The overall goal of the following experiment is to detect IgE mediated drug hypersensitivity by the basophil activation test using well characterized HAP inized drug carrier protein conjugates. This is achieved by first chemically linking a drug via an intrinsic or organo chemically introduced carboxy group to lysine residues of human serum albumin. Secondly, the conjugates are physical chemically characterized to determine the exact coupling degree by performing high performance size exclusion chromatography or H-P-S-E-C, sequential refractive index and ultraviolet detection.
Next, the well characterized drug conjugates are used to stimulate basophils of drug hypersensitive donors. Ultimately, the basophil activation test or BAT can be used to evaluate IgE mediated mechanisms in drug hypersensitivity. My name is Michael Steiner from the Allergy Research Group at the University of Salzburg in Austria.
Today we would like to introduce a technique to you which has the implication of measuring the involvement of drug specific IgE E antibodies in drug hypersensitivity reactions. Here we show the procedure with two widely used representatives of nonsteroidal anti-inflammatory drugs, namely propone and diclofenac. However, the method has the potential to be used for routine diagnosis of immediate type drug allergies.
In general, Add 10 microliters of a freshly prepared N ethyl, N prime, three dimethyl amino propyl, carbo ide hydrochloride, or EDC stock solution to the propafenone or PP sample. Vortex the sample for one minute. Next, add 16.9 microliters of human serum albumin or HSA solution with a concentration of 118 milligrams per milliliter to the sample, and then incubate the sample for two hours at room temperature while shaking at 300 rounds per minute.
After shaking is completed, dialyze the samples three times against two liters of PBS for several hours each at four degrees Celsius. Then centrifuge the samples for 10 minutes at room temperature at 14, 000 times G, and collect the S supernatant containing the NSAIDs conjugated to HSA. Check the supernatant for proteins by performing an SDS page using 12%gels.
Loading about 12 micrograms of HSA conjugate into one gel slot for the analysis of the coupling rate of the NSAID conjugates by high performance size exclusion chromatography or H-P-S-E-C. First dilute the HSA standard sample to a concentration of one milligram per milliliter in demineralized water. For detector calibration, inject 50 microliters of HSA standard solution.
Then using a 7.8 by 300 millimeter TSK gel, G 2000 SSW XL column, and an online coupled refractive index or RI and UV detector system. Calculate the RI constant case sub RI and the UV constant K sub UV for the detector. Using these two formulas, dilute PP derivative standards for H-P-S-E-C in demineralized water in five, 10, and 30 in animal amounts for ease of injection, and then calculate mean values for DN by DC of the drug and DA by DC of the drug for both standards.
Using these two formulas now inject the drug conjugates onto the H-P-S-E-C and determine their RI and UV peak areas. Then using the same two equations, calculate the concentration of the PP derivatives and the HSA for the unknowns First label, seven flow cytometry vials depending on their content. Unstained control, negative control, positive control and drug conjugates.
Next, pipette 50 microliters of basophil stimulation buffer into the vials reserved for the unstained and negative control. Add 50 microliters of the anti FC epsilon R one solution from the flow two cast kit into the positive control vial. Now add 0.02 to 20 micrograms per milliliter of the PP conjugate to the drug conjugate vials in a one to 10 dilution series, and then add 100 microliters of stimulation buffer and 50 microliters of the patient's EDTA whole blood to each tube.
Mix the samples carefully. Then pipette 20 microliters of the flow. Two cast staining reagent containing PE labeled anti CCR three antibodies and ZI labeled anti CD 63 antibodies to each of the drug conjugate and positive and negative control tubes and mix.
Again, incubate the samples for 45 minutes in a 37 degree Celsius water bath. Then to stop the reaction, place the samples on ice for five minutes. Next, lice the erythrocytes by adding two milliliters flow two cast lysing reagent to each vial.
Incubate the samples in the dark for 10 minutes at room temperature. Then after pelleting the samples for five minutes at 500 times G at room temperature. Carefully decant the supernatant re suspend the cells in 500 microliters flow to cast wash buffer, and store the samples on ice until flow cytometry.
Use the unstained sample to adjust the flow cytometer voltage settings. Then gate the cells predicted as lymphocytes within the side scatter forward scatter plot. Similar to this sample figure, basophils are first identified as CCR three high side scatter low.
Similar to this scatterplot. After gating the cells into the fit EPE plot activated basophils can be further identified by their expression of FIT E labeled CD 63 positivity. After setting the cutoff to a maximum of 2.5%of basophils as CD 63 high in the negative control, a sample can be considered positive for basophil activation if greater than 5%Basophils are CD 63 high and the MFI of the sample divided by the MFI of the negative control exceeds two.
This method evaluates bat as a beneficial tool to detect IgE dependent drug hypersensitivity protein. Conjugates of NSAIDs are used for activation of basophils by H-P-S-E-C with RI and UV detection aggregation. State coupling degree and effective yield of conjugates are determined as depicted in this figure.
43.5%of the DF conjugates remained monomeric with a coupling degree of 5.0 DF to HSA for the aggregates. A coupling degree of 9.5 was determined as depicted here. The PPP conjugate was shown to consist of 68.5%monomers with a coupling degree of 21.2.
The coupling degree of the aggregates was 27.9. In total, the determined coupling degree of DF conjugates was 7.6 df, and that of PPP conjugates was 23.2. BAT performed with conjugates under optimized conditions allows investigations of IgE dependent reactions in NSAID hypersensitivity.
As shown here, only the PP conjugate was able to trigger basophil activation visualized by an upregulation. In CD 63 surface expression. 20.6%of basophils were activated as characterized by a log in fluorescence intensity, and the MFI increased by a factor of 4.9 from 3 86 for the negative control to 1893.
In contrast, using the DF conjugate, only 3.1%of the basophils were activated, which was below the 5%cutoff. Also, the MFI increased only by a factor of 1.1 from 197 for the negative control to 210. The assay validity is evidenced by one less than 5%basophil activation in the negative control.
Two, a log shift in fluorescence intensity of a basophil subpopulation and three greater than 50%activated basophils for the positive control. After watching this video, you should have a good understanding of how to perform basophil activation tests, and most importantly, how to differentiate IgE from non IgE-mediated drug hypersensitivity reactions. Furthermore, you should have an understanding of how to expand the applicability of the basophil activation test for drug allergies beyond its already established use in pollen food and insect Eno.Allergies.
