U.S. patent number 3,882,225 [Application Number 04/782,467] was granted by the patent office on 1975-05-06 for direct agglutination immunological reagent.
This patent grant is currently assigned to Miles Laboratories, Inc.. Invention is credited to Marion Cook Fetter, Shradha Nand, Virendra Patel.
United States Patent |
3,882,225 |
Patel , et al. |
May 6, 1975 |
Direct agglutination immunological reagent
Abstract
Immunological reagents capable of direct agglutination with
antigens are prepared by pre-polymerizing the antibody to form an
aggregate and then coupling this aggregate to a microbial cell
carrier particle with a chemical coupling agent. The
pre-polymerization of the antibody can be accomplished by the use
of the same or another coupling agent. The use of the microbial
cell carrier particle allows the reagents formed to be used as
immunological indicator particles in slide tests which permit a
quick read-out of test results. The reagent formed may be prepared
and shipped in dry form and then reconstituted with liquid for
use.
Inventors: |
Patel; Virendra (Baroda,
IN), Fetter; Marion Cook (Elkhart, IN), Nand;
Shradha (Elkhart, IN) |
Assignee: |
Miles Laboratories, Inc.
(Elkhart, IN)
|
Family
ID: |
25126143 |
Appl.
No.: |
04/782,467 |
Filed: |
December 9, 1968 |
Current U.S.
Class: |
436/519;
435/7.32 |
Current CPC
Class: |
G01N
33/554 (20130101) |
Current International
Class: |
G01N
33/554 (20060101); G01n 031/00 (); G01n 031/02 ();
G01n 033/10 () |
Field of
Search: |
;424/8,11,12 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Pressman, J. Immunol., Vol. 44, pp. 101-105, 1942. .
Gittens, Biochimica Et Biophysica Aeta, Vol. 66, pp. 237-249, 1963.
.
Ling, J. Med. Lab. Tech., pp. 94-101, Nov., 1960. .
Chemical Abstracts, Vol. 62, Item 5731b, 1965..
|
Primary Examiner: Rosen; Sam
Claims
What is claimed is:
1. An immunological indicator reagent capable of detecting an
antigen selected from the group consisting of bovine serum albumin,
diptheria toxoid, tetanus toxoid, and C-reactive protein,
comprising a pre-polymerized aggregate of an antibody coupled to a
bacterial cell with a coupling agent said; bacterial cell being of
uniform shape and size selected from the group consisting of E.
coli, B. subtilis, L. leichmannii, B. pumilus and P. fragi said
coupling agent being selected from the group consisting of bis
diazobenzidine, bis diazobenzidine disulfonic acid, acrolein,
difluorodinitrobenzene, dicyclohexylcarbodiimide, cyanuric
chloride, 2,7-diaminofluorene, toluene diisocyanate,
dichlorotriazine, difluorodinitrodiphenyl sulfone,
tetrazo-p-phenylenediamine, glutaraldehyde and
N-5-butyl-5-methylisoxazolium perchlorate.
2. A reagent as in claim 16 capable of detecting bovine serum
albumin comprising the pre-polymerized aggregate of an antibody
covalently bound to E. coli with acrolein
3. A reagent as in claim 1 capable of detecting diptheria toxoid
comprising the pre-polymerized aggregate of an antibody covalently
bound to E. coli with bis diazobenzidine.
4. A reagent as in claim 1 capable of detecting tetanus toxoid
comprising the pre-polymerized aggregate of an antibody covalently
bound to E. coli with bis diazobenzidine.
5. A reagent as in claim capable of detecting C-reactive protein
comprising the capable of detecting C-reactive protein comprising
the pre-polymerized aggregate of an antibody covalently bound to E.
coli with bis diazobenzidine.
6. A reagent as in claim 1 capable of detecting bovine serum
albumin comprising the pre-polymerized aggregate of an antibody
covalently bound to E. coli with 2,7-diaminofluorene.
Description
BACKGROUND OF THE INVENTION
This invention relates to immunological reagents which are in the
form of indicator particles which are capable of direct
agglutination with antigens. More particularly, it relates to
immunological indicators wherein a pre-polymerized aggregate of the
antibody to an antigen selected for detection is bound to a
microbial cell carrier particle through a coupling agent.
