Standard methods for Apis mellifera venom research

Figures & data

Figure 1. Bee venom collector Beewhisper 5.0 is based on the electric stimulation protocol (http://www.beewhisper.com). In (A) it is shown how the equipment is put in front of the hive entrance. After completion of the collection cycle, the venom that has been deposited on the glass slide (B) can be scraped down.

Figure 2. Manufactured device used to extract venom from individual honey bees; 1 = cork; 2 = piston; 3 = glass container; 4 = acrylic tube; smaller arrow = plastic; larger arrows = copper wire.

Figure 3. Steps of dissection and separation of the venom reservoirs from the sting apparatus. (A) Pull venom reservoir together with sting apparatus out of the bee's body using a fine-tipped tweezers; (B) with the two structures completely removed from the bee’s body, but still stuck together, use two tweezers (or one micro scissors and one tweezers) to separate one from another.

Table 1. ‘Pros and cons’ to help selecting the appropriate method for the determination of hyaluronidase activity.

Method	Pros	Cons
Capillary Zone Electrophoresis	- Based on the determination of three of the HA degradation products (disaccharide, tetrasaccharide and hexasaccharide). - Very good precision, accuracy and linearity in the HYASE activity range from 16.4 to 124.2 U. - It allows evaluating the HYASE activity originating from different sources. - Rapid test, small sample volume, small amount of buffers used and relative low cost of analysis. - Recommended for specific studies on HYASE activity involving HA degradation products. - Fully compatible with the turbidimetric and viscosimetric methods. - No HYASE purification procedure required.	- Requires specific equipment. - The capillary should be thoroughly washed after each analysis. - Sample dilution is required when HYASE concentration exceeds the operating range. - More complex method compared to the turbidimetric and viscosimetric methods, especially when larger numbers of samples should be analyzed.
Turbidimetry	- Very simple, rapid and reproducible method for the determination of HYASE activity and of 10 to 200 µg of isolated acid mucopolysaccharides. - Based on the determining of the intact, non-digested HA molecule. - Works with common and inexpensive reagents. - Very useful for analysis of numerous samples (for instance: chromatography fractions). - It can be down-scaled to smaller volumes (microplate level). - Requires no specific equipment (only spectrophotometer).	- If the acid mucopolysaccharide is dissolved in distilled water instead of in salt solution, much less turbidity develops on addition of CTAB. - Low specificity.
Viscosimetry	- High sensitivity and reproducibility. - Based on the decrease in viscosity of the substrate HA by the HYASE activity. - Preferred method for studying the kinetics HA degradation and screening of HYASE inhibitors.	- Requires specific equipment. - Not recommended for analysis of large number of samples.

Table 2. ‘Pros and cons’ to help selecting the appropriate method for the determination of PLA2 activity.

Method	Pros	Cons
Colorimetry – method 1	- Works with common and inexpensive reagents. - Simple, fast and sensitive test (30–50 ng of enzyme). - Very useful for analysis of numerous samples (for instance: chromatography fractions, where PLA2 is the major component). - Requires no specific equipment (only spectrophotometer).	- Low specificity (other enzymes which catalyze hydrolysis of phospholipids and produce acids will also be detected). - For quantitative results in absolute units it must be considered that the pH indicator may inhibit some phospholipases. - The pH of dye solution may sometimes need to be readjusted (when a change in absorbance is observed).
Colorimetry – method 2	- Simple, fast and sensitive test that is done at physiological pH. - Works with common reagents, though a little more expensive than those from method 1. - Recommended for diluted samples (1- 5 µg/ml) in 96 well microplates assays.	- Alternative to method 1, with the same disadvantages. - Requires a microplate reader.
Spectrophotometry	- Rapid test that works common and inexpensive reagents. - Recommended to use with purified PLA2 samples. - Useful for kinetic studies of PLA2 inhibitors (for instance in the context of inflammatory processes).	- Requires N2 atmosphere to prepare aqueous phosphatidylcholine substrate. - Lipoxygenase from soybean is used as the coupling enzyme. - A clear assay medium is required, because any turbidity due to the phospholipase, lipoxygenase, or phospholipid substrate can interfere with measurements.
Fluorimetry	- Rapid, simple and very sensitive NanoDrop assay (10 ng of PLA2 render optimal enzyme activity). - It uses a non-fluorescence molecule having fluorescence characteristics by virtue of its chemical nature that do not interfere with the reducing enzymatic activity of PLA2. - It is specific for PLA2 activity. - This assay can be employed for screening PLA2 activity from various sources in either purified or crude form and for analysis of numerous samples. - Also useful for screening of library of compounds targeting inhibition of PLA2.	- Requires specific equipment (NanoDrop fluorospectrometer). - More expensive than the other methods described here.

