Method and apparatus for inspecting a specimen by optical detection of antibody/antigen sensitized carriers

In a method and an apparatus wherein carriers sensitized by an antibody or an antigen are caused to react to a specimen sample and the condensation of the carriers caused by the antigen-antibody reaction is optically detected by the use of flow cytometry, thereby measuring the antigen or antibody in the specimen sample, a plurality of kinds of carriers differing in optical characteristic are sensitized by different kinds of antibodies or antigens, respectively, whereby a plurality of kinds of antigens or antibodies in the specimen sample are measured at a time.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to the field of antigen-antibody reaction 
measurement using a carrier such as latex which is utilized in the field 
of immunology. 
2. Related Background Art 
Heretofore, as an immunity inspecting method, use has been made of a method 
in which a suspension comprising a mixture of a carrier such as latex 
sensitized by a predetermined antibody and a specimen sample is prepared 
and where an antigen to be specified is contained in the specimen sample, 
an antigen-antibody reaction occurs between the antigen and the sensitized 
antibody and carrier particles are coupled together and the presence of 
the antigen in the specimen sample or the amount of the antigen is 
measured from the condensed state of the carrier. In that case, a method 
of discriminating the condensed state of the carrier has been carried out 
by measuring the absorbance of the suspension including the carrier or the 
degree of light scattering of the suspension. Particularly, by the use of 
flow cytometry, that is, by wrapping the suspension in sheath liquid, 
hydrodynamically converging it, causing individual carriers to flow to an 
inspecting position in succession, applying a light beam to the carrier at 
the inspecting position and judging the size of the carrier from the 
intensity of scattered light, it has been possible to judge the condensed 
state of the individual carriers and calculate the presence of the antigen 
or the amount of the antigen and accomplish highly accurate measurement. A 
specific example of it is described, for example, in U.S. Pat. No. 
4,521,521. However, in the above-described example of the prior art, only 
a carrier sensitized by one kind of antibody can be used and only the 
inspection of one kind of antigen can be effected at a time, and this has 
formed a hindrance in enhancing the efficiency during mass medical 
examination. 
As an example of the method of solving this problem, a method in which a 
plurality of kinds of fine particles differing in the kind of fluorescence 
or the particle diameter are used to measure a plurality of kinds of 
antigen-antibody reactions at a time is described in Japanese Laid-Open 
Patent Application No. 62-81567. According to this method, discrimination 
between the kinds of the fine particles is done by the fluorescence 
wavelength or a combination of the fluorescence wavelength and the 
particle diameter. If the particle diameter is only made to differ for a 
plurality of kinds of particles, the kinds of the particles cannot be 
distinguished in the case of a condensed state of the same size and 
therefore, it is necessary to distinguish between them by the 
fluorescence. However, the intensity of the fluorescence which occurs is 
weak and therefore, it has been difficult to reliably discriminate between 
the kinds of the particles from the fluorescence. Further, with an optical 
system for detecting scattered light, a precise optical system for 
detecting the weak fluorescence has been separately necessary. 
SUMMARY OF THE INVENTION 
It is an object of the present invention to provide a specimen inspecting 
method and a specimen inspecting apparatus which can simply measure the 
presence or the amount of a plurality of kinds of particular antigens or 
antibodies in a specimen at a time by the use of a plurality of kinds of 
carriers differing in optical characteristic. 
It is another object of the present invention to provide a specimen 
inspecting method and a specimen inspecting apparatus which can reliably 
measure the presence or the amount of a plurality of kinds of particular 
antigens or antibodies in a specimen at a time by a simple construction 
without the use of fluorescence.

DESCRIPTION OF THE PREFERRED EMBODIMENT 
In the present embodiment, a plurality of kinds of latexes differing in 
absorbance are used as a plurality of kinds of carriers. However, this is 
not restrictive, but use may also be made, for example, of inorganic 
oxides such as silica, silica-alumina and alumina, mineral powder, metals, 
blood cells, staphylococci, cell-wall pieces, liposome, etc., and these 
may be combined to prepare a plurality of kinds of carriers differing in 
absorbance. To sensitize the carriers by an antibody, there are known a 
method of causing the antibody to be physically adsorbed, and a method of 
causing the antibody to be chemically adsorbed by the utilization of the 
functional groups on the carriers. 
As an example, a sample fluid in which a specimen sample such as man's 
serum is added to a mixture of a plurality of kinds of latexes A, B and C 
sensitized by three kinds of antibodies a, b and c, respectively and is 
adjusted to an appropriate reaction time and concentration is put into the 
sample fluid container 16 of FIG. 1(A). A specific example of adjusting 
the sample fluid is described, for example, in the aforementioned Japanese 
Laid-Open Patent Application No. 62-81567. 