The prior art indicates many difficulties in making immunological
reagents in the form of indicator particles which are capable of
direct agglutination with antigens. One difficulty has been that
the antibodies necessary to sensitize the carrier particles have
become inactivated when chemically coupled to said carrier
particles. This effect was apparently due to the high level of
reactivity which most reactive carrier particles exhibit prior to
the chemical coupling with the antibody and to steric hindrance
once the antibody has been coupled. In those reagents made by
physical adsorption of the antibody onto the carrier particle a
continuing problem has exhibited itself in that the antibody
leached from the surface of the carrier particle during testing and
combined with the antigen being detected and, by preferentially
reacting with such antigen thereby lower the observed concentration
of the antigen. Both of these difficulties led to inaccurate test
results.
Another problem which the prior art presents is in the continuing
use of erythrocytes of red blood cells as carrier particles. The
problem which red cells incur in that there are wide variations in
the nature of different batches of red blood cells, even when
withdrawn from the same test animal. It has been found that the
surface characteristics, size, and suitability of the red blood
cells as carrier particles all vary with respect to the condition,
age, medication and previous past history of the test animals from
which the red blood cells are withdrawn. This causes difficulty in
arriving at standardized immunological reagent indicator particles.
An inherent difficulty in the use of red blood cells is that their
large size and their surface characteristics limit the type of
testing in which they can be used to generally those techniques in
which the reaction is run in a test tube, which can only be
properly read after a long period of time. Moreover, since red
blood cells cannot be used in a slide-type test, the reaction time
cannot be shortened by employing such a test. The size and surface
characteristics also have been found to limit the sensitivity of
the resulting test. Olovikov, A.M.: POLIKONDENSIROVANNYI
SUSPENZIONNYI ANTITELO IMMUNOSORBENT I EGO ISPOLZOVANIE V REAKTSII
AGGLUTINATSII DLYA OPREDELENIYA SODERZHANIYA ANTIGENOV
(Poly-condensed Antibody Immunosorbent Suspension And Its Use In
The Agglutination Reaction For The Determination Of The Antigen
Content): Dokl. Adad. Nauk SSSR 1964 158 (5) 1202 - 5 mentions the
problem of the disadvantage of the use of erythrocytes as carrier
particles.
In review, red blood cells in the forms used in the prior art do
not lend themselves to the construction of sensitive slide-type
agglutination tests due to their fragility, size, weight, and
surface characteristics as well as the difficulty in obtaining such
cells.
It has now been discovered that immunological reagents capable of
direct agglutination with antigens can be prepared in a
reproducible and highly active form by pre-polymerizing an antibody
and then chemically coupling this polymerized aggregate to a
microbial cell carrier particle. The pre -polymerization step forms
an aggregate in which at least some of the active sites of the
antibody are exposed at the external surfaces thereof. The use of
the microbial cell as a carrier particle provides a uniformly sized
particle which is of size and weight characteristics capable of
forming an immunological reagent which will allow slide
agglutination test results to be determined in a short time
period.
It is therefore an object of the present invention to provide an
immunological indicator reagent capable of direct agglutination
with an antigen wherein a pre-polymerized aggregate is chemically
attached to a microbial cell carrier particle.
Yet another object of the present invention is to provide a process
for making an immunological indicator reagent capable of direct
agglutination with an antigen.
SUMMARY OF THE INVENTION
The immunological indicator reagent of the present invention is
formed by polymerizing an antibody with a coupling agent to form an
aggregate of said antibody and then reacting this aggregate with a
reactive microbial cell carrier particle. The antibody is obtained
in the form of the gamma globulin fraction of immune serum from
animals injected with various antigens. The microbial cell is
rendered reactive by reacting the same with a polyfunctional
coupling agent. One of the functional groups of the coupling agent
reacts with the surface of the carrier particle, leaving a
nonbounded reactive group on the exterior surface of said carrier
particle. The pre-polymerized aggregate of said antibody is then
reacted with the reactive carrier particle in order to form a
covalent chemical bond therebetween. These reactions are generally
carried out in a fluid such as buffered liquid which can be
maintained at optimum conditions.
After the immunological indicator is formed it can be reduced to a
dry form, if desired, by separating the fluid medium therefrom.
This dry indicator may be later reconstituted with a liquid or the
test sample for use in testing.
