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The determination of the purity of pollen extracts
Review of Palaeobotany and Palynology Elsevier Publishing C o m p a n y , A m s t e r d a m - Printed in T h e N e t h e r l a n d s
THE DETERMINATION OF THE PURITY OF POLLEN EXTRACTS F. H. M 1 L N E R AND C. A. F R A S E R
Bencard Allergy Unit, Beecham Research Laboratories, Brentford (Great Britain)
(Received October 28, 1966)
SUMMARY
The purity of pollen extracts is dependent fundamentally on the integrity of the pollen from which they are prepared. The most likely contaminants are other kinds of pollen, moulds, dust and plant debris. These contaminants can be avoided by proper methods of collection. The present investigation embraced the microscopical examination of pollen: (a) from the visual and morphological point of view, and (b) from particlesize differences. Examination of extracts containing known proportions of other pollen contaminants embraced gel diffusion methods, immuno-electrophoresis, comparative skin tests, infra-red spectrography, ultra-violet spectrography, and the fractionation using Sephadex columns.
INTRODUCTION
Although the title of this paper suggests analytical methods for the examination of pollen extracts, the factors on which the purity of such extracts depend are of necessity concerned with the integrity of the pollen from which the extracts are prepared. Various factors may contribute to contamination of pollen during collection: (1) pollen of other plants adhering to the specimens before being taken to the pollinarium; (2) cross contamination when more than one kind of pollen is being collected at the same time; (3) dust particles; (4) dried powdered foliage, petals, plant hairs, etc.; and (5) mould mycellium and spores if the pollen has been collected under damp conditons. Contamination from all of these causes can be avoided if proper collecting techniques are employed. It is always wise to check ones own pollen after collection but it is essential to be able to assess the purity of pollen obtained from sources not under ones own control. Rev. Palaeobotan. Palynol., 4 (1967) 277-286
277
In order to determine the purity of the starting materials (dry pollen) several methods have been investigated in our control laboratories. (A) On dry pollen: (1) Microscopic examination: (a) Visual (b) By particle size (B) On extracts: (2) Gel diffusion (3) Immuno-electrophoresis (4) Comparative skin tests (5) Infra-red spectrography (6) Ultra-violet spectrography (7) Sephadex columns
EXAMINATION OF DRY POLLEN
Visual examination using the microscope By this visual method it is possible to detect: (a) the presence of mould spores and mycellium; (b) the presence of plant debris (crushed leaves and petals) and plant hairs; and (c) the presence of morphologically different kinds of pollen.
Measurement of particle size, using the microscope While the visual method can enable many contaminants to be identified it is very tedious in estimating the quantity of the contamination. The particle size method depends on obtaining the mean diameter of the pollen and its contaminant and, using a Shearing eyepiece, to scan fields and, measuring the diameter of all of the particles in each field, to assess the proportion of the contaminant. Obviously, this method cannot be used when there is no significant difference in the mean diameter between the required pollen and that of its contaminant. The nearer the diameter of two pollen grains to each other, the less sensitive is the method and the greater the number of observations required. When, however, there is a large difference the quantitative amount of contamination on a particle basis can readily be determined. The weight for weight basis of the contamination can be calculated from the relative sizes of the particles concerned. The method is very much more delicate when the particle size of the contaminant is less than that of the required pollen. As a model, plane-tree pollen (Platanus hybrida) mixed with various amounts of timothy grass pollen (Phleum pratense) was examined. In the final experimental model 90 ~ of the pollen grains were of timothy 278
Rev. Palaeobotan. Palynol., 4 (1967) 277-286
grass and 10% of plane tree. On a weight for weight basis this corresponds with 2.5 g of plane-tree pollen in 97.5 g of timothy grass pollen. From the measurement of the particle size of 100 grains of pure plane and timothy pollen estimates of the mean particle diameter and particle size distribution for each kind of pollen were obtained. The average diameter of plane-tree pollen grains is 22/z and that of timothy grass pollen 37.5 #. A statistical analysis using these experimentally obtained values showed that the counting of about sixty grains of pollen will be sufficient to reach a decision, at a 95 % confidence level on a 10 % particle/particle contamination of timothy pollen with plane pollen. A mixture of timothy pollen contaminated with plane pollen (10 % particle/ particle) was counted in batches of 10 particles and the cumulative frequency of contaminant particles plotted against the number of batch observations on a chart pre-prepared with the boundary lines as calculated from the theoretical model (i.e., sequential test). It was seen from the chart that the contaminant level of 10 % particle/particle ratio is easily detected, as predicted from the theoretical model (Fig. 1).
