FR2570189A1 - IMMUNOENZYMOLOGICAL ASSAY PROCESS AND MEANS FOR IMPLEMENTING IT - Google Patents

Abstract

THE INVENTION RELATES TO A PROCESS FOR IMMUNOENZYMOLOGICAL ASSESSMENT CHARACTERIZED BY THE FACT THAT A BIOCHEMICAL TREATMENT OF THE SUBJECTED SURFACE ARE SUBJECTED TO A SPECTROPHOTOMETER ALREADY OPTICALLY WORKED AND CAPABLE OF GIVING A PRECISE OPTICAL DENSITY MEASUREMENT, A BIOCHEMICAL TREATMENT OF THE SURFACE APPENDANT IMMUNOENZYMATIC REACTION AND TO BE USED AS A SUPPORT FOR THE UNROLLING OF THIS, THEN TO BE CARRIED OUT DIRECTLY ON THE TANK SUCH TREATED TO MEASURE OPTICAL DENSITY. THE MEANS FOR ITS IMPLEMENTATION CONSIST OF A SPECTROPHOTOMETER TANK WHOSE WALLS ARE COVERED WITH ANTIBODIES CAPABLE OF BINDING ITS ANTIGEN IN AN IMMUNOENZYMA REACTION.

Description

 So-called immunoenzymological assay methods currently represent one of the most promising areas of biological analysis and diagnosis.

They consist, briefly, in combining the advantages of the antigen-antibody reaction, very specific, and the measurement of enzymatic activities, very sensitive and which dispenses with the recourse to radioactive isotopes. These methods fall into one of two types:
1. The competition
- The antigen to be assayed (Ag) is fixed at
the antibody (Ac) and forms the complex (C). I1 is
competed with an antigen-enzyme conjugate
(Age). After incubation, the separation is carried out
free conjugate and attached to the antibody and we measure
the enzymatic activity of either fraction.
amount of enzyme associated with the antibody (CE) is therefore
a decreasing function of the amount of antigen to
dose

 A good method of separating the free conjugate from that linked to the antibody consists in fixing the antibody in advance on a solid support.

2. Les techniques "sandwich"
On dispose de 2 anticorps (au moins), l'un
d'entre eux est fixé à un support solide Ac1f, l'autre
est conjugué à l'enzyme Ac2E.L'antigène réagit avec l'un
et l'autre. anticorps, réalisant un pont entre eux ; le
complexe ternaire est appelé sandwich

- A variant of this process consists in having an antigen attached to a solid support (Agf) and an antibody-enzyme conjugate (AcE). The antigen to be assayed competes with the fixed antigen for binding to the labeled antibody. The enzymatic activity associated with the solid phase (CfE) is measured at the end of the incubation. Again, this activity is a decreasing function of the amount of antigen

2. Sandwich techniques
There are 2 antibodies (at least), one
of them is attached to a solid Ac1f support, the other
is conjugated with the enzyme Ac2E.The antigen reacts with one
and the other. antibodies, bridging them; the
ternary complex is called sandwich

We measure the associated enzymatic activity
to the solid support CfE which then presents itself as a function
increasing the amount of antigen to be assayed.

These processes are well known and have given rise to numerous commercial achievements. The state of the question is well summarized by numerous works such as "Enzyme-Immunoassay, Edward T. Maggio, 1980, CRC Press,
Inc. USA "and" Enzyme-Immunoassay, Eiji Ishikawa, MD, Ph.D.,
Tadashi Kawai, MD, Ph.D., Kiyoshi Miyai, MD Ph.D., 1981,
Igaku-Shoin, Tokyo-New-York ".

 These processes, and in particular those which have given rise to a commercial implementation, are characterized by the fact that one of the partners of the reaction (either antigen or antibody) is fixed on a solid phase, and that at the end of incubation, part of the enzymatic activity is associated with the solid phase on which it is measured.

The most used enzymes are
- alkaline phosphatase
- ss -galactosidase
- peroxidase
- acetylcholinesterase.

The most used solid phases are
- powders
- balls
- tubes
- plates.

