Method and device for assaying immunologically reactive substances of clinical interest

A method and device of quantitatively assaying an immunologically reactive substance of clinical interest, wherein the method includes the steps of grafting an immunologically active substance onto natural or synthetic microparticles, agglutinating the microparticles in a liquid medium in the presence of an immunologically reactive substance of clinical interest, and optically measuring the agglutinated substances to determine the assay of the immunological reactive substance of clinical interest. The device employed for carrying out the above method includes a first series of tubes which contain at least one freeze-dried calibration range of the substance to be assayed, a second series of tubes which contain an immunological active substance acting as the assaying agent, and a third series of small tubes containing a freeze-dried specimen of the dilution solution of the calibration range.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a method of assaying immunologically 
reactive substances of clinical interest. It also relates to a device for 
implementing this method. 
2. Description of the Prior Art 
Immunologically reactive substances means chiefly all the antigens 
(including the haptens) and the antibodies (monoclonal or polyclonal) 
produced by cell fusion, or by natural or induced immunization. 
Immunologically reactive substances of clinical interest means chiefly all 
the antigen or antibody molecules whose assay in a biological specimen of 
human or animal origin may be of interest in medical diagnostics, clinical 
research or the monitoring of a pathological process or of a therapy which 
is employed. 
The immunological phenomenon of which use is made in these immunoassay 
methods is shown in FIG. 1. 
The most widely employed assay methods based on the use of the curve in 
FIG. 1 are: 
radial immunodiffusion, 
radioimmunology, immunoenzymology, immunofluorescence; and 
nephelometry. 
Furthermore, some attempts at quantitative versions of the "latex 
immunotest", namely the agglutination of synthetic microspheres, have been 
described over a number of years. Until now, these techniques, which have 
been employed widely and for a long time (J. M. Singer and C. M. Plotz, 
The Latex fixation test, American Journal of Medicine 21, 888 (1956)), 
because of their simplicity and their low cost, were only qualitative 
(strip) or semiquantitative (well) in their form. The qualitative (strip) 
or semiquantitative forms (strip--sheet--well) of the Latex immunotest 
continue, furthermore, to be widely employed in clinical diagnostics at 
the present time and are the subject of many patents and publications, 
especially in the field of virology and microbiology (R. F. Khabbaz, H. C. 
Standford et al, 1985, Measurement of amikacin in serum by a Latex 
agglutination inhibition test, Journal of Clinical Microbiology, 22: 
699-701, U.S. Pat. No. 3,488,156, European Patent Application 186 946). In 
these screening or detection techniques, the antigen or antibody is fixed 
on synthetic microspheres and the absence or presence of agglutination by 
the corresponding antigen or antibody is assessed in a tube or in a well, 
visually or by turbidimetry. 
The few quantitative attempts at a Latex immunotest which are proposed in 
the literature (for example J. P. Ripoll, A. M. Roch, G. A. Quash and J. 
Grange, 1980, Journal of Immunological Methods 33, 159-173, European 
Patent Applications 189 389 and 5978) involve substantially three stages: 
Stage I: Stage of fixing the antigen or the antibody on a micrometer of 
synthetic polymer, between 0.01 micrometer and 5 micrometers in size. The 
fixing is performed by any known means, which are identical with those 
described in the strip techniques (for example U.S. Pat. No. 4,217,338). 
Stage II: Stage of immunological agglutination of the synthetic 
microspheres carrying the antigen or the antibody. This stage II of the 
method, namely the immunological agglutination of the synthetic 
microspheres, should aim at two objectives: 
(1) the maximum reduction in the nonspecific agglutination of the 
particles; nonspecific agglutination means the agglutination of the 
particles which is due to weak interactions (of the hydrogen bond, ionic 
or van der Waals type), between the microspheres or the proteins which are 
fixed on the latter, 
(2) the maximum increase in specific (immunological) agglutination of the 
particles. 
During this stage, substantially chemical or biochemical means are employed 
to stabilize the particles, in order to reduce nonspecific agglutination 
to a minimum. 
During this stage, means are also employed in order to increase the 
specific agglutination of the particles, and consequently the accuracy of 
the assay. 
To reduce the nonspecific agglutination of the particles, U.S. Pat. No. 
4,329,152 proposes to stabilize them by fixing bovine albumin made 
electronegative at a pH which is substantially equal to 10. The highly 
basic pH imposed in this manner on the reactant and on the calibration 
ranges is incompatible with survival of the proteins, and makes it a 
reactant which is difficult to employ. Furthermore, the reliability of the 
method is reduced thereby, because buffer solutions at pH values as basic 
as this are highly unstable. Furthermore, the bovine albumin fixed by 
hydrophobic bonding separates progressively from the particles at pH 10, 
and progressively loses its stabilizing capacity. 
Unfortunately, the chaotropic agents which are sometimes employed to reduce 
the nonspecific agglutination of the particles have the disadvantage of 
weakening or breaking the antigen-antibody bonds. 
