FR2624613A1 - Microspheric support for the immunoenzymatic, fluorescent, chemiluminescent or radioactive detection of microdetermined quantities of substances in biological media - Google Patents

Abstract

System for the immunoenzymatic or radioimmunological detection of microdetermined quantities of substances from a drop of whole blood, serum or any biological fluid. The invention, which relates to the field of biological tests, makes it possible in numerous cases to have a biological information of primary importance in a few minutes. It relates to an immunological detection or assay system consisting of microspheres in the form of a combination of aluminium, hydroxyapatite, acrylic fibre and magnetite. These microspheres, 100 microns in diameter, are coated (activated) with substances which remove the disadvantages (steric hindrance, background noise, brittleness) of the other existing solid phases and provide an improved yield, an improved detectability by the multiplication of the area of contact and a high concentration of bound immunological material. Upon contact with small quantities of whole blood (50 microlitres) or serum (25 microlitres) or any biological fluid, this system rapidly provides a response. Its main applications will be the detection of tumour markers, antigens and antibodies of viral disorders, hormonology-endocrinology, microbular infections, allergology, enzymology and membrane receptors.

Description

 The present invention relates to a solid phase useful for carrying out rapid biological tests using the principle of the antigen-antibody reaction to reveal by coloring a substrate, the presence of substances in infinitesimal quantities (below nanogram); The purpose of these biological tests is to detect a number of conditions involving an agent (the antigen) and the reaction of the organism to that agent, ie, the revelation and assay of antibodies against this agent, from a drop of any liquid coming from the human body (blood, serum, plasma, urine, saliva, tears, cerebrospinal fluid, urethral humors, vaginal fluid, breast milk, liquids collected during surgery or on organs-to graft to detect AIDS for example), or even fragments of solids any previously solubilized or simply stirred with a little water.

 There are immunoenzymatic detection devices using antigens or antibodies but requiring on the one hand staff for blood sampling, manipulations and on the other hand using a complex system, expensive and especially very long (3 hours) because of the repeated cycles following incubation periods of half an hour between each phase.

The device according to the invention makes it possible to remedy these drawbacks and to make available to patients, medioles, small health centers,
Transfusion, Emergency Services, etc ... a whole range of rapid tests, since this device that requires only a drop of liquid to analyze, allows a diagnosis in the next few minutes by reading the antigen-antibody reaction revealed by a color reaction or by measurement of radioactivity (RIA) linked to radioactive labeling.

 To detect the presence of an antigen or antibody in a biological fluid, the corresponding antigen or antibody must be fixed on a solid phase, bringing the biological fluid into contact with this solid phase. If the antigen is present in this liquid, it is fixed by one of its epitopes to the antibody fixed on the solid phase, then it is revealed, in a second step, by a labeled antibody (enzyme + dye or radioactivity ). If the antibody is present in the biological fluid, it binds by its ends Fab (NH2) to the antigen previously fixed on the solid phase, then is revealed by a second labeled antibody.

 The diagram reproduced in FIG. 1 of the attached drawings illustrates the reactions which have just been described.

This technique, however, has the following disadvantages
- fragility of the binding of antibodies or antigens on the solid phase, whether by physical bonds (electrostatic, Van der Vaals forces, hydrogen or hydrophobic) or by chemical bonds (covalent - COOH, -SH or NH2)
- steric hindrance on the solid phase the accumulation of immunological material on this phase causes electrostatic promiscuous reactions (rejection of the material to be assayed, biasing the assay) by molecular and electrostatic configurations, which forbid promiscuity, as shown Figure 2 of the accompanying drawings
background noise (bottom): the lower limit of the assay technique: below a certain concentration, the response of this technique is always the same, as illustrated by, for example, Figure 3 of the drawings. appended
- reading limits due to weak staining or RIA counting
poor performance: A fixed antigen-antibody reaction to be assayed can not fall below 10 nanograms per milliliter
- need for agitation to bring the fixed material in contact with its correspondent in the test liquid;
- Too much distance of the fixed material relative to its corresponding in the test liquid, as shown in the diagram shown in Figure 4 of the accompanying drawings; the wells of a plate have a volume of 0.25 ml so that 0.25 ml of microbeads are introduced therein, the bringing into contact is facilitated.
- Last disadvantage: these solid phases require many manipulations, multiple washes and can not be mobilized (96-well plates or tubes).

The curve shown in FIG. 5 of the appended drawings shows the losses of immunological material as a function of the washes.

 The object of the present invention is to remedy these drawbacks by providing a mobile solid phase consisting of microspheres with a mean diameter of 100 microns which can withstand on their surface solid immunological material which is stable in a medium. wide range of pH and temperature, which avoid annoyance and steric hindrance, help to reduce background noise, amplify the reading of low dosages, increase the efficiency of the antigen-antibody reaction, significantly reduce the stirring and reading time, and finally zero to the fixed material distance - material to be dosed.