>16
Pollen- g r a i n r a t i o 10 plane t r e e 9 0 t i m o t h y grass
o
m 14 -613 o_ 11
Significantly more particles
t_ 9 7
"6 6 Significantly particles
~ 4 z / -e- - - - e . - .
-~---
-e ....
i
i
I
I
I
I
1
2
3
4
5
6
Pure t i m o t h y pollen e.... ~- ---.~
I
I
I
I
7
B
9
10
Number of botches
-4
-5 Fig.1. G r a p h of cumulative frequency of occurrence of small grains (plane-tree pollen) plotted against the n u m b e r of field counts. (N.B, One batch = one field count = 10 pollen grains).
Rev. Palaeobotan. Palynol., 4 (1967) 277-286
279
Pure timothy pollen can be seen to follow the boundary line into the region of “no difference”, whilst the contaminated pollen crosses the boundary line into the region “of difference” from pure timothy pollen.
EXAMINATION
OF POLLEN
EXTRACTS
The examination of dry pollen microscopically, whether for particle size difference or for morphological differences, is very tedious and has obvious limitations. For instance, morphologically different kinds of pollen of the same particle size cannot be differentiated by methods depending on particle size and morphologically similar pollen types of similar particle size cannot be differentiated by either of these methods. Differentiation of such pollen types is often important from the clinical point of view and might also be desirable from the botanical viewpoint. Grass pollen grains, which are of great importance clinically, cannot be differentiated visually or physically. There are several ways in which pollen extracts may be examined to detect contamination of one pollen with another but most of the methods are inadequate to differentiate the closely related proteins which are the main distinguishing factors. This difficulty disappears when immunological reactions are used.
Inmunodif~sicn
in gels
If rabbits are injected with suspensions of whole pollen grains (of known
TABLE
I
SMALLEST
AMOUNT
OF POLLEN
THAT
Test pollen
Lowest level of detection
Cocksfoot grass
100 pg/ml
Elm tree Fescue grass
250 250
Plane tree Plantain
250 dml
Rye grass
250
Timothy grass
250 pgid
280
fig/ml pg/ml
100 pg/ml ,ug/ml
CAN
BE DETECTED
BY THE OUCHTERLONY
Level of detection in other pollen
METHOD
Partial cross reactions with fescue, rye and timothy none cocksfoot, rye and timothy none none cocksfoot, fescue and timothy cocksfoot, fescue and rye
Rev. Palaeobotan. Palynol., 4 (1967) 277-286
purity) in complete Freunds adjuvant, a very good antibody response is obtained and the serum so produced, when mixed wittrthe appropriate pollen extract at the correct concentration, will give an antigen/antibody precipitate. When this reaction is carried out in agar gels, by the method described by OUCHTERLONY (1949), one or more precipitation lines appear in the gel. In closely related kinds of pollen, such as grass pollen, some of the lines are common to all kinds of grass pollen thus indicating some common antigens but each has its own specific lines as well, so that it is possible to detect a contamination of one pollen with another or several others, even though they be closely related, so long as the corresponding specific antisera are available. Table I gives examples of the smallest amount of contamination that can be readily detected by this method. It will be seen that the soluble antigens from 100-250/~g of pollen/ml of extract can be detected by this method and that the lowest contamination of one pollen with another that can be detected with certainty is 1.0~o. At the lowest sensitivity therefore (i.e., 250 /~g/ml) 1 ~o of contamination can be detected if an extract of 2.5 g of the contaminated pollen with 100 ml of buffered saline (pH 7.0) is used. It is also seen that the extractive from 100-250 #g of pollen in 1 ml of buffered saline can be used to detect and identify the pollen by the Ouchterlony method of gel diffusion. Since a maximum of 0.1 ml of such an extract is used in the test it follows that quantities of pollen as small as 10-25/~g can be detected and identified. The pollen grains used to illustrate these findings were those of cocksfoot grass (Dactylis glomerata), elm tree (Ulmus procera), fescue grass (Festuca pratensis), plane tree (Platanus hybrida), plantain (Plantago lanceolata), rye grass (Lolium perenne), and timothy grass (Phleum pratense). It is to be observed that in a mixture of timothy grass 94 parts with cocksfoot, elm, fescue, plane, plantain, and rye grass each at 1 ~ the presence of each of the six kinds of contaminating pollen could be detected and the contaminants identified by the use of the appropriate antisera in Ouchterlony gel diffusion plates of suitable design.
