Method and apparatus for the measurement of antigens and antibodies

A method of the quantitative measurement of antigens and antibodies by reacting antibody- or antigen-sensitized insoluble carrier particles with a corresponding antigen or antibody or a mixture thereof in a sample and irradiating the reaction mixture with light of a specific wavelength to measure the absorbance or percent absorption of the reaction mixture, and an apparatus for use therein.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to a method and apparatus for the measurement of 
antigens and antibodies. More particularly, this invention relates to a 
method of the quantitative measurement of antigens and antibodies by 
supporting an antibody or an antigen on insoluble carrier particles having 
minute particle diameters to sensitize the insoluble carrier particles, 
then reacting the sensitized carrier with a corresponding antigen, 
antibody or mixture thereof and irradiating the reaction mixture with 
light of a specific wavelength to measure the absorbence or percent 
absorption of the reaction mixture, and an apparatus for use therein. 
2. Description of the Prior Art 
There is a continuing need for rapid, accurate, qualitative and 
quantitative determinations of biologically active substances, e.g., 
antigens, antibodies, at extremely low concentrations. Today, there is a 
wide need for determining the presence of drugs in body fluids. In 
addition, in medical diagnosis, it is frequently important to know the 
presence of various substances which are synthesized naturally by the body 
or ingested. 
Heretofore it has been known to detect antibodies or antigens 
semiquantitatively by reacting latex particles on which an antibody or an 
antigen has been supported with a corresponding antigen or antibody on a 
glass plate and observing visually the agglutination. 
In recent years, it was proposed in the following articles to 
quantitatively determine antigens and antibodies using the above-mentioned 
latex particles by supporting an antibody or antigen on the latex 
particles to sensitize them, reacting the supported antibody or antigen 
with a corresponding antigen or antibody to be determined to agglutinate 
the latex particles, and measuring the rate of decrease in turbidity of 
the supernatant of the latex by means of visible lights for the 
determination of the antigen or antibody utilizing the agglutination 
phenomena of the latex reagent: 
(A) CROATICA CHEMICA ACTA, 42, (1970), p.p. 457-466; and 
(B) European Journal of Biochemistry, Vol. 20, No. 4, (1971), p.p. 558-560. 
Since the method of the above proposal utilizes the measurement of rate of 
decrease in turbidity to determine the antigen or antibody, it is 
necessary to use an antibody- or antigen-sensitized latex of an extremely 
low concentration, for example, in the range of 0.007 to 0.028%, to carry 
out the reaction of the latex and the antigen or antibody in a stationary 
state, to remove any impurity capable of affecting the turbidity from the 
sample, and the like. As a result, the above-mentioned method is 
disadvantageous in that the rate of the antigen-antibody reaction is 
inevitably decreased, both the precision and the reproducibility are 
insufficient for the determination technique of antigens or antibodies, 
and that the removal of impurities sometimes requires extremely 
complicated operations. Accordingly it is difficult to apply the above 
method to the determination of such antigens as fibrinogen (Fg), human 
chorionic gonadotropin (hCG) and the like, since they require complicated 
procedures for the preparation of their reagents and they are difficult to 
cause reproducible agglutination reactions if they are present in blood or 
urine which additionally contains various other substances capable of 
adversely affecting the reaction. 
Also in the article, 
(C) Immunochemistry, Vol. 12, p.p. 349-351 (1975) 
it was proposed to determine quantitatively antibodies and antigens by 
irradiating the above-mentioned agglutinated latex particles with a laser 
beam and measuring the change in width or spectral lines of the scattered 
light of the laser beam in order to determine the mean diffusion constant 
(D) which gives an indication of the Brownian motion of the agglutinated 
particles which in turn is inversely proportional to the size of the 
agglutinated particles. Also in this method, since the antibody- or 
antigen-sensitized latex is used in an extremely low concentration, for 
example, as low as 0.001%, the rate of the antigen-antibody reaction is so 
decreased that both the precision and the reproducibility become poor. In 
addition, this method is also disadvantageous in that it requires 
complicated calculation using the technique of spectrum analysis which in 
turn requires complicated operations, and that any impurity in the sample 
must be removed prior to the measurement. Accordingly, this method has not 
been put into practice as well. The above paper C also describes that 
determination by the turbidity method as reported in the foregoing paper A 
gives extremely imprecise results (FIG. 2 on page 350 of the same). 
SUMMARY OF THE INVENTION 
Accordingly, it is an object of the invention to provide a method and 
apparatus for the rapid determination of an antibody and/or antigen in a 
sample to be tested with high precision and good reproducibility. 
It is another object of this invention to provide a method and apparatus 
for rapidly detecting whether the concentration of an antibody or antigen 
in a sample is higher or lower than a certain level, using an extremely 
small amount of the sample. 
It is a still another object of this invention to provide a method and 
apparatus for determining an extremely slight amount of an antigen and/or 
antibody which could heretofore be determined practically only by 
radioimmunoassay (RIA), with a precision equal to or higher than that of 
RIA and much more rapidly and safely. 
It is a further object of this invention to provide a method for the 
quantitative determination of antigens capable of determining not only 
multivalent antigens but incomplete antigens such as, for example, 
haptens. 
It is a still further object of this invention to provide a method for 
determining antibodies and/or antigens using not only the agglutination 
reaction of the antibodies and/or antigens but the agglutination 
inhibition reaction thereof. 
Briefly, these and other objects and advantages of this invention, as will 
hereinafter be made clear from the ensuring discussion, can be attained by 
supporting an antibody or an antigen on insoluble carrier particles with 
an average diameter of not greater than 1.6 microns to sensitize the 
insoluble carrier particles, reacting the supported antibody and/or 
antigen with a corresponding antigen or antibody or a mixture thereof to 
be determined in a liquid medium and irradiating the resulting reaction 
mixture with light having a wavelength in the range of 0.6 to 2.4 microns 
and longer than the average diameter of said carrier particles by a factor 
of at least 1.1 to measure the absorbance or percent absorption of the 
reaction mixture (exclusive of the case where the reaction mixture is 
irradiated with the light having a wavelength longer than the average 
diameter of the carrier particles by a factor of at least 1.5 to measure 
the absorbance of the reaction mixture).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
As previously described, the prior art method wherein the degree of 
agglutination caused by bringing antibody- or antigen-sensitized latex 
particles into contact with a sample containing an antigen or an antibody 
is measured by the rate of decrease in turbidity of the supernatant of the 
latex, involves various disadvantages such as poor precision and 
reproducibility, since the reaction has to be carried out in a standing 
state with an extremely dilute latex. 
Also in this prior art method, it is necessary to previously remove any 
impurity in the sample which may affect the turbidity. 
Thus, it is a matter of course that in order to determine an antigen and/or 
an antibody in a sample with high precision and good reproducibility, an 
antibody- or antigen-sensitized insoluble carrier, for example, latex 
particles should desirably be contacted at as high a concentration as 
possible with the sample which contains an antigen and/or antibody capable 
of reacting with the supported antibody or antigen and in order to 
accelerate the antigen-antibody reaction caused thereby, this reaction 
should desirably be carried out under agitation, not in a standing state. 
We have now found that, in order to carry out an antigen-antibody reaction 
between an antibody or antigen supported on insoluble carrier particles 
and a corresponding antigen or antibody in a sample at as high a 
concentration of the latex as possible under non-standing conditions and 
at the same time to detect quantitatively the degree of this reaction, it 
is remarkably effective: 
(1) to use an insoluble carrier having an average particle diameter of not 
greater than 1.6 microns, 
(2) to irradiate the antigen-antibody reaction mixture with light having a 
wavelength in the range of 0.6 to 2.4 microns and longer than the average 
diameter of the carrier particles by a factor of at least 1.1; and 
(3) to measure the absorbance or percent absorption of the reaction 
mixture. 
