Note: Descriptions are shown in the official language in which they were submitted.
2012~3~
-- 1 --
ENZYM~IC ASSAY KIT A~ E~Q~L~EEI~c~BLE T--Q WHOI.E ~EI~
The pre~ent invention relates to a kit and to an
enzymatic a~ay method u~ing thi~ kit for a~saying endo-
genous enzymes characteri~tic of cell popuIations or sub-
05 population~. The en~ymatic a~ay method and the corres-
ponding kit are al o intended for the assay o the cell~
them3elves via a~say of their endogenou~ enzy~es. These
as~ays are applicable to diagnosis
The method according t;o the invention con~ists in
specifically immobilizing a cell population by immunocap~
ture on a solid support and then in a~aying an endo-
genous enzyme ~acteristic of this p~ation or of a o~l
subpopulation by u~ing a ~ubstrate appropriate for th~o
enzyme and, if appropriate, the nece~sary cofactors or
auxiliary ~ubstance~
According to the pre~ent invsntion, in the de~-
cription and the claim~ which follow, the term "endo-
genou~ enzyme" is under~tood a~ meaning an enzyme which
i~ contained in the cytopl~m of the cell and whose ~ub-
~trate is a low-molecular molecule capable of penetratin~
inside the cell, where it will be tran~formed by the
enzyme, thereby releasing into the medium a colored or
fluorescent reaction product which permit~ the final
mea~urement of a signal proportional to the acti~ity of
the enzyme.
Cell immunocapture i~ a proce s which con~ist~ in
retaining the cells on a ~olid support by binding between
one or more surface antigens of the~e cell~ and one or
more monoclonal antibodies specific for thi~ antigen or
these antigens.
Knowledge of cell ~urface antigen~ or marker~ has
made enormou~ ad~nce~ with the development o~ lymphocyte
hybridization and the discov~ry of monoclonal antibodies
by KOEHLER and MI~STEIN (Nature, 1975, 2~, 495~497)-
particular, monoclonal antibodie~ have made it possible
` 2~2~3~
to reveal and analyze surface markers or membrane anti-
gens of cells of the widest possible variety of origins.
These markers (or antigens) can be of different kinds:
proteins, glycoproteins or glycolipids. The characteri-
05 zations sought therefore apply mainly to tissue or organmarkers, to markers of states o~ differentiation or
activation of normal cells and to the identification or
typing of normal or cancerous cells. A particularly
important field of application is the study of the cell
lines of hemopoiesis (erythrocyte, megakaryocyte, granu-
locyte, monocyte, lymphocyte).
Thus, for example, monoclonal antibodies have
made it possible to specify the respective surface
characteristics of T and B lymphocyte~. The correspon~
ding markers, by themselves or in combination, identify
stages of differentiation and functional specialization
of the lymphocyte~. By international convention, the
surface markers of human leukocytes have been classified
in differentiation groups or differentiation classes (CD)
defined by the IUIS-WHO subcommittee, 1~84, and described
in Bulletin of the World Health Organization, 1984, 62
(5), 813-815.
Monoclonal antibodies are now irreplaceable tools
of clinical biology applied to cell analyses.
Cell counting methods exist which utilize the
labeling of their surface antigens, but these methods are
often lengthy, laborious and dificult to carry out and
their results are sometimes random.
One group of methods for the measurement of anti-
gens is based on quantitati~e evalua-tion o the surface
markers of the overall cell population These methods
make it possible to measure the antigens either by direct
labeling or by indirect labPling, the latter most fre-
quently being carried out in two, thr-ee or four steps.
In all cases, the reagent employed in the last labeling
2~ 2~
step carries a probe which is either of isotopic charac-
ter, for example iodine 125~ for an assay of the radio-
immunometric type (BROW et al., J Immunol. Methods,
1979, 31, 201; STOCKER and HEVSSER, J. Immunol. Methods,
05 1979, 26, 87-95), or an enzyme for an assay of the enzyme
immunometric type, most frequently peroxidase, alkaline
phosphatase or beta-galactosidase (VAN LEUVEN et al., J~
Immunol. Methods, 19789 23, 109-116; MORRIS, Transplan-
tation, l9B3, 36(6), 719; BAUMGARTEN, J. Immunol.
