Latex polymer sensitized with steroid-serum albumin conjugate, preparation and test therewith

Latex reagents for quantitative assay of a variety of steroid hormones or metabolites thereof which are contained in body fluid or excreted fluid of human beings comprise latex particles which are immunologically sensitized with a conjugate selected from a variety of steroid-serum albumin conjugates in which the steroid to be detected is bonded to serum albumin in a very small ratio ranging from 0.5 to 7 molecules per 1 molecule of the serum albumin used. Sensitization of latex particles with such a conjugate is performed using a limited amount dependent on the bonding ratio of steroid molecules per molecule of serum albumin in the conjugate and the particle size of the latex used. The latex reagents show very high sensitivity amounting to 0.2-0.04 nmole steroid equivalent/ml.

BACKGROUND OF THE INVENTION 
It has been known that the quantity of steroid hormones and/or metabolites 
thereof contained in human body fluid or excreted fluid is correlated with 
physiological and pathological states of human beings. Therefore 
quantitative analysis of steroid hormones and/or metabolites thereof is 
useful for purposes of diagnosis and clinical examination. 
However, the quantity of steroid hormones and metabolites thereof contained 
in human body fluid or excreted fluid is generally small, and the 
variation in quantity of steroid hormones and metabolites thereof due to a 
specific physiological or pathological states is also small. 
Consequently quantitative analysis of steroid hormones and/or metabolites 
thereof requires extremely high accuracy. 
Recent developments in analytical instruments make it possible to conduct 
extremely accurate quantitative analysis. Such instruments are highly 
elaborate, and installation and maintainance of such instruments involve 
high costs in general. Furthermore, manipulation of such instruments is 
not easy for persons who are not skilled, and often requires troublesome 
and time-consuming preliminary treatment of samples to be analyzed. 
Therefore it is significant to provide a means for enabling accurate and 
rapid quantitative detection of steroid hormones and/or metabolites 
thereof without necessitating special training and special instruments. 
It has been known that quantitative detection of steroids can be 
accomplished by immunological methods. One of the typical immunological 
methods utilizes the specificity of the antigen-antibody reaction and 
visibility of the agglutination and agglutination inhibition reaction 
thereof. More specifically, in the typical prior art, the type of steroid 
to be detected is bonded to protein to give a steroid-protein conjugate, 
then a reagent is prepared having latex particles sensitized with the 
conjugate and antiserum is prepared by injecting the conjugate into a 
mammal. The thus obtained antiserum may react with the steroid to be 
detected and also may react with the reagent. Therefore if a given 
quantity of a test sample of human body fluid or excreted fluid is 
collected and mixed with a given quantity of said antiserum, a reaction 
takes place therebetween. Then a given quantity of the reagent is added to 
the liquid mixture of the test sample and the antiserum. If an excess 
amount of antiserum remains in the fluid mixture of said test sample and 
the antiserum, antigen-antibody reaction between the remaining antiserum 
and the reagent can be observed as an agglutination reaction, and if the 
antiserum is just neutralized with the steroid contained in the test 
sample or an excess amount of steroid remains in the fluid mixture no 
antigen-antibody reaction occurs, and this can be observed as and 
agglutination inhibition reaction. Therefore, if serially diluted test 
samples are tested with the antiserum, which is used at a predetermined 
titer, and the reagent, quantitative assay of the steroid and/or 
metabolites thereof can be performed. 
One of such prior art methods is disclosed in Japanese Early Opened Patent 
application Publication No. 51-112513 (Filing Date: Mar. 25, 1975; 
Inventor: Osamu Kanemitsu; Applicant: Asahi Kasei Kogyo Kabushiki Kaisha; 
Date of Laying-Open: Oct. 5, 1976). This first prior art publication 
refers to a method for immunological quantitative assay of 
dehydroepiandrosterone sulphate (which is an intermediate in the 
biosynthesis of a sex hormone), but this prior art adds nothing new to the 
aforementioned typical prior art. Though these methods of the prior art 
provide useful diagnostic testing methods, it has been strongly desired to 
increase the sensitivity of such immunological quantitative assay, since 
this makes it possible to detect slighter variations of steroid quantity 
and to provide increased accuracy for conventional use. 
Meanwhile, human body fluid or excreted fluid usually contains many kinds 
of compounds besides steroids, and the amounts of these compounds are 
usually far greater than the steroid content. Some of these compounds, for 
example, protein and saccharide, affect immunological assay, and it is 
necessary to remove them or dilute the body fluid or excreted fluid to be 
tested to the extent where no interference is caused. Removal of the 
interfering compounds requires a complicated process, and in clinical 
analysis which requires simplicity and rapidity, it is necessary to dilute 
the fluid to be tested. This makes it necessary to increase the 
sensitivity of such immunological assay. 
Adjustment of sensitivity is referred to in the Japanese Early Opened 
Patent application Publication No. 50-123819 (Filing Date: Mar. 14, 1974; 
Inventor: Hideaki Manita et al; Applicant: Teikoku Zoki Seiyaku Kabushiki 
Kaisha; Date of Laying-Open: Sept. 29, 1975). 
This second prior art publication discloses a method for immunological 
assay of steroids contained in human body fluid or excreted fluid which 
utilizes an antibody obtainable from a mammal immunized with steroid 
conjugated with antigenic protein having free amino group and sensitized 
carriers wherein carriers are sensitized with the steroid conjugated with 
protein which is different from the antigenic protein. In this second 
prior art method, adjustment of sensitivity can be achieved by varying the 
amount of the steroid-protein conjugate for sensitizing latex particles in 
the reagent and by varying the concentration of the antiserum. Though the 
descriptions in the second prior art publication are not necessarily 
clear, even if they may suggest that a smaller amount of excess antiserum 
can agglutinate sensitized latex particles when the latex particles are 
sensitized with a smaller amount of the steroid-protein conjugate, this 
approach may not give a significant improvement of sensitivity for the 
reason mentioned below. Originally, latex particles have a tendency to 
show non-specific agglutination when a proper stabilizing agent is not 
added thereto, and this non-specific agglutination cannot be distinguished 
from specific agglutination due to the antigen-antibody reaction. The 
steroid-protein conjugate used for sensitization of latex particles may 
act as a stabilizing agent for eliminating this non-specific agglutination 
of the latex particles proper. Accordingly the adjustment of sensitivity 
in the second prior art method is limited to a certain extent where 
non-specific agglutination is eliminated, and it is not possible to 
increase sensitivity beyond the limitation. 
An approach to overcoming this limitation is considered to use the 
techniques disclosed in Japanese Patent Publication No. 49-11407 (Filing 
Date: Dec. 29, 1970; Inventor: Tadamitsu Sudo; Applicant: Teikoku Zoki 
Seiyaku Kabushiki Kaisha; Publication Date: Mar. 16, 1974) and Japanese 
Early Opened Patent application Publication No. 50-82230 (Filing Date: 
Nov. 29, 1973; Inventor: Tadamitsu Sudo et al; Applicant: Teikoku Zoki 
Seiyaku Kabushiki Kaisha; Date of Laying-Open: July 3, 1975). In these 
third and fourth prior art publications, it is disclosed that latex 
particles can be stabilized by making latex particles absorb an 
immunologically inert protein before or after the latex particles are 
sensitized with steroid-protein conjugate or antibody. Combining the 
second prior art publication and the third or fourth prior art 
publication, there is a possibility of obtaining sensitized latex 
particles having increased sensitivity with the latex particles being 
sensitized with a smaller amount of steroid-protein conjugate and being 
stabilized with an immunologically inert protein. It has been found, 
however, that this stabilization of latex particles by the inert protein 
tends to affect the immunologically specific agglutination. 