The immunological indicator particles made by the above process are
than comprised of a microbial cell carrier particle having bound to
at least a portion of the external surface thereof a
pre-polymerized aggregate of the antibody. The aggregate is bound
to the surface through covalent bonds formed by the reaction with
the residues of coupling agent molecules which were first reacted
with said carrier particles. As an alternative the coupling agent
can be added to a mixtue of the carrier particles and the
pre-polymerized aggregate so that it contacts both simultaneously.
These indicator particles have sizes of about from 0.2 to 1.5
microns in the smallest dimension and up to about 5 microns in the
largest dimension. The microbial cell carrier particles can be
rendered stable by reaction with a preservative such as
formaldehyde prior to contact with the coupling agent. In a like
manner the indicator particles can be rendered more visually
distinguishable from any given background by staining the microbial
cells with a suitable dye.
The microbial cell usable as carrier particles can be any of these
from the group of bacterial, fungal, protozoological or viral
cells. These cells can be any self-reproducing micro-organism which
is propagated with or without dependence upon other organisms. Both
gram positive and gram negative bacterial cells can be used. Fungal
cells and protozoological cells can likewise be employed, as can
viral particles. These are generally unicellular organisms which
are occasionally joined in clumps or aggregates. The cells may be
used in this form provided their aggregate size does not form a
carrier particle which is so large that the test system formed with
it will not properly agglutinate in the presence of a substance
which is homologous to the antigenic substance bound to the
aggregated cells. Aggregate sizes greater than 100 microns cause
this difficulty.
The preferred microbial cells are bacterial cells or aggregates
thereof which are of uniform shape and size and have maximum
external dimensions in one direction of up to about 5 microns.
While not preferred, a mixture of different but uniform cells may
be used. For these bacteria the usable microbial cells include
those in Division I of the Vegetable Kingdom, including Classes I,
II, and III, Order I. The Class III, Order I microbial cells
include the intracellular viral particles which have maximum
dimensions of about 0.2 micron.
Reference may be had to Bergey's Manual of Determinative
Bacteriology By R. S. Breed, E. G. D. Murray, N. R. Smith, 7th
Edition, 1947, The Williams and Wilkins Company, for a complete
listing of usable bacterial cells. Particularly useful are the
bacteria of Class II, Suborder II, Family IV (Pseudomonadaceous)
and Class II, Order IV, Family IV (Enterobacteriaceae). All Tribes
I - V are considered to represent preferred microbial cells for the
purpose of this invention. Also Class II, Order IV, Families V
(Brucellaceae), X (Lactobacillaceae) and XIII (Bacillaceae) are
considered preferred. Both Orders I and II of Class III organisms
can be employed where smaller particle sizes of about 0.2 micron or
under are desired. Particularly, the Order II Virales are of small
dimension which limits their usefulness.
Escherichia coli is a specially preferred bacterial cell for
purposes of this invention. Another specially preferred microbial
cell is the commonly available yeast, Saccharomyces cerevisae. The
yeast growth phases of the fungal cells are also preferred for use
as carrier particles.
Other preferred microbial cells are Bacillus subtilis,
Lactobacillus leichmannii, Bacillus pumilus, and Pseudomonas
fragii.
These microbial cells can be obtained by properly culturing a
starter culture of each of them in a nutrient medium. The cells can
then be harvested at their maximum growth point.
The antibodies which can be pre-polymerized according to the
present invention and employed to make the novel immunological
reagent can be any of the antibodies produced in any animals.
Antibodies are believed to be chemically similar since they are
gamma globulin molecules modified in such a way that they have at
two positions antigenic receptor sites for the antigens for which
they are immunological counterparts. Hence, the main surface of the
antibodies consists of roughly the same proteinaceous groups which
are present in pure gamma globulin molecules. The antibodies can
thus be those produced in animals injected with any of the
following exemplary materials obtained from various animals: serum
albumins, myoglobins, hemoglobins, ovalbumins, serum alpha, beta
and gamma globulins, beta-lipoproteins, blood group substances A
and B, and human transferrins, and all hormones, including insulin
and human chorionic gonadotropin (HCG). Enzymes constitute another
class of antigenic materials which can be employed, since
antibodies are formed in many animals when such materials are
injected. Such enzymes include but are not limited to the
following: diastase, maltase, zymase, amylase, and invertase.