Other stabilizing methods which are highly complicated and which cannot be 
employed in an industrial reactant make use of mixtures of heavy and light 
particles (ex: European Patent 163 312) and of centrifuging operations. 
In order to promote the specific agglutination of the particles, that is to 
say immunological agglutination, the methods described employ conventional 
agitation, at a constant temperature in a water bath at 37.degree. C. or 
40.degree. C., for periods ranging from one half hour to one hour. Such 
periods of incubation at 37.degree. C., in addition to promoting the 
undesirable nonspecific agglutination of the particles, demand strict and 
painstaking stopwatch timing of the initial introduction of the reactant 
into each tube in the series, and of the process of interrupting the 
reaction in the series of tubes. 
An increase in the specific (immunological) agglutination of the particles 
is sometimes practiced by adding to the reaction medium additives such as, 
for example, dextrose, known under the trademark "Dextran" or polyethylene 
glycol. The disadvantage of these substances is that frequently they are 
to a large extent hydrophobic, and therefore they attach themselves to the 
Fc fragments of the antibodies fixed on the microparticles, fragments 
which are themselves highly hydrophobic, thus producing a nonimmunological 
agglutination of the particles, and thereby inducing interferences and, 
ipso facto, false positive reactions. 
Stage III: Stage of reading the result. 
At the present time the techniques of reading the result of the 
agglutination in quantitative latex immunotests are: 
particle counting, 
opacimetry, also known as turbidimetry (sometimes carried out in the 
visible or the infrared, in most cases in the near infrared), 
laser nephelometry, and 
centrifugal analysis. 
Laser nephelometry measures the light scattered by aggregates of latex 
particles. This technique, described, for example, in the following 
article: J. Grange, A. M. Roch and G. A. Quash, 1977, Journal of 
Immunological Methods 18, 326-375, introduces the disadvantage of 
requiring a costly and sophisticated reading apparatus. 
Particle counting, described, for example, in the following article: C. G. 
Magnusson, P. L. Masson, Journal of Allergy Clin. and Immunol. 70: 326, 
1982, also requires a complex and costly apparatus, accessible to only a 
few laboratories. 
Opacimetry in the visible, also called turbidimetry in the visible, 
measures the light transmitted by the particle suspension, that is to say 
the light which is neither absorbed by the particles nor scattered by the 
latter. The opacimetry or turbidimetry of the latex immunotests, whether 
qualitative or quantitative, must be performed at a wavelength which lies 
fairly close to the particle size. Validity of the turbidimetric 
measurement calls for highly dilute suspensions, in order to enable the 
desired screening effect to be seen and, as indicated in the Certificate 
of Addition no 78/28,250 to french patent no 77/25,049, in order to reduce 
the absorption of light by the particles to a minimum. 
Depending on the particle size, turbidimetric (or opacimetric) measurements 
in the visible of the latex immunotests described in the literature 
(example: A. M. Bernard, R. R. Lauwerys, 1982, clinica Chemica Acta 119, 
335-339) employ various wavelengths in the visible, for example 360, 400, 
450 and, much more widely, between 600 and 750 nanometres. The precision 
of the turbidimetric results is mediocre because the particle suspensions 
must be very dilute in order, as already stated, to produce the desired 
screeen effect variations and in order to reduce the absorption by the 
latices to a minimum. Furthermore, in order to increase the precision of 
the result, reading cells in which the optical path is long, of the order 
of two to four centimeters, must be generally employed in these 
techniques. 
In order to employ more concentrated latex suspensions and thus to increase 
the precision of the assays, and in order to be free from interference by 
the absorption of the latices in the visible or in the ultraviolet, French 
Patent FR-A-77/25,049 and its Certificate of Addition FR-A-78/28250 
recommend infrared opacimetry for particle reading. In point of fact, 
latices no longer absorb light in the infrared. The disadvantage of this 
technique is that it calls for costly and sophisticated apparatus for 
infrared opacimetric reading. 
SUMMARY OF THE INVENTION 
The primary objective of the present invention is therefore to overcome the 
numerous disadvantages of the stages II and III emphasized above. 
With respect to the Stage II techniques discussed above, one of the 
objectives of the present invention is to eliminate the nonspecific 
agglutination of the particles and simultaneously to increase the specific 
immunological agglutination of the particles, without introducing all the 
disadvantages described above, and, what is more, with a considerable 
shortening of the duration of the operation. 
Another object of the present invention is the implementation of an 
agglutination method and apparatus which is simple to use, and very fast. 
Another object of the present invention is the implementation of a method 
of reading the result which is also simple and very fast, and within the 
reach of any laboratory. The objectives of the present invention are 
reached by a method for assaying immunologically reactive substances of 
clinical interest of the type in which: 
an immunologically active substance acting as the assay reactant is grafted 
beforehand onto the microparticules, 
the said microparticles are agglutinated by means of the immunologically 
reactive substance of clinical interest, 
the result produced by the agglutination reaction, which is compared with a 
calibration range, is measured by reading. 