Japanese Early Opened Patent application Publication No. 48-49918 (Filing 
Date: Oct. 26, 1972; Inventor: John Anthony Coppola et al; Applicant: 
American Cyanamid Company; Date of Laying-Open: July 14, 1973) discloses a 
method for preparation of antibody which has excellent specificity against 
progesterone and applicability of the antibody for radioimmunoassay and an 
agglutination test for determination of the concentration of progesterone 
in test samples. However, this fifth prior art publication does not 
generally refer to improvement of sensitivity for detection of a variety 
of steroids. 
Moreover, in this fifth prior art publication, it is apparently stated that 
the steroid (hydroxyprogesterone)-protein conjugate has 15-40 molecules of 
steroid per molecule of protein, and this conjugate is used not only for 
preparation of antibody but also for sensitization of latex particles. 
Also, in the second prior art publication (Japanese Early Opened Patent 
application Publication No. 50-123819), 
estriol-16.alpha.-glucuronide--rabbit serum albumin conjugate is used for 
sensitization of latex particles, and the bonding ratio of steroid 
glucuronide to protein ranges from 27 to 30 moles per mole of protein 
used. 
As will be seen from the foregoing, steroid-protein conjugates in which a 
relatively large number of molecules of steroid are bonded per molecule of 
protein have been used for sensitization of latex particles in the prior 
art (hereinafter the number of molecules of steroid which are bonded to a 
molecule of protein is simply referred to as the steroid bonding number). 
The inventors have considered that further improvement of sensitivity would 
not be expected through conventional approaches, and the inventors' 
attempts have been concentrated on improvement of the conjugate proper. As 
a result, the inventors have found that when a steroid-serum albumin 
conjugate is prepared so that the steroid bonding number falls within a 
range of 0.5-7.0, and then latex particles are sensitized with such 
conjugate, the thus obtained sensitized latex particles show extremely 
high sensitivity, and that this is applicable irrespective of the sort of 
steroid and serum albumin used. As far as the inventors know, there is no 
reference which refers to decreasing steroid bonding number for increasing 
sensitivity of steroid detection. 
SUMMARY OF THE INVENTION 
Therefore, one of the primary objects of this invention is to provide a 
method for preparation of a latex polymer sensitized with steroid-serm 
albumin conjugate having increased steroid detection sensitivity in which 
the steroid for preparation of the conjugate is bonded to serum albumin in 
a ratio ranging from 0.5 to 7 mole to 1 mole of serum albumin. 
Another object of this invention is to provide a method for preparation of 
a latex reagent which may detect steroids with a high sensitivity although 
the latex particles are sensitized only with steroid-serum albumin 
conjugate without stabilizing the latex particles with immunologically 
inert protein. 
A further object of this invention is to provide a method for preparation 
of a polymer latex sensitized with steroid-serum albumin conjugate for 
detecting steroids with increased sensitivity wherein the latex polymer is 
selected from the group consisting of styrene polymer, butadiene polymer 
and styrene-butadiene copolymer. 
A still further object of this invention is to provide a method for 
preparation of a latex polymer sensitized with steroid-serum albumin 
conjugate having increased steroid detection sensitivity wherein the 
steroid for preparation of the conjugate is selected from the group 
consisting of the steroid to be detected, metabolites thereof and 
synthetic steroids having similar structure thereto, and the serum albumin 
for preparation of the conjugate is selected from the group consisting of 
bovine serum albumin, equine serum albumin, sheep serum albumin, rabbit 
serum albumin and human serum albumin. 
Another primary object of this invention is to provide a latex reagent 
having high sensitivity for steroid detection wherein latex particles are 
sensitized with steroid-serum albumin conjugate having a relatively small 
steroid bonding number. 
These and other objects and features of this invention will become apparent 
with reference to the following detailed description of this invention. 
DETAILED DESCRIPTION OF THE INVENTION 
First the general and basic aspects of the present invention will be 
described and then discussion will enter into the details thereof. 
Steroid 
Generally speaking, steroids which can be utilized in the present invention 
are the following: 
follicular hormone (estrogen), 
corpus luteum hormone (progestrone), 
male sex hormone (for example, 17-ketosteroid), 
adrenocortical hormone (for example, 17-hydroxycorticosteroid), and 
steroids which may be a variety of metabolites of the steroid hormones 
mentioned above 
These steroids when used for preparing conjugates with serum albumin are 
preferably in the states in which they are found in human body fluid or 
excreted fluid, for example in the state of steroid-glucuronide conjugates 
or steroid-sulphate conjugates. 
However, any natural or synthesized steroids may be utilized provided they 
have sufficient immunological cross-reactivity with any specific steroid 
hormone or metabolite thereof to be detected in human body fluid or 
excreted fluid and provided they have a functional group in which they may 
be chemically bonded to serum albumin. For instance, steroid-hemisuccinate 
or steroid-(O-carboxymethyl)oxime may be utilized as a synthetic steroid. 
Serum Albumin 
Any refined serum albumin which is commercially available may be utilized, 
for example, 
______________________________________ 
Bovine Serum Albumin (BSA) 
Equine Serum Albumin (ESA) 
Sheep Serum Albumin (SSA) 
Rabbit Serum Albumin (RSA) 
Human Serum Albumin (HSA) 
______________________________________ 
The codes shown in parentheses above are the initials of the respective 
serum albumins, and hereinafter these codes are utilized for simplicity. 
Especially BSA or RSA is preferable among them. 
Latex Particles 
The following latex particles are commercially available and may 
conveniently be utilized in the present invention: 
Polystyrene Latex Particles 
Polybutadiene Latex Particles 
Styrene-Butadiene Copolymer Latex Particles 
It is important that the latex particles do not have reactive radicals and 
they must be inert in the chemical and immunological senses. Polystyrene 
latex particles are especially preferable among them. 
The particle size may be 0.05 .mu.m-1.0 .mu.m and preferably 0.2 .mu.m-0.8 
.mu.m. 
Preparation Of Conjugate 
The following methods are known and may adequately be utilized in the 
present invention for preparing a steroid-serum albumin conjugate which is 
utilized for sensitizing latex particles and for obtaining antiserum or 
antibody. 
Carbodiimide method (cf. Gross et al: Immunochemistry, Vol. 5, page 55, 
1968), 
Acid Chloride method (cf. Erlanger et al: Journal of Biological Chemistry, 
Vol. 228, page 713, 1957), 
Mixed Acid Anhydride method (ditto), and 
Isocyanate method (Goodfriend et al: Canadian Journal of Biochemistry and 
Physiology, Vol. 36, page 1177, 1958) 
An example of preparation of steroid-serum albumin conjugate by the Mixed 
Acid Anhydride method is shown hereunder. 
Ten moles of steroid-glucuronide, steroid-hemisuccinate or 
steriod-(O-carboxymethyl)oxime per mole of serum albumin to be used were 
dissolved in dimethylformamide. To this solution, the same number of moles 
of tri-n-butylamine as of the steroid were added as an auxiliary activator 
and the mixture was agitated. Then the same number of moles of 
isobutylchloroformate as of the steroid were further added as an activator 
to the thus obtained mixed solution and the mixture was agitated. The thus 
obtained mixed solution was referred to as liquid A. 
