Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
-- 1 --
RAN 4093/59
Acid phosphatases represent a heterogenous group of
enzymes contalning many isoenzymes, each specific for one
type of tissue. As a result of this tissue specificity,
determination of a tissue specific isoenzyme in serum is a
valuable aid in identifying abnormalities in that tissue.
10 It has been demonstrated in a number of scientific commu-
nicatlons that serum prostatic acid phosphates (PAP~ iso-
enzyme levels increase in association with the presence of
prostatic carcinoma [R.M. Townsend, Ann. Clin. and l.ab.
Sci. Vol., 7, No. 3, pp 254-261, May-June 1977].
The measurement or determination of PAP isoenzyme
levels in serum is complicated, however, by the presences
of other aci~ phospha^tase isoenzymes from tissue of non-
prostatic origin. In Eact significant levels of non-prosta~
20 tic acid phosphatase isoenzymes are normally present in ^the
serum [Li, C. et al., J. I,ab. Clin. Med. 82, 446-460 (1973)].
As a resul^t of a variety of sources for producing elevated
levels of acid phosphatase isoenzymes, there is the need
for an assay for PAP isoenzyrne, which is specific for -that
25 iSoenzyme-
Prior art approaches to determining the amount o:E PAPin serum has been directed toward substrate specificity
for and chemical inhibition of PAP. These approaches have
30 not been very satisfactory because even the most specific
substrate or inhibitor reacts with isoenzymes other than
PAP. As a r,esult these prior art approaches produce false
positive or nega-tive test results with the accompanying
high possibility of improper -therapy.
Klt/ 29.4.82
-- 2 --
Several immunochemical assay methods for PAP have been
reported. Choe, B.l~., et al in Proc. Soc. Exper. Biol. and
Med. 162, 396-400 (1979) has reported an assay system for
the measurement of enzyme activity as well as radioactivity
of I-labeled PAP in ammonium sulfate precipitated
antigen-antibody complexes. By this me-thod PAP is deter-
mined by measurement of the ammonium sulfate precipitated
antigen-antibody complexes. In this method, however, there
is a high background from acid phosphatases due to the
10 nonspecific nature of amrnonium sulfate precipitation.
In an attempt to resolve the problem of interference
from other isoenzymes in assays for individual organ spe-
cific isoenzymes of human alkaline phosphate, a double-
1~ antibody immunoenzyme assay procedure was reported inSussman et al., J. Biol. Chem. 243, 160 (l968) and in Choe
et al., Clin. Chem. 26, 1854-1859 (1980). As reported in
both of these references the specific antibody to the de-
sired isoenzyme was reacted with the test sample and then
20 in a subsequent step the antibody-isoenzyme complex was
reacted with a second soluble antibody ~an-ti-~-globulin~.
After centrifugation the precipitate was tested for residual
isoenzyme activity.
In order for the Sussman and Choe procedures to be
accurate, however, it is necessary for -the second soluble
antibody to reac-t with the antibody-isoenzyme complex
in such a manner as to precipitate completely the iso-
enzyme to be determined. This reaction is dependent on such
30 factors as ternperature, pH, ionic strength, time, protein
concentration and the like. These factors are difficult
to control in an assay procedure, and thus the Sussman and
Choe procedures have limited usefulness in accurately de-
termining the specific amount of isoenzyme.
The present invention relates to an immunochemical
process for determining the amount of PAP isoenzyme in a
biological fluid sample. Elevated levels of PAP isoenzyme
., .
in serum indicate prostatic carcinoma.
The immunochemical process of this invention employs
a double-antibody immunoenzyme assay wherein one of the
antibodies is an insolublized antibody constituted as a
reaction product o-f antibody and amylose. The process
comprises incubating a test sample with a first antibody
which selectively binds immunologically the isoenzyme of the
sample and an insolubilized second antibody which selecti-
lO vely binds immunologically with the first antibody. The~esulting immunochemical reactivities provide insoluble
immunocomplexes which are easily isolated. After isolation
the immunocomplexes are measured for the isoenzyme activi-
ty contained therein.