Methods, 1906, 94, 91-98).
It ~ill be noted that the enzymes used in such
methods are analytical reagents introduced during the
assay in the form of a conjugate with an antibody which
recognizes an antigen exposed on the outside of the cell
membrane. These enzymes cannot therefore be confused
with the endogenous enzymes of the cell, which form part
of its natural enzymatic equipment.
These methods are rather inconvenient, laboriouæ
and risky to apply because of the need to wash and cen-
trifuge the cell material many times, it is sometimesnecessary to take a sample of the colored medium resul-
ting from the enzyme reaction in order to carry out the
final spectrophotometric measurement; lastly, chemical
fixation of the cells, which is used in most cases,
causes irreversible destruction of certain antigens which
are particularly sen~itive to the customary chemical
fixative~ such as glutaraldehyde or methanol (DROVER et
al., J. Immunol. Methods, 1986, 90, 275-281).
Cell i~munocapture on a solid support is des-
cribed in patent application WO 86/02091, in which the
object is to remove undesirable cells from samples of
bone marrow intended for transplants. In said patent
application, cell capture is effected on floating
microbeads and requires that the antibody used be fixed
to the solid support by a complex macromolecular struc-
~0129~
- 4
ture, called a network-relay, which i~ capable of en-
suring a preferential orientation of the antibody rela-
tive to that of the corresponding cell antigen. Said
patent application gives no indication of an a~plication
~ of the technique to the quantitative assay of a cell
enzyme.
Cell immunocapture i8 also de~cribed in patent
application W0 84/03151 for an analytical application.
In said patent application, the object i~ to identify the
tissue groups to which the examined cells belong (this
operation generally being called HLA typing). Cell cap-
ture is effected by means of antibodie~ arranged accor-
ding to a particular geometry on very specialized sup-
port~ (microscope cover glasses). The results are
obtained simply by visual observation of the support and
produce "all or nothing" responses.
Thus the cell immunocapture systems described
hitherto do not lead to analytical applications permit-
ting the quantitative determination of a marker charac~ *ic
of the cell, and in particular a constituent enzyme of
the cell.
The assay method forming the subject of the
present invention has considerable advantages over all
the tPchniques known and used in the prior art, since it
permits quantitative mea3urement of any endogenous enzyme
of a cell population. Thi~ assay is performed on cells
which have not undergone any chemical or physical inter-
vention at the moment of their specific capture and which
are therefore in their state of physiological integrity.
Furthermore, the assay method according to the invention
ha~ the characteristics of very high specificity which
are inherent in the double recognition systems involving
two different specific markers carried by the sa~e cell,
one being an antigen selected for immunocapture of the
3~ cells which it is desired to analyze, and the other being
~ 2~3~
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an endogenous enzyme present in the cells which have been
captured. This method is simple, rapid and reproducible.
It is totally suitable for the analysis of a large number
of samples, which enables it to be used for diagnostic
05 purposes in clinical biology laboratories handlin8 these
large numbers.
The present invention thus relates to a kit -for
assaying at leas~ one endogenous enzyme characteristic of
a cell population or subpopulation7 said kit comprising
the following components:
a) a solid support to which one or more mono-
clonal antibodies are fixed by covalent bonding or physi-
cal adsorption, said monoclonal antibodieR being directed
against surface antigens of the cell population examined,
and being intended for immobilization, on the support, o~
the cells which include those of the subpopulation pos-
sessing the enzyme to be assayed;
b) a developer for the endogenous enzyme, namely
one or more solutions providing the rea~ent~ (substrate
and, if appropriate, chromogen) necessary for developin~
the activity of the enzyme, and, if appropriate, a
reagent which improves the permeability of the cell mem-
brane; and
c) a ~uffer solution intended for washing of the
2S solid support.