Antigenic mmaterials of either pathological or natural organisms
exemplified by Trichinella antigen, tuberculin purified protein
derivatives, toxins such as diphtheria toxoid and tetanus toxoid
can be used also.
The coupling agents which can be employed for forming the
pre-polymerized aggregate of the antibody and for attaching the
aggregate to the microbial cell carrier particle are, in general,
those polyfunctional compounds having two or more of the reactive
groups: azo, sulfonic acid, fluoro groups activated by nitro
groups, azide, imine, and reactive chloro groups such as chloro
groups attached to a ring having appropriate resonance structures.
These reactive groups are capable of reacting with the primary
amino, sulfhydryl (mercapto), carboxylic and hydroxyl groups in the
materials constituting the surfaces of the carrier particles and
the proteinaceous materials constituting the antibody
aggregates.
A representative list of such coupling agents is:
bis-diazobenzidine, bis-diazobenzidine disulfonic acid,
tetraazo-p-phenylenediamine, difluorodinitrobenzene,
difluorodinitrodiphenyl sulfone, a carbodiimide, toluene
diisocyanate, cyanuric chloride, dichloro-s-triazine,
N-t-butyl-5-methylisoxazolium perchlorate, a dialdehyde, an alpha,
beta-unsaturated aldehyde, and mixtures thereof. Some of these
coupling agents, notably the cyanurating agents, the aldehydes and
the alpha, beta-unsaturated aldehydes, are capable of preserving
the microbial cells at the same time as they couple to groups on
the cell surfaces so that these cells are then stabilized against
lysis. Thus, when such coupling agents are used, no separate
stabilization or preservation treatment is necessary. When
N-t-butyl-5-methylisoxazolium perchlorate is used, the surface to
which it is to be coupled must first be treated with a
succinylation reagent such as succinic anhydride.
The carbodiimides which can be employed are, among others, the
following: N,N'-dicyclohexyl carbodiimide; 1ethyl-3-(3-dimethyl
aminopropyl) carbodiimide hydrochloride; and 1-cyclohexyl-3
(2-morpholinyl-(4)-ethyl carbodiimide) metho-p-toluene sulfonate. A
specific difluorodinitrobenzene which can be employed is
1,3-difluoro-4, 6-dinitrobenzene, and a specific
difluorodinitrodiphenyl sulfone, which can be employed is
p,p'-difluoro-m, m'-dinitrodiphenylsulfone.
The dialdehyde coupling agents are aliphatic dialdehydes having at
least one methylene group separating the carbonyl groups. Examples
of these dialdehydes are glutaraldehyde, propanedial
(malonaldehyde) and butanedial (succinaldehyde).
The alpha, beta-unsaturated aldehydes can be any compound having a
formula of the type: ##SPC1##
wherein any one of R.sub.1 or R.sub.2 can be hydrogen or a methyl
group. Representative of this aldehyde are: acrolein, methacrolein,
and 2-butenal crotonaldehydel. Of these acrolein is preferred.
It is also possible to convert the aldehyde reactivity remaining on
the surfaces of the cells after treatment with dialdehydes or the
unsaturated aldehydes to amino groups by further treating the cells
with compounds having one of the following formulas:
H.sub.2 N -- Ar -- NH.sub.2,
H.sub.2 N -- Ar -- OH,
H.sub.2 N -- NH -- Ar -- NH.sub.2,
or
H.sub.2 N -- NH -- Ar -- OH
where Ar is selected from the group of phenyl, biphenyl, stabilized
heterocyclic ring, polynuclear hydrocarbon structures, polynuclear
heterocyclic rings, triphenylmethane or substituted groups and
mixtures thereof. Generally all of the polyfunctional compounds so
defined have at least one diazotizable amino group and at least one
amino, hydroxyl, or hydrazino group. The diazotizable group can be
activated by nitrous acid for ultimate use. When desired, many of
these polyfunctional converting compounds can be used in their
water soluble forms by using the acid salts thereof. Sulfonic acid
salts and hydrochloride salts are of utility in this regard. The
sulfonic acid groups can be activated by phosphorus
pentachloride.