The assay method according to the invention is characterized in that the 
agglutination stage is performed by subjecting the grafted microparticles 
to a periodic motion at a frequency of between 4 and 40 Hertz, and with an 
amplitude ranging from a few millimeters to a few centimeters. 
It has been found, in fact, that when the frequency is less than 4 Hertz, 
the advantage of the method was reduced because the duration of the 
agglutination stage is increased in order to produce a usable result and 
because the required effect on the nonspecific agglutination becomes 
mediocre; on the other hand, when the frequency is higher than 40 Hertz, 
breakup of the aggregates (immunocomplexes) formed by the agglutinated 
microparticles is produced. As it is apparent from the above description, 
in the agglutination stage (stage II of the method), in order to overcome 
all the disadvantages of the chemical methods described above, aiming at 
reducing the nonspecific agglutination of the particles, or increasing the 
immunological agglutination thereof, the agglutination means used 
according to the invention employs a mechanical method which 
simultaneously meets both these objectives. This mechanical method is 
based on the observation that nonspecific agglutination, which is 
associated with weak interactions between the particles (bonds of ionic, 
hydrogen or Van der Waals type) is rendered impossible or is greatly 
inhibited by a periodic motion imparted to these, of very low amplitude 
and very high frequency, whatever the trajectory of the periodic motion 
(for example and conveniently rectilinear, circular or ovalized). It has 
also been shown that a method of this kind markedly promotes the specific 
(immunological) agglutination of the microparticles carrying the 
corresponding antigen or antibody, and hence permits a rapid specific 
reaction which, moreover, is carried out at ambient temperature. 
The present invention also proposes a method for assaying immunologically 
reactive substances of clinical interest of the type in which: 
an immunologically active substance acting as the assay reactant is grafted 
beforehand onto the microparticles, 
the microparticles are agglutinated by means of the immunologically 
reactive substance of clinical interest, and 
the result produced by the agglutination reaction, which is compared with a 
calibration range, is measured by reading, characterized in that the stage 
of reading the result is performed: 
(1) firstly, by determination of the absorption maximum by the particles of 
ultraviolet and visible light of a suspension in a liquid medium of 
nonagglutinated microparticles, the determination being performed by 
obtaining the ultraviolet and visible absorption spectrum of the said 
suspension, the wavelength corresponding to the absorption maximum being 
called .lambda.max, 
(2) then by reading the result, by means of ultraviolet and visible 
absorption spectrophotometry, operating in the vicinity of the said 
wavelength .lambda.max, determined earlier, after the immunological 
agglutination reaction and a dilution of the incubation medium performed 
in a known manner. 
It is surprising, especially when the microparticles are latices, that it 
is possible to perform this stage of reading using ultraviolet and visible 
absorption of the latex particles, given that the above mentioned 
Certificate of Addition FR-A-78/28,250 describes this phenomenon as a 
major disadvantage. It seems that, in the method of reading according to 
the invention, it is the actual absorption of the ultraviolet and visible 
light by the particles themselves which is utilized, and this constitutes 
a new step. In fact, the turbidimetric (opacimetric) methods measure only 
an "apparent absorption" of the particles suspension, that is to say 
actually the light which is not absorbed by the particles. 
In fact, it has been observed, and this is one of the characteristics of 
the invention, that a microparticle capable of absorbing ultraviolet and 
visible light and whose size can vary between 0.01 and 5 micrometers, of 
synthetic polymer, whatever its size and its chemical nature may be (for 
example: polystyrene, polyvinyltoluene, acrylonitrile, styrene-butadiene, 
acrylonitrile-acrylic acid copolymers, acrylic ester, 
polyvinyl-butadiene), has a characteristic maximum of absorption of 
ultraviolet and visible light with an extinction coefficient which depends 
on the wavelength. In addition, it has been shown, and this observation is 
employed in the method of reading according to the invention, that if a 
measurement is carried out on a suspension in a liquid medium of these 
nonagglutinated microparticles, using absorption spectrophotometry at the 
wavelength .lambda.max corresponding to the maximum absorption of 
ultraviolet and visible light by the nonagglutinated microparticles 
(.lambda.max being characteristic of a given microparticle), the result is 
not at all or very weakly effected by the presence of aggregates (or 
agglutinates) of these same microparticles in the medium, because the 
aggregates of these microparticles, whatever their size, no longer absorb 
ultraviolet and visible light at this .lambda.max, their absorption maxima 
being far from the .lambda.max defined earlier. 
