Method for quantitatively determining the degree of agglutination of particles

A method and apparatus for quantitatively determining a degree of agglutination of a suspension of particles or the presence of the agglutination thereof is disclosed, wherein a liquid which contains agglutinated clots, otherwise a substance or substances to be about to agglutinate is made to slowly transfer through a small tube, in course of which agglutinated clots and non-agglutinated particles separate from each other in the liquid, when a degree of agglutination of particles and the concentration of the substance to be tested herewith can be determined quantitatively through the detection of difference in the optical properties of both the accumulation layer of agglutinated clots and the suspension layer of non-agglutinated particles, or the change in the optical properties of either of the above two layers, in particular the suspension layer.

BACKGROUND OF THE INVENTION 
This invention relates to a method and apparatus for quantatively 
determining a degree of agglutination of particles with the help of 
optical means and more particularly is concerned with the optical 
measurement of the state of agglutination of particles while making them 
agglutinate through the antigen-antibody reaction by using sensitized 
particles being coated with an emulsion containing either antigen or 
antibody. 
The cognition of the state or existence of the agglutination in a 
suspension of particles is helpful to grasp the chemical or mechanical 
properties, stability, and reactivity of it. 
In particular, the agglutination reaction--in which sensitized particles 
such as latex, bentonite, or kaolin being coated with an emulsion 
containing either antigen or antibody are made to react with the other 
inversely corresponding antibody or antigen being possibly contained in a 
liquid to be tested--is being put to practical use as a simple and easy 
measuring method of immunochemical ingredients. Best of all, the 
agglutination reaction carried on in latex composed of polystyrene and the 
like is serviceable especially at the time of measuring various sorts of 
proteins and hormones including RF (rheumatoid factor), CRP (c-reactive 
protein), and others because of the detecting sensitivity of high grade 
with an excellent specificity. 
For the time being, however, the measurement of the agglutination reaction 
of such a kind of latex is made as a rule depending on the decision of the 
presence of the latex which has agglutinated through the antigen-antibody 
reaction in making the sensitized latex liquid react with the liquid to be 
tested usually on slide glass or plate, on the basis of eye measurement, 
otherwise on some occasion by the use of optical means. The results of 
measurement in both cases mentioned above may be stated as having done 
throughout in a qualitative way. Especially, in the case of eye 
measurement, it is ready to cause each time the obscurity in deciding the 
presence of the agglutination. 
Further, in the case of resorting to the optical measuring method, and 
aiming at the quantitative measurement at that, it is necessary to prepare 
a series of test objects having been diluted in stages, observe the 
presence of the agglutination about each sample out of the series while 
manipulating the reaction, and take a measure to indicate the quantity of 
antigen or antibody by detecting which stage of dilution forms the 
boundary layer discriminating the state of agglutination from the state of 
non-agglutination. Such being the case, the procedure as mentioned above 
has such a marked imperfection that needs much labor and time or a great 
deal of latex reagent at the time of measuring even one sample. 
On the other hand, there is a way of measuring the light transmissivity of 
the liquid in the cell by means of a light of wave length belonging to the 
near infra-red region with the object of quantatively determining a degree 
of agglutination. In this case, quite a hard point to settle lies in that 
inasmuch as the clots of agglutinated latex and the other particles of 
non-agglutinated latex coexist within the observation area of the example, 
the measurement of the transmissivity, for example, cannot help being done 
on the mixed system of the clots of agglutinated latex and the particles 
of non-agglutinated latex, as a result bringing about the drop in its 
sensitivity. It makes no difference even if some scattered light would be 
used. This method has another defect that the reproducibity of the 
measurement done is not good because the agglutinated clots are scattering 
distributed. 
Further, there is also a method for quantitatively determining a degree of 
agglutination in which the agglutination of latex is made to carry on in a 
suitable vessel, the agglutinated clots are made separated from the 
non-agglutinated particles of latex by means of the centrifugal 
precipitation of the reactant liquid, and then the transmissibity of its 
supernatant liquid comes to be measured. In this case, however, the 
provision of a centrifugal separator and a relative operational process 
are required, all the more the measurement becoming complicated. 