Examples of the polyfunctional compounds are the following:
2,7-diaminofluorene; 2,5-diaminofluorene; benzidine . HCl;
3,3'-diamino benzidine; o-tolidine; hydrazine. HCl;
3,3'-dimethoxybenzidine dihydrochloride; o-dianisidine;
p-rosaniline chloride and acetate; thionin; basic fuchsin;
safranin-O, amino group substituted triphenyl methane dyes,
p-hydrazine benzene sulfonic acid. Some of these compounds have
only two functional groups while others have a greater number of
functional groups, in which case there are two or more reactive
functional groups available on the surface of the particles for
reaction with the subsequently added biological materials. In the
above formulas the reactive groups in excess of the two required
groups are included in the substituted Ar groups.
The term "antibodies" as used herein denotes immunological
materials produced as a result of antigenic stimulation in animals.
The term "immunological counterpart" as used herein denotes either
an antigen or an antibody which reacts specifically with the
corresponding antibody or antigen, respectively.
As mentioned above the microbial cells can be colored by a dye or
stain in order to improve the visual distinction of the final
immunological reagents from the surrounding background. Stains such
as hematoxylin, fuchsin and crystal violet can be used for this
purpose. Another optimal preparation treatment for the microbial
cells is washing with an organic solvent such as alcohol, ether,
etc. to remove any polysaccharide or wax layers which may be
present and which might interfere with the reaction between the
microbial cells and the coupling agents.
The immunological reagent indicator particles produced according to
the present invention are capable of direct agglutination with the
antigen which is the immunological counterpart to the antibody used
in making the indicator particles. The indicator particles are
mixed with a fluid sample containing such antigen, and the
particles are then observed to determine whether or not an
agglutination or a nonagglutination pattern results. The occurrence
of an agglutination pattern denotes the presence of the antigen
tested for, while the absence of an agglutination pattern denotes
the absence of that antigen.
While the primary capability of the immunological reagent of the
present invention is to allow direct testing for antigens, this
same reagent can be used for detecting the presence of the antibody
itself in a fluid sample. For example, when testing for the
presence of an antibody, a quantity of the antigen thereto can be
added to the test medium prior to or along with the indicator
particles of the present invention which consist of the microbial
cells coupled to the pre-polymerized antibody aggregate. If the
fluid sample contains the antibody, the antigen added to the
testing medium will preferentially react with this antibody and the
antigen will thus be unable to agglutinate with the immunological
reagent. Hence, the agglutination which would otherwise occur is
inhibited, and the pattern which appears is said to be a
nonagglutination pattern.
The agglutination testing and the inhibition of agglutination
testing above referred to are preferably carried out by a slide
agglutination method in which the test reagents are mixed with a
fluid sample on a flat glass surface and the resulting pattern
observed after a short time period. A smooth milk-like consistency
is an indication of a nonagglutination pattern whereas an
agglutination pattern is denoted by a number of clumps or
floccules. If desired, microbial cells large enough in size and
weight characteristics can be employed so that the agglutination
reaction can be carried out according to a micro-titrator method
wherein the immunological reagent is placed in each of a series of
wells formed in a row in the upper surface of a plastic or other
suitable plate. The linear arrangement of the wells allows serial
dilution of the fluid sample used in the testing.
The serial dilution is carried out by first placing one drop of a
diluent in each of the wells in the row and then adding a one drop
volume of the fluid sample having an initial dilution of, for
example, 1:5 to the first well using a loop or spiral calibrated to
hold one drop of fluid. The loop is submerged in the first well,
mixed and one drop of the fluid is withdrawn and is then mixed with
the diluent in the second well. The dilution of the first well is
then 1:10 and the dilution of the second well is 1:20. This process
of serial dilution is repeated until all of the wells in the row
have been treated, thus producing a series of antigen dilutions in
each well differing by a factor of one-half from the adjacent
earlier treated well.
To perform an inhibition of agglutination test, serial dilutions of
a test sample are made in each of the wells in a row, and fixed
amounts of the antibody added to each of the wells, followed by one
drop of a suspension of the immunological reagent of the present
invention. In such inhibition of agglutination testing the
dilutions of the test sample are controlled so that the
concentration of the antigen therein lies in the mid-portion of the
serial dilutions made. This procedure allows an identification of
the concentration of the antigen in the fluid sample and hence a
semi-quantitative determination thereof. The same semi-quantitative
determination can be made by using a direct agglutination procedure
with the micro-titrator method.