It was discovered that proteins such as immunoglobu-
lin (IgG), enzymes and the like can be coupled to amylose,
a solid support, to pr~ovide an insolubilized protein such
as IyG-amylose for use as immunoadsorbants. It was disco-
20 vered that a protein such as for example IgG insolubilizedby a reaction with amylose provides advantages over protein
insolubilized by other solid supports. In particularly the
advantages are that the resulting IgG-amylose can be pro-
cluced by a simple reaction. There is a large surface area
26 provided by amylose for binding the IgG; and the resulting
reaction product of IgG-a~ylose can readily be centrifu-
ged at low speeds but yet remain in suspension long enough
for the reaction in an immunological assay to be comple-
ted.
The process of the present invention has substantial
advantages over procedures used in the prior art in that
said process is very specific, rapid, highly accurate and
reproducible, providing a speciflc isola-tion of PAP iso-
35 enzyme from sera containing non-prostatic isoenzymes and
then speci~ic measurement of the amount of PAP isoenzyme
isolated.
' ;
a,
In particularly, the process of the present invention
is advantageous in that the resulting immunocomplexes of -the
reaction mixture are quickly precipitated and made availa-
ble for measurement of the isoenzyme activity. Further-
rnore the reaction mixture producing the immunocomplexes isindependent of such factors as temperature, pH, ionic
strength, time, protein concentration and the like. Since
these factors are absent, the process of the present in-
vention eliminates these factors as possible sources of
10 error and provides, -thereby, greater accuracy in deter-
mining specific amount of isoenzyme.
For the invention processes it is possible to use
as a test sample a sample of any biological fluid, e.g.
15 whole blood, plasma, sera, lymph bile, urine, spinal fluid,
sputum, sweat and the like as well as stool excretions of
hurnans or other animals. It is possible also to use fluid
preparations of human or other animal tlssue such as
skeletal muscle, heart, ]cidney, lungs, brain, bone marrow,
20 skin, and the like. Biological fluids are preferredO The
preferred biological fluid for a test sample in this inven-
-tion processes is human serum.
Particularly the present invention relates to a pro-
~5 cess for determining the amount of PAP isoenzyme in abiological fluld sample which may contain other non-pros-
tatic isoenzymes or other protenacious substances. rrhe
process of the present invention is achieved by isolating
the specific PAP isoenzyme of interest and then measuring
30 the enzymatic activity of the isolated PAP isoenzyme.
The isolation of the PAP isoenzyme is accomplished
by employing two different antibodies, a first antibody
ancl a second an-tibody, which are reacted in a reaction
35 mix-ture with the biological fluid sample, followed by
separation of immunochemical complexes containin~ the PAP
isoenzyme from the reaction mixture.
~ 5 --
In accordance wi-th this invention the first antibody
can be any antibody which is scluble ln the reaction mix-
ture and which selectively binds the PAP isoenzyme wi-thout
significantly inhibiting the enzymatic activi-ty of the PAP
isoenzyme. The first antibody can be prepared in -the conven-
tional manner by injection of a purified PAP isoenzyme
as antigen into an animal and bleeding the animal to obtain
the sera containing the antibody. Any of the conventional
means which are known in the art can be used for obtaining
10 and purifying PAP isoenzyme and for injecting the puri-
fied PAP isoenzyme as antigen into anirnals to obtain antl-
PAP serum containing the first antibody. The anti-PAP serum
need not be purified and may be used directly in the pro-
cess of this invention. The anti-PAP serum ob-tained by use
15 f purified PAP isoenzyme does not react with acid phos-
phatase from any tissue other than prostatic tissue and
does not significantly inhibi-t the enzymatic activity oE
the YAP isoenzyme to which it immunologically binds. The
result is that the first antibody contained in the anti-PAP
20 serum will specifically bind immunologically only PAP iso-
enzyme of a test sample, while at the same time not affec-
ting the enzymatic activity of the PAP isoenzyme.
