1 both BCA (Preferal, Bactimos, Vertalec and Mycotal) and microBCA (Supresivit, Binab, Dipel and Vectobac) method.
² Dubois.
³ Carbohydrate contents were determined for pellet and supernatant separate. This value is an average, but both were comparable.

1.1.2 Testing of coating-characteristics and cross-reactivity

Coating characteristics were tested by determining IgG concentrations against the different allergen-extracts. In most cases strong reactions were found against most or all of the 8 allergen-preparations. The presence of cross-reactivity was determined by IgG-inhibition experiments with the different allergens. Cross-reactivity was noted between Supresivit and Bactimos, Binab and Supresivit, Mycotal and Preferal, and Vertalec, Vectobac and Mycotal.

1.1.3 Specific IgE testing

Plates were coated overnight at 4° c with 100m l of a 10m g/ml solution of allergen in PBS (pH=7,0). Samples were diluted 10x in PBS with Tween20 (0,05%) and gelatine (2g/l) (PBTG) and 100m l was pipetted into 2 wells. On each plate blanks for all 8 allergens (100 ml PBTG) and a positive control for Vertalec (100 ml of a 10x diluted pooled serum) were included. After incubation on a shaking platform at 37° C for 2 hrs. plates were washed with PBS with Tween20 (0,05%; PBT) and 100m l of a monoclonal mouse-antibody against human IgE (Moa HuIgE, CLB art.M1294; 1:16.000) was pipetted in each well. Plates were left shaking for another hour, washed and incubated again with a biotinylated monoclonal rabbit-antibody against murine IgG (Ram/BIO, DAKO art. E0354; 1:5.000). After 1 hr. plates were washed again and incubated with peroxidized avidine (AV/PO, DAKO art. P0364; 1:2000) for an hour. After the last washing plates were incubated with ortho-phenylenediamine dihydro-chloride (OPD) with 0,05% H₂O₂. After 30min. the reaction was stopped with 50m l HCl and the optical density (OD) was read at 492nm. Results were calculated as follows. First, a daily average blank was calculated for each allergen (n=9 for Vertalec, n=16 for all other allergens) and subtracted from the corresponding sample-OD’s resulting in an an adjusted OD (OD*). Second, if the OD* of both wells was > 0,05 the sample was decided to be positive (+). Alternatively, if the OD* of both wells was <= 0,05 the sample was negative. If only one of the OD*’s was > 0,05 the sample was retested and the new result was used. All positive samples as well as a subset of negative samples were retested to assess the reproducibility of the test.

1.1.4 Retesting of baseline and follow-up samples on the same plate

Samples of individuals for which both baseline and follow-up sample were available and for which at least one of these had been found positive previously were retested with both samples on the same plate.

1.1.5 Total IgE

Plates were coated overnight at 4° C with 100m l of a 1:4000 solution of a monoclonal mouse-antibody against human IgE (Moa HuIgE, CLB art.M1294) in PBS (pH=7,0). Samples were diluted 25x in PBTG and pipetted in 4 wells in a serial 2-fold dilution (25x, 50x, 100x, 200x). On each plate blanks and a standard were included in 10 2-fold dilutions (0,012….6,25kU/l). After incubation on a shaking platform for 2 hrs plates were washed with PBT and in each well 100m l of a monoclonal rabbit-antibody against human IgE (Raa HuIgE, DAKO art.A094; 1:10.000) was pipetted. Plates were left shaking for another hour at 37° C, washed and incubated again with a peroxidized monoclonal horse-antibody against rabbit-IgG (Hoa RaIgG/PO, CLB art. M1234; 1:10.000) for an hour. After the last washing they were incubated with ortho-phenylenediamine dihydro-chloride (OPD) with 0,05% H₂O₂. After 30min. the reaction was stopped with 50m l HCl and the optical density (OD) was read at 492nm. Results were calculated as follows. Concentrations were interpolated from the standard-curve insofar as OD*’s were within a range of 0,1 to 3,4. The resulting concentration was further dichotomized into a positive (IgE > 100 kU/l) and negative result (IgE <= 100 kU/l).

1.2 Results

1.2.1 Specific and total IgE

Prevalence rates of specific sensitisation (OD* > 0.05) against biological pesticides were highest for Vertalec, Vectobac and Mycotal (table 2). Retesting did not always confirm initial results, possibly because most of the samples were only marginally positive (see appendix; tables 7A-I and graphs 2A-I). Moreover a slight but significant decrease in the OD492* for a positive pooled control on Vertalec was noticed (nov/dec 1998 OD* = 0.089; feb/april 1999 OD* = 0.079), making interpretation of differences between marginally positive baseline and follow-up samples analyzed in different periods even more difficult (appendix; table 8A-I, graphs 3A-I). This could not be resolved by adjusting for the result of the pooled serum and therefore samples of all individuals for which both baseline and follow-up samples were available and which had tested positive either at baseline or at follow-up were retested on the same plate. If necessary this was done on the complete panel of allergens (9 individuals, 18 samples) but in most cases only Vertalec, Mycotal and Vectobac had to be used (70 individuals, 140 samples). Results are given in table 3A-I and for the allergens Vertalec, Mycotal and Vectobac in graphs 1A-C. An overview of the layout off all relevant datafiles (dBase/SAS) is included in the appendix.

Table 2
Number of positive results on specific allergens and total IgE