The term "cell" as used in the present descrip~
tion and in the claims which follow encompasses human
cells or animal cells and especially blood cell3~ in-
cluding the platelets.
The assay method according to the invention
applies to whole cells, i.e. non-lyzed cells.
These cells have not undergone any physical or
chemical intervention at the moment of their immuno-
capture and they are used in a state of complete physio-
logical inte~rity. This situation constitutes the be~t
~01%~3~
guarantee of integrity of the antigen used for capture
and of the endogenous enzyme chosen as the assay target.
As the solid support it is possible to u~e any
device suitable for handling cell suspensions, and pre-
05 ferably tubes, particulate magnetic supports or rigid orflexible microtiter plates made of polyethylene, poly-
styrene, polyvinyl chloride or nitrocellulose, which con-
tain microwells. The monoclonal antibodies intended for
immobilization of the cells can be fixed to the solid
supports either by covalent chemical bonding or by
physical adsorption according to the classical techni~ueæ
well known to those skilled in the art, such as the tech-
nigues described by STOCKER and HEUSSER, J. Immunol.
Methods, 1979, vol. 26, p. 87-95. Advantageously, the
support can be saturated with a protein beforehand.
According to the invention, the monoclonal anti-
body or antibodies fixed to the solid support must permit
immunocapture of the cPll population which includes the
cell population or populations containing the enzyme to
be assayed. When this population consists of human
cells, the preferred monoclonal antibodies for immuno-
capture are the anti-class I HLA antibodies which are
specific for the common part of the HLA-A, -B and -C
antigens present on the leukocytes and numerous other
cell lines of the organism. Likewise~ monoclonal anti-
bodies specific for the antigens of the differentiation
classes which ha~e been determined on the leukocytes are
preferred. Of these antibodies, the one called S-class
I, marketed by BIOSYS, is particularly preferred.
In other cases where the cells ~xamined are human
cells and in all cases where these cells are not human
cells, monoclonal antibodies appropriate to the type of
cells examined can also be used for immunocapture accor-
ding to the invention.
The substrate for the endogenous enzyme to be
2~:l2~3~
assayed and the reagents are chosen so that the final
product of the reaction or reaction qe~uence caused by
the enzyme, involving these substances, is:
- either a colored or fluorescent s~bstance which dif-
05 fuses into the liquid medium surrounding the cells andwhich is the ob~ect of the final spectrophotometric or,
respectively, fluorimetric measurement,
- or an insoluble colored substance which deposits on
the cells and the walls to which they are fixed, and
which can ke the object either of photometric measure-
ment by reflection or of visual evaluation, if approp-
riate against a scale of standard shades.
Thus it is not necessary to use a chromogen if
the substrate alone makes it possible to obtain a colored
or fluorescent substance.
A~ an additional component, the assay kit con-
tains a buffer solution intended ~or washing of the solid
support after immobilization of the cell~.
As other additional components, the assay kit can
also contain the samples necessary for standardization
and ~uality con-trol of the assay
The present invention further relates to a method
of assaying the endogenous enzymes of a cell population
or subpopulation, said method consiæting in:
- immobilizing the cell population, which includes the
subpopulation containing the endogenous enzyme, on a
solid support using one or more monoclonal antibodies
fixed to said support beforehand by covalent bonding or
by physical adsorption and capable of recognizing an
antigen present on the surface of the cells;
- observing an incubation period to allow immunocapture;
- washing the solid support to remove the non-immobilized
cells,
- adding the reagent or reagents (substrate and, if
appropriate, chromogen) necessary for developing the
%~ ~ 2~
activity of the endogenous enzyMe; and
- reading the results by measuring the light signals
(coloration or fluorescence) with reference, if approp-
riate, to a standard scale.