Also, a sheath fluid such as distilled water or physiological saline 
solution is put into a sheath fluid container 14. The sample fluid 
container 16 and the sheath fluid container 14 each are pressurized by a 
pressurizing mechanism, not shown. In a flow cell 4, the sample fluid is 
wrapped in the sheath fluid and converged into a fine flow by the laminar 
sheath flow principle, and passes through the substantially central 
portion of the through-flow portion in the flow cell 4. At this time, the 
individual latexes contained in the sample fluid are separated and flow 
one particle by one or one lump by one. A laser light emitted from a laser 
source 1 is converged into an arbitrary shape by a set of cylindrical 
lenses 2 and 3 whose directions of bus line are the direction of the 
through-flow portion and orthogonal to the direction of the through-flow 
portion and is applied to the flow of the latexes. The shape of the light 
beam applied to the latexes should preferably be an elliptical shape 
oblong relative to the flow. This is for the purpose of causing the light 
beam to be applied to the latexes with a uniform strength even if the 
positions of the flows of the individual latexes fluctuate. 
When the light beam is applied to the latexes, scattered lights are 
produced. Of the scattered lights, forward scattered light produced in the 
forward direction of the optical path is received by a condensing lens 5 
and a photodetector 6. In order to prevent the applied light beam from 
directly entering the photodetector 6, a light-absorbing stopper 17 is 
provided in the optical path forwardly of the condensing lens 5, and the 
direct light from the irradiating light source and the transmitted light 
transmitted through the carriers are removed. Thus, only the scattered 
light from the carriers can be received. Where the transmitted light 
transmitted through the carriers is necessary as will be described later, 
the stopper 17 is obliquely provided as a reflective small mirror in the 
optical path as shown in FIG. 1(B), and the transmitted light can be 
obtained by the light reflected by the small mirror 17 being received by a 
photodetector 18. Alternatively, an apertured mirror 22 may be obliquely 
provided as shown in FIG. 1(C) and scattered light reflected thereby may 
be received by the condensing lens 5 and the photodetector 6 and 
transmitted light transmitted through the aperture may be received by a 
photodetector 18. 
Also, of said scattered lights, laterally scattered light produced in the 
lateral direction orthogonal to the optical path is condensed by a 
condensing lens 7, is reflected by a dichroic mirror 8 and is received by 
a photodetector 11. Generally, the direction in which the laterally 
scattered light is received is often the orthogonal direction as in the 
present embodiment, whereas this is not restrictive, but may be, for 
example, a direction of 45 degrees or a direction of 60 degrees. Also, 
where the latexes are fluorescence-dyed, weak fluorescence produced with 
the scattered lights is received and therefore, of the fluorescence 
condensed by the condensing lens 7 and passed through the dichroic mirror 
8, green fluorescence is detected by a set of dichroic mirror 9 and a 
photodetector 12 and red fluorescence is detected by a set of total 
reflection mirror 10 and photodetector 13. Although not shown in FIG. 
1(A), a band-pass filter for passing only a light of the detected 
wavelength range therethrough is installed short of each photodetector. 
When the signals of the photodetectors 6, 11, 12 and 13 and the 
aforedescribed transmitted light are to be detected, the signal of the 
photodetector 18 is input to a calculation circuit 15, where calculation 
of particle analysis is effected. 
In the sample fluid container 16, as previously described, a plurality of 
kinds of latexes A, B and C of different absorbances sensitized by the 
particular antibodies a, b and c, respectively, are mixedly present and a 
sample in which a specimen sample (for example, man's serum) is added 
thereto and the reaction time and concentration are appropriately adjusted 
is contained as a sample fluid. Of these latexes A, B and C, those of the 
same kind are equal in both of absorbance and size. The sizes of the 
latexes of different kinds may be the same or different from each other. 
Where an antigen uniquely coupled to the antibodies a, b and c sensitized 
by the respective latexes is contained in the serum, an antigen-antibody 
reaction occurs and the latexes of the same kind are coupled to each other 
through the antigen to form condensed lumps. When the antigen desired is 
not present, condensation does not occur and the latexes remain as single 
pieces. 
FIG. 2 shows the construction of a more preferable form of the specimen 
inspecting apparatus of the present invention. In FIG. 2, reference 
numerals similar to those in FIG. 1(A) designate similar members. In FIG. 
1, the plurality of kinds of latexes A, B and C differing in absorbance 
are mixedly put into a sample fluid container, but as shown in FIG. 2, 
latexes A, B and C of different kinds are discretely put into different 
sample fluid containers 19, 20 and 21, and by applying a pressure in 
common to these sample fluid containers 19, 20 and 21, the latexes forced 
out merge at the through-flow portion and flow through the flow cell 4 one 
particle by one or one lump by one. Thus, the latexes do not mix together 
until immediately before they flow into the measuring portion and 
therefore, the possibility of the respective antigen-antibody reactions 
influencing each other can be minimized. 