Furthermore, it has been shown that a spectrophotometric measurement of 
ultraviolet and visible absorption of a suspension in a liquid medium of 
the said nonagglutinated (monomeric) microparticles obeys the Beer-Lambert 
law, and does so even at very high particle concentrations, and this gives 
it a clear advantage in relation to the principle of turbidimetric 
measurements, which calls for very dilute suspensions. 
Advantageously, in practice: 
the trajectory of the periodic motion may be of any kind, but is preferably 
circular, rectilinear or ovalized, 
the agglutination stage is performed at ambient temperature, 
the duration of the periodic motion imparted to the microparticles during 
the agglutination stage is a function of the frequency of the vibration, 
of the trajectory and of the amplitude of the motion. It is advantageously 
10 minutes for a frequency of 20 hertz and a periodic circular motion 4 
millimeters in diameter, 
the microparticles suitable for the spectrophotometric reading of 
ultraviolet and visible absorption consist of natural or synthetic 
materials, of any absorbing nature, especially polystyrene, 
polyvinyltoluene, acrylonitrile, styrene-butadiene, acrylonitrile-acrylic 
acid copolymers, acrylic ester and polyvinylbutadiene copolymers, and 
preferably polystyrene, 
the microparticles are of any shape, but are preferably in the form of 
microspheres, 
the diameter of the natural or synthetic microspheres is between 0.01 and 5 
micrometers, and preferably close to 0.8 micrometers, 
the microparticles consist of an absorbing but colorless material, 
it is also possible to use absorbing microparticles which are colorless. In 
this case, the spectrophotometric reading of the result by ultraviolet and 
visible absorption spectrophotometry is performed either at a wavelength 
corresponding to the absorption maximum of the material of the 
nonagglutinated particles, or at the wavelength corresponding to the 
absorption maximum of the color of the particle in question. 
The invention also relates to a device for implementing the method 
characterized in that it comprises at least: 
a first series of tubes containing at least one freeze-dried calibration 
range of the substance to be assayed, 
a second series of tubes containing an immunologically active substance and 
acting as the assaying agent, grafted onto the microparticles, the 
suspension referred to as "reactant" being in an liquid phase ready for 
use, the concentration in weight of the microparticles being comprised 
between 0.1 and 10 per cent 
a third series of tubes containing a freeze-dried specimen of the dilution 
solution of the calibration range, 
The manner in which the invention may be implemented and the advantages 
which stem therefrom will become more apparent from the example of 
embodiment which follows, given by way of guidance and without any 
limitation, in support of the attached figures.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The example which follows corresponds to the assay of antigens by means of 
antibodies grafted onto microspheres. It is obvious that it would be 
equally possible to assay antibodies by means of antigens by following the 
same procedure but by replacing the antigens by the antibodies and vice 
versa. 
Before the assay as such, the antibodies are first grafted onto graded 
synthetic microspheres, also called a latex. These are made, for example, 
of polystyrene, polyvinyltoluene, acrylonitrile, styrene-butadiene, 
acrylonitrile-acrylic acid copolymer, acrylic ester, polyvinyl butadiene, 
and the like. For example, the particles of latices K 140, K 109, K 160, 
.psi.512, .psi.513, .psi.480, .psi.502 (--COOH) or .psi.169, .psi.181 
(--CONH2), sold under these trade names by Rhone-Poulenc, are very highly 
suitable for the method. 
The grafting of the antibodies onto the synthetic microspheres is performed 
in a known manner, that is to say by ionic, hydrophobic or covalent 
fixation. 
When use is made of attachments of an ionic or hydrophobic type between the 
antibodies and the latex microspheres, these attachments, which are 
produced in a known manner merely by bringing the protein and the support 
together, lead to phenomena of detachment of the proteins from the support 
(also referred to as salting out). One of the reasons for the salting-out 
phenomena is that continual exchanges take place between the population of 
antibodies of the immuno-gamma-globulins G type fixed in this manner and 
the population of the said immuno-gamma-globulins G which is frequently 
present in the specimen to be analyzed. 
Advantageously, the covalent fixation methods are more reliable and are 
preferable in this type of method of immunoassay. Methods of covalent 
fixation of antibody or of antigen onto the synthetic microspheres, or 
latex, are well known, so that there is no need to describe them here 
further in detail. 
In the method according to the invention, when a fixation of the covalent 
type is employed, use is advantageously made of a fixation method which 
has never been described in an application of this type, and which yields 
good results. 
According to this coupling method, the functional radicals present on the 
microspheres and called into action are chiefly the following: --COOH, 
--CONH2, --CONH--NH2 and --COOCH3. 
These radicals are converted into acyl azides (--CON3) by the method which 
will be described, the azide reacting with the --NH2, --SH or --OH 
radicals of the biological molecules (antigens or antibodies) which it is 
intended to graft. 