05 Thus the developing of the endogenous enzyme to be
assayed, which belongs to the immobilized cell Population
or to one of its subpopulation~s, is effected direct by
means of a substrate specific for this endog~nous enzyme,
after which the actuaI assay of the endogenous enzyme i~
performed by photometric measurement by transmi~sion or
reflection, or measurement of the fluorescencs emission.
The assay kit and method according to the inven-
tion are particularly simple because, when preprepared
immunocapture supports are available 7 they involve only
the solution or solutions containing the specific re-
agents necessary for ~easuring the activity of the enzyme
(substrate and, if appropriate, chromogen).
The assay kit and the enzymatic method according
to the invention are preferably applied to the assay of
the endogenous enzyme~ of the for~ed elem~nt~ of human
blood, especially the leukocytes and more particularly
the granulocytæs.
The assay kit and the enzymatic method according
to the invention make it possible to measure signals
(absorbed or emitted light) which depend both on the
number of cells present in the cell population examined
and on the concentratlon of the endogenous enzyme
measured in these cells. Measurement of the e signals
permits quantitative evaluakion of the activity of the
molecules of this enzyme which are contained in the cells
of the cell population or subpopulation examined.
One application of the invention becomes apparent
if a microtiter plate is chosen as the solid support.
The assay kit and the enzymatic method according to the
invention can then advantageously be used for the assay,
2~2~
on a single plate, of a series of endo~enous enzymes
characteristic of various sub,populations making up the
cell population examined. For thi~ application, it i8
possible on the one hand to take ready-to-u~e microtiter
05 plates to which one or more monoclonal antibodie~ capable
of retaining all the cells of the population examined
have been fixed beforehand, and on the other hand to have
a series of substrates which are each specific for an
endogenous enzyme characteristic of one of the subpopu-
lations to be evaluated. Thus, on one and the samesupport, it is possible to perform the ~uantitative a~say
of all the endogenous enzymes necessary for characteriza~
tion of the chosen subpopulations.
A oase which ma~ be mentioned as an application
of the invention is that of the human leukocytes, for
which there are in particular the subpopulation of cell~
called polynuclear leukocytes or granulocytes.
The presence of human granulocytes can be evalua-
ted in blood or in urine. Thus, in the case of urinary
infections such a~ cystitis or pyelonephritis7 it i~ par-
ticularly valuable to have a simple method of assaying
the granulocytes in urine.
The assay kit and the enzymatic method accordin~
to the invention can be u~ed for assaying granulocyte~.
In this case, specific immobilization of the granulocytes
in the sample examined i~ effected on a solid support and
the assay of an endogenous enzyme characteristic of the
granulocytes is performed using an appropriate substrate.
Myeloperoxidase is an endogenous enzyme of granulocyte~
and its assay is performed u~ing hydrogen peroxide as the
substrate for the peroxidase.
Specific immobilization of the granulscytes is
effected using anti-CD1~ monoclonal antibodies fixed to a
solid support such as, for example, tha wall~ of the
microwells of microtiter plates
2~.2~3~
-- 10 --
The plates prepared in this way can be lyophi-
lized and ~tored, preferably at 4~C. This step can be
carried out on the industrial scale and i-t is thus
possible to have ready-to-use plates for the assay kits
05 which can be applied to the granulocytes in the ~ample of
blood or urine examined.
The samples containing the cell~ to be aæsayed,
which originate from blood or from any appropriate bio-
logical fluid - normal or pathological - in par-ticular
urine, can be used as such or after preparation, especi-
ally after concentration.
Aliquots of the appropriate cell suspen~ion are
brought into contact with the solid support, for example
in tubes containing the microparticulate immunocapture
support or in the microwells of a microtiter plate pre-
pared beforehand. After washing, the solution forming
part of the assay kit and containing the substrate
specific for the endogenous enzyme characteristic of the
target cell population is added.