The process for making the immunological indicator of the present
invention involves three major steps, each of which can be carried
out in any of a number of buffered liquid media. The first and last
steps are preferably carried out at room temperature, and the
second is preferably carried out at 4.degree. C. The first step is
to form a polymerized aggregate of the antibody to the antigen
which is to be detected, and this is accomplished by reacting a
coupling agent with the antibody. Next, the microbial cell is made
reactive to the polymerized aggregate of the antibody by reacting
the cell with a coupling agent which can, but need not necessarily,
be the same coupling agent employed in the first step.
In the second step the microbial cells are dispersed in a fluid
medium at a 1 to 10 percent suspension concentration. Short times
of less than 1 hour can be used for this step when conducted at
room temperature (20.degree. to 25.degree.C.).
When a preservative agent is employed, the microbial cells are
pre-treated therewith prior to carrying out the second step. As
aforementioned the preservative agent used is preferably
formaldehyde. In a like manner, when a stain is employed, the
microbial cells are treated therewith prior to reacting the
coupling agent with the cells in the second step.
In the third step the polymerized aggregate of the antibody is
placed in contact with the reactive carrier particles for a time
period sufficient to allow a coupling reaction to take place
therebetween. Upon completion of the third step the immunological
indicator formed can be separated from the suspending liquid and
dried. This separation and drying is preferably accomplished by a
lyophilization method in which volumes of several drops of a
suspension of the immunological indicator are placed in containers
and cooled to -40.degree.C. under a vacuum. This method can be
carried out in standard commercial lyophilizers in a short time.
The dried reagent can then be packaged under a low humidity
condition and later reconstituted for use.
DESCRIPTION OF THE PREFERRED EMBODIMENT
Bovine serum albumin (BSA) is injected into a rabbit to produce
anti-BSA globulin. This antibody-containing globulin is separated
from the other serum proteins by DEAE-cellulose (diethylaminoethyl
cellulose) chromatography.
To the separated globulins 1 ml. of a 1:80 dilution of 0.024 M
bis-diazobenzidine (BDB) in 0.15 M phosphate buffer, pH 7.3 is
added to form the pre-polymerized antibody aggregate. The reaction
can be carried out at room temperature (22.degree.C) with
occasional shaking until a faint turbidity develops.
E. coli cells are cultured in advance, separated from the growth
medium and formalinized in order to preserve the same. Then BDB is
coupled to the formalinized E. coli cells at 4.degree.C. and these
cells are then washed with saline in order to remove al unreacted
BDB. The pre-polymerized antibody aggregate is then added to the
washed BDB cells and the mixture is incubated at 22.degree. C. for
thirty minutes, after which it is washed with saline. The
sensitized cells are then resuspended in 1 percent normal rabbit
serum (NRS) to protect the cells during a subsequent
lyophilization. The cells as thus constituted are usable for
testing in the manner described. Preferably, the concentration of
the immunological indicator is adjusted to the optimum level for
each particular test situation.
The above objects and description are further detailed in the
examples which are to be construed as illustrative only and are not
to be limitative of the invention.
EXAMPLE 1
An immunological indicator reagent was made by pre-polymerizing the
globulin fraction from a rabbit which had been injected with BSA.
This antibody aggregate was then coupled to reactive microbial cell
carrier particles in order to form an immunological reagent for
detecting BSA.
DEAE-cellulose chromatography was employed in the standard manner
to separate the antibody containing globulin fraction from the
other serum proteins of rabbit anti-BSA serum. Two and one-half mg.
of the purified globulins were then dissolved in 1 ml. of 0.15 M
sodium phosphate-potassium phosphate buffer, pH 7.3 which was made
up by adding 808 ml. of 0.15 M Na.sub.2 HPO.sub.4.H.sub.2 O to 192
ml. of 0.15 M KH.sub.2 PO.sub.4. To this mixture was added 1 ml. of
a 1:80 dilution of 0.024 M BDB in the 0.15 M phosphate buffer, pH
7.3 (resulting in a 0.0003 M BDB concentration). The resulting
mixture was incubated at 22.degree.C. for 30 minutes with
occasional shaking at which time a faint turbidity was noticed,
indicating the presence of the pre-polymerized antibody
aggregate.