In addition to the above, there is still more known a method for detecting 
the presence of the agglutination through the measurement of the viscosity 
with regard to common latexes in the field other than the immunochemistry. 
This method also involves various problems such as the consumption of a 
comparatively great deal of samples, the intricacy in the temperature 
control and working curves, and so on, therefore being not able to expect 
the exactitude of the measurement. 
SUMMARY OF THE INVENTION 
It is an object of this invention is to eliminate the above-mentioned 
defects and provide a method for quantitatively determining a degree of 
agglutination of a suspension of latex, or, more generally, common 
particles with the objective reproducibility of the agglutination reaction 
or the state of agglutination thereof. Another object of this invention is 
to provide a method by which the measurement can be performed 
automatically, and an apparatus which is materialized in a simple 
construction for applying the same with ease. A further object of this 
invention is to provide a method for quantitatively measuring the 
immunochemical ingredients on the basis of the antigen-antibody reaction, 
and an apparatus on which the above measurement is to be performed 
automatically and continuously. 
These objects of the invention will be achieved by observing and measuring 
the difference in the optical properties between the both layers of the 
suspension or the change in optical properties of either layer thereof 
which has been separated into agglutinated clots and non-agglutinated 
particles while the suspension is made to transfer slowly through a small 
tube by taking advantage of the transfer characteristic of the suspension 
flowing through the small tube. 
The other objects than the above will become obvious from a reading the 
description in detail given hereinafter.

DETAILED DESCRIPTION 
Description will be now directed to the details of the invention with 
reference to the accompanying drawings while taking the case of adding a 
test sample, which contains antibody or antigen as a substance to be 
tested, to a sensitized latex which is used as a suspension of particles. 
Incidentally, the case mentioned above implies that the suspension of 
particles is used in the capacity of reagent. Contrary to this, it is also 
possible to detect the substance to be tested which is contained in the 
suspension of particles by the use of a different kind of reagent. The 
example of the latter case, however, was omitted in the following for the 
simplification of explanation. 
The flow velocity of the fluid flowing through a small tube is more rapid 
in the vicinity of the center of the section, and becomes slower the more 
it nears toward the wall of the tube, under the influence of the viscosity 
and the friction between the tube wall and the fluid. It is known that 
when the flow velocity is small and the laminar flow is formed, the 
distribution of the flow velocity within a circular straight tube takes a 
form of parabola, as shown in FIG. 1, wherein let the radius of the tube 
be "a" and the maximum flow velocity be V.sub.M, then the flow velocity V 
at the spot remote from the center of the tube by a distance r is equal to 
V.sub.M (1-r.sup.2 /a.sup.2), and the average flow velocity V is equal to 
1/2V.sub.M. 
Now, when a latex reagent which is sensitized with antigen or antibody is 
allowed to react with a test sample containing antibody or antigen in the 
interior of a reaction vessel, a prescribed quantity of the reactant 
liquid (30) thus created is sucked into a small tube (1), as shown in FIG. 
2, being sandwiched in between the front and rear air layers (2), and the 
vacuole (3) of the reactant liquid (30) is made to transfer at a low speed 
in the direction of the arrow, then the distribution of the flow velocity 
of the reactant in the central part of the vacuole (3) becomes 
approximately as shown in FIG. 1, when the vacuole has a more rapid speed 
the more it nears toward the central part. However, there are generated 
vortexes at the front and rear of the vacuole (3) contacting with the air 
layers (2), so that the liquid close to the tube wall is caught up into 
the central part at the meniscus part (3b) of the rear, thereby moving on 
forward at a speed more than the average flow velocity. 
