Note: Descriptions are shown in the official language in which they were submitted.
~LZ1~3~29
PARTICULATE LIGAND ASSA~ - METHODS AND PRODUCTS
This invention relates to methods and products for
analyzing materials and detecting specific compounds in
mixtures of what may be ehemically similar compounds.
More particularly, this invention relates to methods and
products for analysis of specific ligands or other com-
pounds by a ligand assay which is much simpler, l~ss
expensive, as specific and often more accurate than the
cumbersome radioimmunoassay, enzyme-immunoassay or other
assay techniques previously known.
Progress in the study of bioehemistry has been advanced
considerably by the diseovery of radioim~lunoassay teehni-
ques, which permit analysis for specific proteins and
other ligands in a melange of biologic materials, and
determination of the concentration of that substance with
very high aceuracy. See Thorell et al,- Radioimmunoassay
and Related Techniques (C.V. Mosby, 1978~.
The typical radioimmunoassay (RIA) techniques depend on
competitive binding of the compound to be measured (ligand)
and a radiolabelled ligand with an antibody or other
receptor whieh specifically binds that ligand. Thus, for
example, radioimmunoassay analysis for a partieular ligand
usually initially involves radiolabelling a sample of that
compound, and obtaining an antibody or other binder which
speeifically binds to that compound. The antibody is
usually obtained by injecting the ligand as an immunogen
into an animal such as a rabbit, and utilizing the anti-
sera produced by the animal.
Having these basic reagents, samples containing unknown
amounts of the ligandcan be analyzed by mixing the sample,
the radiolabelled ligand, and the ligand binder together,
preferably for a period of time sufficient to permit
~j 'Ly,.
,~, ,.
2~
~ 2 --
equilibrium to be at-tained in the reaction between the
radiolabelled ligand, the ligan~(if any) in the sample to
be analyzed, and the binder for ligand. The ligand bin-
der, and its bound (labelled and unlabelled) ligand are
then separated from the unbound (labelled and unlabelled)
ligand, e.g. by precipitation of the binder, and the radio-
activity of the bound material is counted by a radiation
detecting apparatus. The higher the level of (unlabelled)
ligand in the sample to be analyzed, the more of that un-
labelled ligand will be bound to the binder. Thus theunknown concentration of the ligand in the sample to be
analyzedvaries inversely with the radioactivity detected
in the hound fraction.
While these techniques have been substantial improvements
over previously available methods, they are ex-tremely cum-
bersome, re~uiring radioactive reactants and typically
large, expensive radioactivity counting devices, and
large amounts of time to get the samples collected, counted
and to interpret the results. Further, the use of radio-
activity requires a balance between the desired sensitivityand the functionality of the compounds to be detected. For
example, in order to obtain the maximum sensitivity, the
radioactivity may be increased, so that even small amounts
of bound radioactive materials can be detected. However,
high radioactivity tends ~o degrade the reactants and in-
terfere with the chemistry involved. Further, radioactive
reagents, in addition to posing radiation hazards, break
down and thus do not have great stability.
The cumbersomeness of RIA techni~ues have in part led to
other immunoassay techniques, such as the enzymatic ap-
proaches, wherein the ligand is bound to an enzyme. See,
e.g., Engvall, "Enzyme Immunoassay ELISA and EMIT", 70
Methods in ~nzymology 419 (1980). For example, an enzyme-
bound ligand may be mixed with an unknown amount of un-
bound ligand ~rom the sample to be analyzed. The ligandwhich is bound to the enzyme is analyzed by contact with
~<~
~2~29
reactants,and observing a reaction, or measuring the rate
of a reaction, which is catalyzed by that enzyme. As in
RIA, the higher the amount o~ bound l:igand after incuba-
tion, the lower the concentration o~ the unbound ligand
in the sample.