On the other hand, serum albumin was dissolved in deionized water, and 
after adjusting its pH to 8.0-10.0 with N NaOH, dimethylformamide was 
added thereto. The thus obtained solution was referred to as liquid B. The 
quantity of dimethylformamide in liquid B was determined so that the total 
quantity of dimethylformamide in liquids A and B was the same as that of 
the deionized water in liquid B. 
Then liquid A was added dropwise to liquid B over one and a half hours with 
agitation, and after its pH was adjusted to 8.0-10.0 with N NaOH, the 
mixed liquid was agitated for three and a half hours whereby steroid-serum 
albumin conjugate was synthesized. The thus obtained mixed liquid was 
dialyzed against water, and then acetone was added thereto in the ratio of 
two parts of acetone per one part of the dialyzed mixed liquid and they 
were homogeneously mixed. Adding N HCl to the mixed liquid to precipitate 
the conjugate, the precipitated conjugate was centrifugally separated. The 
proper quantity of water was added to the separated precipitate, and N 
NaOH was added thereto for adjusting its pH to 8. The thus obtained 
solution was subjected to dialysis against water for removing acetone 
whereby the conjugate was taken up. The process for preparation of the 
conjugate was carried out at low temperature (about 6.degree. C.). 
The steroid bonding number in the conjugate can be determined by known 
methods, for example, the ultraviolet absorption method and the 
dinitrophenylation method (see Test 1). 
Sensitization Of Latex Particles 
A suspension of latex particles was prepared by washing and/or diluting 
latex particles with a suitable buffer solution (pH 7.2-8.6), and the 
steroid-serum albumin conjugate which was prepared in a proper 
concentration was added thereto and the mixture was kept at 37.degree. C. 
for 2 hours with agitation to obtain a suspension of sensitized latex 
particles. The suspension was centrifuged and the precipitate was 
separated, and then washed with the buffer solution. The centrifugation 
and washing were repeated several times, and the thus obtained precipitate 
was resuspended in the buffer solution to obtain a suspension of latex 
particles sensitized with the conjugate. The quantity of the conjugate 
utilized for sensitizing the latex particles may be limited within a range 
within which the following requirements are fulfilled: 
(1) The sensitized latex particles finally obtained do not show 
non-specific agglutination due to the nature of the latex particles 
proper. 
(2) Specific agglutination due to the antigen-antibody reaction can occur. 
(3) When antibody is neutralized with a sufficient amount of steroid hapten 
and then the suspension of the sensitized latex particles is added 
thereto, the resulting mixed liquid remains a homogeneous suspension 
without any agglutination. 
As will be mentioned hereunder, the amount of conjugate used for 
sensitization of latex particles (hereinafter referred to as the 
sensitizing quantity) is varied depending on the size of the latex 
particles and the steroid bonding number in the conjugate to be used. 
Latex particles having different sizes, 0.234 .mu.m and 0.721 .mu.m were 
respectively sensitized with a conjugate in which 
estriol-16.alpha.-glucuronide was bonded to RSA in the ratio of 2.0 
molecules per molecule of RSA in accordance with the process mentioned 
above. The minimum sensitizing quantities in the respective latex 
particles for eliminating non-specific agglutination were 72 mg and 18 mg 
per gram of latex particles. In general the smaller particles tend to 
require larger sensitizing quantities. 
Latex particles having a particle size of 0.234 .mu.m were sensitized with 
estriol-16.alpha.-glucuronide conjugate having a steroid bonding number of 
15.1 using different sensitizing quantities. The minimum sensitizing 
quantity for eliminating non-specific agglutination was 54 mg per gram of 
the latex particles. It should be noted that the minimum sensitizing 
quantity in the latter case was relatively small compared with that in the 
former case (72 mg/g) in which latex particles were sensitized with the 
conjugate having a steroid bonding number of 2.0 (see Table 1). 
It should also be noted that sensitization of latex particles can be 
performed by dialysis, ultrafiltration or combination of any one of them 
with the aforementioned method. Also sensitized latex particles can be 
freeze dried. 
Preparation Of Antibody 
Antibody may be prepared by conventional methods, for example, antiserum or 
antibody separated therefrom may be obtainable in such a manner that 
steroid-serum albumin conjugate is first prepared by reacting steroid in 
the state found in human body fluid or excreted fluid, or steroid having 
immunological cross-reactivity therewith, to serum albumin, and then the 
thus obtained conjugate is non-orally dosed (e.g. injected) to a mammal 
which has serum foreign to the serum used to prepare the conjugate for 
immunizing the mammal from which antiserum is to be collected. Antiserum 
thus obtained may be utilized as original antibody. Of course, the 
antibody may be separated from the antiserum. 
It should be noted that the steroid to be detected may react as a hapten 
with the antibody, and the conjugate for sensitizing the latex particles 
also may react therewith. 
Steroid Detection 
The conventional method is utilized except that latex reagent prepared in 
accordance with the present invention is used. The detection method 
comprises the following two processes: 
(1) A given quantity of human body fluid or excreted fluid either undiluted 
or properly diluted is taken out as a test sample, and a given quantity of 
antibody is added and mixed with the test sample to produce an 
antigen-antibody reaction between the steroid which is the hapten 
contained in the test sample and the antibody (neutralization of 
antibody). 
(2) A given quantity of the mixture of the test sample and the antibody is 
dropped on a glass slide and a given quantity of the latex reagent is 
added thereto and mixed with an elongated small rod, and the mixed liquid 
on the glass slide is observed a few minutes later to determine whether an 
agglutination reaction has occurred or not. 
That is, if the antibody is not fully neutralized by the steroid contained 
in the test sample and consequently an excess amount of the antibody 
remains after the first process, then antigen-antibody reaction occurs 
between the latex reagent and the remaining antibody and agglutination can 
be observed in the second process. In case the antibody is fully 
neutralized with the steroid contained in the test sample (including the 
case in which an excess amount of steroid remains in the first process), 
no antigen-antibody reaction occurs and agglutination cannot be observed, 
that is, agglutination inhibition reaction is observed in the second 
process. 
It will be understood that the concentration of the test samples, latex 
reagent and the antibody can be properly determined depending upon the 
anticipated or reference concentration of steroid hormone and/or its 
metabolite to be tested in the test sample. The agglutination reaction in 
the second process may be terminated at the latest within 5 minutes.

Having described the general and basic aspects of the latex reagent, method 
for preparation thereof and the method for detecting steroids using the 
latex reagent in accordance with the present invention, now the invention 
will be further described in detail based on a number of exemplifying 
tests and examples of the present invention. 
TEST 1 
I. Material Preparation 
(1) Conjugate Preparation 
Using estriol-16.alpha.-glucuronide (Sigma Company) and RSA (INC 
Pharmaceuticals Company), nine kinds of conjugate which were different in 
steroid bonding number were prepared as follows: 
Liquid A Preparation 
Ten milligrams of estriol-16.alpha.-glucuronide were dissolved in 1 ml of 
dimethylformamide (Wako Junyaku Kogyo Kabushiki Kaisha). To this solution, 
5.1 .mu.l of tri-n-butylamine (Tokyo Kasei Kogyo Kabushiki Kaisha) were 
added and the solution was agitated. Then, 2.8 .mu.l of 
isobutylchloroformate (Aldrich Chemical Company) were added and the 
solution was agitated. 