For example, the latices .psi.169 and .psi.181 are first treated with 
hydrazine in solution and then with the vapor of acidic sodium nitrite, 
under mild conditions, before being brought into contact with a 
poly-L-lysine-antibody mixture. The carboxylated latices .psi.480 or 
.psi.502 are carboxymethylated with acidic methanol, and then treated with 
hydrazine in solution, and then with vapor of acidic sodium nitrite under 
mild conditions, before being grafted in an appropriate ratio with a 
poly-L-lysine-antibody mixture. 
EXAMPLE 
Grafting of human anti-immuno-gamma-globulin G antibodies and human 
anti-B2-microglobulin onto microspheres of polystyrene .psi.480 and 502 
(Rhone-Poulenc) with diameters of 0.8 and 0.85 micrometers respectively. 
This grafting takes place in accordance with various stages, summarized 
below: 
methylation of the latex carboxyl groups: esterification of the free 
carboxyls of the latices is performed in a bath of acidified methanol for 
a minimum of three days and a maximum of ten days, the saturation plateau 
being generally at an average of five days, 
vigorous rinsing with water, 
treatment of the carboxymethylated latices with hydrazine for 1 to 15 hours 
with stirring at 20.degree. C., 
vigorous rinsing with water at 0.degree. C., 
treatment with vapor of sodium nitrite acidified with hydrochloricacid 
(mild conditions); the constituents of the mixture and the mixture being 
prepared ad hoc and the reaction being performed at 0.degree. C., 
vigorous rinsing with a coupling buffer, 
grafting of the antibody-poly-L-lysine mixture; the poly-L-lysine coupled 
to the antibody-latex in this manner has a saturation effect on the 
grafting sites (--CON3), and stabilizes the reactant sterically during its 
storage; the proportions of antibody and poly-L-lysine vary depending on 
the desired enrichment in antibody on the synthetic microsphere and 
depending on whether it is intended to employ the right-hand or left-hand 
portion in relation to the equivalence point of the curve shown in FIG. 1; 
it should be noted that the method according to the invention permits the 
use both of the right-hand portion and of the left-hand portion of the 
said curve, 
desorption of the antibodies fixed by ionic bonding (3M KCl for 15 
minutes), and by hydrophobic bounding (KSCN 1M, for 10 minutes) 
rinsing 
storage. 
It is obvious that when the functional groups of the particles are --CONH2 
(example 169 and 181) or --COOCH3, the hydrazine stage represents the 
first stage of the grafting process. 
The following stage corresponds to the microsphere agglutination stage and 
this can be done according to the following manner. The latex microspheres 
are subjected to a periodic circular motion at a frequency of 20 hertz and 
with an amplitude of 4 mm, this being for a period of ten minutes. This 
stage is performed at ambient temperature, resulting in a significant 
reduction in the apparatus (no water bath or heating apparatus of any 
other type). Performed in this manner, this agglutination stage permits a 
large decrease in the nonspecific agglutination of the particles. In fact, 
its efficiency for the nonspecific agglutination of the particles is very 
high: when one of the methods described earlier, that is to say at 
37.degree. C., for one half hour in a water bath with conventional 
agitation, is employed, nonspecific agglutination is of the order of 30 to 
40%. The nonspecific agglutination according to the method described in 
the invention varies only from 2 to 5%. 
Furthermore, this method does not require the particles to be stabilized 
with bovine albumin or any other ionic agent made electronegative or 
electropositive at highly basic or highly acidic pH values, which are 
incompatible with the storage of the reactants. 
Consequently, the method makes it therefore possible to maintain the 
reactant and the calibration ranges at physiological pH values (near 7), 
thus ensuring very good integrity and excellent storage of the 
constituents. It should be noted that another advantage which exists lies 
in avoiding the use of proteinic stabilizing substances, as permitted by 
the method. In fact, proteinic stabilizing substances, such as albumin, 
are a source of bacterial contamination, and this is very inconvenient in 
an industrial reactant which needs to be stored for several months. 
Furthermore, the above method permits a marked increase in the specific 
immunological agglutination of the particles simultaneously with a very 
marked decrease in the nonspecific agglutination of the particles, and 
this is also an advantage of the invention. 
A saturation plateau in the immunological agglutination of the microspheres 
is quickly reached in approximately ten minutes. Such a short incubation 
period, in addition to being highly advantageous for the user who obtains 
an assay result extremely rapidly, does not, furthermore, involve any 
particularly painstaking stopwatch timing during the initial addition of 
the reactant or during the stage when the reaction is stopped. 
Furthermore, the efficiency of the method according to the invention in 
respect of the specific agglutination is such that the agitation may be 
performed at ambient temperature, and, as already said, this avoids the 
use of heating apparatus or of water baths, which is an undoubted and 
appreciable gain in convenience. 