Other variations are described by Engvall, supra. As
also pointed out, enzyme-immunoassay techniques are limited
in this application, and suffer from a number o~ serious
disadvantages. These include the necessity of ~aving
large amounts of pure antigen or antibody in order to
effectively label with enzymes; the adverse effects on
the enzymes caused by competitive enzymes and/or inhibi-
tors in common biological sample fluids such as serum,
urine, etc.; difficulties in identifying enzymes which
are readily detectable at nanogram levels and have the
requisite availability, cost and shelf life; etc. See
Engvall, supra, 70 Immunochemical Techniques at 423-25.
Other techniques which have been developed include
agglutination techniques, where a specific reagent, such
as an antibody, is bound to inert particles and mixed
with one or more dilutions of samples to be tested. The
coated particles are crosslinked or otherwise aggregated
by a complementary antibody or multivalent antigen. See,
e~g., Von Schulthess et al, "Detection of Agglutination
Reactions Using Anisotropic Li~ht Scattering * * *" 17
Molecular Immunology, 81-92 (1980). However these methods
involve specialized light scattering devices or other
devices necessary for use of turbidimetric techniques to
measure the number of agglutinated particles. Von Schulthess
et al, supra; Masson et al, I'Particle Counting Immuno-
assay", 74 Methods in Enzymology 106 (1981); Cohen et al,
"Immunoassay by Light Scattering Spectroscopy", 12
_munochemistry 349 (1975); Blume et al, "Application of
Dif~erential Light Scattering to the Latex Agglutination
35 Assay for Rheumatoid Factor", 21 Clin. Chem. 1234 (1925);
z~
Hoigne et al, "Serologic Studies on Serum of Drug
Allergies With a Polystyrene Latex Technique Measured by
Nephelometry", 13 Acta Aller~olegia 364 (1959, and Grange
et al, "Nephelometric Assay of Antigens and Antibodies
With Latex Particles", 18 J. Imm. Methods 365-75 (1977).
It is accordin~ly an object of the present invention to
provide a method o~ analysis which is as sensitivie as,
or preferably more sensitive than, previous immunoassay
techni~ues, but without xe~uiring tagging of liga~ds or
antibodies with radioactive materials, enzymes, fluoro-
chromes or other tags.
It is a further ob]ect of the invention to pro~ide a
method o~ analysis which is at least as sensitive as
prior immunoassay techniques, but does not require the
complex, time-consuming manipulative steps of previous
immunoassay techniques.
It is a further object of the invention to provide a
method of analysis which is at least as sensitive as
radioimmunoassay techniques, but which does not require
the expensive and complicated apparatus which is required
to utilize radioimmunoassay techniques.
It is a further object of the invention to provide immuno-
assay reactants and products which can be used to analyze
accurately for minute quantitites of ligand by persons
with little or no technical training.
It is a further object of the present invention to pro-
vide methods ana products for particulate immunoassay
which do not require sophisticated particle analyzers or
other complicated apparatus in order to o~tain accurate
analyses.
In accordance with one aspect o~ the present invention
/i
~%~L8~29
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there is provided a method of analyzing a sample ~or a
ligand, comprising incubating the sample with a binder,
the binder being a material which specifically binds to
the ligand and which binder has a plurality of binding
sites for the ligand, and with particles of predetermined
size bearing a predetermined amount of ligand on their
outer surface, passing the mixture through a filter hav-
ing apertures which are larger than the ligand bearing
particles but smaller than aggregates of the ligand
bearing particles, and analyzing at least one of the
group of ~a) the particles which pass through the filter
and (b~ the particles which do not pass through the
filter.
In accordance with a ~urther aspect o:E the present inven-
tion there is p.rovided an assay kit for analysis of a
ligand, comprising binder particles which comprise par-
ticles of predetermined size bearing on their outside
surface a binder material which specifically binds to ~he
ligand, competitive binding particles comprising particles
of a predetermlned size bearing a predetermined amount of
ligand on their outer surface, and a filter having aper-
tures which are larger than the competitive binding par-
ticles but smaller than aggregates of competitive binder
particles with binder particles.
The above and other objects and advantages, which will be
apparent to the person skilled in the art from the present
disclosure or practice of the invention as disclosed here-
in, are achieved b~ use of the methods and products des-
cribed more particularly below.
