EPA-650/2-74-044
April 1974
Environmental Protection Technology Series
I
55
o
\
ID
O
iji^ijjjliljft^^^^^^^*^!:^^^^
-------
EPA-650/2-74-044
ISOLATION OF HAYFEYER ANTIGENS
FROM SHORT RAGWEED POLLEN
by
Robert M. Flora
VVorthington Biochemical Corporation
Freehold, New Jersey 07728
Contract No. 68-02-0566
Program Element No. 1AA010
EPA Project Officer. Eva Wittgenstein
Chemistry and Physics Laboratory
National Environmental Research Center
Research Triangle Park, North Carolina 27711
Prepared for
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
April 1974
-------
This report has been reviewed by the Environmental Protection Agency
and approved for publication. Approval does not signify that the
contents necessarily reflect the views and policies of the Agency,
nor does mention of trade names or commercial products constitute
endorsement or recommendation for use.
11
-------
-1-
Abstract: Short ragweed pollen has been processed to yield antigens
associated with hayfever allergy. Antigens K and Ra3 have not been
processed to purity but were collected and retained as by-products
during the preparation of antigen E. Antigen E has been examined
by physical and immunological means and found to meet high
standards of purity.
Introduction: A fixed-price contract for $40, 000 was established
for Worthington Biochemical Corporation to use accepted, published
methods for isolating antigens from short ragweed pollen. The
basic process uses extraction, salt precipitation, and column
chromatography. Process steps are monitored by testing samples
with antisera specific for the individual antigens and by polyacrylamide
disc gel electrophoresis. Worthington Biochemical Corporation has
operated this process several times prior to this run for the Environ-
mental Protection Agency, and was fortunate.in encountering no
difficulties this time. The process behaved normally and gave yields
which fell into the anticipated range.
Materials and Methods
Short ragweed pollen, Lot 18-15-72, consisting of 20 x 1 kg bottles
was provided by EPA to the contractor in November of 1972. Informa-
tion about the handling or drying of the pollen or of its crop year was
not provided. The pollen was held in cold storage from the time of
arrival until processed.
All chemicals utilized were of reagent grade and suppliers were
the following; tris (hydroxymethyl) amino methane (THAM), Fisher
Scientific; ethyl ether, sodium chloride, sodium hydroxide and
hydrochloric acid, J. T. Baker; ammonium sulfate, J. T. Baker
or Mallinckrodt. Column chromatographic media were: Sephadex
G-25 and G-100, and DEAE Sephadex A-50, Pharmacia Fine Chemicals;
Celite 545, Johns-Manville.
Antisera were from the following sources; anti-antigen E, lot 92008A,
and anti-whole ragweed, 1022P, N. I. H. Research Reagents distributed
by the National Institute of Allergy and Infectious Diseases; anti-antigen
K, 2KA, Dr. T. P. King; anti-antigen Ra3, 2KA, Dr. L. Goodfriend.
Immunodiffusion plates were from Kallestad Laboratories. Immuno-
electrophoresis was performed in a thin film agarose medium using
films and equipment supplied by Analytical Chemists Incorporated.
Disc electrophoresis in polyacrylamide gel and micro-Kjeldahl analyses
were, conducted in a standard manner by the Worthington Quality Control
Department.
-------
-2-
Isolation Procedures
*
Purification of antigen E was based upon methods published by T. P.
King and co-workers. ^« ^« •*'
The substitution of DEAE Sephadex A- 50 for TEAE cellulose and later
DEAE cellulose was a significant difference between the process
described by King and the procedure followed by Worthington. One
deviation from the technical proposal was required; it was
not feasible to carry out recycling gel filtration over Sephadex G-100
because available optical monitoring units could not handle high
absorbancies encountered at the scale of this work. An additional,
final step not in the original technical proposal was incorporated as
an ammonium sulfate gradient/Celite 545 chromatographic procedure
more recently described by
All chromatographic work was monitored by appropriate immunological
techniques, absorbancy at 280nm, and in latter stages by disc gel
electrophoresis.