PAP isoenzyme employed as antigen to raise the first
25 antibody, can be obtained from any suitable biological fluid
such as prostatic flu:id, klood serum or any suitable bio-
logically fresh prostatic tissue. The preferred source of
this isoenzyme is animal prostatic fluid or tissue, espe-
cially preferred is human prostatic fluid or tissue.
Purification of PAP isoenzyme to a state of high
puri-ty before using them for raising the firs-t antibody
is most advisable in order to diminish the presence of
nonspecific antibodies. The PAP isoenzyrne may be purified
35 by any conventional purification procedure recogni~ed in
-the art for such purposes. The preferred purification pro-
cedure encompasses conventional art recognized procedures
such as affinity chromatography or electrophoresis.
-- 6 --
The degree of high purlty of the PAP isoenzyme used
to raise the first antibody as well as the specificity
of the resulting antibody can be determined by currently
acceptable practices in the art such as by immunodiffusion
or electrophoretic techniques. The preferred method for
purity of the PAP isoenzyme is acrylamide gel electropho-
resis.
The first antibody may be produced in any animal species
lO recognized in -the art. The animal species include espe-
cially vertebrates, e.q. pig, cattle, dog, donkey, horse,
goat, rabbit, rat and the like. Among these animals,
mammals such as donkey, sheep and goat are preferred. The
most preferred are goats.
In one preferred aspect of the invention, the first
antibody is produced by immunizing goats, a first animal
species, by conven-tional immunological techniques with
purified PAP isoenzyme obtained from human prostatic tissue
20 and obtaining goat anti-PAP, the first antibody.
The amount of first antibody used in the -test sample
i5 an amount sufficient to bind the PAP isoenzyme of the
sample. For best results the Eirst antibody is added to -the
25 sample in an amount in excess of that amount normally
re~uired to bind the PAP isoenzyme. The determination of
the amount of the first antibody needed for each sample
can be determined by well recognized procedures in this
art.
The test sample is treated with an insolubilized second
an-tibody, after adding the firs-t antibody. The time of
adding the insolubilized second antibody after adding the
~irst antibody is not critical, however it is preferred
35 to add the insolubilized second antibody at least 15 minu-
-tes after adding -the first antibody.
5~
-- 7
In accordance wlth this invention there is provi-
ded an insolubillzed second antibody by coupling -the se-
cond antibody to amylose by an amine linkdge~ The second
antibody of the insolubilized second antibody can be any
antibody which will selectively bind the first antibody
without inhibiting the enzymatic activity of the isoen~yme
bound by -the first antibody. The second antibody of -the
insolubilized second antibody rnay be produced by any method
recoynized in the art in any animal species, o-ther than the
10 animal species used to produce the first antibody. That is
the second antibody is prepared by immunization of an animal
different than the one in which the specific firs-t an-ti-
body is prepared with a gamma globulin from the blood of
the host species used for the first antibody preparation.
15 Thus the second antibody will be lmmunoreactive for the
first antibody and will complex with it~
The amylose utilized to form the insolubilized second
antibody can be a homopolymer of D-glucose. The D-gluco-
20 pyranose units in amylose are substantially in (l-~) a-
glucosidic linkages. The molecular weight of amylose varies
de~ending on the source of the amylose, extending from an
ave~age molecular weight of about lO0,000 to about
1, 1()0, 000 .
In accordance with the process of the present invention
amylose from any source or of any known molecular weight
may be employed to provide the reaction produc-t oE anti-
body and a~ylose. If desired the amylose may be purified
30 by any conventional method before prividing the reaction pro-
duct of antibody and amylose. The degree of purity however
is not critical but it is preferred that the amylose have
a purity of 50% to 100%.
The method of attachment of the second antibody or
other protein via an amine linkage to amylose -to produce
the insolubilized second antibody may be by any conven-
tional method of coupling polysaccharides -to proteins.