According to the invention, it has thus been verified by experiment that 
nonspecific agglutination, which is associated with weak interactions 
between the particles (bonding of the ionic, hydrogen or van der Waals 
type) is made impossible or is highly inhibited by a periodic motion 
imparted to it, of very low amplitude and very high frequency, whatever 
the trajectory of the periodic motion (for example, and conveniently, 
rectilinear, circular or ovalized). It has also been verified that a 
method such as this markedly promotes the specific (immunological) 
agglutination of the synthetic microspheres carrying the antigen or the 
antibody, in the presence of the corresponding antigen or antibody, and 
thus permits a specific reaction which is rapid and is performed at 
ambient temperature. 
When the agglutination stage has been performed, the incubation medium 
containing the said microspheres is diluted with water to a volume of 
around 1 to 4 milliliters. 
This dilution can be performed by either water, saline solution or any 
suitable buffer solution. 
This volume corresponds, in fact, to that of a conventional 
spectrophotometry cell 1.times.1 cm in size. 
The reading and measurement stage is then performed. This can be done 
according in the following manner. In fact, before the agglutination stage 
as such, the ultraviolet and visible absorption spectrum of an aqueous 
suspension of the colorless nonagglutinated polystyrene microspheres is 
obtained in order to determine the absorption maximum of ultraviolet and 
visible light by these particles; the wavelength representing this 
absorption maximum is called .lambda.max. It has been shown that the 
absorption maximum .lambda.max characteristic of a given natural or 
synthetic microsphere is identical, whether the said microsphere is 
uncoated or coated with an antibody or antigen molecule. By way of 
example, FIG. 2 shows the absorption maximum in the ultraviolet and 
visible region of a concentrated suspension of microspheres of colorless 
polystyrene with a diameter of 0.8 micrometers, the spectrum being 
identical when the microspheres are uncoated or coated with an antibody or 
antigen. However, we can notice that the peak of absorption maximum is a 
bit flattened because of the interference with the Mie law (intensity of 
the scattered light forward). 
With this wavelength .lambda.max determined in this manner, after the 
immunological agglutination stage, reading of the result by means of 
ultraviolet and visible absorption spectrophotometry is performed by 
working in the vicinity of the said wavelength .lambda.max, determined 
earlier. In the example described, .lambda.max has a value of 380 
nanometers. 
Experience shows that the results obtained in this manner according to the 
invention, by ultraviolet and visible absorption spectrophotometry, show 
only the absorption of the monomers of the microspheres, the peak 
amplitude (degree of absorption--optical density--percentage transmission) 
being proportional to the concentration of the monomeric microspheres in 
the medium (Beer-Lambert law). 
Now, it is already known in an assay of this type that, once the 
immunological agglutination reaction is complete, the concentration of 
residual nonagglutinated monomers is proportional to the logarithm of the 
initial concentration of antigen in the medium. 
The result of the reading of the reaction by ultraviolet and visible 
absorption spectrophotometry in the vicinity of the .lambda.max of 
absorption of the monomers of the nonagglutinated microspheres is thus 
also proportional to the logarithm of the initial concentration of antigen 
in the medium. 
It should be noted that this method can be employed whatever the nature of 
the polymer (especially those listed earlier), and whatever the size of 
the particles or their color. When the latex particles are colored, the 
reading can be taken either at the max of the particle or at the 
.lambda.max of its color. In fact, in the case of the colored latices, the 
ultraviolet and visible absorption spectra show two peaks, one 
corresponding to the substance (independent of the color), the other 
corresponding to the colorant of this substance. 
This is illustrated in FIG. 5 which shows two peaks in the absorption 
spectrum of a suspension of red polystyrene microparticles 0.8 micrometers 
in diameter. The first peak, at 380 nm, corresponds to the absorption 
maximum of the substance of the microparticles. The second peak, at 560 nm 
for the red microparticles, corresponds to absorption maximum of the 
colorant of this substance. 
This phenomenon can be used to obtain an additive effect. This is 
illustrated in FIG. 6 which shows the ultraviolet and visible absorption 
spectra of a suspension colorless and yellow polystyrene microspheres 0.8 
micrometers in diameter. 
The concentration of the microspheres is the same for the two spectra. 
The spectrum of the colourless microspheres show a peak at 380 nm, which is 
the absorption maximum of the substance of the microspheres. 
The spectrum of the yellow microspheres also show a peak at 380 nm, but 
this peak is higher than the latter. It is known that the absorption 
maximum of a yellow colorant is at about 380 nm. Thus, in this case, there 
is an additive effect between the absorption maximum of the substance of 
the microspheres and the absorption maximum of the colorant of the 
substance. 
This additive effect allows increasing in accuracy of the assay. 