1. Defatting of pollen with ethyl ether
The 20 kg of pollen was suspended in 60 liters of ethyl ether,
slurried manually then mechanically over a period of 30 to 60
minutes followed by transfer to filtration apparatus for solvent
removal. The pollen cake was resuspended in 50 liters of
ethyl ether and stirred mechanically for 30 minutes, again
transferred to filter funnels and the cakes washed extensively
with ether until the filtrate was pale yellow. The washed pollen
was spread and air-dried until the odor of ether was no longer
detectable. The defatting operation was conducted at ambient
temperature; all subsequent operations were at 4°C.
2. Extraction of the pollen antigens
The ether extracted dried pollen was suspended in 100 liters of
cold deionized water and stirred mechanically overnight (16-18 hrs).
The aqueous extract was collected by filtration, the pH adjusted to
7. 0 with 1. ON NH40H and brought to cold saturation with ammonium
sulfate, 690g /liter. The pollen cake was resuspended in 80 liters
of cold deionized water, stirred overnight, filtered, and the second
aqueous extract treated as described above. Twice more the pollen
cake was extracted with water, 60 liters each time, the extracts
neutralized and brought to saturation with ammonium sulfate.
-------
-3-
2. x Extraction of the pollen antigens (cont'd. )
The aqueous extractible, ammonium sulfate precipitable material
was collected by centrifugation, dissolved in water, all four
extracts combined, pH adjusted to 7. 0, and the solution clarified.
3. Column I; Sephadex G-25
The 9500 ml solution of crude ragweed antigens was desalted and
freed of considerable pigment by passage over a 40 x 100 cm bed
of Sephadex G-25 equilibrated and eluted with 0. 025M Tris-Cl
buffer, pH 7. 9. All fractions containing appreciable protein and
preceding the elution of the ammonium sulfate were pooled.
4. Columns Ha and lib; DEAE Sephadex A-50
The crude ragweed protein after desalting was applied directly
to two in-parallel columns, 21. 5 x 100 cm. , of DEAE Sephadex
A-50 equilibrated with 0. 025M Tris-Cl buffer, pH 7.9, followed
by washing with the equilibrating buffer until a volume of approx-
imately 60 liters for each column had been collected. Antigens
Ra3, Ra5, and BPAP were eluted with the equilibration buffer
and these pools were precipitated with ammonium sulfate, dissolved,
dialyzed, and lyophilized as the side fractions from columns Ila and
lib. Antigen E and accompanying antigen K were eluted by applica-
tion of a non-linear gradient derived from 24 liters, each vessel, of
equilibration buffer and 0. 05M Tris-Cl, 0.25m NaCl, pH 7. 9.
Fractions containing antigens E and K were combined, precipitated
with ammonium sulfate, collected and redissolved in minimal
volume and dialyzed against column III buffer.
5. Columns Ilia. Illb and IIIc; Sephadex G-100
The antigen E and K solution in 0. 05m Tris-Cl, 0. 4M ammonium
sulfate, pH 7.4, (approximately 1200 ml) was applied to a column
bed, 21. 5 x 100 cm. of Sephadex G-100 packed with the same
buffer. Fractions containing antigen E essentially free of antigen K
were pooled and held. Fractions containing antigen E contaminated
with antigen K were pooled, concentrated, and passed over an
identical column, Illb, of Sephadex G-100. Again antigen E was
pooled and held separately from the antigen E and K containing
fractions. A third pass over G-100, column IIIc, was performed.
Main pools of antigen E from columns Ilia, Illb and IIIc were
-------
-4-
5. x Columns Ilia, Illb and IIIc; Sephadex G-100 (cont'd. )
combined. Leading fractions to the main E pools were combined,
precipitated with ammonium sulfate, redissolved, dialyzed,
clarified and lyophilized as "leading side fractions", columns
Illabc. Trailing side fractions, not subject to repeat passage
over the Sephadex G-100 columns, were treated in a similar
manner and were lyophilized as "trailing side fractions"
columns Illabc and should contain the bulk of antigen K.