55~
-- 8 --
I-t is a preferred embodiment of the present invention that
the second antibody or other protein be coupled covalen~ly
to activated amylose through the use of a conventional
Schif~'s base reaction [Guthrie, R.D. Adv. Carbohydrate
Chem. 16:101, 1961].
In accordance with the preferred procedure, amylose
containing a plurality of D-glucopyranose units is activa-
ted by an oxidizing agent such as sodium periodate (NaI04)
10 which oxidizes the alpha-1,4-linked glucose unit to pro-
vide amylose-dialdehyde. The resulting amylose-dialdehyde
then reacts with the primary amino group of the added
second antibody or protein to form a Schiff base or aldi-
mine. The Schiff base is reacted with a reducing agent to
15 provide a primary amino linkage between the protein and
amylose. Among the conventional reducing agen-ts for use in
this reaction it is generally preferred to u-tilize a redu-
ciny agent such as sodium borohydride (NaBH4). The pro-
cedure to prepare a second antibody-amylose product is
20 illustrated in the following reaction scheme. In the reac-
tion scheme n represen-ts a plurality of D-glucopyranose
l~nits.
g
Re~ction Scheme for
Amylose/Protein Coupling
f~ CH20H
Asnylose ~ ~
-J\~O)t ~/~--
. ~
n .
NalO4
.
~It
Amylos Jldehyde ~
-J\I ~-
~ O
. , ~ N~t2
~, protein
CH20H CH20H
(,41nylG5 ~ro~cin) ~ 2
--o~ /1~
. . HOf\ /~OH 0~ ~f
I'' ' I
I protcin protein
S~hi~f base
. ~aldimine~
. . NaBH,~,
. .' ~, ' . .
~ , C~t20H
Amyl05 protein ~/~ \~
1, . ` .'~0~ ~/~0--
Ho \ jP
. I
pro~cin
-- 10 --
In forming the insolubilized antibody, the antibody
is reacted with amylose in -the aforesaid manner -to couple
the antibody to the amylose via an amine linkage. In pre-
paring the insolubilized antibody the amounts of amylose
and an-tibody are not critical. However, from about l to
about 200 parts by weight of amylose per part by weight
of antibody is generally used. It is preferred that about
8 to about 20 parts by weight of amylose per par-t by weight
oE an-tibody be used.
In accordance wi-th this invention, once the immuno-
complexes are formed after adding the second an-tibody, the
immunocomplexes may be separated from the sample solution
or mixture by any conventional means known in -the art and
15 the resulting precipitate may be measured for PAP enzyma-
tic activity. In accordance with this invention it is the
precipitate which is measured for enzymatic activity.
Arnong the conventional means available for separating
20 the precipitate from the sarnple solu-tion are conventional
fi1tration and chromatographic methods. The most preferred
methvd for separating the precipitate from the sample solu-
tion is centrifugation followed by decanting the super-
natant and measurement of the precipitate, which permits
25 determination of the amount of PAP in the sample.
The measuremen-t or determination of the activity of
PAP isoenzyme isola-ted from the test sample may be
accomplished by any conventional methods recognized in
30 the art. Among these methods are included colorimetric
methods, as described, for example in "Methoden der enzyma-
tischen Analyse", edited by H.U. Bergmeyer, 3rd edition
(1974), Vo]. l, page 145 et seq, as well as fluorometric
methods.
The invention also relates to a diagnostic test kit
system for ~uantitatively determining the presence of PAP
; isoenzyme in a biolo~ical fluid. The constituents of the test
s~s
kit system are: (a) a first container contalning an antibody
capable of selectively binding immunologically with PAP
isoenzyme, (b) a second container containing a second anti-
body insolubilized by being coupled to amylose by an amine
linkage and capable of selectively reacting immunologically
with the antibody contained in the first container, and (c)
a substrate reagent capable of determining the enzymatic
activity of PAP isoenzyme.