In an immunoenzymatic reaction, the revelation of the enzymatic activity is generally a colored reaction which develops on contact with the solid phase. This situation has so far remained hardly compatible with the measurement of optical density. The solutions adapted to this problem are - or, the separation of the solid phase from the medium
incubation by decantation, centrifugation or transfer
by suction - or, for undivided solid phases (walls of
tubes or cells), we developed spectrophoto
special meters. Optical density measurement in
cells of the culture plates is imprecise, even if the
tanks are flat-bottomed, due to the curvature of the meniscus.

où x = paramètre arbitraire
Io(x) = intensité de lumière incidente l(x) = chemin optique
L'invention résulte de l'abord du problème par une voie inverse : partant d'un spectrophotomètre du commerce, fonctionnant avec cuves optiquement travaillées, donc capables de donner une mesure de densité optique précise, on fait subir un traitement biochimique de surface approprié qui la rende apte à subir la réaction immunoenzymatique.Celle-ci se déroule alors dans le support même qui servira à la mesure de densité optique. De plus, en adoptant les cuves à deux faces parallèles, on rend le résultat linéaire vis-b-vis de la concentration en produit formé.It is also used in round tubes
Indeed, it results from BEER law
LAMBERT that the optical density is no longer proportional to the concentration as soon as the optical path is no longer uniform, as the following relationships show
uniform path ::
DO = Log I = lC
I where
DO = optical density
10 = incident light intensity
I = intensity of transmitted light = = extinction coefficient characteristic of
the substance
1 = optical path
variable path

where x = arbitrary parameter
Io (x) = incident light intensity l (x) = optical path
The invention results from the approach to the problem by a reverse route: starting from a commercial spectrophotometer, operating with optically worked cells, therefore capable of giving a precise measurement of optical density, an appropriate biochemical surface treatment is carried out which makes it able to undergo the immunoenzymatic reaction. This takes place in the same support which will be used for the measurement of optical density. In addition, by adopting the tanks with two parallel faces, the result is linear vis-a-vis the concentration of product formed.

 Among the spectrophotometer tanks which can be used according to the invention, mention will be made of the single-use commercial plastic tanks such as those which are found on the market for example under the names - "Ratiolab", ref. 45/104 P - "C M L", ref. SMCU 1 - "OSI", ref. 12.100.5.

These cells generally have an optical path of 1 cm.

 The tanks are covered with antibodies (or antigen) by any known method such as for example according to the method described in patent application No. 8305618.

The following nonlimiting example is given as an illustration of the invention - Determination of microglobulin ss2
a) Preparation of the reagents
- Tanks coated with a first monoclonal antibody
antip2 microglobulin (process according to request from
patent mentioned above) - Conjugated: second anti-2 monoclonal antibody
microglobulin linked to laB galactosidase according to
Ishikawa (reference recalled above).

b) Procedure
50 til standard solution of ss 2 microglobulin +
1 ml of conjugate (second monoclonal antibody
antip2-microglobulin linked to ss galactosidase)
diluted in 10 2 molar phosphate buffer,
0.15 M NaCl (pH 7.2) -albumin (1 g / l) -Tween
(1 g / l).

 4 h incubation. at room temperature.

3 washes in 10 2 molar phosphate buffer,
0.15 M NaCl (pH 7.2) -albumin (1 g / l) -Tween
(1 g / l).

Incubation of the lag galactosidase substrate
orthonitrophenyl-8 -D-galactoside for
revelation of the reaction = 1 ml per tank,
for 1 h at 200C.

 Reading of the D.O. at 410 nm.

The corresponding curve is established. This
figure in the accompanying drawing. We use this
so-called standard curve to assess the
title of any solution of unknown title
enp 2-microglobulin depending on the value of the
optical density obtained by using a
tank according to the invention.

 It goes without saying that the present invention has been described for purely explanatory and in no way limitative and that any useful modifications may be made thereto without departing from its scope.

Claims (4)

 1. Immunoenzymological assay method, characterized in that a spectrophotometer cell, already optically worked and capable of giving a precise measurement of optical density, is subjected to an appropriate biochemical surface treatment making it capable of undergoing a reaction immunoenzymatic and thus serve as a support for the progress thereof, then to proceed directly to the cell thus treated with a measurement of optical density.  2. As a new industrial product, a spectrophotometer tank suitable for biochemical measurements, characterized in that its walls are covered with antibodies capable of fixing its antigen in an immunoenzymatic reaction or vice versa and suitable in the process according to claim 1.  3. Tank according to claim 2, characterized in that it comprises at least two parallel faces.  4. Cell according to claim 3, characterized in that it has an optical path of 1 cm.