6. Column IV; DEAE Sephadex A-50
The combined antigen E pool from the previous step was
dialyzed against equilibration buffer, 0. 025M Tris-Cl, pH 7. 9,
prior to application to a 10 x 100 cm column bed of DEAE
Sephadex A-50. Elution was effected by ^developing a linear
gradient, 10 liters each vessel, of the equilibrating buffer and
0.025M Tris-Cl, 0.4m Nad, pH 7. 9. Fractions containing
the substitative portion of the antigen E were combined. Leading
and trailing side pools were also made; these were dialyzed and
lyophilized as side fractions, column IV.
7. Column V; Cel'le 545/Ammonium Sulfate Gradient
On the finding that antigen E from column IV had not attained the
desired state of purity, an additional chromatographic procedure
was employed. A trial run with 5 per cent of the material
demonstrated that the ammonium sulfate gradient procedure would
further purify antigen E. The bulk of antigen E from column IV
in 0. 05M Tris-Cl, pH 8. 0, was precipitated by 0. 7 saturation of
ammonium sulfate in the presence of Celite 545. The resulting
protein precipitate-Celite slurry was packed into a 5 x 50 cm
column over a small plug of Celite. The column was washed with
0. 7 saturation of ammonium sulfate following by a gradient,
2 liters each vessel of decreasing ammonium sulfate between the
limits of the original conditions down to the simple buffer, 0. 05M
Tris-Cl, pH 8.0.
The pool of fractions containing the antigen E from column V was
extensively dialyzed until free of salt, including the small amount
from the trial run and the entire E pool concentrated approximately
five-fold in an Amicon ultrafilter device with a 10, 000 molecular
weight cut-off membrane to a final antigen E concentration of 12 mg/
ml.
-------
-5-
x Antigen K obtained from column V was precipitated by ammonium
sulfate, redissolved, dialyzed and lyophilized, and provided as
one of the by-product materials.
Antigen E obtained in bulk from the processing described was
subjected to final contractor testing as described below. On
the determination that it met the required specifications of
quality it was prepared for final lyophilization, dispensed
into brown 50cc bottles and lyophilized. Fifty-four bottles
containing 100 mg. of antigen E were delivered and one
bottle, of approximately 50 mg, was retained by the contractor.
Results;
Characterization of Antigen E, Lot 53H382
«
I Immunological (Micro-Ouchterlony) Analysis
A. Employing specific anti-antigen E serum:
Antigen E at concentrations ranging from 1200 to
12 ug/ml vs. anti-antigen E serum (#92008)
exhibits a single continuous precipitin line
establishing the identity of the material as
antigen E. Refer to (Plate I).
B. Employing an anti-whole ragweed serum:
Antigen E at concentrations ranging from 1200 to
12 ug/ml vs. anti-whole ragweed serum (#1022P)
exhibits again a single continuous precipitin line
(Plate II). At the highest level, 1200 ug/ml,
another diffuse line is visible. Based upon the
level of the sample which shows the contaminant
line relative to the concentration of the sample
applied it is estimated that the contaminant does
not exceed a level of 40 ug/ml or 3. 3% of the
antigen E. The indication from immunoelectro-
phoresis is that this contaminant is antigen K.
It should be noted that the specification calls
for the antigen concentration to be over the
range 10 to 200 ug/ml. Where concentrations of
the antigen from 12 to 400 ug/ml are used, a
contaminant line is not observed.
-------
-6-
II Immunoelectrophoresis
Antigen E exhibits a triphasic precipitin line against both
anti-antigen E and anti-whole ragweed sera, correlating
with the three bands visible in disc polyacrylamide gel
electrophoresis. At the level of 1Z mg/ml a distinct arc
is observed against the anti-whole ragweed serum and a
somewhat less distinct line against anti-antigen K serum.
At 4 mg/ml the contaminant line is considerably diminished
against the anti-whole ragweed serum and almost imper-
ceptible vs. the anti-antigen K serum. Negative results
were obtained with anti-antigen Ra3 serum.