The basic idea of a method of applying a light beam to the sample fluid 
flowing in the above-described apparatus and measuring a plurality of 
kinds of antigen-antibody reactions at a time from the strength of the 
forward scattered light and the strength of the laterally scattered light 
will now be described with reference to FIGS. 3 to 5. 
FIGS. 3 to 5 show an example when measurement data are indicated by a 
cytogram with the strength of the laterally scattered light and the 
strength of the forward scattered light plotted as the vertical axis and 
the horizontal axis, respectively, where three kinds of latexes A, B and C 
differing in absorbance are used, and the ranges encircled by dotted lines 
indicate the ranges in which the measurement data as plotted collect. 
It is generally known that when a light is applied to minute particles such 
as latexes, the information of the optical characteristic such as the 
absorbance of the particles is chiefly included in the strength of the 
laterally scattered light and the information of the particle size is 
chiefly included in the strength of the forward scattered light. 
Consequently, the difference in absorbance between the latexes appears in 
the strength of the laterally scattered light and therefore, the 
distinction between the kinds of the latexes is judged by the latexes 
being separated in the direction of the vertical axis of the cytogram. 
Also, the larger the particle diameter of the particles to be examined, 
the greater is the strength of the forward scattered light and therefore, 
when an antigen-antibody reaction occurs and condensed lumps of the 
latexes are formed, the apparent latex diameter increases and therefore, 
the strength of the forward scattered light becomes great and the range 
widens in the direction of the horizontal axis of the cytogram. FIG. 3 
shows the distribution when the desired antigen is not present in the 
serum and the antigen-antibody reaction does not occur at all, and by the 
kinds A, B and C of the latexes, plotting is effected with the latexes 
separated into narrow ranges indicated by the dotted lines of groups I, II 
and III. FIG. 4 shows the cytogram when antigens uniquely coupled to three 
kinds of antibodies a, b and c are all present, and since the latexes are 
condensed and the number of particle lumps of large size is increased, 
plotting is effected in the range widening in the direction of the 
horizontal direction in the distribution graph, as indicated by the dotted 
lines of groups I, II and III. FIG. 5 is the cytogram when only the latex 
C causes an antigen-antibody reaction and condensed lumps are produced, 
and only the group III widens in the direction of the horizontal 
direction. That is, it can be judged that the antigens a and b are not 
present in the specimen sample and only the antigen c is present. Thus, 
discrete antigen-antibody reactions appear separately on the distribution 
graph and therefore, the presence of a plurality of antigens can be judged 
by one measurement at a time. Also, by seeing the size of the particle 
lumps, a rough amount of antigen can be grasped. 
In the present embodiment, three kinds of latexes differing in absorbance 
are used, but it is also possible to measure four or more kinds at a time, 
and if the number of kinds is two, distinction can be made more clearly. 
In the present embodiment, the latexes are sensitized by the antibody, but 
conversely, the latexes may be sensitized by an antigen and inspection may 
be done with a sample to be examined including an antibody added thereto, 
whereby it is also possible to discriminate a particular antibody. 
Further, in the present embodiment, the difference in absorbance between 
the carriers is discriminated from the laterally scattered light output, 
but the laterally scattered light includes the information of optical 
characteristics such as the degree of light transmission which is in close 
relation with absorbance, the degree of light refraction and the degree of 
light reflection of the carrier surface. Consequently, even if use is made 
of a plurality of kinds of carriers differing in these optical 
characteristics, similar measurement can be accomplished by an apparatus 
construction similar to that described above. 
In the foregoing, description has been made of the basic idea of the method 
of detecting a plurality of kinds of antigen-antibody reactions at one 
time by the use of carriers differing in absorbance. Strictly, however, 
the clear output as shown in FIGS. 3 to 5 is not provided. This is 
considered to be because the strength of the forward scattered light and 
the strength of the laterally scattered light do not have entirely 
discrete types of information, but have a correlation therebetween. 