Immuno-electrophoresis Preliminary experiments on the possibility of using immuno-electrophoresis in determining the identity and purity of pollen extracts has been undertaken in our laboratories. The precipitation patterns obtained are much more complex than those seen in the corresponding gel-diffusion experiments and much more work is needed to compare the two methods before it can be said that one is better than the
Rev. Palaeobotan.Palynol.,4 (1967) 277-286
281
other. Although the electrophoretic method is more discerning the complexity of the patterns may render the method more difficult to interpret and, therefore, to use as a routine procedure. Skin test reactions as a means o f detecting contamination o f one pollen with another
In the measurement of skin test reactions we have laid down the following procedure: In the case of prick tests the a r m is marked at intervals of not less than 1 inch with the allergens to be used using a ball point instrument. A drop of the solution to be investigated is placed on the skin of the patient (inner aspect of the forearm) gently rubbed using a clean glass rod or the applicator attached to the stopper of the prick test solution bottle. A prick is made through the drop of solution to be tested by applying a needle or other sharp pointed instrument at right angles to the skin and applying pressure until the skin is felt just to have been pierced. After 5 min, the superfluous extract is removed using a fresh piece of cotton wool for each site. Exactly 15 min after making the prick, the raised wheal, where present, is outlined using a ball point instrument. A control is always made using the extracting fluid alone. A piece of cellotape 1 inch (2.5 cm) wide is placed on the arm so as to cover the outlines of the reactions and the identifying marks which were made on the skin. The cellotape is gently rubbed so that it picks up a true impression of all the ball point pen marks on the skin. It is then peeled off and stuck to a piece of graph paper marked in square millimetres. In this way, a permanent record of the sizes of the skin test reactions can be made and the areas-can be measured by counting the number of millimetre squares enclosed in each area.
TABLE II SKIN TESTS ON
48
TIMOTHY POLLEN SENSITIVE PATIENTS
Conttol
Plane pollen Timothy pollen
25,000 25,000 25,000 25,000 25,000 25,000 25,000 25,000 2,500 1,000 500 250 100 50 25 0
Nil
Areas (mm2) Maximum Minimum
97 9
57 5
64 5
64 5
29 2
34 3
30 1
26 0
10 0
Total (mm2)
1,524
1,091
877
924
658
577
530
413
95
282
Rev. Palaeobotan. Palynol., 4 (1967) 277-286
Investigations in our own Allergy Unit have demonstrated that in order to obtain a statistically significant differentiation between two batches of extract of the same allergen it is necessary to use 25 sensitive volunteers (details will follow in a paper to be published elsewhere). However, we endeavour to obtain results on larger numbers of volunteers whenever possible. When these methods were applied to mixtures of timothy grass (Phleum pratense)-pollen extract and plane-tree (Platanus hybrida)-pollen extract the results shown in Table II were obtained. These figures on statistical analysis at a 95 70 confidence level indicate that one part by weight of grass pollen can be detected in 1,000 parts by weight of planetree pollen. When the relative sizes of the pollen grains is taken into account it is demonstrable that 1 grain of timothy pollen in 4,000 grains of plane-tree pollen can be detected by this method. It is quite evident, however, that this method can only be used when patients are available who are sensitive to the contaminating pollen but not to the pollen the purity of which is under investigation. Thus although this is a very accurate method it is, as well as being very tedious, of very limited application.
Infra-red speetography of pollen grains In these experiments actual pollen grains were compressed at 1 70 in a halide disc (KBr) and the infra-red absorption spectrum was recorded. At low resolution using a Perkins-Elmer 137 grating spectrometer the differences between grass pollen, tree pollen and flower pollen was characteristic. However, the differences in spectra between different kinds of grass pollen or different kinds of tree pollen was insufficient to determine the degree of contamination of one kind of grass pollen with another or the degree of contamination of one kind of tree pollen with another. When, however, slow scan high resolution spectra were obtained using the Unicam SP 100 spectrometer a much greater differentiation was possible and this method is being further investigated to determine its accuracy as a method of detecting low contamination of one kind of grass pollen with another or one kind of tree pollen with another.
Ultra-violet absorbtion spectra of pollen extracts The possibility of using the Beckman Model D.B. ultra-violet recording spectrophotometer as a method of identification of pollen extracts and therefore as a method of detecting "other" pollen impurities in pollen extracts, was investigated.
Rev. Palaeobotan.Palynol., 4 (1967) 277-286
283
o7[ 0.6
~3
0..5
5 u 0.4 0 0.3 0.2
/
0.1
/
%0-
~
............