The time re~uired for immobilization of the cells
is preferably less than or equal to 15 minute~ During
this time, the solid support can be centrifuged to
improve the immobilization of the cells. The solid
support, for example the tube or microtiter plate, is
then washed to remove the unfixed cells
The appearance of a colored or fluorescent pro-
duct is brought about by adding, to the ~olid support to
which the cell population carrying the endogenous enzyme
to be a~sayed has been fixed, a solution containing the
substrate for the enzyme and, if necessary, one or more
auxiliary reagents such that the reaction product which
is finally obtained is either a colored product ~oluble
in the medium, or an insoluble colored product, or a
soluble fluore~cent product, a~ explained earlier. The
light signal coming ~rom the sample~ treated in this way
~0~2~3~
is then measured with the equipment appropriate to each
case, i.e. a tranæmi.ssion or reflection photometer or,
respectively, a fluorimeter~ When the solid support is a
microtiter plate, the light æignal can be read æe~uen-
05 tially in all the wells of one and the æame plate bymeans of automated readeræ commonly used in biology
laboratorie~, æuch asl fvr example, the Titertek plate
reader or the Fluoro~can plate reader for the spectro-
photometric or, respectively, fluorometric reading~.
The reagentæ used to develop the endogenous
peroxidase or the endogenous myeloperoxidase contain
hydrogen peroxide, which is the substrate for the enzyme,
and an appropriate chromogen, for example orthophenylene-
diamine or 2,2 -azino-bis(3-ethylbenzothiazoline-6-
sulfonic) acid, or ABTS, to give a final reaction product
which i9 colored and soluble in the medium, or elæe 3,3'-
diaminobenzidine, 3-amino-9-ethylcarbazole or 4-chloro-
alpha-naphthol to give an inæoluble final reaction pro-
duct, or else parahydroxyphenylpropionic acid to give a
fluorescent reaction product which i3 soluble in the
medium.
In a preferred form, the kit according to the
invention for assaying myeloperoxidase characteristic of
the granulocytes compriseæ:
a) a microtiter plate in whose well~ one or more
anti-granulocyte monoclonal antibodies have been fixed;
bl) a solution containing hydrogen peroxide,
which is the substrate for the enzyme~ in an appropriate
buffer; and
b2) a solut.ion containing the chromo~en used to
develop the expression of the activity of the enzyme and,
if appropriate, a reagent which improves the permeability
of the cell membrane.
In another preferred form, the kit according to
the invention for asæaying myeloperoxidaæe characteristic
2~2~3~
of the granulocytes comprises, as the solid support,
tubes or alternatively particu:Late magnetic supports to
which one or more anti-granulocyte monoclonal antibodies
have been fixed.
05 The results of the assay of the endogenous en-
zymes according to the invention can be expressed accor-
ding to any procedure appropriate to the examination
carried out. More particularly, these results can he
expressed as the total activity of a particular endo-
genous enzyme present in a given volume of the sample
examined (for example per microliter of blood).
The activity of a particular endogenous en~yme in
the sample examined will preferably be determined u~ing a
standard scale consisting of appropriate cells or prepa-
rations containing the endogenous enzyme to be assayed,which will have been calibrated beforehand by a known
reference method. These standards will preferably
consist either of cells identical in their o~igin to the
cells which are to form the subject of the assay, or of
cells of established cell lines containing the de~ired
endogenous enzyme, or of cell-free preparations contain-
ing the chosen enzyme.
These standards are then treated in exactly the
same way as the samples ~o be examined. The resulting
signals are used to build up a standard scale against
which the signal~ measured with the samples to be
examined are compared. The subse~uent calculations are
conventional.
The enzymatic assay method according to the
invention is simple, rapid and reproducible. Its use is
totally suitable for the analysis of a large number of
samples. For an understanding o~ its advantages compared
with the other methods described, the various steps
should be analyzed.