Ill Ultraviolet Spectrum
A scan (Fig. 2) of the antigen E over the wavelength range
400 to 2ZO nm indicates the preparation possesses an
adsorptivity which is typical of protein in general, z°"'250 =
2.40, and characteristic of antigen E in particular. Freedom
from significant pigment contamination is indicated by the
adsorbancy, 280'320 = 22.7. Both ratios exceed the contract
specifications, which are, 280/250^- 2.2 and 280/320^ 15.
IV Disc Electrophoresis in Polyacrylamide Gel
Antigen E obtained exhibits three bands on disc gel electro-
phoresis. A densitometric scan (Fig. 3) indicates the bands
to be equi-spaced and in the approximate ratio of 15:60:25.
Since antigen E may contain as many as four electrophoretically
separable components, designated A-D, this finding is compatible
with published reports. Preparations conducted over the past 18
months at Worthington Biochemical Corporation have been
observed to favor the isolation of more of the A-form and lesser
of the D-form of antigen E. Photo 1.
Comparison of lot 53H382 with lot 1KA delivered previously to
the EPA reflects a greater degree of antigen E homogeneity
as evidenced by freedom from the uppermost band (not in the
equi-spaced pattern) and a lesser amount of leading edge
smearing. Photo 2.
Admixture of a standard antigen E preparation, XPTA, and
lot 53H382 can be reconciled with the following. The standard,
primarily forms B and C, with a trace of D band, co-electro-
phoresised with lot 53H382, some A but mainly B, and C bands,
gives the pattern of four visible bands as expected, A, mainly
B, C, and a trace of D. Photo 3.
-------
-7-
IV Disc Electrophoresis in Polyacrylamide Gel (cont'd. )
X
The side fraction (Column V; llOmg) containing antigen K is shown
compared to a standard antigen K, 3BA. Photo 4.
V Nitrogen Content
Micro-Kjeldahl analysis has determined that the nitrogen content
of the antigen E is 15.4%, within the specification range of 15-17%.
Description of Fractions Obtained as By-products, Lot 53H38Z
Antigens K and Ra3 were not obtained in high states of purity and
thus have not been subject to critical analysis. The following
fractions obtained as by-products of the Antigen E preparation
have been delivered as lyophilized bulks of pooled fractions and
are listed with a general description.
1. Side Fractions - Column II; 354 g. Known to contain antigen
Ra3 and suspected to contain in addition antigens Ra5 and BPAR.
Relatively crude.
2. Leading Side Fractions - Columns Ilia, IHb and IIIc;
8.6 g. Contains impure antigen E.
3. Trailing Side Fractions - Columns Ilia, Illb and IIIc;
6.5 g. Contains impure antigen K.
4. Leading Side Fractions - Column IV; 3. 0 g. Contains
some impure antigen E.
5. Trailing Side Fractions - Column IV; 2.4 g. Contains
some antigen E and antigen K.
6. Side Fractions - Column V; 110 mg. Contains relatively
pure antigen K.
References
1. King, T. P., Norman, P. S., and Connell, J. T. Biochem. ^,
458 (1964).
2. King, T. P., Norman, P. S., and Lichtenstein, L. M. Biochem.
6, 1992 (1967).
3. King, T. P. Biochem. U_, 367 (1972).
-------
\,
.":.-)..•, ,•.-.;, ••-' •,.'.--..-. •>"-•., ', ' '.-.- -•- . .• .. •'
PLATE I
Mlcro-Ouchterlony analysis employing anti-
antigen E serum, 92008A, vs. antigen E,
53H382.
Center well: Antt-antlgen E serum
Wells 1-5= 1200, ^00, 120, AO, and 12 pg/ml
antigen E.
PLATE I I
Mlcro-Ouchterlony analysis employing anti-
whole ragweed serum, 1022P, vs. antigen E,
53H382.
Center well: Ant I -whole ragweed serum
Wells 1-5: 1200, 1»00, 120, 1*0, and 12 yg/ml
antigen E
.'••.-.••• •:.!-!!f:;!i!:-i!fi-!-!:;-!-!::;:'.: Hfijigjigp!!*?!.