The reason is that the degree of the antigen-antibody reaction in the 
presence of the sensitized insoluble carrier particles correlates very 
closely to the intensity of the transmitted light. It is apparent that the 
degree of the antigen-antibody reaction also correlates to the amount (or 
concentration) of the antibody and/or antigen in the sample as long as the 
reaction is carried out under specifically determined conditions. The 
above-mentioned method according to this invention, therefore, enables 
rapid determination of an antibody and/or antigen in a sample with an 
extremely high precision by a technique quite different from the 
measurement of turbidity or mean diffusion constant as in the prior art 
methods. The light of a wavelength in the range of 0.6 to 2.4 microns 
which is used in this invention is in the near infrared region or in a 
portion of the visible region which is closely adjacent to the near 
infrared region. Of these regions, the light used in accordance with the 
invention has preferably a wavelength in the near infrared region of 0.8 
to 1.8 microns, more preferably 0.9 to 1.4 microns. 
Heretofore the technique of spectrum analysis using light in the infrared 
region of a wavelength of at least 2.5 microns or light in the ultraviolet 
region of a wavelength of not greater than 0.4 micron is known as a method 
for investigating molecular structures or characteristics thereof. The 
light in the near infrared or the adjacent visible region which is used in 
this invention and which may hereinafter be referred to as "light in the 
near infrared region" for the sake of convenience, however, have 
heretofore been considered to have only limited uses and therefore 
attracted little attention. 
According to our investigation, it has been found that the above-mentioned 
light in the near infrared region in principle possesses eligibility as 
the light to be used in this invention, since it is transmitted very well 
by aqueous media such as water, aqueous solutions and the like which are 
used most generally as the basal media for the antigen- or 
antibody-containing samples such as water, sera, urine, salt solutions, 
etc., as well as, as the basal media for the above-mentioned latices, and 
among the near infrared light, particularly rays of wavelengths of 0.8 to 
1.4 microns and those of 1.53 to 1.88 microns are absorbed by the aqueous 
media only to a very little extent. In addition it has been found that, 
when the reaction mixture obtained by reacting the foregoing antibody- or 
antigen-sensitized insoluble carrier particles having an average diameter 
of not greater than 1.6 microns, preferably 0.1 to 1 micron with an 
antigen and/or antibody in a sample to cause agglutination is irradiated 
with a light in the above-mentioned near infrared region having a 
wavelength longer than the average diameter of the carrier by a factor of 
at least 1.1, preferably at least 1.5 in accordance with the invention, 
the absorbance or percent absorption of the reaction mixture correlates 
very closely to the degree of the agglutination resulting from the 
antigen-antibody reaction. As previously mentioned, this invention 
excludes the case where the reaction mixture is irradiated with the light 
having a wavelength longer than the average diameter of the carrier 
particles by a factor of at least 1.5 to measure the absorbance of the 
reaction mixture. 
The term "percent absorption" used herein is defined by the equation: 
##EQU1## 
wherein S represents the percent absorption, I.sub.o represents the 
intensity of the transmitted light when the cell contains the same system 
as the reaction mixture, except for the absence of the antigen and/or 
antibody to be measured, and I represents the intensity of the transmitted 
light when the cell contains the reaction mixture. 
As is apparent from the above definition, the percent absorption used 
herein may be referred to in another way as the percentage of attenuated 
or not transmitted light. Since the percent absorption correlates to 
absorbance (A) which can be measured by means of a conventional 
spectrophotometer, for example, for use in infrared spectrometry, it may 
be expressed in terms of absorbance for the sake of convenience. 
In the infrared spectrometry, absorbance (A) is defined by the equation: 
EQU A=log I.sub.o /I (2) 
wherein I.sub.o and I have the same meanings as in Equation 1. Thus, it is 
possible to determine antigens and antibodies by the measurement of either 
parameter of percent absorption defined by Equation 1 or absorbance by 
Equation 2. In either course, the results will coincide with an acceptable 
deviation, as long as the measurement is carried out properly. 
In brief the above-mentioned percent absorption (S) or absorbance (A) 
relates to the relative ratio of I.sub.o /I. If the basal medium of the 
sample is a transparent liquid medium, the measurement of I.sub.o may 
conveniently be performed with only the suspension containing the 
antibody- or antigen-sensitized insoluble carrier particles, said 
suspension having been diluted with, for example, water to the same 
concentration as that in the mixture. 
By the way, percent transmission spectrum in the range of 0.6 to 2.4 
microns of a water layer 1 mm in thickness is shown in FIG. 1, wherein the 
abscissa indicates the wavelength of light and the ordinate the percent 
transmission of the light. It can be seen from FIG. 1 that the lights of 
wavelengths in the range of 0.6 to 1.4 microns are transmitted by water 
without substantial absorption by the water which is employed most widely 
as the basal media for latices and samples, and that the lights of 
wavelengths in the range of 1.53 to 1.88 microns are also considerably 
transmitted by water so that light of a wavelength in these ranges can be 
utilized in principle in the practice of this invention. Also, it is 
apparent from FIG. 1 that the lights of wavelengths in the range of 2.1 to 
2.35 microns are also transmitted by water in the order of 20%, and 
therefore it should be understood that the rays of such wavelengths can be 
used in conjunction with a highly sensitive photometer, although they are 
rather not preferred. 
FIG. 2 shows the relationship between the absorbance of a polystyrene latex 
(1% solids content by weight) in the ordinate and the wavelength of light 
in microns in the abscissa when a cell of 2 mm in thickness is used. In 
FIG. 2, Curve A denotes the change in absorbance of a polystyrene latex in 
which the average diameter of the particles is 0.481 micron and Curve B 
denotes that of a polystyrene latex in which the average diameter is 0.804 
micron. In the determination of absorbance, the latex was diluted for the 
convenience of the measurement, and the absorbance of the latex was 
evaluated by multiplying the actually obtained value of absorbance by the 
dilution factor. 
As will be understood from FIG. 2, the absorbance of the latex is so 
significantly increased with the lights of wavelengths less than 0.6 
micron that it is quite difficult to measure the change in percent 
absorption of an antigen-antibody reaction mixture using a light of such a 
wavelength, whereas with the lights of wavelengths of at least 0.8 micron, 
particularly at least 1 micron, the absorbance of the latex itself is 
relatively small so that light of a wavelength of at least 0.8 micron, 
preferably at least 1 micron is suitable for the above-mentioned 
measurement of percent absorption of such reaction mixture. 
When Curve A is compared with Curve B in FIG. 2, it is recognized that the 
absorbance of the polystyrene latex increases with increasing average 
diameter of the polystyrene particles. Accordingly it would also be 
understood that those latex particles having an excessively large average 
diameter are unfavorable for the method of this invention. 
In accordance with our investigation, it has been found that the insoluble 
carrier particles useful for this invention must have an average particle 
diameter of not greater than 1.6 microns and that those latex particles 
having an average diameter of 0.1 to 1 micron, preferably 0.2 to 0.8 
micron are suitable. 
FIG. 3 shows the relationship between the change in percent absorption of 
an antigen-antibody reaction mixture in the ordinate and the wavelength of 
light in microns in the abscissa at various reaction time when the 
antigen-antibody reaction is carried out in exactly the same manner as in 
Example 1 except that a polystyrene latex with an average particle 
diameter of 0.234 micron is used. In FIG. 3, Curves C, D and E denote the 
percent absorption of the reaction mixture after the antigen-antibody 
reaction is carried out for 3, 10 and 20 minutes, respectively. 
As can be seen from FIG. 3, when the percent absorption of the 
antigen-antibody reaction mixture is determined with a light of a 
wavelength less than 0.6 micron, in the wavelength region of about 0.6 to 
0.4 micron the progress of the reaction (i.e., the reaction time) does not 
correspond to the percent absorption, and in the wavelength region of not 
greater than about 0.4 micron the absorbance does not appreciably vary 
with the progress of the reaction. On the other hand, with a light of a 
wavelength of at least about 0.75 micron, the percent absorption of the 
reaction mixture gives a significantly good correlation to the reaction 
time or progress of the reaction. The dotted line sections in Curves C, D 
and E in FIG. 3 indicate that in these wavelength regions the percent 
absorption cannot be determined accurately even with an increased slit 
width, since the absorption by water is much higher in these regions. 