When all the components of the diagnostic test kit
system are used in accordance with the process of the inven-
tion as provided herein, one skilled in the art would be
able to determine the enzymatic activity of PAP isoenzymes
in a biological fluid sample containing non-prostatic iso-
1~ enzymes. The test kit would be suitable for clinics, hos-
pitals, laboratories, and individual physicians having
a need to determine the amount of PAP isoenzymes in biolo-
~ical fluid samples.
The following examples further illustrate the inven-
tion but are not intended to restrict the invention in
scope or spirit.
~5
- 12 -
Example 1
_urification of PAP:
Part A
100 grams of frozen human prostate tissue is placed
lnto an homogenizing solution containing 0.05 M ethylene-
diam,ine tetracetic acid (E~TA) and 0.01% by weigh-t Tween
~0 at fiC. The frozen -tissue .is allowed to thaw in the
10 homogenizing solution -for approximately 1/2 hour. After
thawing, the tissue is homogenized in a blender until no
lurnps are apparent in the resulting mixture, the homoge-
nation being performed with the mixture in an ice bath and
for no longer than 20 seconds. Thereafter the resulting
15 homogenized material is stirred overnight at 4C. The
material is then centrifuged for 1 hour at 4~C at 16,300 Xg.
The resulting supernatant approximately 240 ml is dialyzed
aya:i.nst 3 changes (4.0~1it.ers per change) of 0.005 M Sodium~
Phosphate buffer pH 6.3 containing 0.9% NaCl. Dialysis in-
20 creased the original volume from about 240 ml to about260 ml.
'I.'he resultlng dialyzed material is then placed into a
1000 ml lyophilizing flask, prepared for lyophilization by
25 fas~ freezing over dry ice and acetone, and -then lyophi-
lized overnic,~ht. The resulting lyophilized material is then
reconstituted with 20.0 ml of 0.005 ~ Sodium Phosphate buf-
fer pH 6.3. containing 0.9% NaCl to provide the reconsti-
tuted material for Part B below.
Part B
An LKB column was previously packed with 100 ml of
Con-A Sepharose 4B gel preswollen in the aforementioned
buffer, and the gel is allowed to settle in the column
35 without a flow rate. The resulting packed column was then
washed with about ~00 ml of the aforementioned buffer.
The reconstituted material from Part A above is put
. .
- 13 -
on the LKB column as prepared above. Once the material is
on the column, the material is allowed to sit on the column
for about 3 hours. 800 ml of the aforementioned buffer is
passed through the LKB column at a flow rate of 40.0 ml
per hour. Thereafter 500 ml of a 0.0 to 0.3 M Mannose
gradient, prepared separately in 0.005 M sodium phosphate
pH 6.3 with 0.9% NaCl buffer and then filtered through
charcoal, was passed through the LKB column, with 10.0
ml fractions collected at a flow rate of 40.0 ml per hour
10 (a -total of 50 fractions collected). Thereafter 500 ml of
1.0 M Mannose was passed through the LKB column and a to-
tal of 50 fractions collec-ted in the same manner. All the
resulting fractions were tested for protein and PAP acti-
vity. Fractions containing PAP acticity with specific acti-
vity ~ 2000 Sigma units/mg protein were pooled, dialyzed
against 0.005 M sodiumphosphate buffer pH 6.3 with 0.9/O
NaCl, and concentrated using an Amicon filter system, to
provide a 4.0 ml concentrated dialyzate for Part C below.
Part C
7.5% acrylamide gel is prepared using a Buchler prepara-
tive system. The following buffers were also prepared for
~uchler system: 0.035 M ~3-alanine in 0.014 M acetic acid
or the upper buffer of the system and 0.14 M ~-alanine
25 in 0.056 M acetic acid for the lower and elution buffer of
the system.
