Utilization of commercial plasma administered to the index case
An aliquot of the commercial plasma that had been administered to the index case described previously, which apparently contributed to a fatal anaphylactic reaction was available for analysis. This sample was aliquoted into 10 mL and 2 mL vials and stored at -80oC.
Indirect ELISA to detect BSA-specific IgE
This protocol was adapted from Gershwin et al. 2012 with modifications. ELISA plates were coated with 1 ug/well BSA in bicarbonate-carbonate buffer pH 9.6 overnight. ELISA wells were blocked with 0.5% rabbit serum albumin (RSA) in coating buffer. Each well in a 96 well plate was filled with 200 µL of 0.5% RSA for blocking. The plates were incubated at 4oC overnight. After incubation concluded, the wells were washed with 300 µL of PBSt (PBS + 0.1% Tween-20) by an electronic plate washer 3 times. Plates were coated and blocked in bulk and stored at 4˚ C until use within one week. The commercial plasma that was administered to the index case was used as a positive control. Unvaccinated horse serum was used as a negative control. PBS was used as a blank to establish background absorbance. Two wells were designated as the positive control, two as the negative control and two as the blank. Undiluted serum samples (100 µL) were added to each remaining well in duplicate and incubated at room temperature for 2 hours. A standard curve was not available and optical density (OD) values were used to analyze the data.
Plates were washed as described above. Anti-horse IgE horse radish peroxidase (HRP)10 was diluted out to 1:500 with wash buffer, added to each well (100 µL) and incubated for 1 hour at room temperature. The plates were washed again. Tetramethylbenzidine (TMB) (100 µL) was added to each well and left to incubate for 30 minutes at room temperature protected from the light. After 30 minutes 100 µL of sulfuric acid (2 M SO2H4) was added to each well to terminate the reaction. Plates were read with an electronic plate reader at 450 nm and recorded to obtain the optical density.
All horse samples including 0, 14, 30, 90, 180, 270, and 365 day post-vaccination, if available, were tested by way of ELISA for the presence of BSA-specific IgE. All horses classified as a reactor were tested by ELISA. Plasma from the plasma donor samples from commercial source were all tested by ELISA. All plates contained the same positive control, negative control and blanks. The OD value for blank wells was averaged for each plate and then subtracted from each sample well to correct for background absorbance. The adjusted duplicate sample OD values were then averaged together for each horse.

Statistical analysis

Statistical analysis was completed with Prism (v.8) on the BSA-specific IgE ELISA data. Horses in the healthy vaccine group had their post-vaccine Ig OD measurements compared with baseline values. Means were determined for each timepoint for all age groups. Differences between groups at specific timepoints were determined with t-tests. Significance was defined as a p -value <0.05. Data was then compiled in Microsoft Excel as a spreadsheet function.
RESULTS

ELISA BSA-specific IgE

The OD value for the positive control mean across all plates was 0.340. The OD value for the negative control mean across all plates was 0.062. The OD value for the blank control mean across all plates was 0.071. The OD value for the positive control mean was significantly different from the negative control mean (p value <0.001). The OD value for the negative control mean was significantly different from the OD value blank control mean (p value = 0.008).

Healthy Horses

The mean OD value +/- standard deviation for day 0 BSA-specific IgE in all healthy horses (0.108 +/- 0.089) was not significantly different when compared to the mean OD value +/- standard deviation (SD) for 14 days post-vaccination BSA-specific IgE in all healthy horses (0.124 +/- 0.118) (p value = 0.43) (Figure 1). The mean OD value +/- SD for day 0 BSA-specific IgE in all healthy horses (0.108 +/- 0.089) was significantly higher when compared to the mean OD value +/- SD for 180 days post-vaccination BSA-specific IgE in all healthy horses (0.073 +/- 0.069) (p value = 0.007) (Figure 1).
When comparing the fold change between day 0 BSA-specific IgE OD values and 14 days post-vaccination BSA-specific IgE OD values, 63% of horses between the ages of 3 and 9 years had a fold increase greater than 1.5 (Figure 2 and Table 1). The percentage of horses displaying a fold change greater than 1.5 decreased with age across age groups. Only 29% displayed a fold change greater than 1.5 in the 10-14 year old age group, 0% in the 15-19 year old age group and 5.6% in the 20 -32 year old age group (Table 1).
The mean BSA-specific IgE OD values +/- SD at time points 0, 14, 30, 90, 180, 270 and 365 days after vaccination were compared across age groups (Table 1). The mean BSA-specific IgE OD value +/- SD for horses 3-9 years of age at day 0 was 0.072 +/- 0.086 and at 14 days post-vaccination had increased to 0.125 +/- 0.09 (p-value = 0.013) (Table 1). The mean BSA-specific IgE OD value +/- SD decreased to 0.070 +/- 0.070 at 180 days post-vaccination with days 0 and 180 being significantly different (p-value = 0.035) (Table 1). All other timepoints compared to day 0 within this age group were not significantly different. When comparing all other age groups’ mean BSA-specific IgE OD values at 14 days after vaccination and day 0, the differences were not significant. When comparing all other age groups’ mean BSA-specific IgE OD values at day 0 and 180 days after vaccination, the differences were significant for age groups 10-14 years (p value = 0.001) and 20-32 years (p value = 0.028). In all of these age groups, the mean BSA-specific IgE OD values decreased at 180 days post-vaccination compared to time 0 and 14 days post-vaccination (Table 1). No other time points in any age group were significantly different when compared to day 0.
Anamnestic response of BSA-specific IgE expression in response to annual vaccination in healthy horses was most common in younger horses, aged horses rarely demonstrated a > 1.5 fold change in BSA-specific IgE expression within 14 days of annual booster vaccination (Table 1).
Among young adult horses that were evaluated over the course of a year, two-thirds of the horses demonstrated a greater than 1.5 fold increase in BSA-specific IgE expression without adverse event (Figure 2). Among horses greater than 10 years of age, it was uncommon to have a marked increase in BSA-specific IgE expressed following booster vaccination (Table 1).