As can be seen hereinafter from Example 4, when a polystyrene latex having 
an average particle diameter of 0.804 micron is used, a good correlation 
is established between the percent absorption of the antigen-antibody 
reaction mixture and the concentration of the antigen by the use of light 
of a wavelength longer than the average diameter by a factor of at least 
about 1.1, e.g., a wavelength of about 1.2 microns, as long as the 
concentration of the antigen in a sample is not greater than 0.6 .mu.g/ml 
(0.6.times.10.sup.-6 g/ml). In this case, therefore, the quantitative 
determination according to this invention can be performed by irradiation 
with light having a wavelength of 1.2 microns or longer. In the case of 
those latex particles having a relatively small average diameter, however, 
as can be seen from FIG. 3, it is advantageous to use a near infrared 
light having a wavelength in the range of about 0.8 to 1.4 microns, 
preferably 1 to 1.4 microns and longer than the average diameter of the 
carrier by a factor of at least 2. 
In accordance with one embodiment of this invention, it is desirable to 
irradiate a reaction mixture resulting from a carrier of a specific 
particle size on which an antibody or antigen is supported and an antigen 
or antibody or a mixture thereof in a sample solution with light of an 
appropriate wavelength in the range of 0.6 to 2.4 microns in order to 
preliminarily detect a wavelength region of applied light in which a 
quantitative correlation is established between the concentration of the 
particular antigen or antibody or a mixture thereof (including the 
reaction product) in the sample solution and the absorbance or percent 
absorption of the reaction mixture, and subsequently to effect the 
measurement of absorbance or percent absorption with light of a specific 
wavelength in this region. 
Thus, in accordance with the invention, it is possible to determine the 
amount or concentration of an antigen and/or an antibody in a sample by 
using insoluble carrier particles having an average diameter of not 
greater than 1.6 microns, preferably in the range of 0.1 to 1.0 micron, 
more preferably 0.2 to 0.8 micron and most preferably in the range of 0.3 
to 0.6 micron, supporting an antibody or an antigen on the carrier (i.e., 
sensitizing the carrier with the antibody or antigen), reacting the 
sensitized carrier with the antigen and/or antibody in the sample, and 
measuring the absorbance or percent absorption of the reaction mixture 
with light of a wavelength in the range of 0.6 to 2.4 microns, preferably 
0.6 to 1.4 microns, more preferably 0.8 to 1.4 microns and most preferably 
1 to 1.4 microns. As previously mentioned, the light to be applied should 
have a wavelength longer than the average diameter of the insoluble 
carrier particles by a factor of at least 1.1, preferably at least 1.5, 
with the proviso that this invention excludes the case where the reaction 
mixture is irradiated with light having a wavelength longer than the 
average diameter of the carrier particles by a factor of at least 1.5 to 
measure the absorbance of the reaction mixture. 
The insoluble carrier particles useful for this invention include those 
organic polymer microparticles which are substantially insoluble in the 
particular liquid medium used for the measurement according to the 
invention and which have an average diameter within the above-mentioned 
range, such as, for example, latices of organic polymers such as 
polystyrene and styrene-butadiene copolymer obtained by emulsion 
polymerization; dispersed coccal bacteria such as staphylococci and 
streptococci, Bacillus prodigiosus, rickettsia, cell membrane fragments, 
etc.; as well as microparticles of inorganic oxides such as silica, 
silica-alumina and alumina, and finely pulverized minerals, metals and the 
like. 
In accordance with the invention, an antibody or antigen which is reactive 
with the antigen and/or antibody in a sample to be determined is supported 
on the above-mentioned insoluble carrier particles such as, for example, 
latex particles (i.e., to sensitize the carrier). For this purpose, the 
antibody or antigen may be physically and/or chemically adsorbed on the 
carrier. Antibodies consist of proteins, whereas antigens are composed of 
one member selected from various substances such as, for example, 
proteins, polypeptides, steroids, polysaccharides, lipids, pollen, dust 
and the like. 
There have already been proposed a number of methods for supporting these 
antibodies or antigens, particularly antibodies on insoluble carrier 
particles. 
When an incomplete antigen, particularly a hapten is supported on insoluble 
carriers, it is advantageous to chemically modify the carriers with, for 
example, a coupling agent and subsequently adsorb the antigen chemically 
on the modified carriers. 
If the carrier is a latex of a high molecular substance which has a 
functional group such as, e.g., sulfo, amino, or carboxyl or its reactive 
derivative group, an antigen and/or antibody can directly be adsorbed 
chemically on such latex. 
As the liquid medium useful for this invention, water is the most 
preferably, although a mixture of water and a water-miscible organic 
solvent can be used. Suitable water-miscible organic solvents include 
alcohols such as methanol, ethanol, etc. and ketones such as acetone. 
Contrary to the known prior art methods which utilize the measurement of 
turbidity or the measurement of mean diffusion constant with a laser beam, 
the method according to this invention provides conditions that enable the 
insoluble carrier particles sensitized with an antibody or an antigen to 
react with a corresponding antigen and/or antibody as activity as 
possible. 
On this account, in accordance with the invention, the insoluble carrier 
particles, for example, latex particles, which are sensitized with an 
antibody or an antigen (hereinafter referred to as "sensitized carrier 
particles") may be used as a suspension having a concentration of not less 
than 0.05% by weight, preferably in the range of 0.05 to 1%, more 
preferably 0.2 to 0.6%. 
When the concentration of the sensitized carrier particles is much too 
high, as is apparent from FIG. 2, the transmittance of the suspension 
itself is so decreased that the measurement of absorbance or percent 
absorption according to the invention is made difficult. However, in the 
concentration range in which such a measurement of absorbance or percent 
absorption is possible, higher concentration of the sensitized carrier 
particles in the suspension is preferred, whereby it is possible to 
increase the sensitivity of the quantitative determination of antigens and 
antibodies. 
In accordance with the invention, also contrary to the prior art methods, 
the sensitized carrier particles and and antigen- and/or 
antibody-containing sample are reacted under non-standing conditions. 
For this purpose, the reaction may be advantageously carrier out under 
agitation. Since the reaction is generally carried out in a thin cell, the 
agitation is conveniently effected for example, by moving a rod vertically 
or transversely in the cell. Of course, the sensitized carrier particles 
and the sample may be reacted outside the cell for a certain period of 
time under predetermined conditions and thereafter the reaction mixture is 
placed in the cell for the measurement of absorbance or percent 
absorption. However, in order to make the reaction conditions 
reproducible, particularly with respect to reaction time in every 
measurement, the sensitized carrier particles and the sample may be 
reacted under predetermined non-standing conditions directly in a cell 
which has been set in a spectrophotometer, whereby more accurate 
determination can be achieved by measuring the absorbance or percent 
absorption. 
Immediately after a prescribed period of reaction time or by measuring the 
time required to reach a predetermined value of absorbance or percent 
absorption while the reaction is continued under substantially fixed 
conditions. 
In this way, the present invention not only makes it possible to determine 
such a concentration of an antigen and/or antibody in a sample that could 
heretofore be obserbed visually in a semiquantitative manner, but enables 
the determination of an antigen and/or antibody in such a trace amount 
that could heretofore be determined only by radioimmunoassay (RIA), with a 
precision equivalent to or higher than that of the RIA method. 
In order to determine an antigen and/or antibody in a sample containing an 
unknown amount of the antigen and/or antibody in accordance with the 
invention, a set of dilute standard samples are prepared from a standard 
sample containing a definite amount of the same antigen and/or antibody by 
diluting it by various factors. Each of the dilute and undiluted standard 
samples is reacted under predetermined conditions with insoluble carrier 
particles sensitized with a definite amount of the corresponding antibody 
or antigen in accordance with the invention, and the absorbance or percent 
absorption of each reaction mixture is determined to prepare a standard 
curve for the particular combination of the antigen and/or antibody with 
the sensitized carrier particles, which indicates the relationship between 
the amount (concentration) of the antigen or antibody in the standard 
sample and the absorbance or percent absorption (this type of standard 
curve being hereinafter referred to as "Standard Curve A" for 
convenience). Subsequently, an unknown sample to be tested is reacted with 
the same sensitized carrier particles as that used in the preparation of 
the standard curve under substantially the same conditions as in the 
preparation of the standard curve, and the absorbance or percent 
absorption of the reaction mixture is measured. 