When the sensitized latex particles (4) will not agglutinate, then those 
particles transfer for the most part along the liquid current in the 
direction of the arrow, the particles which have moved on toward the front 
part of the vacuole (3) are caught up into the vortexes at the front 
meniscus part (3a) to shift to the side of the tube wall, and after that 
turn relatively backward. Consequently, the state of suspension becomes 
uniform almost throughout the whole vacuole, and present the behavior as 
if the reactant liquid were transferring while being merely mixed by 
stirring. 
On the contrary, when the agglutination reaction proceeds and a good many 
agglutinated clots (5) . . . come to grow, then several phenomena distinct 
from the above are perceived as follows: 
When the reactant liquid (30) in which the agglutinated clots (5) are 
growing, or the reactant liquid (30) which contains the agglutinated clots 
(5) from the beginning, is made to be sucked into the small tube (1) and 
subsequently to move on slowly forward (FIG. 3(a)), then the reactant 
liquid (30) in the vacuole (3) transfers while being stirred similarly to 
the case of FIG. 2, but the distribution of the agglutinated clots (5) . . 
. will not become uniform, and it is perceived that the agglutinated clots 
(5) . . . go to concentrate and accumulate on the front part of the 
vacuole (3), as shown in FIG. 3(b) and (c). This is for the reason that 
the agglutinated clots (5) of latex entwine each other easily to 
agglutinate, wherein the agglutinated clots (5) . . . which have ridden on 
the rapid flow in the center of the small tube (1) collide while rotating 
with the other agglutinated clots (5) . . . on the way, grow to the larger 
agglutinated clots, and finally reach the front part of the vacuole (3) 
(FIG. 3(b)). The agglutinated clots (5) . . . having reached the front 
part of the vacuole (3) come to accumulate thereat while losing their 
rapid speed without being caught up into the vortexes at the meniscus part 
(3a) of the front because of their largeness in sige and 
agglutinativeness. Against and to these agglutinated and accumulated clots 
strike and adhere the newly following agglutinated clots from behind to 
form a larger aggregate of agglutinated clots at the front part (FIG. 
3(c)). 
If the vacuole (3) is made to transfer in a state of having extended the 
length of the small tube (1) so as to take more hours in its shifting 
movement, the agglutinated clots (5) . . . come to gather consecutively at 
the front part. In the interim state until the complete separation, the 
concentration gradient of the agglutinated clots (5) . . . is already 
generated along the progressive direction of the vacuole (3), where the 
concentration is higher the more it lies near the front part of the 
vacuole (FIG. 3(b)), finally the reactant liquid coming to be separated 
into two layers (FIG. 3(c)). The front layer is an aggregate or an 
accumulation layer (51) of agglutinated clots (5) . . . . The rear layer 
is a suspension layer (41) of non-agglutinated particles (4) of latex. 
With the lapse of a proper time of agglutination, the accumulation layer 
(51) of agglutinated clots in the front becomes thicker and thicker as the 
agglutination reaction proceeds with the increasingly high degree of 
agglutination, in proportion to which a large number of latex particles 
(4) is consumed for it. Consequently, in the rear, the suspension layer 
(41) composed of non-agglutinated particles (4) of latex lowers its 
concentration correspondingly. 
This invention aims at optically measuring the concentration of the 
reactant liquid (30) in the vacuole (3) composed of both the accumulation 
layer (51) of agglutinated clots and the suspension layer (41) of 
non-agglutinated particles of latex, and subsequently quantitatively 
determining a degree of agglutination at the time of the agglutination 
reaction of latex through the observation of the behavior of the change in 
the concentration to be obtained in the foregoing procedure. Referring 
further to an embodiment of this invention, the quantitative determination 
of a degree of agglutination is performed also here by optically measuring 
the concentration of the suspension layer (41) in the rear part of the 
vacuole. 