The same quantity of liquid A was prepared in 9 vessels. 
Liquid B Preparation 
RSA was dissolved in deionized water as shown in Table 3 to obtain RSA 
solutions having different concentrations. Adding N NaOH to the respective 
solutions, the pH was adjusted to 8.0-10.0. To these solutions different 
amounts of dimethylformamide were added, and 9 kinds of liquid B in which 
different amounts of RSA were contained were prepared. The amount of 
dimethylformamide contained in each of liquids B was adjusted so that the 
total amount of dimethylformamide contained in liquids A and B was equal 
to the amount of deionized water contained in liquid B. 
Conjugate Preparation 
One sample of liquid A was added dropwise to each sample of the liquid B 
over one and a half hours with agitation, by adding N NaOH to the 
respective solutions the pH was adjusted to 8.0-10.0, and each of these 
solutions was further agitated for 3 hours. Thus and 9 kinds of 
estriol-16.alpha.-glucuronide-RSA conjugate solutions were obtained. 
Each of these solutions was subjected to dialysis against deionized water. 
To the dialyzed solutions acetone was respectively added in the ratio of 2 
parts acetone per 1 part of the solution, and after suficient agitation N 
HCl was respectively added to precipitate the conjugate. The precipitated 
conjugate in each of the solutions was separated by centrifugation for 5 
minutes at 10,000 r.p.m. To each of the separated conjugates deionized 
water was added, and after adjusting its pH to about 8 with N NaOH, each 
of these solutions was dialyzed against deionized water to remove acetone. 
Thus 9 kinds of conjugates having different steroid bonding numbers (see 
Table 3, Samples Nos. 1-9) were obtained. The processes of preparation of 
the conjugate were carried out at low temperature (6.degree. C.). 
The respective steroid bonding numbers in the conjugates are shown in Table 
3. Measurement of steroid bonding number will be described later on. 
Conjugate Latex Particles Preparation 
Minimum sensitizing quantities with respect to the conjugates of samples 
Nos. 1-9 for eliminating non-specific agglutination of the obtainable 
conjugate latex particles were determined as follows: 
Dissolving 5.0, 5.2, 5.4 and 5.6 mg of sample No. 1 (conjugate) in Table 3 
respectively in 40 ml of 40 mM veronal buffer solution containing 150 mM 
NaCl, 4 kinds of conjugate solutions were prepared. Then each of the 4 
kinds of conjugate solutions, 1 ml of a 10% suspension of the latex 
particles (Dow Chemical Co.) having a particle size of 0.234 .mu.m was 
added, and they were kept at 37.degree. C. for 2 hours. The thus obtained 
sensitized latex particle suspensions were each subjected to 
centrifugation at 4,000 r.p.m. for 20 minutes to precipitate the 
sensitized latex particles. The separated precipitates were each suspended 
in 20 ml of the same veronal buffer solution and were centrifuged under 
the same condition mentioned above to separate the sensitized latex 
particles. This last mentioned process was respectively repeated once 
again, and the thus obtained precipitates were each resuspended in 5 ml of 
the same veronal buffer solution, and 4 kinds of sensitized latex particle 
suspensions having different sensitizing quantities were thus obtained. 
On glass slides, 0.1 ml of each of the sensitized latex particle 
suspensions was respectively dropped to which 0.1 ml of the antiserum 
which was neutralized with an excess amount of 
estriol-16.alpha.-glucuronide was respectively added and mixed. Several 
minutes later, the presence of non-specific agglutination in the 
respective mixed drops was observed. 
Non-specific agglutination was observed in the droplets for the conjugate 
sensitized latex particle suspensions having sensitizing quantities of 5.0 
mg and 5.2 mg of the sample No. 1, and was not observed in those prepared 
using 5.4 mg and 5.6 mg of sample No. 1. Thus the minimum sensitizing 
quantity of sample No. 1 was determined as 5.4 mg per 0.1 g of latex 
particles. 
Like tests were conducted with respect to samples No. 2-No. 9 to determine 
the respective minimum sensitizing quantities. The results are shown in 
Table 1. 
TABLE 1 
______________________________________ 
Sample No. 
1 2 3 4 5 6 7 8 9 
______________________________________ 
estriol-16.alpha.- 
15.1 10.7 7.4 5.1 3.2 2.0 0.9 0.5 0.3 
glucuronide 
bonding 
number 
(per 1 
molecule of 
RSA) 
minimum 5.4 5.9 6.3 6.5 7.0 7.2 7.4 7.4 7.4 
sensitizing 
quantity 
(mg/0.1 g 
latex 
particles) 
______________________________________ 
Using the respective minimum sensitizing quantities of the samples No. 
1-No. 9, sensitized latex particle suspensions of the samples No. 1-No. 9 
were respectively prepared. 
II. Measurement of Steroid Bonding Number and Steroid Detection Sensitivity 
(1) Steroid Bonding Number Measurement 
Steroid bonding number measurement was carried out by the method of 
Erlanger et al utilizing ultraviolet absorption (Journal of Biological 
Chemistry, Vol. 234, page 1090, 1959) and by Sanger's method utilizing 
dinitrophenylation (Biochemical Journal, Vol. 39, page 507, 1945). 
(i) Measurement by Ultraviolet Absorption 
Estriol-16.alpha.-glucuronide and RSA absorb in the same spectral region, 
i.e., with a maximum at 278 nm, and the absorption maximum of the 
conjugate thereof can be measured as the sum of the absorption maxima of 
the respective components. Accordingly the absorbance derived from 
estriol-16.alpha.-glucuronide in the conjugate can be obtained by 
subtracting the absorbance of RSA from that of the conjugate. Comparing 
the thus obtained absorbance derived from estriol-16.alpha.-glucuronide in 
the conjugate with the absorbances of a variety known concentrations of 
estriol-16.alpha.-glucuronide, the estriol-16.alpha.-glucuronide bonding 
number was obtained. 
(ii) Measurement by Dinitrophenylation 
Lysine radicals (Lys) in the conjugate and in the serum albumin used for 
preparing the conjugate were respectively dinitrophenylated with 
dinitrofluorobenzene (Wako Junyaku Kogyo Kabushiki Kaisha) to produce 
dinitrophenylated conjugate and dinitrophenylated serum albumin. Then 
these dinitrophenylated products were respectively hydrolyzed at 
110.degree. C. to obtain 24 hours for free dinitrophenyllysine. Thus two 
kinds of solutions containing dinitrophenyllysine were obtained, one 
derived from the conjugate and the other derived from serum albumin. On 
the other hand, commercially available dinitrophenyllysine (Tokyo Kasei 
Kogyo Kabushiki Kaisha) was obtained as a reference substance and serially 
diluted solutions thereof were prepared. The former two kinds of 
dinitrophenyllysine containing solutions and serially diluted reference 
dinitrophenyllysine solutions, were subjected to colorimetric 
determination at 390 nm, and the steroid bonding number in the conjugate 
was calculated from the values of the optical density thereof. 
(2) Measurement of Steroid Detection Sensitivity 
In general, steroid detection sensitivity was measured in such a manner 
that first the titer of the original antibody was determined with 
reference steroid solution, and then the titer of the respective samples 
No. 1-No. 9 was determined with reference to the titer of the original 
antibody. The details thereof are mentioned below. 