1.0 ml of 40% sucrose and 3 drops of methyl green
dye are added to the 4.0 ml concentrated dialysate from
30 Part B to provide a mixture. The mixture is then placed
onto the 7.5% acrylamide gel of the Buchler system and
electrophoresis is started (1000 volts, 200 milliamps for
approximately 16 hours). The mixture is eluted using the
lower/elution buffer, with 10.0 ml fractions being coliec-
35 ted. The resulting fractions are tested for PAP and proteinactivity. Fractions containing PAP activity are pooled and
dialyzed against 0.01 M citrate buffer pH 6.0 overnight
with two changes to provide the highly purified PAP for use
:::
: ~ .
5~
- 14 -
in -the lmmun.ization procedure of Example 2. The highly
purified PAP may be stored a-t -20~c.
Example 2
Immunization of ~oats with purified PAP to produce first
.. . .
antibody:
-
In orcler to prepare the immunogen for immunizing
10 goats, the highly purified PAP in 0.01 M citrate bufferpH 6.0 obtained from the procedure of Example 1 is mixed
with an equal volume of complete Freund's adjuvant (a
mineral oil suspension contai.ning killed M-tu~erculosis
bacilli) to provide 0.5 mg PAP/ml of resulting mixture.
15 The resulting mixture is homogenized to produce an aqueous/
oil emulsion which constitutes the immunogen. Goats were
immunized for three consecutive weeks wlth an injection
of 2 ml of immunogen subcutaneously into the auxillary
regions at two sites, each site receiving 1 ml of the immu-
20 nogen. Two secondary injections with incomplete Freund'sadjuvant were given at two week intervals after the primary
injectlons, followed by additional secondary injections with
:incomplete Freund's adjuvant at monthly intervals for se-
veral months. The goats were bled every one to two wee}cs
following immunization to provide antiserum.
Example_3
_eparation of Glubulin Fraction of Donkey_Anti Goa
30 I~G Serum
The globulin fraction of donkey anti goat IgG serum
for use in coupling to amylose is prepared using the follo-
wing ingredients and procedure:
- 15 -
Item Inqredient Quantity
_
a) Donkey anti Goat IgG serum
obtained commercially 500 ml
5 b) Satura-ted ammonium sulfate
solution 550 ml
prepared by adding 500 g of
solid ammonium sulfate (granular)
to 500 ml of deionized water and
stirring vigorously at room
temperature for one hour. Store
at 4C and allow crystals to
settle out.
c) 0.01 M Sodium Phosphate buffer
pH 7.4 approx. 14 liters
d) 0.01 M sodium carbonate-
hicarbonate 5 liters
buffer,~pH 9.5, prepared
hy dissolving 1.1 g of
sodium carbonate and 2.5 g sodium
bicarbonate in approximately 3.5
liters of deionized water, stirring
for 5-10 minutes and bringing
volume to 4 liters with deionized
26 water.
PROCED~RE:
1. 500 ml of donkey anti goat IgG serum is placed in a
suitable vessel at 4C on a magnetic stir plate. There is
30 then added to the vessel slowly with moderate stirring
300 ml of saturated ammonium sulfate (i-tem b) to bring the
contents of the vessel to 37.5% saturation. The vessel is
allowed to stir for one hour at 4C, and thereaf-ter -the
precipitated antibody is collected as a pellet by centri-
35 fugation at 1200 x g for 30 minutes at 4C. The pellet isdissolved in approximately 350 ml of phosphate buffer (item
c) and the volurne is brought to original 500 ml wi-th the
same buffer. Thereaf-ter 250 ml of satura-ted ammonium sulfa-te
-
~ 16 -
(item b) is added slowly with moderate stirring at 4C
to bring the resulting mixture to 33% saturation. The
mixture is allowed to stir for one hour at 4C. The pre-
cipitated antibody from the mixture is obtained as a pellet
after centrifugation at 1200 xg for 30 minutes at 4C. The
pellet is dissolved in approximately 200 ml of the phos-
phate buffer, transfered to a dialysis bag and dialyzed
against the phosphate buffer at 4C. The buffer is changed
three times (4 liters per change) after a minimum of four
10 hours dialysis each time. Then the resulting dialysate
is dialyzed against one 4 liter change of carbonate-bi-
carbonate buffer (item d). The protein concentration of
this final dialysate is determined by the ~owry method and
diluted to 12 mg/rnl in a final volume of 800 ml to provide
15 the globulin fraction of donkey anti goat IgG serum ready
for coupling to the activated amylose obtained in Example
4 below.