The amount (or concentration) of the antigen and/or antibody in the unknown 
sample can be determined by comparing the value of absorbance or percent 
absorption thus obtained with Standard Curve A. 
Alternatively, in the preparation of a standard curve like that described 
in the above, another type of standard curve which indicates the 
relationship between the amount (or concentration) of the antigen or 
antibody in the standard sample and the reaction time required to reach a 
predetermined value of absorbance or percent absorption may be prepared 
(this type of standard curve being hereinafter referred to as "Standard 
Curve B" for convenience). Also in this case, an unknown sample is reacted 
with the same sensitized carrier particles under substantially the same 
conditions as in the preparation of the standard curve, and the amount (or 
concentration) of the antigen and/or antibody in the unknown sample can be 
determined by reading the time required to reach the predetermined value 
of absorbance or percent absorption. 
Thus, in accordance with the invention, the amount or concentration of an 
antigen and/or an antibody in an unknown sample may be determined by way 
of, either 
(A) the measurement of absorbance or percent absorption of the unknown 
sample (using Standard Curve A for calibration), or 
(B) the measurement of the rate of reaction, or the reaction time required 
for the absorbance or percent absorption to reach a certain value (using 
Standard Curve B for calibration). 
As described previously, the above method (A) is useful as a determined 
system with a significantly high precision, not only when the 
concentration of an antigen and/or antibody in an unknown sample is 
relatively high, but even if it is so low that it could heretofore be 
determined only by the RIA method. On the other hand, the above method (B) 
wherein the reaction rate is measured is suitable for determining a 
relatively large amount (concentration) of an antigen and/or antibody in 
an unknown sample, but it is advantageous in that the measurement is quite 
simple. 
According to our investigation, Standard Curve A as described above gives 
generally a gentle S-shaped curve rather than a straight line, but no 
unfavorable effect is found on the precision of the determination. 
The reason why the curve assumes the S-shape as described above is presumed 
by us to be that the rate of reaction takes part in this shape at low 
concentrations of the antigen and/or antibody, whereas the saturation of 
active sites in the carrier takes part at higher concentrations. 
It is possible, of course, to enlarge the linear portion in the S-shape 
curve by the selecting the conditions appropriately in the preparation of 
the standard curve, and apply substantially only this portion to the 
determination of unknown samples. 
As stated above, the present invention is characterized in that the 
sensitized carrier particles at as high a concentration as possible are 
contacted and reacted with a sample. 
Therefore, the cell for use in measuring the absorbance or percent 
absorption of the reaction mixture should have a thickness less than that 
of a cell for use in visible spectroscopic analysis, and, for example, 
those cells having thickness in the range of 0.5 to 10 mm, particularly 1 
to 5 mm are preferred. 
In order to effect a highly sensitive determination of a trace amount of an 
antigen or antibody which has heretofore been subjected to the RIA method, 
it is particularly advantageous: 
(a) to use an antigen or antibody having as high an equilibrium constant as 
possible, 
(b) to use latex particles, particularly with an average diameter of 0.2 to 
0.8 micron, the size distribution of which should be as narrow as 
possible, 
(c) to measure the absorbance or percent absorption with light of a 
wavelength of 1.0 to 1.4 microns 
(d) to select a relatively long reaction time, for example, in the range of 
1 to 3 hours, and 
(e) to increase the concentration of the sensitized latex carrier as long 
as the absorbance or percent absorption is measurable at the 
concentration. 
Also, in order to determine an unknown sample accurately in a relatively 
short time by the measurement of reaction rate (using Standard Curve B), 
it is advantageous, 
(f) to use latex particles having a relatively large average diameter, 
(g) to increase the concentration of the carrier particles in the latex as 
long as the measurement of absorbance or percent absorption is possible at 
the concentration, and 
(h) to use a relatively short period of reaction time, for example, in the 
range of 5 seconds to 10 minutes, preferably 10 seconds to 3 minutes. 
In this case, when the time required for the absorbance or percent 
absorption to reach a predetermined value is plotted as the ordinate and 
the concentration as the abscissa, both on a log scale, the resulting 
Standard Curve B will give a straight line to advantage. 
The present invention is described in the above with respect to the 
determination of an antigen and/or antibody in a sample by applying the 
latex agglutination phenomenon caused by contacting the antigen and/or 
antibody in the sample with sensitized carrier particles (i.e., LA 
system). 
The method according to the invention is also suitable for the 
determination of a sample using its inhibitory action against the 
above-mentioned agglutination reaction (i.e., LI system). 
Incomplete antigen such as, for example, haptens can be determined by 
applying the method according to the invention to the LI system. 
In this case, for instance, an antigen may be supported on the insoluble 
carrier particles used in this invention, the sensitized carrier particles 
are reacted competitively with a given amount of an antibody which has 
been reacted with an antigen of a known concentration (i.e., a standard 
antigen solution), and the absorbance or percent absorption of the 
resulting reaction mixture is measured. The above procedure is repeated at 
various concentrations of the standard antigen solutions to prepare 
Standard Curve C. Subsequently, an unknown sample is reacted with the same 
antibody of the definite concentration, and the resulting reaction mixture 
is the reacted with the sensitized carrier. These reactions should be 
carried out under substantially the same conditions as in the preparation 
of Standard Curve C. The absorbance or percent absorption of the final 
reaction mixture with the sensitized carrier particles is measured and 
compared with the standard curve (C) to determine the amount 
(concentration) of the antigen in the unknown sample. 
Following the procedure of the above-mentioned LI system except that a 
certain antibody is supported on the insoluble carrier particles, an 
antibody in an unknown sample can be determined by the LI system. In 
addition, it is possible, if desired, to support both an antigen and an 
antibody of different species on the insoluble carrier particles and 
determine an antigen and an antibody in an unknown sample. 
Thus, in accordance with the invention, the quantitative determination of a 
wide variety of antigens and/or antibodies are possible, for example, 
(1) blood examination of subjects or blood donors which is indispensable 
for emergency operations, for example, detection of blood group 
substances, the Au- or HB-antigen or other contaminants in the blood, or 
determination of fibrin/fibrinogen degradation products (FDP) which is 
recently regarded as useful in the convalescent control for kidney 
transplantation or renal failure patients, 
(2) determination of human chorionic gonadotropin (hCG) which is regarded 
as significantly important in the pregnancy diagnosis or the convalescent 
control of chorioepithelioma, 
(3) determination of hCG, or urinary estriol glucuronide which is a 
metabolite of follicular hormone, said determination being required for 
monitoring pregnancy, 
(4) determination of oxytocin in blood which is considered to be a uterine 
contraction inducer, 
(5) determination of certain adrenal cortical hormones such as corticoids 
and aldosterone, or adrenocorticotropic hormones (ACTH), 
(6) determination of insulin for diabetics, or determination of follicle 
stimulating hormone, luteinizing hormone, estrogens, corpus luteum 
hormone, etc., 
(7) determination of gastrin or secretin which is a gastrointestinal 
hormone, 
(8) detection and determination of an antibody in the body fluid of 
patients with allergy, syphilis, or hemolytic streptococcicosis, rubella, 
antoimmune diseases such as collagen disease and other infection diseases, 
and the like. 
The present invention may be adopted, of course, for the qualitative or 
semi-quantitative measurement of these antigens and/or antibodies. 
In accordance with another aspect of this invention, there is provided a 
novel apparatus for measuring antigens and antibodies which can be used in 
the above-mentioned method of this invention. 
The apparatus according to the invention involves 
(a) insoluble carrier particles for supporting an antibody or antigen, said 
carrier particles having an average diameter of not greater than 1.6 
microns, 
(b) an absorption cell for holding a reaction mixture obtained by reacting 
the antibody or antigen supported on the insoluble carrier and a 
corresponding antigen and/or antibody in a liquid medium, said cell having 
a thickness in the range of 0.5 to 10 mm, 
(c) a photometer for the measurement of absorbance or percent absorption 
equipped with an irradiation unit capable of applying light of a 
particular wavelength in the range of 0.6 to 2.4 microns. 