BRIEF DESCRIPTION OF THE DR~WINGS
Figure 1 is a ~raphical representation of a standard
curve obtained b~ utilizing the present invention in
connection with analvsis of various concentrations of
~2~
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penicillin G in a phosphate buf~er.
Figure 2 is a graphical representation of a standard curve
obtained by utili~ing the present invention to measure
various concentrations o~ penicillin G in milk.
DESC~IPTION OF THE PREFERRED EMBODIMENTS
Prior particulate immunoassay techniques have not solved
the problems which have prevented the utilization of im-
munoassay techniques by anyone not having access to com-
plex, sensitive equipment, such as the blood cell counter
of Masson et al, the photocolorimeter/nephelometer of
Hoigne et al, the spectrophotometer, Photogoniocliffuso-
meter, differential spectro~luorimeter and particle coun-
ter of Grange et al, supra, etc. This fact sharply
restricts the use of particle immunoassay techni~ues, and
prevents their use in many areas where they could be most
useful.
For example, one problem which has occurred is the inges-
tion by humans o~ antibiotics contained in milk. Dairy
farmers use antibiotics, typically B-lactam antibiotics
such as deri.vatives of penicillins and cephalosporins, to
treat a variety of disorders, such as mastitis, in their
cows. When those antibiotics show up in the milk, inges-
tion by humans can lead to reduced ef~ectiveness of those
antibiotics in treating human disorders, and sometimes
can precipitate allergic reactions. In addition, antibi-
otics in milk inhibit the production of biologically
derived milk products such as cheese, yogurt, etc.
Accordingly, the FDA has set stringent standards for levels
o~ antibiotics which can be contained in milk. As a prac-
tical matter, if milk contains more than 0.01 InternationalUnit of B-lactam antibiotic per ml (equal to about 6 ng/ml
for penicillin G), it is unacceptable and cannot be sold
2~
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for human consumption.
Typically, at present, milk collected by tank trucks from
different farms is tested ~or antibiotic content at the
processing centre. This testing, using RIA or similarl~
cumbersome techniques or even slower microbiological
inhibition techniques, should be performed before the
truck is unloaded, to prevent contamination of large vol-
umes of previously unloaded milk. The loss of time is a
large financial burden on the trucker, and rejection of
the whole truck load of milk is an even larger burden on
the farmers whose milk filled the truck and on the milk
cooperative or dairy. If the contaminated milk, perhaps
from one herd or even from one cow, could be detected and
prevented from being loaded into the tank truck, this
substantial loss could be prevented. However, the truck
driver or the farmer can hardly be expected to carry with
him or operate a chemical laboratory, including scintil-
lation or radiation counters for RIA analysis, or the
sensitive blood cell counters, etc. required for previous
particulate techniques.
The present invention is ideal for this and other similar
situations. The present invention provides a simple,
rapid, accurate test, which can be used in minutes by the
milk collection centre, to let the trucker rapidly unload
and get back to work without waiting hours for test
results. It provides a simple~ rapid, accurate test
which is so straightforward that truckers and farmers can
easily utilize it to prevent contaminated milk from being
collected, or to determine when a cow which has been
treated with antibiotics can be put back into production.
Other similarly advantageous uses will be readily appa-
rent to the person skilled in the art.
In accordance with one preferred embodiment of the present
invention, a ligand-binding protein or other material
~aZ~ 29
-- 8 ~
(binder) is immobilized on a particle of defined
dimensions, preferably a plastic particle having a sphe-
rical shape and defined uniform diameter~ The binder can
be an antibody, an enzyme, or any protein or other
material which speci~ically binds to the ligand o~ in-
terest. A known amount of the ligand to be determined is
preferably also immobilized which may be on a similar
particle. The amounts of immobilized binder, immobilized
ligand, and particles are preferably adjusted so that, in
the absence of some type of binding inhibition, each of
the binder-bearing particles will aggregate with one or
more of the ligand-bearing particles, when mixed together
for a reasonable time tincubated).