In practical implementation of the method, it is advantageous to choose a 
synthetic polymer microsphere whose nature, size or color has an 
ultraviolet and visible absorption maximum at wavelengths which do not 
interfere with obstructing wavelengths, especially at 280 nanometers, 
corresponding to the protein absorption. Advantageously, the method 
according to the invention employs, for example, carboxylated microspheres 
of colorless polystyrene with a diameter of the order of 0.8 micrometers, 
whose ultraviolet and visible absorption maximum is at about 380 
nanometers, as already stated. Furthermore, this wavelength does not, in 
fact, interfere, and thus not with the protein absorption maximum situated 
at 280 nanometers. This wavelength of 380 nanometers may be selected by 
means of any spectrophotometer or photometer, regardless of whether an 
ultraviolet or visible lamp is employed, bearing in mind that the value of 
380 nanometers is situated on the borderline between the ultraviolet and 
the visible. Furthermore, the reading at 380 nanometers in the visible is 
within the range of any laboratory photometric or spectrophotometric 
apparatus, resulting in a major simplification of the apparatus required 
for the assay method. 
Furthermore, experience shows that the absorption maximum .lambda.max is 
characteristic of the deagglutinated microspheres and that agglutinates of 
these same microspheres no longer absorb at this maximum, even when they 
are very small in size. It has been observed, in fact, that aggregates of 
synthetic microspheres of polymers have an absorption maximum which is 
characteristic of their size. It has also been observed that the 
agglutinates have an absorption maximum which is proportionately shifted 
towards longer wavelengths the larger their size. FIG. 3 shows (arrows) 
the absorption maximum of colorless microspheres of antibody polystyrene 
(0.8 micrometers in diameter), nonagglutinated, and the absorption maxima 
of the agglutinates of increasing size of these same microspheres. It 
shows: 
on one hand, the absorption of the residual microspheres not agglutinated 
by the antigen (at .lambda.max), and 
on the other hand, the absorption of the aggregates of increasing size, 
induced by increasing antigen concentrations. 
From the preceding it clearly follows that if the reading of a solution 
containing a mixture of monomeric microspheres and of their agglutinates 
is performed by ultraviolet and visible absorption spectrophotometry, 
operating at a wavelength which corresponds to the .lambda.max of 
absorption of the nonagglutinated (monomeric) microspheres, then the 
measurement performed in this manner will relate to and hence will detect 
only the monomers of the particles, because only they absorb at this 
maximum. 
This novel reading method, according to the invention, has many advantages 
when compared with the state of the art described earlier: 
it does not require the use of very sophisticated and costly apparatus such 
as particle counters, laser nephelometers or infrared opacimeters, 
in contrast to turbidimetric techniques in the visible, it can be employed 
with very high particle concentrations in the incubation medium, and this 
endows the assay with very high precision, which is impossible to obtain 
in visible or near infrared turbidimetry (opacimetry). By way of example, 
Table I below gives the optical densities obtained by spectrophotometric 
reading of ultraviolet and visible absorption of the result produced by 
the agglutination of colorless microspheres of antibody polystyrene 0.8 
micrometers in diameter, for two very low concentrations of the 
corresponding antigen 
TABLE I 
______________________________________ 
Initial concentration of antigen 
10 ng/ml 1 .mu.g/ml 
optical density, UV and visible absorption at 
1.3 0.5 
380 nm 
______________________________________ 
The 380 nm wavelength corresponds to the maximum of the absorption of 
ultraviolet and visible light by the nonagglutinated monomeric 
microparticles, 
the degree of precision permitted by the reading method according to the 
invention also permits the use of any spectrophotometer and the use of 
reading cells of a commonplace model (1.times.1 cm). 
The results given in the above Table correspond to a measurement performed 
by means of a conventional reading cell with a 1 cm optical path. An 
optical path of 4 centimeters, which is difficult to achieve in practice, 
would be needed to obtain the same precision using turbidimetry. 
In addition to an absorption spectrophotometer of a common type, the device 
for implementing the method described above comprises a compartmented 
casing and a means suitable for imparting a vibrational motion to the 
solution containing the microspheres. 
The compartmental casing, illustrated in FIG. 7, in the form of a 
rectangular parallelepiped, is 13.times.16.times.4 cm in size and is made 
of plastic or of stiff cardboard. It has four compartments: 
(1) the first compartment (1) contains a plurality of horizontal and 
parallel haemolysis tubes (4), which are closed and labelled (marked with 
the antigen concentration), and containing 2 to 4 calibration ranges in 
freeze-dried form, 
(2) the second compartment (2) also contains a plurality of haemolysis 
tubes (5), but larger in size, horizontal and parallel, and containing the 
reactant ready for use, in this case the antibodies grafted onto the 
microspheres, immersed in an appropriate diluent. In fact, the diluent 
should ensure the preservation of the antibodies, their stability with 
time and their satisfactory integrity. 
These tubes are four in number per standard range, and contain 2.5 
milliliters of reactant. The concentration in weight of the microparticles 
is comprised between 0.1 and 10 percent and preferably between 1 and 5 
percent. 
FNT *It is surprising and one main advantage of the invention, that we could 
proceed with such high concentrations, which were only used with visual 
qualitative processes readings on glass strips, as described for example 
in U.S. Pat. No. 4,542,103. 