Immobilization of the cells on the solid ~upport
2~12~
- 13 -
is the stage of the assay which usually present~ the mo~t
difficulties or which is the most critical to carry out.
The means often used i8 chemical fixation of the cells
with glutaraldehyde or methanol in cups which may or may
05 not have been treated with poly-L-lysine (VAN LEUVEN F.
et al , J. Immunol. Methods, 1978, 23~ 109). However,
chemical fixations performed in this way can reduce or
even suppress the desired specific detection or, conver-
sely~ can induce false-positive labeling of cells, which
is a very serious disadvantage (DROVER and MARSHALL, J.
Immunol. Methods, 1986, 90, 275-281).
Furthermore, the chemical fi~ation method has to
be carried out in several steps: centrifugation of the
cells, preparation of the fixative mixture, fixation and
then washing of the fixed cells several times.
Drying of the cells at 37C, optionally followed
by fixation with methanol in the microwells, has also
been proposed (BAUMGARTEN, J. Immunol. Methods, 1986, 94,
91-98). Actually, drying of the cells at 37C can de-
grade certain fragile antigens which might be useful forimmunocapture of the cellss as well as the endogenous
enzymes to be assayed.
Furthermore, the reproducibility of this method
is doubtful; in fact, the settling of the cell~ in the
assay microwells and the drying of the cells can vary
from one experiment to the next. Finally, this assay is
lengthy to perform because the cell drying st2p alone
takes more than 2 hours.
The immobilization of lymphocyte populations ha~
also been achieved by using polyclonal antibodies adsor-
bed in microwells (STOCKER and HEUSSER, J. Immunol.
Methods, 1979, 26, 87-95). This method makes it possible
to immobilize cells foreign to the single population
which it is desired to analyze; this also represents a
certain disadvantage.
2~ 3~
- 14 -
The use of highly specific and related monoclonal
antibodies adsorbed on or fixed to the ~olid support, and
especially in the assay wells, in the tubes or on the
particulate support, permits exclusive capture of the
05 desired cells, the other non-retained cell population~
being removed in the course of the washes carried out.
Furthermore, no chemical or physical agent modifies the
characteristics of the antigens in this ~tep because the
various operations for chemical or phy~ical fixation of
the cells to the support are omitted.
Thus, according to the present invention, it has
been found that the immobilization of cells by monoclonal
antibodies is a method which makes it possible to simpli-
fy the step for immobilizing the cells carrying the
enzyme to be assayed, while at the ~ame time making the
results more reliable.
The method according to the invention, which com
prises the use of a procedure for immunocapture of whole
cells without physical or chemical intervention on the
cells, and the measurement, in all or some of these
cells, of the activity of an endogenous enzyme by using
its specific substrate, is the first method to permit the
quantitative assay of the chosen endogenous enzymes on
the cells themselves.
According to the invention, the direct labeling
of immunologically immobilized cells permits:
- a saving of reagents,
- an improved reliability through a reduction in the
number of steps and manipulation3;
- a time saving; and
- the possibility of treating large numbers of samplss at
the same time, exclusively with the use of conventional
equipment and apparatuses.
The time required to immobilize the cell popula-
tion or cell subpopulation to be assayed is short. It is
20:~2~
- 15 -
less than or equal to 15 minutes in the case of the assay
of granulocytes in the blood or urine. Likewisel the
period for assay of the activity of the endogenous enzyme
is less than or equal to 1~ minutes.
05 After the solid support has been wa~hed, the
actual assay is performed by using conventional appara-
tuses to observe a signal which is precise and simple to
measure: light absorptlon or emission.
Thus, overall, the method according to the inven-
tion has numerous advantages: it is rapid, reliable,
economic and simple.
It has been verified that the signals recorded
(photometric measurements) make it possible to obtain
satisfactory uniform ~tandard curves as a function of the
number of cells used, under the customary handling con-
ditions.