During the test the particles after mixing are expose~ to
a filter having a defined pore size which is larger than,
but close to, the coated particle size, so that aggregated
particles will normally not pass through the filter. For
example, where the particles have a diameter of 0.3 ~m, a
filter having a pore size of 0.4 ~m might be used.
Where the proportions of materials are adjusted so that
essentially all of the coated particles will aggregate
upon mixing, the presence of ligand in a sample to be
tested can be determined by admixing the sample with the
binder-bearing particles, prior to or at the same time
those particles are mixed with the ligand-bearing partic-
les. The ligand (if any) in that sample competes ~or
binding sites on the binder-bearing particles with the
ligand immobilizer or the ligand-bearing particles.
Where the ligand ~rom the sample is bound to the binding
sites o~ the binder-bearing particles, the ligand-bearing
particles are prevented from aggregating with the binder
particles. Thus when the mixture of particles is there-
after subjected to ~iltration through the filter having a
controlled pore size, there is a very substantial increase
in the amount of unaggregated particles which pass through
.j~ ,,
i A . _
9 ~8~2~
g
the filter. The number o~ such unaggregated particles
can be suffici~nt to be visible to the naked eye, and
this visibility can also be enhanced, e~g., by selection
of the size, colour, optical density, fluorescence or
other properties of ~he particles. For example, the par-
ticles can also have enzymes attached to the particle
surface, and which enzymes can catalyze a colour change
on a substrate, thus making the results highly visible in
another way.
Thus the lab technician, ~armer, truck driver, etc. can
see after only a short mixing period whether there is
ligand in the sample, wi-thout having to wait to run the
sample through a radiation counter, spectrophotometer,
fluorometer, goniodif~usimeter, etc. to get the results.
In a further preferred embodiment, one of the particles
is larger than the other, and in fact is too large to
pass through the filter pores. This embodiment provides
several advantages over the situation where the two par-
ticles are the same size. In the latter situation, aggre-
gation typically occurs not only between individual par-
ticles to form pairs but also to form larger aggrega~es.
Thus, the blocking of a single binding site does not
automatically give rise to the release of a single ligand-
bearing particle since it still may be held together in
the aggregate by other bonds. The use of substantially
larger binder particles, (containing a very small number
of binding sites) in amounts in excess of the number of
ligand coated particles reduces the number of large
aggregates and increases the number of pairs of ligand-
binder particles, thereby giving rise to the release ofone particle for each blocking event.
In this invention, the amount of unaggregated particles
which pass through the filter is proportional to the
amount of ligand in the sample. Thus visual standards
~2~L8~2~
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can be set showi~g the appearance, e.g., colour, ~luores-
cence or other variable, o;E a standard sample containing
the upper limit of concentration. Then the filtrate from
any given sample can simply be comparedwith the visual
standard to determine whether the sample is or is not
acceptable. Similarly, visual standards for minimum,
maximum and intermediate concentration points within a
desired range can be set up for sample evaluation.
While it is thus not normally necessary to employ addi-
tional instrumentalities in order to obtain quick accurate
determinations of whether or not a ligand falls within a
certain range, it is certainly possible to do so. More-
over, the determination of the concentration oE particles
in the filtrate obtained from the present process is fas-
ter, more accurate and more reproducible than measurementstaken in accordance with the previous approaches to par-
ticulate analysis referred to above. For one thing,
previous approaches measured the entire melange of ag-
gregated and unaggregated product with various techniques
being utilized to distinguish relative amounts of the ag-
gregated from the unaggre~ated particles in that melange.
The present technique physically separates the aggregated
rom the non-aggregated particles, and thus permits the
extremely accurate measurement of only the unaggregated
particles.
A distinct advantaye of the present invention is that it
makes possible a direct or forward aggregation assay by
measurement or analysis of the aggregated particles.