(i) Determination of Titer of Antibody 
Antiserum was obtained from rabbits which were immunized with 
estriol-16.alpha.-glucuronide-BSA conjugate by a proper conventional 
method, and the antiserum was utilized as an original antibody. This 
original antibody was serially diluted with 40 mM veronal buffer solution 
containing 150 mM NaCl. To 0.05 ml of the serially diluted antibody, 0.05 
ml of a solution prepared by dissolving 0.1 nmole of 
estriol-16.alpha.-glucuronide per 1 ml of the buffer solution was 
respectively added and agitated well. Taking dropwise 0.1 ml of the 
respective agitated mixed solutions on glass slides, 0.1 ml of the 
conjugate sensitized latex particle suspension having an 
estriol-16.alpha.-glucuronide bonding number of 2.0 was added to each of 
the droplets on the glass slides and they were agitated well. Observation 
after 3 minutes showed that agglutination reaction was detectable in the 
droplets having 200-fold or less dilution of the original antibody. 
TABLE 2 
______________________________________ 
Dilution of 
antibody .times.50 
.times.100 
.times.200 
.times.300 
.times.400 
Agglutination 
+ + + - - 
______________________________________ 
This means that the original antibody when diluted 200-fold can neutralize 
at least 0.1 nmole of estriol-16.alpha.-glucuronide/ml. 
Thus the titer of this 200-fold antibody was determined to be 0.1 nmole 
estriol-16.alpha.-glucuronide equivalent/ml, and the titer of the original 
antibody to be 20 nmole estriol-16.alpha.-glucuronide equivalent/ml. 
(ii) Determination of Titer of Latex Reagent 
The original antibody was serially diluted with 40 mM veronal buffer 
solution and 0.1 ml of each of the serially diluted antibody samples was 
dropped on glass slides. To these droplets 0.1 ml of each of the samples 
No. 1-No. 9 (see Table 1) was added and agitated well, and the results 
were observed after 3 minutes. Based on the maximum dilution in the 
droplets in which agglutination reaction was observed and the titer of the 
original antibody, the titers of the latex reagent samples were 
calculated. It will be understood therefore that a smaller numerical value 
of the titer means higher detection sensitivity. 
The estriol-16.alpha.-glucuronide bonding number and detection sensitivity 
of respective samples No. 1-No. 9 are shown in Table 3. 
TABLE 3 
__________________________________________________________________________ 
detection 
sensitivity 
Estriol-16.alpha.- 
Estriol- 
glucoronide 16.alpha.- 
preparation condition 
bonding number 
glucuronide 
Estriol-16.alpha.- 
deionized 
ultraviolet 
dinitro- 
equivalent 
sample 
RSA 
glucuronide/RSA 
water 
absorption 
phenylation 
antibody 
No. (mg) 
(mole ratio) 
(ml) method 
method 
(nmole/ml) 
__________________________________________________________________________ 
1 56 25.0 1.4 15.1 17.2 1.60 
2 93 15.0 2.4 10.7 11.9 0.53 
3 140 
10.0 3.0 7.4 8.7 0.13 
4 200 
7.0 5.1 6.2 0.09 
5 311 
4.5 8.0 3.2 3.6 0.05 
6 466 
3.0 12.0 2.0 2.4 0.04 
7 932 
1.5 24.0 0.9 1.5 0.05 
8 1863 
0.75 48.0 0.5 0.7 0.08 
9 2795 
0.5 72.0 0.3 0.5 0.19 
__________________________________________________________________________ 
From this Table 3, it will be noticed that there is a correlation between 
the steroid bonding number and the detection sensitivity of the samples 
No. 1-No. 9. That is, detection sensitivity increases as steroid bonding 
number decreases. However, detection sensitivity reaches the maximum when 
the steroid bonding number is 2.0 (by the ultraviolet absorption method), 
and thereafter detection sensitivity decreases as steroid bonding number 
decreases. 
Therefore, it will be understood that in accordance with the present 
invention excellent sensitivity, over 0.2 nmole 
estriol-16.alpha.-glucuronide equivalent/ml, is achieved, and it is 
remarkably higher than that of the conventional latex reagent which is at 
the level of 2.5 nmole estriol-16.alpha.-glucuronide equivalent/ml. 
This excellent detection sensitivity may be obtained when the steroid 
bonding number falls within a range of about 0.5-8.7 measured by both the 
ultraviolet absorption method and the dinitrophenylation method. Higher 
sensitivity, over 0.1 nmole estriol-16.alpha.-glucuronide equivalent/ml, 
may be achieved when the steroid bonding number falls within a range 
0.7-7.0. 
TEST 2 
A test similar to Test 1 was conducted with respect to latex particles 
sensitized with dehydroepiandrosterone-17-(O-carboxymethyl)oxime-BSA 
conjugate. 
Using dehydroepiandrosterone (Sigma Company) (hereinafter referred to DHEA) 
and BSA (ICN Pharmaceuticals Company), 8 kinds of conjugates having 
different steroid bonding numbers within the range 0.5-18.3 were prepared 
as follows: 
Amounts of 0.75 g of DHEA and 0.69 g of (O-carboxymethyl) hydroxylamine 
hydrochloride (Wako Junyaku Kogyo Kabushiki Kaisha) were dissolved in 20 
ml of ethyl alcohol. 
In 20 ml of ethyl alcohol, 0.75 g of DHEA and 0.69 g of 
(O-carboxymethyl)hydroxylamine hydrochloride (Wako Junyaku Kogyo Kabushiki 
Kaisha) were dissolved and 2 ml of 6.4 M sodium succinate solution added 
thereto to keep it alkaline. The resulting solution was refluxed for an 
hour to promote the reaction, and then Na.sub.2 CO.sub.3 was added 
thereto. The quantity of NaCO.sub.3 to be added was such that the 
concentration of Na.sub.2 CO.sub.3 in the resulting solution amounted to 
5%. 
After wahing the resulting solution with ether, its aqueous layer was 
acidified with concentrated hydrochloric acid and the resulting 
precipitate was separated. This precipitation was recrystallized from 
ethyl alcohol and 0.6 g of DHEA-17-(O-carboxymethyl)oxime (hereinafter 
referred to DHEA-CMO) was obtained. 
Using this DHEA-CMO and BSA in the mole ratios shown in Table 4, 8 kinds of 
conjugate (samples No. 1-No. 8) having different steroid bonding numbers 
were prepared in like manner as in Test 1. The steroid bonding number in 
the samples No. 1-No. 8 was measured by the dinitrophenylation method. The 
results are also shown in Table 4. 
The minimum sensitizing quantity was determined with respect to the samples 
No. 1-No. 8 in like manner as in Test 1. The results are also shown in 
Table 4. 
Using the minimum sensitizing quantity of samples No. 1-No. 8, 8 kinds of 
latex reagents were prepared and the steroid detection sensitivity was 
measured with respect to these samples as follows: 
To 1 ml of a 10% suspension of latex particles (Dow Chemical Company) 
having a particle size of 0.721 .mu.m, 4 ml of 20 mM phosphoric acid 
buffer solution (pH 7.2) containing 150 mM NaCl was added. After 
sufficient agitation, the resulting solution was centrifuged at 4,000 
r.p.m. for 20 minutes. The obtained precipitate was suspended in 5 ml of 
the same buffer solution. The resulting suspension was centrifuged again 
under the same conditions mentioned above and the precipitate was 
separated. The same quantity of such washed latex particles was prepared 
in 8 lots. 