This antibody aggregate was coupled to carrier particles prepared
in the following manner. Five ml. of a 2.5 percent suspension of
formalinized E. coli, previously cultured and preserved, were
washed 3 times with 10 ml. of physiological saline solution and
then resuspended in 3 ml. of the 0.5 M phosphate buffer, pH 7.3 at
4.degree.C. To this mixture was added 10 ml. of 0.0012 M BDB in the
0.15 M phosphate buffer. The resulting mixture was agitated for 30
minutes at 4.degree.C. after which it was washed twice with cold
saline at 4.degree.C. After the last wash the reactive carrier
particles were resuspended in 3 ml. of the 0.15 M phosphate buffer
at room temperature and the above pre-polymerized aggregate
dispersion added thereto. The coupling reaction was allowed to
proceed for 30 minutes at room temperature with occasional mixing,
after which time the preparation was centrifuged and the
immunological indicator recovered and washed three times with
physiological saline, after which it was adjusted to a 4 percent
indicator concentration in saline.
The immunological indicator thus prepared was tested by a direct
slide agglutination method by employing the following
concentrations of BSA: 0.1, 1, 10, and 100 micrograms per ml.
Agglutination patterns were observed for the last three BSA
concentrations. A negative control in which saline alone was
employed gave no agglutination pattern. In an inhibition test which
was conducted using a 10 microgram per ml. solution of BSA in
saline pretreated with a 1:50 dilution of anti-BSA in saline,
inhibition of agglutination was observed.
In a corresponding experiment the same procedure as above set out
was followed except that the antibody was not pre-polymerized. When
the immunological indicator prepared in this manner was reacted
with BSA, no agglutination was seen at any of the concentrations
employed above. However, the thus prepared indicator did react with
a 1:200 dilution of anti-rabbit globulin in saline to provide a
strong agglutination pattern. This showed that although the
antibody molecules had indeed coupled to the microbial cells, their
antibody properties had been denatured or in some manner impaired,
but their antigenic properties due to the protein nature of the
globulin were still active. This would indicate that the antibody
sites of the gamma globulin molecules are inactive when the gamma
globulin molecules are reacted directly with carrier particles
without the pre-polymerization step.
EXAMPLE 2
An immunological indicator capable of detecting diphtheria toxoid
was prepared in the following manner. One ml. of diphtheria
antitoxin, containing 100 AU (antitoxin units International
Standard) in the above 0.15 M phosphate buffer, pH 7.3 was
polymerized with 1 ml. of 0.0006 M BDB in the 0.15 M phosphate
buffer. The subsequent reaction for the preparation of the
pre-polymerized antibody aggregate and the coupling of this
aggregate to reactive E. coli cells was then carried out in the
manner of Example 1 above.
This immunological indicator was tested with the following
concentrations of diphtheria toxoid: 0.01, 0.1, 1 and 10 LF
units/ml. (based on the International AU Standard). Agglutination
patterns were seen with the last three diphtheria toxoid
concentrations while a saline control showed no agglutination.
EXAMPLE 3
An immunological indicator capable of detecting tetanus toxoid was
prepared in the following manner. One ml. of tetanus antitoxin
containing 80 antitoxin units (International Standard AU) in the
above 0.15 M phosphate buffer, pH 7.3 was polymerized with 1 ml. of
0.0006 M BDB in the 0.15 M phosphate buffer. This aggregate was
then allowed to react in the same manner as for Example 1 and was
then coupled to the active E. coli carrier particles in the same
manner as in that example. It was then tested in the same manner as
for the indicator reagent of Example 2 using the same
concentrations of tetanus toxoid instead of the diphtheria toxoid.
Agglutination patterns were observed in the last two concentrations
of the tetanus toxoid and a saline control showed no
agglutination.
EXAMPLE 4
An immunological indicator capable of detecting C-reactive protein
(CRP) by agglutinating therewith was made following the process
generally as in Example 1.
The antibody to the CRP was produced in a goat by injecting an
adjuvant-CRP mixture into the animal and then bleeding the animal
after a sufficient length of time. The antibody containing globulin
was then separated from the other serum proteins by DEAE-cellulose
chromatography. Three mg. of the separated globulins were dissolved
in 1 ml. of the 0.15 M phosphate buffer, pH 7.3 and this solution
then reacted with a 1 ml. of 0.0006 M BDB in the same buffer for 25
minutes at room temperature. A turbidity developed at approximately
this time and the antibody aggregates were then reacted with BDB
treated E. coli cells in the same manner as described in Example
1.