FIGS. 6 to 8 and 12 show the results of the measurement effected on the 
three kinds of latexes A, B and C of the same particle diameter and 
differing in absorbance described in the previous embodiment sensitized by 
discrete antibodies a, b and c. When only the latex A of the three kinds 
of latexes is caused to flow, the histogram of the forward scattered light 
by particles condensed by the antigen-antibody reaction is represented as 
shown in FIG. 6(A). In the figure, the horizontal axis represents the 
strength of the forward scattered light FS received by the photodetector 
6, and the vertical axis represents the number N of particles. Since the 
strength of the forward scattered light depends on the particle diameter, 
the latexes are varied in apparent size by condensation and are indicated 
on the graph while being separated as I.sub.1, I.sub.2 and I.sub.3. The 
numbers of condensed latexes are considered to be one, two and three, 
respectively. FIG. 6(B) is a histogram of the laterally scattered light, 
in which the horizontal axis represents the strength of the laterally 
scattered light received by the photodetector 11 and the vertical axis 
represents the number N of particles. Since the strength of the laterally 
scattered light is varied by the absorbance of particles, the absorbance 
of condensed lumps is varied by condensation and is indicated on the graph 
while being separated as J.sub.1, J.sub.2 and J.sub.3. FIG. 6(C) is a 
cytogram showing these two histograms in a lump. In the figure, the 
horizontal axis represents the strength of the laterally scattered light 
and the vertical axis represents the strength of the forward scattered 
light. I.sub.1 is FIG. 6(A) and J.sub.1 in FIG. 6(B) are by the same 
particle, and likewise, the sets of I.sub.2, J.sub.2 and I.sub.3, J.sub.3 
are by the same lump of particles. Consequently, there appear on the 
cytogram three groups such as a group of particles in which the laterally 
scattered light is J.sub.1 and the forward scattered light is I.sub.1, a 
group of particles in which the laterally scattered light is J.sub.2 and 
the forward scattered light is I.sub.2, and a group of particles in which 
the laterally scattered light is J.sub.3 and the forward scattered light 
is I.sub.3. This can be judged as the particles being in the condensed 
states of one, two and three particles, respectively. The foregoing has 
described the case where the antigen-antibody reaction has occurred and 
particles have been condensed, but when the desired antigen is not present 
and condensation does not occur, I.sub.2, J.sub.2, I.sub.3 and J.sub.3 by 
condensation do not appear on the cytogram, but only I.sub.1 and J.sub.1 
appear. When four or more large condensed lumps are present, they appear 
on a broken line linking the groups of FIG. 6(C) together. By seeing the 
groups appearing on the cytogram as described above, the presence of the 
antigen to be sought after can be detected. 
Likewise, measurement is effected by the use of other kinds of latexes B 
and C having the same particle diameter as said latex A but differing in 
absorbance from said latex A, and when the antigens b and c are present, 
there can be obtained histograms and cytograms as shown in FIGS. 7 and 8, 
respectively. 
FIG. 12 is a cytogram in which the cytograms of FIGS. 6(C), 7(C) and 8(C) 
are arranged into one, and corresponds to FIGS. 3 to 5. In this figure, 
the axes of the strength of the forward scattered light FS and the 
strength of the laterally scattered light SS are replaced with each other. 
In the cytogram of FIG. 12, the group lying on each broken line is the 
information regarding the same antigen (antibody). FIG. 12 shows the 
result when the antigens for three kinds of antibodies a, b and c are all 
present. In FIG. 12, the point of intersection at which FS and SS both 
assume the same value does not substantially exist and therefore, the kind 
and amount of the antigen can be specified. 
It is a popularly used method to effect window processing indicated at W to 
thereby examine the number (count number) of particles present in the 
window. Now, referring to FIG. 12, there are portions in which groups are 
close to one another on the cytogram, and this is because latexes having 
the same size but differing in absorbance, that is, latexes which are 
substantially equal in the strength of the forward scattered light and 
differ in only the strength of the laterally scattered light are selected 
as the latexes A, B and C. This leads to the problem that the window 
processing becomes difficult. 
So, let it be assumed that the latexes A, B and C differ in both absorbance 
and particle diameter. Specifically, by using three kinds of latexes A, B 
and C which are greater in particle diameter as absorbance is greater, 
design is made such that each group appears widely on the cytogram. 
FIG. 9 shows the cytograms by a latex which is small in both particle 
diameter and absorbance, that is, a latex in which the strength of the 
forward scattered light FS is small and the strength of the laterally 
scattered light SS is of a great output, FIG. 10 shows the cytograms by a 
latex in which FS is great and SS is also great, and FIG. 11 shows the 
cytograms by a latex in which FS is great and SS is small. By using a 
combination of such three kinds of latexes A, B and C, the resultant 
cytogram is a cytogram as shown in FIG. 13 wherein respective groups are 
widely separated and the window processing is easy to do. 
In the embodiments described hitherto, the output of the laterally 
scattered light is used to examine the absorbance of the carriers, but the 
information of the optical characteristics such as the absorbance, etc. of 
the carriers is included also in the transmitted light of the light 
applied to the carrier. Consequently, if the strength of the transmitted 
light is measured, the kinds of the carriers can be distinguished also 
from the measured strength of the transmitted light. As previously 
described, in the apparatus of FIG. 1(A) or 2, the forward scattered light 
receiving system is made into a construction as shown in FIG. 1(B) or 
1(C), whereby the transmitted light can be detected. Consequently, by 
effecting an analysis similar to that described above in the calculation 
circuit 15 by the use of the transmitted light instead of the laterally 
scattered light, there can be obtained a substantially similar measurement 
result.