-e...._
peoks in this region
2o
~o
6%
7'0 8'o
40
1oo
Fraction
~io ~2o number
Fig.2. Graph of the optical densities (vertical axis) of 120 consecutive fractions of timothy-grass (Phleum pratense)-pollen extract (horizontal axis) at optical density 280 mtt. A 4 ft. × 6" column of Sephadex G25 was used, the sample size was 30 ml, the fraction size was 23 ml, this was eluted with distilled water.
It was observed that the saline extracts of pollen gave spectra characteristic of protein solutions having maximum absorption in the neighbourhood of 280 m#. The absence of an absorption peak at 260 m/~ was noticeable thus demonstrating the absence of nucleic acids or their degradation products. These results are similar to those of JOHNSON and THORNE (1958). It was decided that the ultra-violet spectra of pollen extracts were so similar as to be of no value in detecting contamination of one kind of pollen with another.
The use of Sephadex G25 When solutions containing molecules of different sizes are submitted to gel filtration using Sephadex gels a resolution can be obtained and the different sizes of molecules can be separated into fractions and examined for optical density. In our experiments a column 4 ft. (122 era) long and 6 inches (15.24 cm) in diameter containing Sephadex G25 was used. 20 ml of an extract prepared from 5 g of pollen and 100 ml of saline extracting fluid was passed through the column and eluted with distilled water. Some 120 fractions each of 10 ml were collected in each case and the optical density at 280 m# of each fraction was measured and plotted on a graph. In the case of grass-pollen extracts the curves were very similar and it was not possible to use the differences between the curves for individual grass-pollen extracts to assess the degree of contamination of one kind of grass pollen with another by treating a mixture of extracts in the Sephadex columns, nor were the differences 284
Rev. Palaeobotan. Palynol., 4 (1967) 277-286
such that the c o n t a m i n a n t could be identified. Tree-pollen extracts gave curves distinguishable from grass-pollen curves b u t n o t sufficiently distinct a m o n g themselves for identification purposes. The same was true of the optical density curves o f other kinds of pollen treated i n this way. This method, therefore, is n o t suitable for detecting c o n t a m i n a t i o n of one pollen with another a n d the results obtained do n o t encourage further investigation of this method. 0.6 0.5
g "o 0.4 -6 u
/L
"~0.3
0.2
No peaks in t~'Fe'g'~o'n". . . . j
0.1
0
40
50
60
70
80
90
i
100 Froction
I
I
110 120 number
Fig.3. Graph of the optical densities (vertical axis) of 120 consecutive fractions of cocksfoot-grass (Dactylisglomerata)-pollen extract (horizontal axis) at optical density 280 m#. A 4 ft. x 6" column of Sephadex G25 was used, the sample size was 30 ml, the fraction size was 23 ml, and for elution distilled water was used. 0.7 O.6 ~
o.5
u 0.4 o 0.3 0.2
0.1 f
0 30
4
5 IO
60,
70,
810
9 I0
, l 1 100 110 120 Froction n u m b e r
Fig.4. Graph of the optical densities (vertical axis) of 120 consecutive fractions of equal parts of the extracts of timothy (Phleum pratense) and cocksfoot (Dactylis glomerata)-pollen extracts (vertical axis) at optical density 280 m/~. A 4 ft. x 6" column of Sephadex G25 was used; the sample size was 30 ml, the fraction size was 23 ml, this was eluted with distilled water.
Rev. Palaeobotan.Palynol., 4 (1967) 277-286
285
CONCLUSIONS Using dry pollen, the particle size differentiation (where it exists) is the most convenient method and is accurate. Pollen contaminants of from 0.1-1.0~o can readily be observed, the higher degree of accuracy being obtained where the great difference in size exists. The morphological method is much more tedious but can be applied where there is a difference in shape but no difference in size of pollen grains. In the case of extracts the gel diffusion and the skin test methods are comparable in detecting 0.2-1.0 ~o of known pollen contaminant. The infra-red and ultra-violet spectrographic methods are of no value and the immuno-electrophoresis and Sephadex column fractionation methods give very complex patterns which need much further investigation before an evaluation of their usefulness or otherwise can be achieved.
ACKNOWLEDGEMENTS We are most grateful to Dr. B. Dybas under whose supervision Miss P. M. Knight and Miss K. V. Mathews carried out the skin tests.