In the E~amples below~ the following termq or
their abbreviations will be used indiscriminately:
BSA: bovine serum albumin
PBS: phosphate buffered saline at pH 7.4
EXAMPLE 1
26 LEUKOCYTES OE HUM~N ~LOO~: ASSAY PERFORM~D QN A
Myeloperoxidase is an enzyme of glycoprotein
character compri~ing a heme structure. This enzyme,
which is abundant in the polynuclear leukocytes of human
blood, is involved ln the bactericidal and antimicrobial
functions of calls (KLEBANOFF S.J., J. Bacteriol., 1968,
95, 2131; DIAMOND et al., J. Clin. Invest., 1580, ~,
908-917).
The assay of myeloperoxidase in the polynuclear
3~ leukocytes, according to the mæthod of the invention,
3~
- 16 -
comprises three steps carried out in ~ucce~sion:
1. separation of the polynuclear leukocytes from whole
blood,
2. capture and selective immobilization of the cella by
05 means of a monoclonal antibody adsorbed beforehand
in the microwells of a microtiter plate, and
3. developing of the activity of the cell enzyme.
a) Preparation of the plate
The plate used i~ a plastic microtiter plate con-
taining 96 microwells, marketed by NUNC (reference
64394). Each microwell receives 200 ~1 of a solution
containing the purified anti-CD15 monoclonal antibody
(called SMY 15a) used to immobilize the polynuclear
leukocytes, i.e. to effect their immunocapture. Thi~
antibody~ marketed by BIOSYS, Compiègne, France~ under
the reference SMY 15a, i5 used at a concentration of 5
.g/ml in a phosphate buffered saline (PBS) at pH 7.4.
The adsorption of the monoclonal antibody i~
effected at 4C for 12 hours. The excess antibody is
removed by turning the plate over.
A solution containing 0.1% of gelatin and 0.3% of
BSA in a phosphate buffered saline is prepared. 250 ~1
of this solution are introduced into each microwell ~o as
; to saturate the surface of the wells with protein, which
takes 1 hour at 37C, the plates are washed 3 times with
phosphate buffered saline. The plates prepared in this
way are stored at 4C.
b) Separation of the polynuclear leukocytes
The blood sample is taken on an anticoagulant
~heparin). 2 ml of MONO-POLY separating medium (FLOW
ref. 16.980-49) are introduced into a 5 ml hemolysis tube
and 2 ml of blood are deposited on top. The tube is then
centrifuged at 400 x g for 40 minutes at laboratory tem-
perature. Two cell suspension rings are formed; the
lower ring contains the polynuclear leukocytes, which are
2~ 2~3~
recovered with a micropipette
c~ Cell immunocapture
Cell capture is effected by means of the adsorbed
anti-CD15 monoclonal antibody
05 100 ~l of the cell suspension, adjusted to 5 x
10~ cells per ml of PBS, are introduced into the micro-
wells of the plate. To improve the fixation of tha cells
on the support, the plate is centrifuged for 3 minutes at
150 x g after a wait of 10 minutes at room temperature.
d) Developing and measurement of the myeloperoxi-
dase
The microwells are emptied by turning the plate
over. TheY are washed twice with 200 ~l of PBS, making
it possible to remove the undesirable cell populations,
such as the monocytes or erythrocytes, which can cause a
~purious absorbance signal. After the second wash, 100
~l of the developing reagent~ prepared for immediate use~
are added to each well.
The developing reagent is obtained in the fol-
lowin~ manner: a 0.1 M citrate buffer i~ prepared by dis-
solving citric acid monohydrate in water to give a 2%
solution and adjusting the pH to ~ by the addition of 7 N
sodium hydroxide solution. A 2% solution of hexadecyl-
trimethylammonium bromide (= CETAB, TOUZART and MATIGNON
T 5650) in the buffer is then prepared. The reagent
renders the cell membranes permeable and improves the
activity of the myeloperoxidase on the ~ub~trate. 30 mg
of orthophenylenediamine dihydrochloride and then 40 ~l
of hydrogen peroxide (substrate for the enzyme) are added
to 20 ml of this solution before use
After incubation for 10 minutes, the absorbance
is measured on a spectrophotometer at 4~0 nm (type 310 C
Titertek Multiskan apparatus - Flow Laboratories).