In the above case, although the vacuole (3) was sandwiched in between the 
air layer (2). (2) from both front and rear sides, respectively, it is 
permissible to use instead of air some other fluid not mingling with the 
reactant liquid, for example, such as this and that inert gases, silicone, 
and the like. As for the reactant liquid (30) to be introduced into the 
small tube (1), it may be the one as of being prepared in advance by 
mingling the latex reagent with the tests object, as mentioned above, or 
else it will do if the latex reagent and the test object are fed 
separately into the small tube to mix each other therein, and are made to 
react with each other while being transferred. 
FIG. 4 shows a schematic drawing of an apparatus for quantitatively 
determining a degree of agglutination by means of the above-described 
method. This apparatus (10) comprises a suction nozzle (11), a small tube 
(1) being connected to the nozzle (11), an optical measuring means (12) 
being provided on the side part of the small tube (1), a pump (13) sucking 
a fluid into the small tube (1), and a waste fluid receptacle (14). 
As a start, when a fixed quantity of the reactant liquid (30) in a reaction 
vessel (15) is sucked by the sucking action of the pump (13) throught the 
nozzle (11), then the nozzle (11) is lifted up out of the reactant liquid 
by a nozzle-driving device (16), and subsequently it sucks up the air. 
In this case, it is a matter of course that some inert gas or silicone can 
be sucked in place of air. The reactant liquid (30) forms a vacuole (3) 
being sandwiched in between air layers (2). (2) from behind and before in 
the interior of the small tube (1) being connected to the nozzle (11), it 
is moved on slowly in the direction of the arrow by the sucking action of 
the pump (13), and it reaches the optical measuring means (12) being 
provided on a part of the system of the small tube (1). This optical 
measuring means (12) comprises a light source (121) and a light detector 
(122) for measuring the transmitted light. During the intervening time, 
the agglutination reaction of the reactant liquid (30) in the vacuole (3) 
is proceeding (or it does not matter if the reaction might be brought to 
completion in a sample container). The agglutinated clots (5) accumulate 
at the front part of the vacuole (3), as shown in FIG. 3, on the back of 
which the non-agglutinated particles (4) of latex become suspended in the 
rear reactant liquid. As described above, the reactant liquid (30) in the 
vacuole (3) shifts while being stirred, so that the non-agglutinated 
particles (4) of latex in the rear suspension layer (41) are distributed 
almost uniformly, the concentration of which corresponds to the degree of 
agglutination of the agglutinated clots of latex. The optical measuring 
means (12) is for use in measuring the concentration of the reactant 
liquid consisting of the accumulation layer (51) and the suspension layer 
(41). 
In this connection, when different kinds of reactant liquids (30) . . . are 
made to be sucked up successively for the purpose of enhancing the 
efficiency of measurement, it is preferable to dip the nozzle (11) in a 
wash bowl (18) to such wash liquid (19) in order to prevent the 
contamination every time of sampling, whereby one to several pieces of 
vacuoles (not shown) are able to be fed into the small tube (1). In the 
figure, reference number (20) indicates a waste liquid. 
When the shifting vacuole (3) is observed by the optical measuring means 
(12) with the aid of transmitted light, then the curves as seen in FIG. 
5(a) are obtained, whereat Steps A . . . denote the transmissivities of 
the respective air layers (2) . . . , and Steps B to F indicate the 
transmissivities of the reactant liquid in the vacuole temporally passing 
the respective steps. 
Out of Steps B to F, the first Steps B shows the transmissivity in the case 
where the reactant liquid did not or does not yet begin the reaction of 
agglutination, that is, in the case where the particles of latex is not at 
all consumed for the reaction of agglutination. This transmissivity is 
uniform trough the whole vacuole. In Step C, the reaction of agglutination 
is proceeding to some extent, and the accumulation of the agglutinated 
clots (5) is perceived faintly at the front part of the vacuole, where the 
transmissivity starts to lower little by little, while on the other hand 
the transmissivity of the rear suspension layer is rising in some measure 
(Corresponding to FIG. 3(a)). Step D shows an sample in which the reaction 
of agglutination is in an advanced stage, and where the accumulated layer 
(51) of agglutinated clots in the front is increasing more and more, while 
the transmissivity of the suspension layer (41) in the rear is arriving 
near to that of the air layer (2) (Corresponding to FIG. 3(b)). Steps E 
and F show separately the transmissivities of the reactant liquid having 
displayed the reaction of agglutination stronger than in the former step. 