The immunological indicator thus prepared was tested against the
following dilutions of commercially available CRP in saline: 1:5,
1:10, 1:100 and 1:1000. Agglutination patterns were observed in the
first two dilutions while a saline control did not show
agglutination. The immunological indicator also agglutinated with a
1:100 dilution of anti-goat serum proteins in saline showing that
the gamma globulin derived antigenic properties of the antibody are
in fact active.
An inhibition test was also performed using both 1:10 and 1:50
dilutions of CRP in saline and employing for the inhibition 0.1 ml.
of CRP antibody suspended in saline. Inhibition of the
agglutination patterns was observed.
EXAMPLE 5
An immunological indicator was prepared to detect BSA by
agglutinating therewith. The pre-polymerized anti-BSA aggregate was
coupled to microbial cells in a 4-stage coupling procedure in which
acrolein was first used to coat the microbial cells with active
aldehyde groups and then o-dianisidine was used to convert the
aldehyde reactivity to an aminogroup activity, after which the
amino groups were reacted with BDB to provide the final reactive
carrier particles. The pre-polymerized antibody -aggregate was then
coupled to said reactive particles. It is also possible to employ a
one stage coupling procedure using only the acrolein, since the
aldehyde groups directly couple to the pre-polymerized antibody
aggregate.
More specifically, E. coli was deantigenated by extracting the
cells with a 1:20 dilution of phosphate buffered saline, pH 7.2 in
a 100.degree.C. water bath for 2.5 to 3 hours. The concentration of
the cells was then adjusted to 2.5 percent in saline, and 2 volumes
of a 4 percent saline solution of freshly distilled acrolein were
added thereto. This mixture was incubated at 22.degree.C. for 72
hours with occasional shaking. The reactive cells were then washed
three times with 250 ml. of saline and the cell concentration
adjusted to 2.5 percent prior to the addition of an equal volume of
0.25 percent o-dianisidine dihydrochloride in water, after which
the mixture was incubated under the same conditions as above.
The prepared cells were then washed 5 times with saline and coupled
to BDB in the manner described in Example 1.
The anti-BSA pre-polymerized aggregate was prepared in the manner
set out in Example 1 and coupled to the reactive carrier particle
as set out in Example 1.
A BSA concentration of 1 microgram per ml. agglutinated this
immunological reagent and a saline control did not.
EXAMPLE 6
The procedure of Example 5 was followed except that
2,7-diaminofluorene dihydrochloride was substituted for the
o-dianisidine dihydrochloride. The same test results were
obtained.
EXAMPLE 7
It is possible to vary the size and surface characteristics of the
carrier particles by coating them with one or more layers of a
proteinaceous material as mentioned above. In this example a
microbial cell is first coated with BSA in order to increase the
size and weight of the base carrier particle.
BSA was used to coat E. coli by the following treatment. Five ml.
of 2.5 percent forminalized E. coli were washed 3 times and then
resuspended in 3 ml. of the 0.15 M phosphate buffer, pH 7.3. Next
10 mg. of BSA dissolved in 2.5 ml. of saline was added together
with 10 ml. of 0.0024 M BDB in the 0.15 M phosphate buffer. The
mixture then placed on a rotary shaker for 30 minutes at room
temperature, centrifuged and washed twice with 10 ml. of saline
prior to resuspending the cells in 10 ml. of a 0.6 percent BSA
solution in saline. After letting the carrier particles stand 30
minutes they were washed again with saline three times and
resuspended to a 2 percent concentration in saline.
An anti-BSA pre-polymerized aggregate was prepared and coupled to
these BSA coated carrier particles by following the procedure of
Example 1. When tested with BSA, agglutination was seen down to a
concentration of 1 microgram per ml. while a saline control showed
no agglutination. Also a strong agglutination pattern was observed
when the immunological indicator was mixed with a 1:100 dilution of
anti-rabbit gamma globulin in saline showing that the antigenic
activity of the pre-polymerized aggregate was not impaired.
A test was also made to determine whether or not the coating layer
of BSA had been effectively covered with the pre-polymerized
antibody aggregate. This check was carried out by testing the
indicator with a 1:100 dilution of anti-BSA in saline, with the
result that no agglutination was found.
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