REFERENCES
JOHNSON, P. and "I~ORNE, H. V., 1958. Grass pollen extracts, I. The general properties of aqueous extracts of rye grass pollen. Intern. Arch. Allergy AppL ImmunoL, 13: 257-275. OUCHTERLONY,0., 1949. Antigen-antibody reactions in gels. Arkiv Kemi Mineral. GeoL KgL Svenska Vetenskapsakad., 26B: 1-9.
286
Rev. Palaeobotan. PalynoL, 4 (1967) 277-286
THE DETERMINATION OF THE PURITY OF POLLEN EXTRACTS F. H. M 1 L N E R AND C. A. F R A S E R
Bencard Allergy Unit, Beecham Research Laboratories, Brentford (Great Britain)
(Received October 28, 1966)
SUMMARY
The purity of pollen extracts is dependent fundamentally on the integrity of the pollen from which they are prepared. The most likely contaminants are other kinds of pollen, moulds, dust and plant debris. These contaminants can be avoided by proper methods of collection. The present investigation embraced the microscopical examination of pollen: (a) from the visual and morphological point of view, and (b) from particlesize differences. Examination of extracts containing known proportions of other pollen contaminants embraced gel diffusion methods, immuno-electrophoresis, comparative skin tests, infra-red spectrography, ultra-violet spectrography, and the fractionation using Sephadex columns.
INTRODUCTION
Although the title of this paper suggests analytical methods for the examination of pollen extracts, the factors on which the purity of such extracts depend are of necessity concerned with the integrity of the pollen from which the extracts are prepared. Various factors may contribute to contamination of pollen during collection: (1) pollen of other plants adhering to the specimens before being taken to the pollinarium; (2) cross contamination when more than one kind of pollen is being collected at the same time; (3) dust particles; (4) dried powdered foliage, petals, plant hairs, etc.; and (5) mould mycellium and spores if the pollen has been collected under damp conditons. Contamination from all of these causes can be avoided if proper collecting techniques are employed. It is always wise to check ones own pollen after collection but it is essential to be able to assess the purity of pollen obtained from sources not under ones own control. Rev. Palaeobotan. Palynol., 4 (1967) 277-286
277
In order to determine the purity of the starting materials (dry pollen) several methods have been investigated in our control laboratories. (A) On dry pollen: (1) Microscopic examination: (a) Visual (b) By particle size (B) On extracts: (2) Gel diffusion (3) Immuno-electrophoresis (4) Comparative skin tests (5) Infra-red spectrography (6) Ultra-violet spectrography (7) Sephadex columns
EXAMINATION OF DRY POLLEN
Visual examination using the microscope By this visual method it is possible to detect: (a) the presence of mould spores and mycellium; (b) the presence of plant debris (crushed leaves and petals) and plant hairs; and (c) the presence of morphologically different kinds of pollen.
Measurement of particle size, using the microscope While the visual method can enable many contaminants to be identified it is very tedious in estimating the quantity of the contamination. The particle size method depends on obtaining the mean diameter of the pollen and its contaminant and, using a Shearing eyepiece, to scan fields and, measuring the diameter of all of the particles in each field, to assess the proportion of the contaminant. Obviously, this method cannot be used when there is no significant difference in the mean diameter between the required pollen and that of its contaminant. The nearer the diameter of two pollen grains to each other, the less sensitive is the method and the greater the number of observations required. When, however, there is a large difference the quantitative amount of contamination on a particle basis can readily be determined. The weight for weight basis of the contamination can be calculated from the relative sizes of the particles concerned. The method is very much more delicate when the particle size of the contaminant is less than that of the required pollen. As a model, plane-tree pollen (Platanus hybrida) mixed with various amounts of timothy grass pollen (Phleum pratense) was examined. In the final experimental model 90 ~ of the pollen grains were of timothy 278
Rev. Palaeobotan. Palynol., 4 (1967) 277-286
grass and 10% of plane tree. On a weight for weight basis this corresponds with 2.5 g of plane-tree pollen in 97.5 g of timothy grass pollen. From the measurement of the particle size of 100 grains of pure plane and timothy pollen estimates of the mean particle diameter and particle size distribution for each kind of pollen were obtained. The average diameter of plane-tree pollen grains is 22/z and that of timothy grass pollen 37.5 #. A statistical analysis using these experimentally obtained values showed that the counting of about sixty grains of pollen will be sufficient to reach a decision, at a 95 % confidence level on a 10 % particle/particle contamination of timothy pollen with plane pollen. A mixture of timothy pollen contaminated with plane pollen (10 % particle/ particle) was counted in batches of 10 particles and the cumulative frequency of contaminant particles plotted against the number of batch observations on a chart pre-prepared with the boundary lines as calculated from the theoretical model (i.e., sequential test). It was seen from the chart that the contaminant level of 10 % particle/particle ratio is easily detected, as predicted from the theoretical model (Fig. 1).