The minimum sensitizing quantities of the respective conjugate samples No. 
1-No. 8 in Table 4 were dissolved in 5 ml of the buffer solution to 
prepare 8 kinds of conjugate solutions having different steroid bonding 
numbers, and these solutions were respectively added to the 8 lots of said 
washed latex particles. After keeping the resulting solutions at 
37.degree. C. for 2 hours, they were each subjected to centrifugation and 
washing with the buffer solution twice. The resulting precipitates were 
each resuspended in 5 ml of the buffer solution and thus were obtained 8 
kinds of DHEA-CMO-BSA sensitized latex particle suspensions (Table 4, 
samples No. 1-No. 8). 
Meanwhile antiserum was obtained from rabbits immunized with DHEA-CMO-ESA, 
and this was utilized as an original antibody. The original antibody was 
serially diluted with 20 mM phosphoric acid buffer solution (pH 7.2) 
containing 150 mM NaCl. An amount of 0.05 ml of each of the diluted 
antibody, solutions was respectively mixed with 0.05 ml of a solution 
prepared by dissolving 0.2 nmole of DHEA per ml of the buffer solution to 
neutralize the antibody therein. On glass slides, 0.1 ml of each of the 
respective neutralized solutions was dropped and to each of these droplets 
0.1 ml of a suspension of latex particles sensitized with the conjugate 
having a DHEA bonding number of 2.4 was respectively added and agitated 
well. Maximum dilution of antibody which enabled detection of 
agglutination within 3 minutes was 50-fold. Therefore the titer of 50-fold 
diluted antibody was determined as 0.2 nmole DHEA equivalent/ml and the 
titer of the original antibody as 10 nmole DHEA equivalent/ml. 
Next, the original antibody was serially diluted with 20 mM phosphoric acid 
buffer solution, and 0.1 ml of each of the diluted antibody solutions was 
respectively mixed with 0.1 ml of the latex reagent samples No. 1-No. 8 on 
glass slides, and the maximum dilution of the antibody which gave 
agglutination within 3 minutes was determined. Respective detection 
sensitivities of latex reagent samples No. 1-No. 8 were calculated in like 
manner as in Test 1 based on the maximum dilution and the titer of the 
original antibody. The calculated titers are also shown in Table 4. 
TABLE 4 
__________________________________________________________________________ 
detection 
DHEA DHEA-CMO-ESA 
sensitivity 
bonding 
sensitizing 
Estriol-16.alpha.- 
preparation number 
quantity glucuronide 
condition dinitro- 
(mg/0.1 g equivalent 
sample 
BSA 
DHEA-CMO/BSA 
phenylation 
latex antibody 
No. (mg) 
(mole ratio) 
method 
particles) 
(nmole/ml) 
__________________________________________________________________________ 
1 71.9 
25.0 18.3 1.2 1.40 
2 120 
15.0 11.0 1.3 0.50 
3 180 
10.0 7.7 1.3 0.15 
4 257 
7.0 5.6 1.5 0.10 
5 400 
4.5 3.6 1.7 0.07 
6 600 
3.0 2.4 1.7 0.05 
7 1200 
1.5 1.2 1.8 0.05 
8 2400 
0.75 0.5 1.8 0.08 
__________________________________________________________________________ 
From Table 4, it will be seen that the highest sensitivity is obtained when 
the DHEA-CMO bonding number falls within the range of 1.2-2.4, and 
sensitivity over 0.2 nmole DHEA equivalent/ml is obtained when the 
DHEA-CMO bonding number falls within the range of 0.5-7.7. 
TEST 3 
Using various kinds of steroids and serum albumins which were not used in 
Tests 1 and 2, similar tests were conducted. The maximum and minimum 
values of steroid bonding number with which higher sensitivity over 0.2 
nmole steroid equivalent/ml is obtainable are shown in Table 5. 
TABLE 5 
______________________________________ 
conjugate for 
sensitizing steroid bonding number 
latex particles minimum maximum 
______________________________________ 
progesterone-11- 0.5 8.0 
hemisuccinate-RSA 
hydrocortisone-21- 
0.5 7.0 
hemisuccinate-HSA 
aldosterone-3- 0.5 7.5 
(o-carboximethyl) 
oxime-ESA 
testosterone-17- 0.5 8.6 
glucoronide-RSA 
pregnanediol-3- 0.5 7.3 
hemisuccinate-ESA 
______________________________________ 
From the foregoing it has been found that regardless of steroid and serum 
albumin used higher sensitivity over 0.2 nmole steroid equivalent/ml is 
obtained when the steroid bonding number of the conjugate falls within the 
range 0.5-7.0. 
TEST 4 
A comparative test for steroid detection sensitivity between the latex 
reagent prepared in accordance with the present invention and that 
prepared by the conventional method was conducted as follows: 
Latex reagent by the conventional method was prepared in the same manner as 
in Test 1 except that 47 mg of RSA (ICN Pharmaceuticals Company) and 1.0 
ml of deionized water were used, and estriol-16.alpha.-glucuronide-RSA 
conjugate was obtained. The steroid bonding number in this conjugate was 
20 by the ultraviolet absorption method referred to in Test 1. Using 
different quantities of this conjugate, latex particles were sensitized in 
like manner as in Test 1 and differently sensitized latex reagents so 
obtained were subjected to a stability test for non-specific agglutination 
in like manner as in Test 1. The minimum sensitizing quantity of the 
conjugate for eliminating non-specific agglutination was determined to be 
5.0 mg per 0.1 g of latex particles. 
Thus a comparative latex reagent (sample No. 10) was prepared by 
sensitizing latex particles with the minimum sensitizing quantity of the 
conjugate. 
Furthermore additional comparative latex reagents (samples Nos. 11-14) were 
prepared in such a manner that latex particles were sensitized with the 
minimum quantities of the conjugates which were differently modified from 
that used for sample No. 10. More specifically the conjugate was modified 
in such a manner that to the same quantity of the conjugate prepared in 4 
lots, different quantities of an aqueous solution of immunologically inert 
RSA were respectively added so that the resultant mole ratio of the 
steroid to the total RSA in the modified conjugates came to be 1, 2, 5 and 
10, and then with respect to these modified conjugates the maximum 
sensitizing quantities were determined in like manner as in the foregoing 
tests. Then using the respective minimum sensitizing quantities of the 4 
kinds of modified conjugates, latex particles were sensitized and the 
additional comparative latex reagents (samples Nos. 11-14) were prepared. 
Using the thus obtained 5 kinds of comparative latex reagent samples Nos. 
10-14 and the latex reagent samples Nos. 6 and 9 in Test 1, a comparative 
test for steroid detection sensitivity was conducted in like manner as in 
Test 1. The results are shown in Table 6. 
TABLE 6 
______________________________________ 
detection 
mixing ratio 
steroid sensitivity 
of inert RSA 
bonding sensitizing 
Estriol-16.alpha.- 
and sample number quantity 
glucuronide 
No. 10 (per (mg/0.1 g 
equivalent 
sample 
(RSA: sample 
1 molecule latex antibody 
No. No. 10) of total RSA) 
particles) 
(nmole/ml) 
______________________________________ 
6* -- 2.0 7.2 0.04 
9* -- 0.3 7.4 0.19 
10 -- 20 5.0 2.5 
11 1 : 1 10 6.0 1.5 
12 3 : 1 5 6.7 1.0 
13 9 : 1 2 7.4 0.5 
14 19 : 1 1 7.5 0.8 
______________________________________ 
(Note) 
*latex particles sensitized with the conjugate in accordance with the 
present invention 
From Table 6, it will be noted that the steroid detection sensitivity of 
sample No. 6 is 62.5 times that of sample No. 10, and that sensitivity of 
the sample No. 9 is 13 times that of sample No. 10 though sample No. 9 has 
the lowest sensitivity among the samples of the present invention. 