The measuring apparatus according to this invention may possess the same 
basic structure as in the prior art photometric apparatus, except for the 
essential structural characteristics as described in (a), (b) and (c). 
Thus, as illustrated in FIG. 4, the basic structure of the apparatus 
according to the invention comprises an irradiation unit comprising light 
source (1) and filter or prism (2); sample cell (3) for holding a sample 
of an antigen-antibody reaction mixture to be measured, and reference cell 
(4) for holding a control sample for compensation; photocells (5) and (6) 
for sensing the intensities of light transmitted by the respective cells 
and transforming them into electric signals; amplifier (7) for amplifying 
the electric signals; and displaying or recording unit (8) for displaying 
or recording the amplified electric signals. 
Light source (1) may be a conventional tungsten lamp and the light emitted 
from light source (1) is monochromatized through filter or prism (2) so as 
to apply a light beam of a specific wavelength in the range of 0.6 to 2.4 
microns, preferably 0.8 to 1.8 microns and more preferably 0.9 to 1.4 
microns to cells (4) and (5). 
Filter or prism (2) is therefore selected from those capable of effectively 
monochromatizing the light of the abovementioned wavelengths. For 
instance, an interference filter of 1,200.+-.50 millimicrons may be used 
as the filter or a quartz or glass prism as the prism. 
The applying light thus monochromatized is converged appropriately through 
a slit or a lens before it is applied to sample cell (3) and reference 
cell (4). 
Sample cell (3) and reference cell (4) may be composed of transparent glass 
or synthetic resin (e.g., acrylic resins) and may generally be a box-shape 
having a rectangular cross section (See FIG. 5). The cell thickness, that 
is, the distance (l) between windows (transmissive windows) (a) and (b), 
respectively, on the side from which the light is applied and on the 
opposite side, may be in the range of 0.5 to 10 mm, preferably 1 to 5 mm. 
The transmissive windows may advantageously possess at least 30% 
transmission, preferably 80% or higher transmission, for lights of 
wavelengths of 0.6 to 2.4 microns. 
In sample cell (3) is placed a reaction mixture prepared by reacting an 
antigen or antibody or a mixture thereof with the corresponding antibody 
and/or antigen supported on insoluble carrier particles in a liquid medium 
in such a manner as previously described with respect to the method of 
this invention. On the other hand, in reference cell (4) is placed a 
control sample prepared by dispersing only the antibody- and/or 
antigen-sensitized insoluble carrier particles in the liquid medium. 
The light beams transmitted by cells (3) and (4), are received by 
photocells (5) and (6), respectively, and transformed into electric 
signals, the respective amplitudes of which are in proportion to the 
respective intensities of light received by the cells. As photocells (5) 
and (6), any type of photocells which function to transduce an intensity 
of light received into an electric signal having a strength proportional 
to the intensity of light may be used. Lead sulfide photoconductive 
element, for example, may be employed to advantage. 
The electric signals thus transformed by the photocells may be amplified by 
amplifier (7) in a conventional manner and displayed or recorded on 
indicator or recorder (8) so as to read them visually. 
If a horological mechanism is incorporated in indicator or recorder (8), it 
is possible to automatically record the absorbance or percent absorption 
after a predetermined period of reaction time, or record the time required 
for the absorbance or percent absorption to reach a predetermined value. 
In preferred embodiment of the apparatus according to the invention, sample 
cell (3) is equipped with an agitator, which may be a mixed rod movable in 
the cell. 
FIG. 6 shows a preferred embodiment of the agitator mechanism for agitating 
mixture 9 of a sample and sensitized carrier particles (e.g., sensitized 
latex) which is held in sample cell 3 used in the apparatus of the 
invention. 
Referring to FIG. 6, L-shaped mixing rod 11 can move up and down to agitate 
mixture 9 in cell 3 by the vertical up and down motion of T-shaped 
hammerheaded connecting rod 14, said connecting rod 14 being contacted at 
its upper flat plate 14' with rotary disc 12 which is driven by eccentric 
shaft 13. FIG. 17 indicates a light-shielding lid, and connecting rod 14 
moves up and down through hollow tube 15 fixed to and through lid 17, 
wherein connecting rod 14 goes down with rotation of eccentric disc 12 and 
is then lifted by the restoring force of spring 16 which is provided 
between lid 17 and upper flat plate 14' of connecting rod 14. 
When sample cell 3 to be used in the photometric apparatus as shown in FIG. 
4 has such a structure, for example, as shown in FIG. 6, cell 3 can be 
placed in the dark which is shielded from sunlight and a mixture of a 
sample and sensitized carrier particles (sensitized latex) introduced in 
cell 3 can be mixed with L-shaped mixing rod 11 while being irradiated 
with near infrared light of a predetermined wavelength, whereby it is 
possible to agitate the mixture without obstruction to the light path of 
the near infrared light. 
Thus, by the use of the above-mentioned apparatus, the antigen-antibody 
reaction between the sample and the sensitized carrier particles can be 
accelerated while being progressed under substantially fixed conditions, 
and in addition, such operation as to stop the reaction immediately after 
a predetermined period of reaction time has passed or to accurately read 
the reaction time elasped by the time the absorbance or percent absorption 
reaches a predetermined value can be extremely readily performed. 
Having generally described this invention, a more complete understanding 
can be obtained by reference to certain examples which are provided herein 
for purpose of illustration only and are not intended to be limiting in 
any manner. 
EXAMPLE 1 
(1) Preparation of an antifibrinogen antibody-sensitized latex 
(anti-Fg-latex) reagent 
To 10 ml of a glycine buffer solution of anti-(human fibrinogen (Fg)) 
antibody (2 mg/ml concentration), 1 ml of a polystyrene latex with an 
average particle diameter of 0.481 micron (Dow Chemical Co., 10% solids 
content by weight) is added, and the mixture is stirred at room 
temperature for 30 minutes, then warmed to 40.degree. C. and stirred for 
an additional 30 minutes at this temperature, and centrifuged (at 12,000 
rpm) for 50 minutes with cooling at 2.degree. to 4.degree. C. 
The precipitates are separated by decantation and the collected anti-Fg 
antibody-sensitized latex particles are suspended in a bovine serum 
albumin solution (0.2 wt. % concentration) to prepare an 
anti-Fg-sensitized latex reagent containing the sensitized latex particles 
at a concentration of 1% by weight. 
(2) Preparation of a standard curve 
A 0.1 ml aliquot of the anti-Fg-latex reagent as prepared in Part (1) is 
placed in a plastic test tube (7 mm inner diameter.times.70 mm long) 
together with 0.3 ml of a standard fibrinogen (Fg) solution (in an 
isotonic sodium chloride solution containing 0.2% by weight bovine serum 
albumin) which contains Fg at a concentration indicated in Table-A below, 
and the mixture is shaken at room temperature for 20 minutes on a 
reciprocal shaker at 200 strokes per minute to effect the antigen-antibody 
reaction. Immediately thereafter, the reaction mixture in the test tube is 
transferred to a glass absorption cell having a thickness of 2 mm and the 
percent absorption of the reaction mixture is measured at a wavelength of 
the applied light of 1.2 microns with an automatic recording 
spectrophotometer (Hitachi Ltd., Model EPS-3; using as a control a 
suspension of 0.1 ml of the anti-Fg-latex reagent diluted with 0.3 ml of 
the isotonic sodium chloride solution containing 0.2% by weight bovine 
serum albumin). The measurements are taken twice with each standard Fg 
solution. The results are summarized in the following Table A. 
Table-A 
______________________________________ 
Concentration of 
standard Fg solution 
% Absorption at 1.2 .mu. 
(.mu.g/ml) First Second Mean 
______________________________________ 
0.1 53.9 56.6 55.3 
0.2 85.4 83.4 84.4 
0.3 91.7 93.2 92.5 
0.4 95.3 95.3 95.3 
0.5 96 96.3 96.2 
______________________________________ 
When the above data are plotted graphically with concentration of standard 
Fg solution as abscissa and percent absorption (mean value) at 1.2 microns 
(in wavelength of the applied light) as ordinate, a standard curve as 
shown in FIG. 7 is obtained. 