(3) the third compartment (3) contains a plurality of closed, 
small-diameter tubes (6) containing a specimen of the dilution solution of 
the calibration range, in freeze-dried form. 
The quantity of freeze-dried dilution solution is advantageously the 
quantity which is necessary to dilute a biological sample, for example the 
quantity necessary to dilute ten times a biological sample which has 
already been diluted with distilled water. A quantity of 0.45 ml or 0.9 ml 
is very suitable. 
(4) the fourth compartment (8) contains at least one flask (7) of dilution 
liquid concentrate. 
The means capable of ensuring the periodic motion of the microspheres at 
high frequency and with low amplitude comprises a motor fixed to a 
verticle shaft actuating a cam. This cam imparts a periodic motion of high 
frequency and low amplitude, and with a circular trajectory, to a tray 
which is itself fixed to a support for parallel tubes. The said tray, of 
elongate overall shape, remains always parallel to its original 
direction.The means capable of ensuring the periodic motion also comprises 
a chassis on which the display of the motor rotation speed and the 
start-up are adjusted. The amplitude of the motion is limited in the 
present case to 4 mm and the frequency displayed is 20 hertz. 
Thus, when an antigen assay of a solution is to be carried out, for example 
the assay of .beta.2 microglobulin in a renal insufficiency serum, 50 
microliters of the serum to be analyzed are first taken and 0.45 ml of 
distilled water is added to it. A 50 microliter sample is taken from the 
solution prepared in this manner and is placed in one of the tubes (6) in 
the compartment (3), made up beforehand with 0.45 ml of distilled water. A 
conventional homogenization is carried out .05 microliters are then taken 
from this tube and are mixed with 50 microliters of the reactant ready for 
use, that is to say the corresponding antibody, grafted onto the 
microspheres and present in one of the tubes (5) of the compartment (2), 
and which has been homogenized beforehand. The in weight-concentration of 
polystyrene microspheres of this reactant is advantageously about 1 
percent. 
The new solution produced in this manner is placed on the support which is 
fixed to the tray, which is preferentially already in motion, means 
ensuring the periodic motion of high frequency and low amplitude. It is 
subjected to this periodic motion for a period of ten minutes, the 
frequency of vibration being 20 hertz and the amplitude 4 mm. When this 
agglutination stage has been performed, the dilution stage takes place. 
The volume obtained changes from 100 microliters to 3.6 ml, that is to say 
to the volume corresponding to the spectrophotometric analysis cell. The 
contents of the tube are then poured into the 1.times.1 cm reading cell 
and a spectrophotometric analysis is carried out at the absorption maximum 
of the nonagglutinated microspheres in the ultraviolet and visible, in 
this case at 380 nanometers, that is to say at the borderline between the 
visible and the ultraviolet. From a practical point of view, the hemolysis 
tubes can be directly placed in the spectrophotometer. Previously, one of 
the calibration ranges present in the compartment (1) of the casing has 
been employed in order to plot the straight line representing the 
relationship between the optical density and the logarithm of the antigen 
concentration. Once the optical density of the solution obtained has been 
determined, it is compared to the calibration curve and the concentration 
of antigens in the initial serum is deduced therefrom in an extremely 
strict manner. 
FIG. 4 is an illustration of an assay of human immuno-gamma-globulins G by 
means of the method and the device according to the invention. In this 
example, it is the portion on the right-hand side of the equivalence point 
of the curve in FIG. 1 which is employed. The coefficient of correlation 
with laser nephelometry which is obtained for this assay for 100 
cephalorachidian liquids and serums is close to 1. 
Table II, which follows, is a Table comparing the method according to the 
invention with a radioimmunotest for the assay of human .beta.2 
microglobulin in normal and renal-insufficency serums and in dialysis 
liquids. 
TABLE II 
______________________________________ 
concentration of .beta.2 microglobulin 
in ug/ml 
______________________________________ 
Method 11.6 5.7 0.7 2.97 2.24 2.13 2.71 2.23 
according to 
the invention 
radio- 13.2 6.7 0.41 2.06 2.12 2.05 2.60 2.30 
immunotest 
______________________________________ 
In addition to the advantages emphasized earlier, the present invention 
gives rise to other advantages, among which there may be mentioned: 
possible in-situ assay, that is to say at the patient's bedside, a thing 
which was hitherto made very difficult because of the bulk of the 
apparatus employed, 
very high speed of implementation and of determination of the results, 
together with very high reliability, which makes this method possible in 
emergency biochemistry or pharmacology, and in following graft survival, 
the feasibility of analysis throughout the usual ambient temperature 
region. 
This invention is therefore particularly suited for many fields of research 
and medicine, especially in cancerology, hormonology, endocrinology, 
clinical biochemistry, toxicology and microbiology. 
Furthermore, it is of great interest in the field of pyretogen control in 
the pharmaceutical industry and in that of haemodialysis.