Therefore, it is clear that the latex reagents of this invention have much 
higher sensitivity than the conventional latex reagent which has rather 
large steroid bonding number. 
Regarding additional comparative latex reagent samples Nos. 11-14, the 
steroid detection sensitivity of the sample No. 13 is the highest among 
them, but the sensitivity is almost 1/12 of that in the sample No. 6 and 
about 2/5 of that in the sample No. 9. Furthermore though these reagent 
samples Nos. 11-14 have considerably small steroid bonding numbers 
comparable to those in the applicant's reagent samples, these reagent 
samples Nos. 11-14 are not necessarily considered to be conventional 
reagents, since there has been no reference, as far as the inventors know, 
which may teach or suggest decreasing the steroid bonding number to such a 
level for increasing sensitivity. In any case, it will be clear that the 
sensitivities of the samples Nos. 11-14 are not comparable to those in the 
samples of the present invention. 
Therefore it will be understood that the present invention enables one to 
detect smaller concentrations of steroid which may not be detected with 
the conventional latex reagent, or alternatively to dilute a liquid to be 
tested for eliminating interference of coexisting substances when the 
liquid contains steroids in higher concentration. In other words the 
present invention makes possible more reliable quantitative assay of 
steroids and also more accurate diagnostic information. 
Furthermore the present invention may provide a latex reagent having 
excellent high sensitivity, and therefore steroid detection can be 
achieved with lesser amounts of antibody or antiserum. Consequently this 
makes it possible to reduce consumption of antibody, and since preparation 
of antibody or antiserum involves very high cost the present invention is 
highly advantageous in from an economic point of view. 
Having described exemplifying tests of the present invention, some examples 
of the present invention will be described for better understanding. 
EXAMPLE 1 
Liquid A was prepared by dissolving 50 mg of estriol-16.alpha.-glucuronide 
(Sigma Company) in 10 ml of dimethylformamide (Wako Junyaku Kogyo 
Kabushiki Kaisha), then 26 .mu.l of tri-n-butylamine (Tokyo Kasei Kogyo 
Kabushiki Kaisha) and then 14 .mu.l of isobutyl-chloroformate (Aldrich 
Chemical Company) were respectively added and agitated well. 
On the other hand 2.33 g of RSA (ICN Pharmaceuticals Company) was dissolved 
in 60 ml of deionized water, 1.0 ml of N NaOH was added and thereafter 50 
ml of dimethylformamide was added thereto whereby liquid B was prepared. 
To this liquid B, previously prepared liquid A was added dropwise and 
agitated for an hour, then 0.1 ml of N NaOH was added and the mixture was 
further agitated for 3 and a half hours. After dialyzing the solution 
against deionized water, two parts of acetone were added per one part of 
the solution, and after thorough agitation N HCl was added to precipitate 
synthesized estriol-16.alpha.-glucuronide-RSA conjugate. The precipitate 
was separated by centrifugation at 10,000 r.p.m. for 5 minutes. 
To the separated precipitate, deionized water was added and its pH was 
adjusted to 8 with N NaOH and then subjected to dialysis against deionized 
water to remove acetone. Thus 1.78 g of the conjugate was obtained (yield 
75%). These processes were carried out at low temperature (6.degree. C.). 
The steroid (estriol-16.alpha.-glucuronide) bonding number measured by the 
ultraviolet absorption method and dinitrophenylation method was 2.2 and 
1.9 respectively. 
Then 76 mg of the conjugate was dissolved in 400 ml of 40 mM veronal buffer 
solution (containing 150 mM NaCl, pH 7.8), and further 10 ml of 
polystyrene latex particle (particle size 0.234 .mu.m) suspension (10% 
concentration) was added and kept at 37.degree. C. for 2 hours for 
sensitization. The sensitized latex suspension was subjected to 
centrifugation at 4,000 r.p.m. for 20 minutes, and the obtained 
precipitate was suspended in 200 ml of the twice said buffer solution and 
then subjected to centrifugation under the same conditions mentioned 
above. The thus obtained precipitate was resuspended in 50 ml of the 
buffer solution, and then sodium azide was added so that the resultant 
concentration thereof came to be 0.1% whereby 50 ml of 
estriol-16.alpha.-glucuronide-RSA sensitized latex particle suspension (2% 
density) was obtained. 
Mixing on a glass slide 0.1 ml of the thus obtained sensitized latex 
particle suspension and 0.1 ml of diluted anti 
estriol-16.alpha.-glucuronide-BSA antibody solution which was prepared in 
like manner as in Test 1 and diluted to have a titer of 0.04 nmole 
estriol-16.alpha.-glucuronide equivalent/ml, agglutination was observed 
within 1-2 minutes. However, when the antibody was diluted to have a titer 
of 0.02 n mole estriol-16.alpha.-glucuronide equivalent/ml, no 
agglutination was observed, and the titer of the latex reagent obtained in 
this example was determined to be 0.04 nmole estriol-16.alpha.-glucuronide 
equivalent/ml. 
EXAMPLE 2 
DHEA-CMO was prepared in like manner as in Test 2. Liquid A was prepared by 
adding 0.5 g of DHEA-CMO to 100 ml of dimethylformamide (Wako Junyaku 
Kogyo Kabushiki Kaisha), then 0.33 ml of tri-n-butylamine (Tokyo Kasei 
Kogyo Kabushiki Kaisha) was added, and further 0.18 ml of 
isobutylchloroformate (Aldrich Chemical Company) was added and the mixture 
was agitated well. 
Meanwhile 18.0 g of BSA (Sigma Company) was added to 600 ml of deionized 
water and further 500 ml of dimethylformamide was added and thus liquid B 
was obtained. 
To this liquid B, previously prepared liquid A was added dropwise, and 
after agitation for one and a half hours its pH was adjusted to 9.0 with N 
NaOH and the mixture further agitated for 3 and a half hours. The thus 
obtained solution was dialyzed against flowing water, then two parts of 
acetone were added per 1 part of the solution. After thorough agitation N 
HCl was added to the solution to precipitate the synthesized conjugate, 
and then the solution was subjected to centrifugation at 5,000 r.p.m. for 
20 minutes. To the separated precipitate, deionized water was added, and 
after its pH was adjusted to about 8 with N NaOH, the solution was 
dialyzed against flowing water to remove acetone whereby 13.0 g of 
DHEA-CMO-BSA conjugate was obtained (yield about 70%). The steroid 
(DHEA-CMO) bonding number of this conjugate measured by the 
dinitrophenylation method was 4.1. 