>16
Pollen- g r a i n r a t i o 10 plane t r e e 9 0 t i m o t h y grass
o
m 14 -613 o_ 11
Significantly more particles
t_ 9 7
"6 6 Significantly particles
~ 4 z / -e- - - - e . - .
-~---
-e ....
i
i
I
I
I
I
1
2
3
4
5
6
Pure t i m o t h y pollen e.... ~- ---.~
I
I
I
I
7
B
9
10
Number of botches
-4
-5 Fig.1. G r a p h of cumulative frequency of occurrence of small grains (plane-tree pollen) plotted against the n u m b e r of field counts. (N.B, One batch = one field count = 10 pollen grains).
Rev. Palaeobotan. Palynol., 4 (1967) 277-286
279
Pure timothy pollen can be seen to follow the boundary line into the region of “no difference”, whilst the contaminated pollen crosses the boundary line into the region “of difference” from pure timothy pollen.
EXAMINATION
OF POLLEN
EXTRACTS
The examination of dry pollen microscopically, whether for particle size difference or for morphological differences, is very tedious and has obvious limitations. For instance, morphologically different kinds of pollen of the same particle size cannot be differentiated by methods depending on particle size and morphologically similar pollen types of similar particle size cannot be differentiated by either of these methods. Differentiation of such pollen types is often important from the clinical point of view and might also be desirable from the botanical viewpoint. Grass pollen grains, which are of great importance clinically, cannot be differentiated visually or physically. There are several ways in which pollen extracts may be examined to detect contamination of one pollen with another but most of the methods are inadequate to differentiate the closely related proteins which are the main distinguishing factors. This difficulty disappears when immunological reactions are used.
Inmunodif~sicn
in gels
If rabbits are injected with suspensions of whole pollen grains (of known
TABLE
I
SMALLEST
AMOUNT
OF POLLEN
THAT
Test pollen
Lowest level of detection
Cocksfoot grass
100 pg/ml
Elm tree Fescue grass
250 250
Plane tree Plantain
250 dml
Rye grass
250
Timothy grass
250 pgid
280
fig/ml pg/ml
100 pg/ml ,ug/ml
CAN
BE DETECTED
BY THE OUCHTERLONY
Level of detection in other pollen
METHOD
Partial cross reactions with fescue, rye and timothy none cocksfoot, rye and timothy none none cocksfoot, fescue and timothy cocksfoot, fescue and rye
Rev. Palaeobotan. Palynol., 4 (1967) 277-286
purity) in complete Freunds adjuvant, a very good antibody response is obtained and the serum so produced, when mixed wittrthe appropriate pollen extract at the correct concentration, will give an antigen/antibody precipitate. When this reaction is carried out in agar gels, by the method described by OUCHTERLONY (1949), one or more precipitation lines appear in the gel. In closely related kinds of pollen, such as grass pollen, some of the lines are common to all kinds of grass pollen thus indicating some common antigens but each has its own specific lines as well, so that it is possible to detect a contamination of one pollen with another or several others, even though they be closely related, so long as the corresponding specific antisera are available. Table I gives examples of the smallest amount of contamination that can be readily detected by this method. It will be seen that the soluble antigens from 100-250/~g of pollen/ml of extract can be detected by this method and that the lowest contamination of one pollen with another that can be detected with certainty is 1.0~o. At the lowest sensitivity therefore (i.e., 250 /~g/ml) 1 ~o of contamination can be detected if an extract of 2.5 g of the contaminated pollen with 100 ml of buffered saline (pH 7.0) is used. It is also seen that the extractive from 100-250 #g of pollen in 1 ml of buffered saline can be used to detect and identify the pollen by the Ouchterlony method of gel diffusion. Since a maximum of 0.1 ml of such an extract is used in the test it follows that quantities of pollen as small as 10-25/~g can be detected and identified. The pollen grains used to illustrate these findings were those of cocksfoot grass (Dactylis glomerata), elm tree (Ulmus procera), fescue grass (Festuca pratensis), plane tree (Platanus hybrida), plantain (Plantago lanceolata), rye grass (Lolium perenne), and timothy grass (Phleum pratense). It is to be observed that in a mixture of timothy grass 94 parts with cocksfoot, elm, fescue, plane, plantain, and rye grass each at 1 ~ the presence of each of the six kinds of contaminating pollen could be detected and the contaminants identified by the use of the appropriate antisera in Ouchterlony gel diffusion plates of suitable design.
