1.1 Source material The pollen was collected from Nawabganj, Rajshahi Town and Rajshahi University ranges of the Rajshahi region, Bangladesh during the pollination period in March to April. These pollen grains were then processed for > 95% purity by sieving through different grades of sieves (100, 200 and 300 mesh/cm2 ). All the samples were analyzed under the microscope which revealed pollen purity varying from 85% to 90%. To remove lipids and irritants of low molecular mass, the pollen sample was defatted with diethyl ether by repeated changes, until the ether becomes colorless. The defatted pollen powder was then completely dried and stored at 40°C in airtight containers until further use.
2.2 Protein extraction The defatted pollen was then used for protein extraction. Proteins were extracted in 0.2M Tris- HCl buffer, pH 7.4 by continuous stirring at 40°C for 24 hours. The extract was clarified by centrifugation at 15,000 x g for 20 min. at 4°C. The supernatant was collected and was subjected to fractional precipitation by solid ammonium sulphate. It was made up to 80% saturation by slow addition of the salt at 40°C. After centrifugation, the precipitate was re-suspended in 0.1M Tris HCl buffer, pH 7.4 and desalted by dialyzing against distilled water for 48 hr at 40°C by frequent changes of the distilled water using dialysis sacs (MW cut off 9 kDa). Finally, the supernatant was passed through a Millipore filter membrane (0.45μm), lyophilized in small aliquots, and stored at -200°C until further use.
2.3 Estimation of protein The protein concentration in the extract, as well as in the various eluted fractions, was estimated by the modified method of Lowry (Lowry et al., 1951). A calibrated solution of bovine serum albumin was used as a standard.
2.4 Gel electrophoresis The protein sample was heated with an equal amount of sample buffer [0.06M Tris HCl (pH 6.8), 1% SDS, 10% sucrose, 0.5% β-mercaptoethanol, 0.01% bromophenol blue] at 100°C for 3 min. 10μl of the sample containing 85μg of protein was loaded in the well of a 12% T mini-gel (8 x 7cm gel) Mini-Protean II slab gel apparatus (Bio-Rad, Hercules, CA, USA) and the gel was run using Laemmli buffer system [1971]) (0.05M Tris, 0.192 M Glycine, 0.1% SDS, pH 8.4) at room temperature for 2 hours 30 min., at 70 V. The molecular mass of the fractions was calculated by calibrating with standard marker protein, lysozyme (14,000 kDa), trypsin inhibitor (20,000 kDa), carbonic anhydrase (29,000 kDa), ovalbumin (45,000 kDa), albumin (BSA, 67,000 kDa) (Pharmacia, Uppsala, Sweden). After electrophoresis, the gel stained with 0.1% Coomassie Brilliant Blue R-250 and destined with methanol: acetic acid: water (4:1:5) mixture.
2.5 Isoelectric focusing (IEF) IEF was performed, as described by Grafin (1990), with precasted, broad-range (3.5 to 9.5) Ampholine PAG plates (Pharmacia, Uppsala, Sweden). Pollen extract was electro-focused on gel along with pI marker (Pharmacia, Uppsala, Sweden) to calibrate the pI of proteins in Litchi pollen extracts. IEF was run for 15 min. at 100 V, followed by another 15 min. at 200 V, and finally at 450 V for 2 hours. The gel was stained with Coomassie Brilliant Blue R-250, as in SDSPAGE.
2.6 Recovery of protein from the gel Protein was eluted from the gel, following the method of Wilson & Goulding (1986). After electrophoresis, one side of the gel (covering 2 lanes) was cut vertically and stained in order to ascertain the banding positions. Only those portions corresponding to the protein bands to be recovered were cut out with a sterile scalpel from the other half of the gel which was not stained. The gel pieces were then transferred into pre-treated dialysis bags filled with electroelution buffer (containing 0.05M Tris and 0.192M Glycine, pH 8.4) so that each gel piece was surrounded by a small amount of buffer with no air bubbles. These dialysis bags were immersed in electrode buffer in an electrophoresis tank of a horizontal gel apparatus. Electric current (120V) was passed through the bags for nearly 3 hours to elude the proteins out of the gel. At the end, the current was reversed for 30 seconds in order to release the protein from the wall of the sac. The buffer containing the eluted proteins was then transferred into a cotton-plugged Eppendorf tube and centrifuged for 2 min. to remove contaminating gel particles. The entire process was repeated 5 times to get sufficient quantities of each fraction for loading in the gel and for further skin tests. The protein in each fraction was quantified and each fraction was again electrophoresed to check its homogeneity.
2.7 Skin prick test The skin tests were performed on patients suffering from nasobronchial allergy as well as healthy volunteers at the Rajshahi Medical College and Rajshahi University students. Each patient was tested by placing 10μl of each allergen; at least 5cm apart on the volar surface of his/ her forearm and each site was then pricked with a disposable hypodermic needle. Negative and positive controls were also performed. The negative control was the buffer saline in which the allergen was resuspended and the positive control was histamine acid phosphate injection diluted with buffered saline to 1:10,000 i.e. 1μg of histamine acid phosphate. The patients were prohibited from using antihistamine, steroid and ephedrine for 48 hrs before the skin prick tests. The skin reactions were read after 15 to 20 min. from the commencement of the test. The test was quantified on the basis of the wheel diameter and graded 1+ to 4+. The skin tests were conducted at the Rajshahi University Medical center and Rajshahi Medical College Hospital, Bangladesh. The patients were selected on the basis of their suffering from respiratory allergic disorders.
2.8 Skin Tests (Practical) A diluted extract of each kind of pollen is applied to a scratch or puncture made on the patient's arm or back or injected under the patient's skin. With a positive reaction, a small, raised, reddened area with a surrounding flush (called a wheal and flare) will appear at the test site. The size of the wheal can provide the physician with an important reaction diagnostic clue. Skin testing remains the most sensitive and least costly diagnostic tool.