Then 68 mg of the thus obtained conjugate was dissolved in 100 ml of 100 mM 
glycine buffer solution (containing 130 mM NaCl and 0.1% of sodium azide, 
pH 8.2), and to this solution 10 ml of a 10% concentration suspension of 
polystyrene latex particles (Dow Chemical Company) was added, and then the 
mixture was dialyzed against said buffer solution at room temperature for 
5 days for sensitization. The thus obtained sensitized latex suspension 
was subjected to centrifugation at 4,000 r.p.m. for 20 minutes, and after 
the precipitate was suspended in 200 ml of said buffer solution the 
suspension was twice subjected to centrifugation and washing with said 
buffer solution under the same conditions. Finally resuspending the 
obtained precipitate in 50 ml of said buffer solution, 50 ml of 2% 
concentration DHEA-CMO-BSA sensitized latex particle suspension was 
obtained. 
Mixing on a glass slide 0.05 ml of 0.1 nmole/ml DHEA with 0.05 ml of 
diluted antibody which was prepared in like manner as in Test 2 and 
diluted with said buffer solution to have a titer of 0.1 nmole DHEA 
equivalent/ml, and then adding 0.1 ml of the eventually obtained 
sensitized latex particle suspension thereto, no agglutination was 
observed even after 10 minutes. When 0.1 ml of the sensitized latex 
particle suspension was added to the mixture of 0.05 ml of 0.02 nmole/ml 
DHEA and 0.05 ml of said diluted antibody, agglutination was observed 
within 2-3 minutes. Through a procedure similar to that of Test 1, the 
titer of the latex reagent obtained in this example was determined to be 
0.05 nmole DHEA equivalent/ml. 
EXAMPLE 3 
Liquid A was prepared by dissolving 1.1 g of testosterone-17-glucuronide 
(Sigma Company) in 200 ml of dimethylformamide (Wako Junyaku Kogyo 
Kabushiki Kaisha), and to the thus obtained solution 0.52 ml of 
tri-n-butylamine (Tokyo Kasei Kogyo Kabushiki Kaisha) and then 0.28 ml of 
isobutylchloroformate (Aldrich Chemical Company) were successively added 
and the mixture was agitated well. 
Liquid B was prepared by dissolving 46.6 g of RSA (Sigma Company) in 1.2 l 
of deionized water, and after its pH was adjusted to 9.0 with N NaOH, 1.0 
l of dimethylformamide was added thereto. In like manner as in Example 2, 
about 39.0 g of testosterone-17-glucuronide-RSA (hereinafter referred to 
as T-17-G-RSA) was obtained (yield about 82%). The steroid (T-17-G) 
bonding number in this conjugate measured by the ultraviolet absorption 
method and the dinitrophenylation method was 2.3 and 2.1 respectively. 
Then 400 ml of 20 mM phosphoric acid buffer solution containing 150 mM NaCl 
(pH 7.2) was added to 100 ml of a 10% concentration suspension of a 
polystyrene latex particles (Dow Chemical Company) having a particle size 
of 0.721 .mu.m, then the suspension was subjected to centrifugation at 
4,000 r.p.m. for 20 minutes. The thus obtained precipitate was suspended 
in 500 ml of said buffer solution and then centrifuging the suspension 
under the same conditions mentioned above, a precipitate was separated. 
Then 500 ml of said buffer solution containing 180 mg of the previously 
obtained conjugate were added to the separated precipitate. After the thus 
obtained suspension was kept at 37.degree. C. for two hours, the 
suspension was subjected to centrifugation under the same conditions, and 
then washed with said buffer solution and the centrifugation process was 
repeated twice. The precipitation obtained was resuspended in 500 ml of 
said buffer solution, and finally sodium azide was added so that the 
eventual concentration thereof came to be 0.1%. Thus 500 ml of 2% 
concentration of T-17-G-RSA sensitized latex particle suspension was 
obtained. 
In like manner as in Test 1, anti T-17-G-RSA antibody was prepared, and 
then it was diluted with said buffer solution to give a titer of 0.05 
nmole T-17-G equivalent/ml. On a slide 0.1 ml of this diluted antibody was 
mixed with 0.1 ml of the eventually obtained latex particle suspension, 
and agglutination was observed within 1-2 minutes. However, when the 
antibody was diluted to give a titer of 0.02 nmole T-17-G equivalent/ml, 
no agglutination was observed within 5 minutes. Thus the latex reagent 
obtained in this example was determined to be 0.05 nmole T-17-G 
equivalent/ml. 
EXAMPLE 4 
Liquid A was prepared by adding a certain number of moles of 
sodiumhydrocortisone-21-hemisuccinate (Sigma Company) to the same number 
of moles of concentrated hydrochloric acid, and then concentrating and 
washing with water, and these concentration and washing processes were 
repeated several times and then the solution was evaporated to dryness, 
and 0.5 g of the thus obtained dried material was dissolved in 100 ml of 
dimethylformamide (Wako Junyaku Kogyo Kabushiki Kaisha), and successively 
0.26 ml of tri-n-butylamine (Tokyo Kasei Kogyo Kabushiki Kaihsa) and 0.14 
ml of isobutylchloroformate (Aldrich Chemical Company). 
Meanwhile liquid B was prepared by dissolving 17.6 g of HSA (Miles 
Laboratories Inc.) in 600 ml of deionized water, and adjusting its pH to 
9.0 with N NaOH and finally adding 500 ml of dimethylformamide. 
Thereafter, in like manner as in Example 2, about 13.6 g of 
hydrocortisone-21-hemisuccinate-HSA (hereinafter referred to as 
HC-21-HS-HSA) was obtained (yield about 75%). The steroid (HC-21-HS) 
bonding number in this conjugate measured by the dinitrophenylation method 
was 3.0. 
Then 200 ml of 40 mM veronal buffer solution containing 150 mM NaCl (pH 
7.8) was added to 50 ml of a 10% concentration suspension of polystyrene 
latex particles (Dow Chemical Company) having a particle size of 0.721 
.mu.m, and the obtained suspension was centrifuged at 4,000 r.p.m. for 20 
minutes. The thus separated precipitate was suspended in 250 ml of said 
buffer solution and then centrifuged under the same conditions mentioned 
above, and the precipitate was collected. Then 250 ml of said buffer 
solution containing 88 mg of the previously obtained conjugate was added 
to the eventually collected precipitation (latex particles), the obtained 
suspension was kept at 37.degree. C. for two hours, and then the 
suspension was centrifuged to collect the precipitate. The precipitate was 
resuspended in 250 ml of said buffer solution and centrifuged under the 
same conditions mentioned above and these processes were repeated once 
again. The thus obtained precipitate was resuspended in 250 ml of said 
buffer solution, and then sodium azide was added thereto so that the 
eventual concentration thereof came to be about 0.1%. Thus 250 ml of about 
2% concentration HC-21-HS-HSA sensitized latex particle suspension was 
obtained. 
Then 0.05 ml of 0.1 nmole hydrocortisone was mixed with 0.05 ml of diluted 
antibody which was prepared in like manner as that in the Test 1 and 
diluted with said buffer solution to give a titer of 0.1 nmole HC-21-HS 
equivalent/ml on a glass slide, and then 0.1 ml of the previously obtained 
sensitized latex particle suspension was mixed therewith. No agglutination 
was observed even after ten minutes. However when 0.05 ml of 0.05 nmole/ml 
hydrocortisone and 0.05 ml of said diluted anti-body was mixed and then 
0.1 ml of said sensitized latex particle suspension was added on a glass 
slide, agglutination was observed within 2-3 minutes. The titer of the 
latex reagent of this example was determined to be nmole 0.05 HC-21-HS 
equivalent/ml.