10 ml) and the radioactivity measured by (^-scintillation spectrom etry (4 min/sample). Data were then expressed as;

= (B-NSB1x100

Bo (Bo-NSB)

where Bq = zero standards

B = sample reading

NSB = non-specific binding

Hence, using the PGD2 standards provided, a curve was plotted and the P G D2 content of the unknown sam ples obtained. Prostanoid values were expressed as ng per 10^ mast cells. To guarantee that the unknown samples met the concentration parameters o f the RIA kit (1-200 pg/ml), prelim inary sam ples w ere tested prior to full assay. If necessary, dilutions w ere com pleted to verify that sample PGD2 levels coincided with the standard curve.