(3) Quantitative determination of Fg in unknown samples 
A sample of blood, urine or fluid in the thoracic cavity (intrapleural 
fluid) is collected from a subject and if the sample is blood, the serum 
or plasma is separated therefrom. A 0.3 ml aliquot of the sample or its 
diluted sample is treated with 0.1 ml of the anti-Fg-latex reagent as 
prepared in Part (1) in exactly the same manner as described in Part (2), 
and the percent absorption is measured in the same manner as described in 
Part (2). Using the standard curve obtained in Part (2), the concentration 
of Fg corresponding to the value of percent absorption is read and the 
results are summarized in Table-B below. 
For the purpose of comparison, Table-B also involves the data obtained in 
accordance with the conventional radioimmunoassay (RIA) method (S. M. 
Ratkey, et al., Brit. J. Haematol. 30, 145-149, 1975) and slide method 
(Fujimaki, Tamura and Takahashi, Rinsho Kagaku (Clinical Science), Vol. 
12, 507, 1976; and Fujimaki, Ikematsu, Takeuchi and Kato, Rinsho Byori 
(Japanese Journal of Clinical Pathology), 21, 973, 1973). 
Table-B 
______________________________________ 
Fg concentration in 
unknown sample (.mu.g/ml) 
Method 
Unknown sample % of 
Dilu- Absorp- 
this 
Subject tion tion inven- RIA Slide 
No. Material factor measured 
tion method 
method 
______________________________________ 
1 Urine .times. 2 
82.7 0.402 0.359 0.5 
2 " .times. 16 
93.1 5.178 5.117 8.0 
3 " .times. 1 
94.1 0.347 0.368 0.5 
4 " " 18.7 0.021 0.024 less 
than 
0.5 
5 " " 2.5 0.003 0.006 less 
than 
0.5 
6 " " 33.1 0.042 0.037 less 
than 
0.5 
7 " " 13.0 0.016 0.011 less 
than 
0.5 
8 " " 32.5 0.041 0.008 less 
than 
0.5 
9 " " 4.5 0.005 0.007 less 
than 
0.5 
10 " " 36.8 0.048 0.072 less 
than 
0.5 
11 Serum .times. 10 
51.0 0.760 0.800 1.0 
12 " " 53.2 0.812 0.863 0.9 
13 " " 69.8 1.317 1.335 1.25 
14 " " 55.1 0.858 0.892 1.0 
15 Fg-free " 71.8 1.400 1.520 2.0 
Plasma 
16 Cancerous 
.times. 640 
93.8 217.12 197.4 320 
intra- 
pleural 
fluid 
______________________________________ 
From the above results, it can been seen that the data of Fg concentration 
obtained by the method of this invention are in significantly close 
agreement with those obtained by the RIA method which is known as the most 
precise determination method of the conventional methods. The correlation 
coefficient between the method of this invention and the RIA method is 
0.999 or higher. 
EXAMPLE 2 
An anti-Fg-sensitized latex reagent (containing 1% by weight latex 
particles) is prepared in the same manner as in Example 1, Part (1), 
except for use of another polystyrene latex having an average particle 
diameter of 0.234 micron (Dow Chemical Co., 10% solids content by weight). 
A 0.1 ml aliquot of the anti-Fg-sensitized latex reagent thus obtained is 
mixed with 0.3 ml of a standard Fg solution (containing 0.5 .mu.g/ml of 
Fg) and shaken at room temperature for 5 minutes on a reciprocal shaker at 
250 strokes per minute to carry out the antigen-antibody reaction. 
Subsequently, the percent absorption of the reaction mixture is measured 
at a wavelength of applied light of 1.2 microns in the same manner as 
described in Example 1, Part (2). In order to confirm the reproducibility 
of the measurement, the same procedure is repeated three more times. The 
results are given in Table-C below. 
Following the above-mentioned measuring test except for use of the serum 
isolated from a blood sample collected from a patient instead of the 
standard Fg solution, the same procedure as described above is repeated 
four times on different days to confirm the reproducibility of the 
measurement for the actual body fluid. The results are also given in the 
following Table-C. 
Table-C 
______________________________________ 
% Absorption 
Measurement Standard Fg 
No. solution Serum 
______________________________________ 
1 73.8 49.1 
2 72.8 47.5 
3 72.6 50.5 
4 71.5 48.3 
Average 72.9 .+-. 1.3 49.05 .+-. 1.55 
Coefficient 
of 1.8% 3.4% 
variance 
______________________________________ 
As is apparent from the results given in Table-C above, the method of this 
invention possesses an excellent reproducibility both in the case of using 
the standard Fg solution sample and in the case of using the actual body 
fluid (serum) sample. 
EXAMPLE 3 
To 5 ml of a glycine buffer solution, 100 mg of silica microparticles 
(prepared in the same manner as described in Example 1 of Japanese Patent 
Laying-Open Publication No. 120497/75) having an average diameter of 0.32 
micron (although containing 15% of those particles having diameters of 0.5 
micron or greater) are added and the mixture is subjected to ultrasonic 
vibrations of 28 KHz for an hour to form a suspension of silica 
microparticles. An anti-Fg-antibody-sensitized silica suspension reagent 
is prepared in the same manner as described in Example 1, Part (1) except 
for use of the silica microparticle-containing suspension thus prepared 
instead of the polystyrene latex with an average diameter of 0.481 micron 
in Example 1, Part (1) and use of another concentration of bovine serum 
albumin solution (0.05% concentration by weight). 
Using the anti-Fg-sensitized silica suspension reagent, the percent 
absorption is measured with each standard Fg solution in the same manner 
as described in Example 1, Part (2). The results are summarized in the 
following Table-D. 
Table-D 
______________________________________ 
Concentration of 
standard Fg solution 
% Absorption 
(.mu.g/ml) at 1.2 .mu. 
______________________________________ 
0.2 6.2 
0.4 25.1 
0.6 48.3 
0.8 65.3 
1.0 76.7 
______________________________________ 
A standard curve is plotted from the above data as in Example 1, Part (2), 
said standard curve being shown in FIG. 8. It can be seen from FIG. 8 that 
a clean linear relationship is established between the concentration of Fg 
and the percent absorption when the concentration of standard Fg solution 
is higher than 0.4 .mu.g/ml. Unknown samples (urine, serum and 
intrapleural fluid) collected from subjects are subjected to the same 
procedure as described in Example 1, Part (3) to determine Fg in the 
unknown samples using the above standard curve. The results are summarized 
in the following Table-E. 
Table-E 
______________________________________ 
Fg concentration 
in unknown 
% sample (.mu.g/ml) 
Unknown sample Absorp- Method 
Subject Dilution tion of this RIA 
No. Material factor measured 
invention 
method 
______________________________________ 
1 Urine .times. 1 
25.0 0.400 0.359 
2 " .times. 10 
38.3 5.000 5.117 
3 " .times. 1 
22.4 0.380 0.368 
4 Serum .times. 1 
63.7 0.770 0.800 
5 " .times. 1 
67.6 0.830 0.892 
6 Cancerous .times. 400 
38.4 200 197.4 
intrapleural 
fluid 
______________________________________ 
EXAMPLE 4 
The percent absorption is measured at wavelengths of applied light of 1.2 
and 1.7 microns in the same manner as desdribed in Example 1, Parts (1) 
and (2), except for use of a polystyrene latex with an average particle 
diameter of 0.804 micron (Dow Chemical Co., 10% solids content by weight) 
instead of the polystyrene latex with an average diameter of 0.481 micron 
(Dow Chemical Co.) and of another concentration of bovine serum albumin 
(0.1% concentration by weight). The results are given in the following 
Table-F. 
Table-F 
______________________________________ 
Concentration of 
standard Fg solution 
% Absorption at 
(.mu.g/ml) 1.2 .mu. 1.7 .mu. 