In each of these latter two steps, with the increasingly advanced 
accumulation of agglutinated clots in the front part is exhibited the 
transmissivity of the suspension layer (41) in the rear to be of the 
higher value than that of the air layer (2) (Corresponding to FIG. 3(c)). 
Further, Steps F' and F" shown in FIG. 5(b) each give the transmissivities 
in the cases where the reactant liquid of the lower degree of 
agglutination is completely divided in two parts, as compared with the 
case of F in FIG. 5(a). 
Now, seeing from the graphs shown in FIG. 5(a) and (b), it is thinkable 
that there may be a variety of methods serviceable for the purpose of 
obtaining a degree of agglutination of the reactant liquid through the 
utilization of the transmitted light as follows: 
To begin with, for example, after the lapse of time sufficient to 
completely divide the reactant liquid in the vacuole into two layers: The 
accumulation layer (51) of agglutinated clots and the suspension layer 
(41) of non-agglutinated particles (Step F in FIG. 5(a), and Steps F' and 
F" in FIG. 5(b)), the output pattern, otherwise the transmissivity of 
either of the accumulation layer (51) of agglutinated clots and the 
suspension layer (41) of non-agglutinated particles is made indicated in a 
displaying device (17) shown in FIG. 4. If adopting the output pattern, it 
is possible to judge not only the unevenness of the graph but also the 
length of duration (time) of the state in question. 
On the other hand, if with the invention of using the transmissivity, it is 
commendable in the present example to take advantage of the 
transmissivities on the side of the suspension layer (41) because the 
values of the transmissivities of the accumulation layer of agglutinated 
clots make little difference among themselves, therefore the resolving 
power here being bad. On the displaying device (17), however, it is also 
possible to indicate the degree of agglutination into which the 
transmissivity has been converted. 
Besides the above, it is also conceivably possible that there may be a 
different method by which the measurement can be made even within a 
certain period of reaction time after the sample starts to react, but not 
until the reactant liquid is completely divided into two layers. In this 
case, it is possible to find a degree of agglutination kinetically also on 
the basis of the observation of the behavior of the change in a plurality 
of transmissivities of the suspension layer (41) measured at regular 
intervals of time, what by arranging the optical measuring means (12) in 
sets of more than one pair of light source (121) and light sensor (122) 
(the case of FIG. 4), or what by making the light source (121) and the 
light sensor (122) move along the small tube (1). Such a measure of 
pluralization or mobilization of the light source (121) and light sensor 
(122) can be applied to the case where the measurement will be performed 
in the complete division of the reactant liquid into two layers. 
The above description is concerned with the case where the reactant liquid 
(30) prepared beforehand is introduced as it is into the small tube (1). 
However, it is also permissible to supply the latex reagent (31) 
constituting the reactant liquid (30) and the test object (32) containing 
antibody or antigen separately into the small tube (1), to make the both 
mix with each other therein, and further to cause the mixture to react 
while shifting therethrough. In this occasion, there are sucked through 
the nozzle (11) successively the latex reagent (31) (latex particles being 
made sensitized with antigen or antibody) in a reagent vessel (21), the 
test sample (32) (liquid to be tested possibly containing antibody or 
antigen) in a test sample container (22), and the wash liquid (19) in the 
wash bowl (18). At this time, however, it must be mindful so as not to 
suck the air between the latex reagent (31) and the test sample (32), but 
so as to suck a sufficient amount of the air in order that a suitable air 
layer is formed between the above both and the wash liquid (19). By doing 
so, the reagent (31) and the test sample (32) mix with each other 
progressively in the upper part of the nozzle (11) and subsequently in the 
small tube (1) connected thereto, going to the form of a vacuole (3) of 
the reactant liquid (30). 