Immuno-electrophoresis Preliminary experiments on the possibility of using immuno-electrophoresis in determining the identity and purity of pollen extracts has been undertaken in our laboratories. The precipitation patterns obtained are much more complex than those seen in the corresponding gel-diffusion experiments and much more work is needed to compare the two methods before it can be said that one is better than the
Rev. Palaeobotan.Palynol.,4 (1967) 277-286
281
other. Although the electrophoretic method is more discerning the complexity of the patterns may render the method more difficult to interpret and, therefore, to use as a routine procedure. Skin test reactions as a means o f detecting contamination o f one pollen with another
In the measurement of skin test reactions we have laid down the following procedure: In the case of prick tests the a r m is marked at intervals of not less than 1 inch with the allergens to be used using a ball point instrument. A drop of the solution to be investigated is placed on the skin of the patient (inner aspect of the forearm) gently rubbed using a clean glass rod or the applicator attached to the stopper of the prick test solution bottle. A prick is made through the drop of solution to be tested by applying a needle or other sharp pointed instrument at right angles to the skin and applying pressure until the skin is felt just to have been pierced. After 5 min, the superfluous extract is removed using a fresh piece of cotton wool for each site. Exactly 15 min after making the prick, the raised wheal, where present, is outlined using a ball point instrument. A control is always made using the extracting fluid alone. A piece of cellotape 1 inch (2.5 cm) wide is placed on the arm so as to cover the outlines of the reactions and the identifying marks which were made on the skin. The cellotape is gently rubbed so that it picks up a true impression of all the ball point pen marks on the skin. It is then peeled off and stuck to a piece of graph paper marked in square millimetres. In this way, a permanent record of the sizes of the skin test reactions can be made and the areas-can be measured by counting the number of millimetre squares enclosed in each area.
TABLE II SKIN TESTS ON
48
TIMOTHY POLLEN SENSITIVE PATIENTS
Conttol
Plane pollen Timothy pollen
25,000 25,000 25,000 25,000 25,000 25,000 25,000 25,000 2,500 1,000 500 250 100 50 25 0
Nil
Areas (mm2) Maximum Minimum
97 9
57 5
64 5
64 5
29 2
34 3
30 1
26 0
10 0
Total (mm2)
1,524
1,091
877
924
658
577
530
413
95
282
Rev. Palaeobotan. Palynol., 4 (1967) 277-286
Investigations in our own Allergy Unit have demonstrated that in order to obtain a statistically significant differentiation between two batches of extract of the same allergen it is necessary to use 25 sensitive volunteers (details will follow in a paper to be published elsewhere). However, we endeavour to obtain results on larger numbers of volunteers whenever possible. When these methods were applied to mixtures of timothy grass (Phleum pratense)-pollen extract and plane-tree (Platanus hybrida)-pollen extract the results shown in Table II were obtained. These figures on statistical analysis at a 95 70 confidence level indicate that one part by weight of grass pollen can be detected in 1,000 parts by weight of planetree pollen. When the relative sizes of the pollen grains is taken into account it is demonstrable that 1 grain of timothy pollen in 4,000 grains of plane-tree pollen can be detected by this method. It is quite evident, however, that this method can only be used when patients are available who are sensitive to the contaminating pollen but not to the pollen the purity of which is under investigation. Thus although this is a very accurate method it is, as well as being very tedious, of very limited application.
Infra-red speetography of pollen grains In these experiments actual pollen grains were compressed at 1 70 in a halide disc (KBr) and the infra-red absorption spectrum was recorded. At low resolution using a Perkins-Elmer 137 grating spectrometer the differences between grass pollen, tree pollen and flower pollen was characteristic. However, the differences in spectra between different kinds of grass pollen or different kinds of tree pollen was insufficient to determine the degree of contamination of one kind of grass pollen with another or the degree of contamination of one kind of tree pollen with another. When, however, slow scan high resolution spectra were obtained using the Unicam SP 100 spectrometer a much greater differentiation was possible and this method is being further investigated to determine its accuracy as a method of detecting low contamination of one kind of grass pollen with another or one kind of tree pollen with another.
Ultra-violet absorbtion spectra of pollen extracts The possibility of using the Beckman Model D.B. ultra-violet recording spectrophotometer as a method of identification of pollen extracts and therefore as a method of detecting "other" pollen impurities in pollen extracts, was investigated.