______________________________________ 
0.2 25.4 17.4 
0.4 44.7 38.1 
0.6 61.9 64.7 
0.8 59.1 74.4 
1.0 44.7 76.1 
______________________________________ 
From the results shown in Table-F, it can be seen that a clear correlation 
is established between the concentration of Fg and the percent absorption 
when the wavelength of the applied light is longer than the average 
diameter of the solid carrier particles (polystyrene latex particles) by a 
factor exceeding 2. 
EXAMPLE 5 
Following the procedure as described in Example 1, Part (1), except that 
the anti-human fibrinogen antibody is replaced by anti-(human chorionic 
gonadotropin antibody (anti-hCG)) and the polystyrene latex with an 
average diameter of 0.481 micron by another polystyrene latex with an 
average particle diameter of 0.234 micron (Dow Chemical Co.), an 
anti-hCG-sensitized latex reagent is prepared. Using the 
anti-hCG-sensitized latex reagent thus obtained, the percent absorption is 
measured at a wavelength of the applied light of 1.2 microns in the same 
manner as described in Example 1, Part (2), except for use of standard hCG 
solutions instead of the standard Fg solutions. The results are summarized 
in the following Table-G. 
Table-G 
______________________________________ 
Concentration of 
standard hCG solution 
% Absorption at 
(IU/ml) 1.2 microns 
______________________________________ 
0.1 10.5 
0.2 25.9 
0.3 32.1 
0.4 45.2 
0.5 56.3 
0.7 76.6 
1.0 89.3 
______________________________________ 
A standard curve is prepared from the above data in the same manner as 
described above. On the other hand, urine samples are collected from 
several subjects and subjected to the determination of urinary hCG in the 
same manner as described in Example 1, Part (3). The results are 
summarized in the following Table-H. 
Table-H 
______________________________________ 
hCG concentration 
in unknown sample 
(IU/ml) 
Unknown samples % Method 
Subject Dilution 
Absorption 
of this RIA 
No. Material factor measured 
invention 
Method 
______________________________________ 
1 Urine .times. 1 
60.2 0.564 0.482 
2 " " 92.4 0.966 1.120 
3 " " 88.9 0.944 0.857 
4 " " 89.8 0.952 0.925 
______________________________________ 
EXAMPLE 6 
In a glass absorption cell having a thickness of 2 mm equipped with an 
L-shaped stirring rod, 0.1 ml of the anti-Fg-sensitized latex reagent as 
prepared in Example 2 and 0.3 ml of one of standard Fg solutions having 
concentrations of Fg as indicated in Table-I below are placed, and the 
percent absorption of the reaction mixture is monitored continuously at a 
wavelength of the applied light of 1.2 microns in the same manner as 
described in Example 1, Part (2) in order to read the time required to 
each 68.4% absorption while the stirring rod is moved up and down 
vertically at a definite speed of 200 vibrations per minute. The results 
are given in the following Table-I. 
Table-I 
______________________________________ 
Concentration of Time required to 
standard Fg solution 
reach 68.4% absorption 
(mg/ml) (sec.) 
______________________________________ 
2 26.9 
4 11.3 
6 6.4 
8 4.7 
10 2.9 
______________________________________ 
The above data are plotted on log-log graph paper with concentration of 
standard Fg solution as abscissa and time required to reach 68.4% 
absorption as ordinate so as to prepare a standard curve. The standard 
curve, as shown in FIG. 9, gives a clean straight line. 
Then, 0.3 ml of an unknown sample (urine, serum or intrapleural fluid) 
collected from a subject and 0.1 ml of the above-mentioned 
anti-Fg-sensitized latex reagent are placed in a glass absorption cell 
having a thickness of 2 mm equipped with an L-shaped stirring rod and the 
time required to reach 68.4% absorption is measured at a wavelength of the 
applied light of 1.2 microns in the same manner as above, whereupon the 
value of Fg concentration corresponding to the length of time measured is 
read from the standard curve as prepared in the above. The results are 
summarized in the following Table-J. 
Table-J 
______________________________________ 
Time Fg concentration 
required 
in unknown 
to reach 
sample (.mu.g/ml) 
Unknown sample 68.4% Method 
Subject Dilution absorption 
of this 
RIA 
No. Material factor (sec.) invention 
Method 
______________________________________ 
1 Urine .times. 1 
9.2 4.5 5.117 
2 Cancerous .times. 50 
11 195 197.4 
intrapleural 
fluid 
3 Serum .times. 1 
25 2.1 2.210 
4 " .times. 1 
20 2.5 2.302 
5 " .times. 1 
3.8 8.6 9.020 
6 " .times. 1 
12 3.7 3.723 
______________________________________ 
EXAMPLE 7 
(1) Preparation of an oxytocin-sensitized latex reagent 
One (1.0) ml of an oxytocin solution at a concentration of 220 IU/ml 
dissolved in aqueous 0.1 N acetic acid solution is mixed with 0.5 ml of a 
polystyrene latex with an average particle diameter of 0.481 micron (Dow 
Chemical Co., 10% solids content by weight), and the mixture is stirred at 
room temperature for 2 hours and then centrifuged (at 12,000 rpm) for 20 
minutes under cooling at 2.degree. to 4.degree. C. The precipitates are 
separated by decantation and the collected oxytocin-sensitized latex 
particles are dispersed in 4 ml of an EDTA-glycine buffer solution 
containing 0.2% by weight bovine serum albumin so as to prepare an 
oxytocin-sensitized latex reagent which contains the latex particles at a 
concentration of 1% by weight. 
(2) Decision of the optimum concentration of oxytocin antiserum 
A 0.1 ml aliquot of the oxytocin-sensitized latex reagent as prepared in 
Part (1) is mixed with 0.1 ml of an isotonic sodium chloride solution and 
0.2 ml of oxytocin antiserum which has been diluted with an isotonic 
sodium chloride solution by a factor indicated in Table-K below. The 
mixture is shaken on a reciprocal shaker at 200 strokes per minute for 12 
minutes and the percent absorption is measured at a wavelength of the 
applied light of 1.2 microns in the same manner as described in Example 1, 
Part (2). The results are given in the following Table-K. 
Table-K 
______________________________________ 
Dilution factor of 
Absorption 
oxytocin antiserum 
at 1.2 .mu. 
______________________________________ 
.times. 20 93.3 
.times. 30 75.9 
.times. 40 59.5 
.times. 50 52.8 
.times. 80 39.7 
.times. 100 31.1 
______________________________________ 
From the above data, it is decided that the optimum concentration of the 
oxytocin antiserum resides in around a dilution factor of about 30. 
(3) Preparation of a standard curve 
In a plastic test tube, 0.2 ml of a solution prepared by diluting the 
oxytocin antiserum as used in Part (2) above by a factor of 30 and 0.1 ml 
of a standard oxytocin solution (dissolved in an aqueous 0.1 N acetic 
acid) at a concentration indicated in Table-L below are placed and 
thoroughly mixed. After the mixture is allowed to stand at room 
temperature for 30 minutes, 0.1 ml of the oxytocin-sensitized latex 
reagent as prepared in Part (1) above is added to the test tube and the 
resulting mixture is shaken on a reciprocal shaker at 200 strokes per 
minute for 12 minutes. The liquid thus obtained is placed in a glass 
absorption cell with a thickness of 2 mm and the percent absorption is 
measured at a wavelength of the applied light of 1.2 microns in the same 
manner as described in Example 1, Part (2). The results are summarized in 
the following Table-L. 
Table-L 
______________________________________ 
Concentration of 
standard oxytocin 
% Absorption 
solution (.mu.IU/ml) 
at 1.2 .mu. .DELTA.D* 
______________________________________ 
2,000 59.8 13.8 
1,500 64.5 9.1 
1,000 67.1 6.5 
500 70.2 3.8 
300 71.8 1.8 
0 73.6 -- 
______________________________________ 
*.DELTA.D = (% absorption at zero concentration of the standard oxytocin 
solution) minus (% absorption at the indicated concentration of the same) 
When the above data are plotted graphically with concentration of standard 
oxytocin solution as abscissa and .DELTA.D as ordinate, a clean linear 
relationship is established as shown in FIG. 10. 
Using the standard curve thus prepared, it is possible to effect the 
determination of oxytocin in the serum samples of pregnant women. 