This embodiment may be desirable when the ligandis multi-
valent and the binder is coated on a particle. This for-
ward aggregation may be assayed by simply resuspending
the aggregated particles after the non-aggregated parti-
cles have been removed into the filtrate. If desired, e.g.
to enhance the analysis of those aggregated particles, they
can then be disagyregated, e.g. by treatment with surface
~2~ Z~
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active agent or other chemical which will break the bonds
between the binder and the ligand or between the particles
and either the li~and or the binder. This technique
differs substantially from the inhibition techni~ues
previously known, such as turbidity measurements and
others which deal with a system containing both aggre-
gated and unaggregated particles, and permits substantial-
ly increased accuracy and ease of operation.
A major advantage of the present method is that the con-
centration of the particles in the filtrate can be
measure<~ instantaneously. Thus there is no need for
Elowing streams or the like for single particle counting
or mathematical or electronic factoring to attempt to
dif~erentiate the single particles from the aggregates,
which are primarily what is measured by turbidometric
techniques.
Preferred particles to be used with the present invention
are small, uniform diameter latex spheres, available in a
variety of diameters, e.g., from Dow Chemical Company, in
the United States, or from Rhone-Poulent in Europe. Other
useful particles includes carboxylated polystyrene, with
or without reactive groups to facilitate reaction with
the binder, such as amino groups, thio groups, carboxyl
groups, or other reactive groups. Butadiene/styrene co-
polymers such as carboxylated styrene butadiene or acrylo-
nitrile butadiene styrene, are also useful. Substituted
and unsubstituted acrylic acid polymers, methacrylic acid
polymers, and v.inyl polymers are also suitable. Inorganic
particles such as silicas, clay, carbons such as activated
3~ charcoal, and other materials on which the binder or
ligand can be immobilized, can be used to advantage with
the present invention. Other useful particulate materials
will be readily apparent to the person skilled in the art.
It is important that the particles all have approximately
9~
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the same diameter, so that they will easily pass through
about the same size ~ilter aperture. Preferably the
particles are about 0.01 - 100 ~m in diameter, more pre-
ferably from about 0.01 to 10 ~m. Most preferabl~ the
size of the particles is about 0.1 to 1.0 ~m in diameter,
and the diameters o~ the various particles do not vary
from the nominal diameters by more than 30%, pre~erably
not more than 15%.
The particles are pre~erably utilized at relatively small
concentrations in the ~luids which carry them, in order
to maximize the free movement of the particles in the
s~stem. Typically a suspension or mixture of from about
0.01% to about 10% b~ weight oE the particles in a ligand
such as a bu:~:Eer solu tlon .
As already indicated, the particles can be made opaque,
or coloured, or fluorescent by adding dyes, pigments or
coatings.
If more specific information than that provided by normal
visual observations is desired/ a variety of additional
techniques can be employed. As indicated, spectrophoto-
metric techniques can provide exact concentrations with
extreme precision, partially because of the fact that it
is only the unaggregated particles ~or aggregated par-
ticles in the case of the direct assay disclosed supra~
which are being measured. Thus extremely simple, battery
operated spectrophotometers, which would not be adequate
with previous particulate assays, can be used to provide
highly accurate results in accordance with the present
invention. It may also be advantageous in some instances
to attach a radioactive tag or a detectable enzyme or
other material to some of the reactants or the particles
used in the present invention. For example, one way the
visual evaluation of the present test can be enhanced is
to bind to some of the reactants or to the particles an
. ~ ,
~218~2~
- 13 -
enzyme or other compound which causes a colour change on
a particular substrate. l~hus for example if the filtrate
of the present invention causes that substrate to undergo
a colour change, it is clear that the filtrate contains
the reactants or particles which bear that enzyme or
compound.
The ligand and binding protein may be chemically bonded
to the latex beads or other particles, but they need not
be. Preferably the beads are coated with a substance to
which the ligand or binding protein will adhere, such as
bovine serum albumin, human serum albumin, etc. So long
as the coating does not interfere with the binding
between the ligand and the binding protein, this method
can be used. This method is preEerred when there is any
risk that chemically binding either of the species would
adversely affect the results. It is possible to adsorb
or absorb the binder and/or the ligand directly onto the
surface of the particle.