Example 4
Activation of Amylose:
~n vrder to couple the globulin fraction of Donkey
25 anti (.oat IgG serum prepared by procedure of Example 3 to
amylose, the amylose is first activated by the following
steps 1-16^
1. Slowly add 20 g of amylose into each of four 1 liter
centrifuge bottles containing 500 ml deionized water.
Stir during and after addition with a magnetic stirrer.
Bring volume to full capacity of bottles with deioni-
-
zed water.
2. Allow amylose to swell in each bottle, with stirring,
at room temperature for one hour.
3. Centrifuge the swollen amylose in each bottle at
2500 xg for 5 minutes.
4. Decant the supernatant from each bottle and save the
- 17 -
pelle-t in each bo-ttle for step 5.
5. Resuspend the pellet in each bottle in 1 liter deioni-
zed water.
6. Repeat steps 3, 4 and 5 twice more for a total of 4
washes.
7. Resuspend pe]le-t in water and leave the pelle-t in the
water overnight at room temperature and the next
day centrifuge the bo-ttles and decant retaining the
final pellets for step 9.
10 ~. Dissolve 25.68 g sodiurn metaperiodate in 700 ml deioni-
~ed water and bring volume to 800 ml.
9. Resuspend the final pellets from step 7 in each of
four bottles with 200 ml of -the 0.15 M sodium meta
periodate produced according to step 8.
15 10. Stir the resuspended pelle-ts of step 9 gently for 2
hours at room temperature.
11. Mix 49.66 g ethylene glycol (liquid) with 300 ml
deionized water. ~ring volume to 400 ml with deionized
water; no centrifugation before addition of ethylene
glycol.
12. At the end of the 2 hours stirring of step 10, add
100 ml of the 2 ~ ethylene glycol prepared in step 11
to each oE the 4 bottles and stir the conten-ts of the
bottles gently for 3b minutes at room temperature.
26 13. Centrifuge the bottles of step 12 at 2500 xg for 5
minu-tes. Decan-t each ~ot-tle and retain the pellets
separately.
14. Resuspend the pellets from step 13 separately in up
to 1 liter (bottle capacity) of carbonate-bicarbonate
buffer (item d of Example 3) and stir 15-30 minutes.
15. Repeat steps 13 and 14 twice more.
16. Decant the supernatants to provide pellets which con-
tain activated amylose ready for coupling to the glo-
bulin fraction of donkey anti goat IgG serum prepa-
red by process of Example 3.
5~
- 18 -
_xample 5
Cou~ling of Globulin Fraction of Donkey Anti Goat I~G
Serurn to Activated Amylose:
The coupling of -the globulin fraction of donkey anti
goat IgG serum (prepared by the process of Example 3) to
activated Amylose (prepared by the process of Example 4)
is accomplished using the following ingredients and pro-
l0 cedure:
Item Ingredient ~uant_ty
1 Activated amylose
(Exarnple 4) 400 ml swelled gel
2 Donkey Anti-goat IgG
(Example 3) 800 ml
3 Phosphate Buffered Saline
(PBS), pH 7.4 approx. 27 liters
20 ~ Sodium borohydride 0.8 g
Bovine Serum Albumin ~.8 g
6 Sodium azide 1.6 g
PROCEDt]RE:
25 1. To each of the four bot-tles containing each a pellet
of activated amylose (20 g dry weight, swelled to
100 ml as prepared in Example 4) is added 200 ml o:E the
donkey anti-goat IgG (as prepared in Example 3) at
12 mg/ml for coupling o:E the amylose to the IgG.
30 2. The coupling is carried out at room temperature, with
stirring for 36-48 hours.
3v The bottles are then centrifuged at 2500 xg for fi.ve
minutes .