EXAMPLE 8 
(1) Preparation of an hCG-sensitized latex reagent 
In 5 ml of 0.05 N hydrochloric acid, 7,900 IU/ml of human chorionic 
gonadotropin (hCG) is dissolved and hydrolyzed at 80.degree. C. for an 
hour. After the solution is subjected to dialysis and subsequent suction 
filtration, the hydrolyzed hCG thus obtained is dissolved in 2 ml of a 
0.05 M borate buffer solution (pH 8.7) and diluted to 10 ml in the total 
volume. 
A 5 ml aliquot of a 2% solution of a polystyrene latex (Dow Chemical Co., 
10% solids content by weight) with an average particle diameter of 0.481 
micron is gradually added to the hydrolyzed hCG solution under stirring. 
The resulting hCG-sensitized latex particles are centrifuged at 13,000 rpm 
for 20 minutes and the sensitized latex particles precipitated are 
separated and suspended in 10 ml of a 0.2% solution of bovine serum 
albumin in the borate buffer solution. The suspension is then centrifuged 
and the collected precipitates are centrifugally washed with the borate 
buffer solution and finally suspended in 10 ml of the buffer solution so 
as to provide an hCG-sensitized latex reagent containing 1% by weight 
latex particles. 
(2) Preparation of a standard curve 
The optimum concentration (i.e., dilution by a factor of 300 in this case) 
of anti-hCG serum is decided in the same manner as described in Example 7, 
Part (2). In a plastic test tube, 0.2 ml of an anti-hCG serum solution 
prepared by diluting the serum with an isotonic sodium chloride solution 
by a factor of 300 and 0.1 ml of a standard hCG solution at a 
concentration indicated in Table-M below are placed and shaken for 10 
minutes. Subsequently 0.1 ml of the hCG-sensitized latex reagent as 
prepared in Part (1) above is added and the mixture is shaken for 10 
minutes on a reciprocal shaker at 200 strokes per minute. The resulting 
liquid is placed in a glass absorption cell with a thickness of 2 mm and 
the percent absorption is measured at a wavelength of the applied light of 
1.0 micron in the same manner as described in the foregoing Example 1, 
Part (2). The results are given in the following Table-M. 
Table-M 
______________________________________ 
Concentration of 
standard hCG solution 
% Absorption 
(IU/ml) at 1.0 .mu. 
______________________________________ 
10 27.6 
1 37.0 
0.1 46.4 
______________________________________ 
When the above data are plotted graphically with logarithm of concentration 
of standard hCG solution as abscissa and percent absorption as ordinate, 
the standard curve prepared gives a clean straight line, as shown in FIG. 
11, in these concentrations at which the measurement is actually carried 
out. Thus, it is possible to effect the determination of hCG in the serum 
samples from patients of chorioepithelioma 
EXAMPLE 9 
In a plastic test tube, 0.1 ml of the anti-Fg-sensitized latex reagent as 
prepared in Example 1, Part (1) (the average diameter of the polystyrene 
latex particles: 0.481 micron; sensitized latex particles content: 1% by 
weight) and 0.3 ml of a standard Fg solution (dissolved in an isotonic 
sodium chloride solution containing 0.5% by weight bovine serum albumin) 
at a concentration indicated in Table-N below are mixed thoroughly and 
then shaken for 3 hours on a reciprocal shaker at 200 storkes per minute. 
Subsequently the percent absorption is measured at a wavelength of the 
applied light of 1.2 microns in the same manner as described in Example 1, 
Part (2). The results are shown in the following Table-N. 
Table-N 
______________________________________ 
Concentration of 
standard Fg solution 
% Absorption 
(ng/ml) at 1.2 .mu. 
______________________________________ 
10 11.5 
20 25 
40 52.6 
60 71.2 
80 80.8 
100 84.3 
______________________________________ 
When a standard curve is prepared on the basis of the above data, a clear 
correlation is found between the concentration of the standard Fg solution 
and the percent absorption. Thus, in accordance with the invention, it is 
possible to determine ultramicro amounts of Fg of the order of ng 
(nanograms)/ml, and such high sensitivity is comparable to that of the RIA 
method. 
EXAMPLE 10 
Anti-Fg-sensitized latex reagents containing the anti-Fg-sensitized latex 
particles at concentrations of 0.75%, 1.0% and 2.0% by weight, 
respectively, are prepared in the same manner as described in Example 1, 
Part (1), except for use of another polystyrene latex with an average 
particle diameter of 0.35 micron. With each anti-Fg-sensitized latex 
reagent thus prepared, a standard curve is prepared in the same manner as 
described in Example 1, Part (2) (wavelength of the applied light 1.2 
microns; shaking time 10 minutes). These standard curves are shown in FIG. 
12, in which Curves a, b and c denote the standard curves obtained at 
concentrations of the anti-Fg-sensitized latex particles of 0.75, 1.0 and 
2.0% by weight, respectively. As is evident from FIG. 12, the detection 
sensitivity for Fg increases as the concentration of the sensitized latex 
particles in the anti-Fg-sensitized latex reagent becomes higher. 
EXAMPLE 11 
An anti-Fg-sensitized latex reagent (concentration of latex particles: 0.5% 
by weight) is prepared in the same manner as described in Example 1, Part 
(1), except for use of another polystyrene latex having an average 
diameter of 0.804 micron. 
A 0.2 ml aliquot of the Anti-Fg-sensitized latex reagent thus obtained is 
mixed with 0.2 ml of each of standard Fg solutions at different 
concentrations indicated in Table-O below, and the mixture is placed in a 
rectangular absorption cell having a thickness of 2 mm and stirred with an 
L-shaped stirring rod moving up and down at a speed of 160 strokes per 
minute in the cell to effect the antigen-antibody reaction. After 3 
minutes, the absorbance of the reaction mixture is measured with light of 
a wavelength of 0.9 micron. The results are summarized in Table-O. 
Table-O 
______________________________________ 
Concentration of 
standard Fg solution 
Absorbance 
(.mu.g/ml) after 3 min. 
______________________________________ 
0.625 0.006 
0.125 0.011 
0.25 0.034 
0.50 0.041 
1.0 0.081 
2.0 0.130 
______________________________________ 
When the above data are plotted graphically with concentration of standard 
Fg solution as abscissa and absorbance after 3 minutes as ordinate, a 
clear linear relationship is obtained as shown in FIG. 13. 
EXAMPLE 12 
An anti-hCG-sensitized latex reagent (containing 0.3% by weight latex 
particles) is prepared in the same manner as described in Example 1, Part 
(1), except that the anti-(human fibrinogen)antibody is replaced by 
anti-(human chorionic gonadotropin)antibody (anti-hCG) and the polystyrene 
latex of an average diameter of 0.481 micron by another polystyrene latex 
of an average diameter of 1.09 microns (Dow Chemical Co.). A 0.2 ml 
aliquot of the resulting anti-hCG-sensitized latex reagent is thoroughly 
mixed with 0.2 ml of each of standard hCG solutions having different 
concentrations indicated in Table-P below, and the mixture is placed in a 
rectangular absorption cell of 2 mm in thickness and stirred therein with 
an L-shaped stirring rod moving up and down at a speed of 160 strokes per 
minute to effect the antigen-antibody reaction. Accurately after 2 
minutes, the absorbance of the reaction mixture is measured at 1.10 
microns in wavelength of the applied light. The results are summarized in 
Table-P. 
Table-P 
______________________________________ 
Concentration of 
standard hCG solution 
Absorbance 
(IU/ml) after 2 min. 
______________________________________ 
0.0625 0.018 
0.125 0.023 
0.25 0.035 
0.5 0.050 
1.0 0.075 
______________________________________ 
When the above data are plotted on log-log graph paper with concentration 
of standard hCG solution as abscissa and absorbance after 2 minutes as 
ordinate, a calibration curve which assumes a clear straight line is 
obtained as shown in FIG. 14. 
Having now fully described the invention, it will be apparent to one of 
ordinary skill in the art that many changes and modifications can be made 
thereto without departing from the spirit or scope of the invention as set 
forth herein.