Rev. Palaeobotan.Palynol., 4 (1967) 277-286
283
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Fig.2. Graph of the optical densities (vertical axis) of 120 consecutive fractions of timothy-grass (Phleum pratense)-pollen extract (horizontal axis) at optical density 280 mtt. A 4 ft. × 6" column of Sephadex G25 was used, the sample size was 30 ml, the fraction size was 23 ml, this was eluted with distilled water.
It was observed that the saline extracts of pollen gave spectra characteristic of protein solutions having maximum absorption in the neighbourhood of 280 m#. The absence of an absorption peak at 260 m/~ was noticeable thus demonstrating the absence of nucleic acids or their degradation products. These results are similar to those of JOHNSON and THORNE (1958). It was decided that the ultra-violet spectra of pollen extracts were so similar as to be of no value in detecting contamination of one kind of pollen with another.
The use of Sephadex G25 When solutions containing molecules of different sizes are submitted to gel filtration using Sephadex gels a resolution can be obtained and the different sizes of molecules can be separated into fractions and examined for optical density. In our experiments a column 4 ft. (122 era) long and 6 inches (15.24 cm) in diameter containing Sephadex G25 was used. 20 ml of an extract prepared from 5 g of pollen and 100 ml of saline extracting fluid was passed through the column and eluted with distilled water. Some 120 fractions each of 10 ml were collected in each case and the optical density at 280 m# of each fraction was measured and plotted on a graph. In the case of grass-pollen extracts the curves were very similar and it was not possible to use the differences between the curves for individual grass-pollen extracts to assess the degree of contamination of one kind of grass pollen with another by treating a mixture of extracts in the Sephadex columns, nor were the differences 284
Rev. Palaeobotan. Palynol., 4 (1967) 277-286
such that the c o n t a m i n a n t could be identified. Tree-pollen extracts gave curves distinguishable from grass-pollen curves b u t n o t sufficiently distinct a m o n g themselves for identification purposes. The same was true of the optical density curves o f other kinds of pollen treated i n this way. This method, therefore, is n o t suitable for detecting c o n t a m i n a t i o n of one pollen with another a n d the results obtained do n o t encourage further investigation of this method. 0.6 0.5
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50
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Fig.3. Graph of the optical densities (vertical axis) of 120 consecutive fractions of cocksfoot-grass (Dactylisglomerata)-pollen extract (horizontal axis) at optical density 280 m#. A 4 ft. x 6" column of Sephadex G25 was used, the sample size was 30 ml, the fraction size was 23 ml, and for elution distilled water was used. 0.7 O.6 ~
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Fig.4. Graph of the optical densities (vertical axis) of 120 consecutive fractions of equal parts of the extracts of timothy (Phleum pratense) and cocksfoot (Dactylis glomerata)-pollen extracts (vertical axis) at optical density 280 m/~. A 4 ft. x 6" column of Sephadex G25 was used; the sample size was 30 ml, the fraction size was 23 ml, this was eluted with distilled water.
Rev. Palaeobotan.Palynol., 4 (1967) 277-286
285
CONCLUSIONS Using dry pollen, the particle size differentiation (where it exists) is the most convenient method and is accurate. Pollen contaminants of from 0.1-1.0~o can readily be observed, the higher degree of accuracy being obtained where the great difference in size exists. The morphological method is much more tedious but can be applied where there is a difference in shape but no difference in size of pollen grains. In the case of extracts the gel diffusion and the skin test methods are comparable in detecting 0.2-1.0 ~o of known pollen contaminant. The infra-red and ultra-violet spectrographic methods are of no value and the immuno-electrophoresis and Sephadex column fractionation methods give very complex patterns which need much further investigation before an evaluation of their usefulness or otherwise can be achieved.
ACKNOWLEDGEMENTS We are most grateful to Dr. B. Dybas under whose supervision Miss P. M. Knight and Miss K. V. Mathews carried out the skin tests.
REFERENCES
JOHNSON, P. and "I~ORNE, H. V., 1958. Grass pollen extracts, I. The general properties of aqueous extracts of rye grass pollen. Intern. Arch. Allergy AppL ImmunoL, 13: 257-275. OUCHTERLONY,0., 1949. Antigen-antibody reactions in gels. Arkiv Kemi Mineral. GeoL KgL Svenska Vetenskapsakad., 26B: 1-9.
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Rev. Palaeobotan. PalynoL, 4 (1967) 277-286