~. The protein of the supernatants from each bottle is
checked for O.D. at 280.
5. To 800 ml of Phosphate buffered saline is added 0.8 g
Sodium borohydride.
6. To each o:E -the four centrifuged bottles of step 3 con-
-- 19 --
taining amylose--pro-tein is added 200 ml o~ the sodium
borohydride solution. The resulting suspensions are
gently stirred for one hour at room tempera-ture.
7. After stirring the suspensions of step 6, -the resulting
reduced amylose-protein gel of each bot~le is centri-
fuged as in step 3 and the supernatant discarded.
. Each bottle of gel is brought to one liter capaci-ty
with phosphate buffered saline and the resuspended
gel is brought to one liter capacity wi-th phosphate
huffered saline and -the resuspended gel is stirred
for 10-20 minutes at room temperature.
9. After stirring, the gel is centrifuge~ as in step 3.
The supernatant is discarded and steps 3 and 9 are
repeated twice.
15 10. Each of the resulting four amylose-prote~n pellets in
each hottle is now resuspended in 200 ml PBS and
transferred with washing to a 2 liter graduate to pro-
vide an amylose-protein suspension.
11. 4.~ g of Bovine Serum albumin and 1.6 y of sodium
azide are added to 100 ml of PBS with s-tirring. This
solution is added to the amylose-protein suspension.
12. The amylose-protein suspension is then brough-t to
5% aqueous suspension by adding PBS to a final volume
of 1600 r~
~5
Example 6
A determination of PAP activity in a biological fluid
sarnple is accomplished by the following procedure:
Part A
200 ~l of -test serum sample or control is pipeted into a
test tube; while for a water blank 200 ~l of distilled water
is added to another test -tube. 20 ~l of goat an-ti-PAP
3S serum obtained by procedure of Example 2 is added to each
tube. The resulting mixtures are mixed and incubated at
room tempera-ture for 15 minutes after addition to the las-t
tube being assayed. Thereafter 200 ~l of a well s-tirred
- 20 -
amylose-protein suspension produced by the procedure of
Example 5 is added to each tube. The tubes are mixed and
allowed to incubate for 15 minutes at room temperature after
addition to the last tube. Thereafter 3.0 ml of 0.9% saline
(9.0 g sodium chloride per 1000 ml of distilled water)
is added to each tube and the tubes are mixed. Each tube
is then centrifuged for 10 minutes at lS00 xy and the su-
pernatant removed from each -tube by decanting, providing
tubes containing a solid phase pellet for use in Part B
10 below.
Part B
The PAP colorime-tric substrate solution is prepa-
red by reconstituting one vial of Worthington sodium thymol-
15 phthalein monophosphate with 41.0 ml of distilled water. Theflask is swirled gently to completely dissolve the sub-
strate. This substrate solution may be stored up -to 60
days at 2-8C or 2 days a-t 1~-26C. This substrate solution
is then equilibrated to room temperature. The -tubes contai-
20 ning the solid phase pellet from Part A are placed into a37C water bath and allowed to equillbrate for 5 minutes.
Thereafter 0.5 ml of the PAP colorimetric substrate solution
is added to each tube containing the solid phase pellet.
Each tube is then mixed and incubated for 30 minutes at
25 37C a~ter addition of the substrate to the first tube
(test tube). At the end ot the 30 minute period, 1.5 ml of
alkali Worthington solution is added to each tube and
each tube is mixed and then centrifu~ed at 1500 xg for 10
minutes. The absorbance of the resulting supernatant in
30 each tube is read at 590 nm in a spectrometer.
Part C
The PAP colorimetric substrate solution as used in
Part B was prepared as follows:
To obtain the concentration of PAP in the samples
or con-trols a standard curve using conventional T~1Orthington
thymolphthalein dilutions are prepared. After substracting
~5~
- 21 -
the absorbance of the water blank from the ahsorbance of the
serum samples and controls, the corrected absorbances are
used to read the concentration of PAP from the standard
curve.