Table 3. ‘Pros and cons’ to help selecting the appropriate method for the determination of protease activity.

Method	Pros	Cons
Non-specific proteolytic activity assay – method 1	- Works with common and inexpensive reagents. - Recommended for initial protease characterization studies in venom samples. - The assays can be performed in the presence of various specific protease inhibitors (serino-, aspartic, metallo- and so on) giving clues about the classification of proteases existing in the sample. - Very useful for analysis of numerous samples (for instance: chromatography fractions). - Requires no specific equipment (only spectrophotometer).	- Low sensitivity - Care should be taken in step 7 of this method (removal of the supernatant), thus avoiding contamination with the precipitate, which can significantly interfere with the reproducibility of the assays.
Non-specific proteolytic activity assay – method 2	- Alternative to method 1, but more sensitive due to chromogenic nature of the substrate Azocoll. - Based on Azocoll hydrolysis yielding soluble, colored peptides in proportion to enzyme concentrations at fixed incubation times. - As it is hydrolyzed by a diversity of proteases, is useful for initial protease characterization studies in venom samples. - Very useful for analysis of numerous samples when performed in microplates.	- Care should be taken with the rigorous Azocoll prewash to eliminate any inhibitory material present in commercial preparations which may interfere with the linearity of the reaction over time. - Due to the non-homogeneity of the substrate solution, the tubes or microplates containing the reaction mixtures should be shaken during all the incubation time to ensure maximum enzyme contact with the substrate.
Serine protease activity	- Simple, fast and specific assay of high sensitivity to detect serine proteases. - Based on that cleavage of a single amide bond of the substrate converts the non-fluorescent bisamide substrate into a highly fluorescent monoamide product. - Very useful in the study of proteolytic enzymes and for identifying inhibitors from different sources - It works with small amounts of enzyme, in range of ng. - Very useful for analysis of numerous samples (for instance: protease purification procedures).	- Requires specific equipment (fluorescence spectrophotometer equipped with a universal digital readout). - More expensive than the other methods here described.

Figure 4. An example of a basophil activation test in a bee venom allergic patient. In panel A single cells are isolated from cell aggregates in a forward scatter plot FCS-H (height) versus FCS-A (area). In B, High IgE positive basophils are selected and analyzed in panel C for their positivity in CD203c (basophil specific). Cells stimulated with buffer (D) and anti-IgE (F) are analyzed as a negative and positive control, respectively. CD203c++ denotes activated basophils, CD203c++CD63+ denotes degranulating cells. In panels F-I cells stimulated with bee venom (F and G) or wasp venom (H and I) are analyzed. It shows clearly a positive dose dependent reaction to bee venom and not wasp venom as expressed by the higher proportion of CD203C++CD63+ degranulating cells.

Figure 5. Basophils selected as in panels A-C in Figure 6, were analyzed for their histamine content or DAO positivity. A and B are the necessary negative and positive controls. C-F is an example of a dose response curve in a bee venom allergic patient showing clearly that DAO positive cells (DAOpos) becoming CD63+ (x-axis) and start to release histamine (DAOrel). The dotted line is the difference between histamine positive and histamine releasing cells.

Figure 6. This figure shows the correct compensation setting as describe in Table 4. A is undercompensated, C is overcompensated and B shows the correct compensation setting.

Table 4. Compensation method.

Tube	1	2	3	4
Basophil type	CD63-DAO-	CD63-DAO+	CD63++DAO+	CD63++DAO-
Explanation	Negative cells	DAO + cells without activation	DAO + and intracellular CD63+ staining without stimulation	DAO and CD63 staining with stimulation
Incubation / activation procedure (see method 2)
Whole blood	200 µl	200 µl	200 µl	200 µl
BAT buffer	200 µl	200 µl	200 µl	–
Anti-IgE (pos. ctl.)	–	–	–	200 µl
Incubation (see method 2)
Membrane staining procedure
CD63	None	None	None	none
IgE/CD203c	20 µl/10 µl	20 µl/10 µl	20 µl/10 µl	20 µl/10 µl
Lyse / fix (see method 2) Wash in PBA with 0.1% Triton X
Intracellular staining procedure (see method 2)
PBS-TX100	100 µl	100 µl	100 µl	100 µl
CD63	–	–	10 µl	10 µl
DAO	–	10 µl	10 µl	10 µl
Population by correct compensation (see Figure 6)	Grey cell in gate A (no staining for DAO and CD63)	Green cells in gate B (DAO + staining alone)	Blue cells in gate C (all basophils are positive for DAO and CD63, note that there is no CD203c up-regulation)	Red cells in gate D (histamine releasing cells)