^^
-------
FIGURE I
THIN FILM AGAROSE IMMUNOELECTROPHORESIS
D
E
2
3
1*
5
6
H
Wells A-D: Antigen E, 53H382, 12 mg/ml ; 2 microliters
In each wel 1 .
Wells E-H: Antigen E, 53H382, k mg/ml; 2 microliters
In each wel 1 .
Trough 1 and 7:
Trough 2 and 6:
Trough 3 and $:
Trough k :
Anti-antigen Ra3 serum, lot 2KA
Anti -antigen K serum, lot 2KA
Anti-whole ragweed serum, #1022P
Anti-antigen E serum, #92008A
(Approximately ^tO microliters serum applied to trough)
-------
FIGURE 2
1.0
0,8 -
0.6 -
CO
I
s
IE
0.4 -
0.2 -
ANTIGEN E, 53x382
GARY KOBEL 15 SCAN
0.80 KG/I! IN 0.01 H TRIS-HCL, PH 7.9
2"0
280
320
WAVELENGTH, NANOMETERS
360
400
-------
FIGURE 3
I
AIITIGEM E, 53H382
DENSIOflETRIC SCAN OF
POLYACRYLAMIDE DISC GEL
ELECTROPKORESIS
FULL SCALE: 2.5 OPTICAL DENSITY
RATE OF SCAN: 2 INCHES EQUALS
1 CM OF DISC GEL TRAVEL
PROTEIN: 40 KICROGRAMS
INCHES
-------
Antigen E, l/>t H382
80 ug 40 ug 24 ug 12 ug
«.
PHOTO 1
Antigen E, 53H382, polyacrylamide disc gel
electrophorests, pH 8.6. Mlcrograms antigen
E applied, left to right, 80, 40, 2^ and 12.
PHOTO 2
Antigen E, lot 1KA and lot 53H382
;/•'; •'::;?"::".''''.' ....,..,
' • ' ' ' ' •
-------
MS 8"^-
Antigen E, left, standard, lot 01A,'right, lot
53H382, and centeV, mixture of 01A and 53H382.
Antigen K, 53H382, a by-product of Column V,
compared with an antigen K standard.
• .': '.r. .1 '^
•
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing}
1. REPORT NO.
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
5. REPORT DATE
Isolation of Hayfever Antigens from Short Ragweed
Pollen
April 26. 1974
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Robert M. Flora
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Worthington Biochemical Corporation
10. PROGRAM ELEMENT NO.
1AA010
11. CONTRACT/GRANT NO.
68-02-0566
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Protection Agency
Chemistry and Physics Laboratory
Research Triangle Park, M. C. 27711
13. TYPE OF REPORT AND PERIOD COVERED
Final. Nov. 72 - Mar. 73
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Contract objectives were to isolate antigens from short ragweed pollen for use as
standard reference materials in the study of aeroallergens and atmospheric proteins.
Antigen E was prepared in pure form while both of the minor antigens, K and Ra3,
were not processed to purity. Twenty kilogram of pollen were defatted and extracted
with water. The antigens were then isolated by salt precipitation and column
chromatography. The process was monitored with electrophoresis and immune precipitin
tests. The final bulk of antigen E obtained was tested for purity and lyophilized
before shipment. Total yield was 5.4g of pure antigen E and approximately 374g of
the side fractions containing K and Ra3. Micro-Ouchterlony tests of antigen E gave
a single precipitin line with anti-antigen E serum. Immunoelectrophoresis resulted
in a triphasic precipitin band corresponding to the three bands obtained in poly-
acrylamide gel electrophoresis. UV absorption scans of the antigen E showed a
typical protein peak at 230 nm wavelength, and micro-Kjeldahl analysts gave a
nitrogen content of 15.4%. Antigens K and Ra3 were not critically tested. All test
results were compatible with published reports and indicated that the antigen E
prepared meets a high degree of purity.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Hayfever
Ragweed Pollen
Aeroallergens
Column Chromatography
Isolation of Antigen E
18. DISTRIBUTION STATEMENT
•
Release Unlimited
19. SECURITY CLASS (This Report)
Unclassified
21. NO. OF PAGES
20. SECURITY CLASS (This page I
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
------- |