Where the ligar.dor binder has multiple functionality, i.e.,
ZO it is "multivalent" or otherwise can bind to more than one
antibody molecule, it is not necessary to attach that
material to a particle in order to obtain effective ag-
gregation and effective measurement. For example, an
antibody, which has at least two binding sites, can be
used to aggregate two particles having the appropriate
antigen.
The invention will be further clarified with reference to
the following example:
EXAMPLE 1
Attachment of Penicillin G Ligand to Lat x Particles
0.1 moles of the sodium salt of penicillin G is dissolved
in distilled water at 4C. This is acidified with lM HCl
29
- 14 -
to precipitate the acid. The precipitate is extracted
into dry chloroform at room temperature, and the chloro-
form i5 then evaporated to yield the dry penicillin G in
acid form.
The resulting product is dissolved in dimethylformamide,
to a concentration of 0.1 molar penicillin G. This
material is put in activated ~orm by admixture with 11%
by volume of 1 molar N-hydroxysuccinimide in DMF and 11
by volume of 1 molar dicyclohexylcarbodiimide and incu-
bated for 24 hours a~ room temperature. After filtrationthrough a Whatman #l filter, the filtrate is activated
penicillin G.
To a solution of bovine serum albumin is added a sodium
phosphate buffer containing 2~ sodium borate (pH 8.2) is
added 50 ~l/ml of the above solution of activated penicil-
lin G, with stirring, at 4C. The stirring at that tem-
perature is continued for 30 minutes. Thereafter the
mixture i5 centrifuged at 10,000 G and the supernatant
fluid is removed, and any precipitate i5 discarded. The
resultant material is dialyzed against distilled water,
with several changes of dialyzate to remove uncoupled
penicillin.
The result is a solution containing a 5.5:1 mole ratio of
conjugate of penicillin to bovine serum albumin containing
4.4 mg/ml of conjugated p~otein. The protein concentra-
tion may then be determined by the Lowry method, and the
amount of penicillin G conjugation estimated, e.g., via
spectral analysis.
Dow Chemical latex beads (#47781) having a diameter of
30 0.305 plus or minus .0084 microns (10% solids) is diluted
to a 2% latex suspension in distilled water, and filtered
through a Whatman ~4 filter.
~ j,
~2: l8~2~
- 15 ~
To one volume of ~he ~SA/Penicillin conjugate described
above, is added 9.6 mg/ml of solid ssA. This is mixed
gently to dissolve the BSA. An equal volume of the 2%
solution of the latex beads prepared as described above
is added. This mixture is then incubated, first in a
sh~king water bath at 37C Eor tw~ hours; then on a ro-
tator at room temperature for one hour, and then over-
night at 40C. The resultant product is washed three
times in an equal volume of O.lM Phosphate Buf~er (pH
6.5). The coated latex product is then resuspended at a
level of 2% by weight in the O.lM phosphate bu~fer.
The end result is a suspension containing 2~ of latex
beads coated with a 5.5 to one conjugate of penicillin G
to BSA in a O.lM solution o~ phosphate buffer containing
9.~ my/ml of unconjugated BS~. The non-conjugated BSA is
added to block further adsorbtlon of proteins to the sur-
face of the particles and also acts as a stabilizer to
keep the coated particles ~rom reacting or self-
agglomerating. Any other non-reactive protein can be
used for this purpose, and in some instances, the stabi-
lizing protein in the solution may be dispensed with
entirely.
EXAMPLE II
Preparation of Protein Binder
.
In this case the protein binder for the Penicillin G
ligand is an antibody raised to penicillin G. Since
antibodies have at least two binding sites, the present
example demonstrates that it is unnecessary to utilize
the double particle embodiment of the present invention,
i.e., the embodiment where the ligand is placed on the
surface of one particle and the binding protein is placed
on another particle. Further, in order to demonstrate
the effectiveness of the present test, a spectrophoto-
meter is utilized to generate exact data regarding the
~218~
- 16 -
concentration of ligand in the sample listed, whereas in
practice a simple visual comparison will normally be
enough to de-termine meeting or failure to meet concantra-
tion limitations.