~or one experiment 7 the absorbances obtained with
50,000 cells are given in Table 1 below:
3 ~
~hE~
CELLS ONLY CELLS CELLS
+ reagents ~ reagents
05 r _ without CETAB with CETAB
¦ Absorbance0.003 0.370 1.545
The presence of CETAB in the developing medium
produces a 4-fold increa~e in the optical denslty due to
the reaction catalyzed by myeloperoxidase, making it
possible to reduce the number of cells required for the
assay.
5 EXAMPLE 2
ASSAY OF MYELQPERQXIDASE IN THE POLYN~lCLEAR
l,EUKOCYTES OF BLOOD AND URINE: A~SAY PERFORME~ ON
This Example is intended to show that the time
required to assay an endogenous enzyme can be reduced,
compared with the conditions described in Example 1, by
modifying the cell immunocapture process. The support
used to capture the cells consists of magnetic beads
known as DYNABEADS. The beads (ref. DYN-llOOl, marketed
by BIOSYS) carry anti-mouso immunoglobulin antibodies on
their surface. Before use, the beads are treated with a
solution of anti-CD15 monoclonal antibodies (ref. SMY
15a - BIOSYS, France) for 12 hours at 4C. This is done
by mixing 25 ~l of the suspension of beads with 0.5 ml of
antibody solution containing 100 ~g~ml of PBS. The beads
are subsequently washed 3 times with PBS and then satu-
rated for 12 hours at 4C with a 0.3% solution of bovine
serum albumin (BSA). The read~-to-use beads are kept at
4~C.
The myeloperoxidase is assayed in a 5 ml hemo-
2012~3 !~
- 19 -
lysis tube, 300 ~1 of PBS, 25 ~1 of the suspension of
beads and 100 ~1 of whole blood taken on lithium hepari-
nate (VACUTAINER tube ref. 606 484) are introduced into
the tube. After 5 minutes of gentle shaking, the beads
05 are separated off by means of a magnet and the cells
fixed to the beads are washed with PBS (5 washes).
The myeloperoxidase is developed with a mixed
reagent containing the substrate (hydrogen peroxide) and
the chromogen (orthophenylenediamine), prepared as in
Example 1, with or without the incorporation o~ CETAB.
The absorbance is measured under conditions identical to
those of Ex~mple 1. The results are given in Table 2.
Measurement of the myeloperoxidases in the
granulocytes present in urine during urinary infections
is an important diagnostic tool for pyelonephritis and
pyuria. It was shown in the second part of the Example
that magnetic particles carrying the same anti-CD15
monoclonal antibody are capable of capturing the poly-
nuclear leukocytes in the urinary fluid. The assay is
performed on 400 ~1 of urinary fluid containing about 2 x
105 granulocytes per ml. The cells are captured with 25
~1 of the suspension of beads and then treated under the
same conditions as in the previous Example. The results
are given in Table 2 below.
~LEæ
ABSORBANCE BEADS + BEADS + BEADS -~ BEADS
developing CELLS CELLS -~ CELLS ~
reagentonly developing developing
reagent reagent
without with
CETAB CETAB
Whole n_ _ _
blood 0.0250.011 0.0323 1.324
. __ ... __ _ _._.
Urine 0.0250.008 0.286 1.115
2~12~3~
- 20 -
According to the method described in thi~
E~ample, the assay was performed direct on the blood
sample and then on the urine sample without the need for
prior separation of the cell population to be analyzed.
05 Furthermore, the incubation period is only 5 minutes.
Thus, by using magnetic bead~ as the solid support, the
assay operations taken as a whole are particularly simple
and rapid.