In the following, the same action to be repeated on exchanging a new test 
sample for the old one on each occasion will enable the continuous 
measurement of a number of test samples. 
FIG. 7 shows also an example where a branch pipe having two suction ports 
is used in the stead of the suction nozzle (11). In this case, the latex 
reagent (31) and the test object (32) are separately sucked and then flow 
together on the way to form the reactant liquid (30). To remark in 
passing, arrangement is to be made for the washing of the suction nozzle 
(11) so as to be able to be done only on the side of the test sample, or 
else, if necessary, on both sides of the agent and the test sample at the 
same time. It need scarcely be said that if a plurality of test sample 
containers (22) are mounted on an intermittently rotating sample stand 
(23), it becomes possible to automatically measure a number of test 
samples continuously. 
In this way, if the latex reagent (31) and the test sample (32) are gotten 
ready to be sucked separately in a non-miscible state, it is feasible to 
save the trouble of mixing and stirring them in advance of sucking, and 
further to fix the time which is taken after the mixing of the reagent and 
the test sample at the confluence point until the beginning of the 
measurement whereby temporal change in the state of agglutination should 
be advantageously grasped with ease. What is more, if adopting such a 
design as shown in FIG. 7 capable of performing the automatic and 
continuous sucking action, it is also possible to promote a splendid 
labor-saving, without the necessity of constantly attending thereupon. 
In all of the above-mentioned examples, it was so devised that the 
transmissivity may be measured in the state where the vacuole (3) is 
slowly shifting through the small tube (1) as it is, but it will do if the 
transmissivity only of the suspension layer (41) is so made as to be 
measured stopping the stream for a while at the time of the measurement. 
On the other hand, in the case of the optical measuring means (12) being 
provided apart from the small tube (1), it will be also at liberty to 
remove the small tube (1) after the stoppage of the stream, mount it on 
the above means, and then submit it to the measurement. 
In the above examples, although description has been made as regards the 
cases of applying the transmissivities, it goes without saying that the 
measurement is able to be carried out also by employing the scattered 
light or the reflected light. As for the small tube (1) for use in this 
invention, it is general a typically cylindrical object, and yet the 
cross-section of it is not always limited to a circle but it can take 
other forms such as an oval, a square or the others. It is further 
allowable to use not only a straight pipe but also a coil-like one being 
wound so as to exhibit somewhat a stirring effect. 
The description heretofore was concerned with the agglutination reaction of 
the latex particles being made sensitized with antigen or antibody. It is 
to be construed, however, that our invention can be applied extensively 
beyond the above scope to various cases, for example, such as a case where 
some mechanical or physical action is added to the suspension of common 
particles, a case where different kinds of reagents or substance to be 
tested are added thereto in order to measure the reactivity, the stability 
or the others, and so on. 
As described above, this invention is such a one as to divide a suspension 
or reactant liquid of particles into two parts: an accumulation layer of 
agglutinated clots and a suspension layer of non-agglutinated particles 
while making it slowly shift through a small tube, to optically measure 
the concentration of the reactant liquid consisting of the accumulation 
layer of agglutinated clots and the suspension layer of non-agglutinated 
particles, and thereby to determine a degree of agglutination of 
particles, in the process of which the measurement can be performed 
quantitatively with an excellent objective reproducibility. 
With the above arrangement, our invention need not dilute one and the same 
sample in stages each time and does not require any complicated operation 
such as the centrifugal separation and others, as a result of which it 
makes possible to continuously measure a great number of samples and that 
at the same time to settle such kinds of problems as the lowering in the 
measurement sensitivity and the badness in the reproducibility as are 
generated at the time of the measurement being conducted in a mixed state 
of the agglutinated clots with the non-agglutinated particles, whereby the 
immunochemical ingredients can be measured with simplicity and exactitude 
through the observation of the agglutination reaction of sensitized latex.