A diluent for antiserum containing the binding compound
(antibody for Penicillin G) is made up of one volume of
6% Dextran T500 (Pharmacia AG.) in normal saline, mixed
with 2 volumes of saline buffered with 0.15M phosphate
buffer (pH 7.2) and containing 4% sucrose and 0.1~ sodium
azide. The antiserum diluent is made up of 0.5~ of the
above mixture in normal mouse serum.
The anti-penicillin G antibody used is a monoclonal anti-
body made by the procedure descrlbed by I~ohler et al,
Nature, Vol. 256, p. 495 (1975); Kohler et al, Eur. J.
Im unol., Vol. 16, p. 511 (1976), which are both incor-
porated herein by reference, in a Balb/C mouse. While
monoclonal antibodies are naturally preferred, any anti-
body which is specific in its binding properties for the
ligand of interest is suitable for use with the present
invention.
EXAMPLE III
_hibition Assay for Penicillin G
In order to determine the effectiveness of the present
method, a series of concentrations of penicillin G in
0.07M phosphate buffer (pH 6.0) solution were tested.
Six samples containing respectively 1 ng/ml, 10 ng/ml,
100 ng/ml, 1000 ng/ml and 10,000 ng/ml of penicillin G
in 50 1 of 0.07M phosphate buffer (pH 6.0) were each
mixed with 50 1 anti-penicillin G antibody in the anti-
sera diluent o~ Example II in a 1 ml tube. After mixing,
the reagents are d~awn into a Z.0 ml syringed fitted w.ith
a 1eur lock connection. The needle is removed from the
syringe, and the syringe is capped. The syringe plunger
,.
~8~2~
- 17 -
is moved back to bring the reagent into the barrel to en-
sure proper mixing. After incubation for five minutes
the needle is re-attached to the syringe and 0.07M phos-
phate buffer (pH 6.0) is slowly drawn up to a 2.0 ml
volume. The syringe is then gently inverted several
times to evenly disperse the latex suspension.
After removing the needle, the syringe is attached to the
inlet port of a 25 mm diameter Swinnex filter adapter
(Millipore Corp.) containing 0.4 micron Nuclepore polycar-
bonate filter, which filter has been pre-wetted with an
0.07 M phosphate buffer (pH 6.0) containing 0.1 mg/ml of
bovine serum albumin.
O~ce the syringe is secured to the Swinnex adaptor a
gentle even pressure is applied to the barrel oE the sy-
ringe. The filtrate is collected from the outlet port ofthe adaptor.
The filtrates are placed in glass cuvettes having a 1 cm
light path and read in a spectrophotometer (Beckman DU)
at a wavelength of 620 nm. Figure 1 shows the curve of
% latex (P~ solids) in the filtrate versus the concentra-
tion of the ligand penicillin G in the sample tested.
EXAMPLE IV
-
Measurement of Penicillin G in Milk
One volume of a sample of raw milk is mixed with 0.5 vol-
umes of a milk clarification solution containing 0.006%
citric acid and 6.0% of disodium salt of eth~lenediamine
tetracetic acid in distilled water. After thorough mix-
ing the solids are removed by filtration through a
Whatman GF/A filter and a 0.22 ~m filter (Millipore Corp).
About 2.0 ml of milk yields roughly 800 ~1 of filtrate.
50 ~1 portions of milk filtrate previously seeded with 1
ng/ml, 10 ng/ml, 100 ng/ml, 1000 ng/ml and 10,000 ng/ml
~.~. .,, J
8~2~9
- 18 -
of penicillin G are mixed with 50 ~l of the coated latex
of Example I and 50 ~l of the anti-penicillin G solution
of Example II, in the manner of Example IV. The spectro-
photometric results are depicted in Figure II of the
drawings.
The specific embodiments described herein are meant to be
exemplary only, and various modifications will be apparent
to those skilled in the art. The claims below are intended
to cover all such modifications.