The subject of the present invention is a mobile solid phase consisting of microbeads-each of which comprises
a matrix having, at its surface, chemical functions capable of securely fixing an activation coating
an activation coating capable of fixing a ligand
a ligand capable of capturing the molecule to be dissolved in a liquid medium.

 According to the present invention, the matrix must satisfy the following criteria: porosity, solidity, reliability (not to cause nonspecific interactions), rigidity and especially must have on its surface chemical functions (-OH, Amide, -CONH2) likely to firmly fix the desired coating (this is activation). Porous glass was discarded, all tests finding very difficult activation. Cellulose has many advantages but is responsible for many nonspecific interactions.

 Polystyrene, despite significant progress in its activation, does not fix more than 100 nanograms of protein per cm2 and has presented during the tests a very bad WRE (Washing Reduce Effect): the necessary washings after immunological protein binding, carry a much of these proteins.

 Some sugars (polysaccharides) apparently interesting by the very large number of -OH groups (6 per molecule) have proved fragile (brittle), not very porous and not rigid.

According to an advantageous embodiment of the microbeads according to the present invention, their matrix is constituted by a combination of aluminum filings, magnetite, acrylic float and hydroxyapatite in substantially equal amounts.
- Aluminum filings provide rigidity, stability to temperature and pH variations, as well as chemical and electrostatic neutrality, thus avoiding unwanted interactions.

 The magnetite filings provide only an easy handling: they make it possible to separate, using a magnet glued against the tube, the microbeads of the liquid of the wash or of the biological liquid to be tested. Its defects (brittle, ionized Fe2o3 ++ unstable, ..) are neutralized by a suitable peripheral coating, which is the acrylic float.

 - The acrylic float brings a precious element: a large number of amide -CONH2 functions that will firmly fix the coating of microbeads.

 - Hydroxyapatite is a crystalline phosphate (Cas (PO4) 2) 3 Ca (OH) 2 of considerable interest: it has on its surface positive ions (Ca ++) and negative ions (PO1 ") allowing a chemical fixation first quality ligand.

 On the other hand, the tests have shown that molecules of very high molecular weight, which pose fixing problems, are so firmly fixed that it is impossible to elute them in spite of a phosphate buffer. M, whereas a normal elution is done with buffers between 0.001 and 0.25 M.

 According to another advantageous embodiment of the microbeads according to the present invention, the activation coating which covers the surface of their matrix and is capable of fixing a suitable ligand is constituted by an activator whose molecule comprises a group -CH = C - Reagent and in particular with glutaraldehyde.

Indeed to allow these microbeads to fix a ligand, it is necessary to activate them for several reasons
the need for an intermediate between the surface of the beads and the molecule to be determined, a solid and stable intermediate reacting on one side with the microbead and on the other side with the molecule to be assayed
- need to space the binding sites to avoid steric hindrance.

 The activation with the best quality, ease / toxicity, instability, is glutaraldehyde activation according to the scheme shown in Figure 9 of the accompanying drawings.

It is sufficient to bind any protein by its amine function -NH2 on the site -CH = C- glutaraldehyde according to the scheme

Protein.

On the other hand, to fix an immunoglobulin by its Fc (COOH) fragment, it is necessary to
- or add a spacer arm ending in -NH2 to bind to -COOH. The best is HMD (hexamethylenediamine) NH2- (CH2) 6 -NH2
- or overactivate the surface of the balls.

 According to yet another advantageous embodiment of the microbeads according to the present invention, these comprise an agent for superactivation of their surface bound to the activating coating agent.

 According to an advantageous arrangement of this embodiment, said overactivation agent consists of protein A for the binding of IgG.

 According to another advantageous arrangement of this embodiment, said overactivation agent is constituted by the G protein for the binding of IgG.

 According to yet another advantageous arrangement of this embodiment, said overactivation agent consists of protein A and protein G attached to the activation coating of the matrix of microbeads.

 According to another advantageous embodiment of the microbeads according to the present invention, the ligand is taken from the group which comprises the antibodies, the antigens, the immunoglobulins, the antigens fixed on appropriate sites of immunoglobulins themselves, fixed according to the invention. case, the activation coating or the overactivation agent carried by the matrix of each of the microbeads.

 In addition to the foregoing, the invention also comprises other provisions, which will emerge from the description which follows.

The invention will be better understood with the aid of the additional description which follows, which refers to the accompanying drawings and to embodiments of the invention of the present invention.
it should be understood, however, that these examples and drawings and the corresponding descriptive parts are given solely by way of illustration of the subject of the invention, of which they in no way constitute a limitation.

 Figure 6 illustrates the composition of microbeads according to the present invention, while Figure 7 illustrates overactivation by A and G proteins of a glutaraldehyde-activated microbead.

 Example 1: production of microbeads.

1.1 Production of the matrix
100 cm3 of water are boiled in a suitable container into which 100 cm 3 of CaCl 2 and 100 cm 3 of Na 2 HPO 4 are poured. We shake gently. CaHPO4 and 2 NaCl brushite are formed. The water is discarded and allowed to cool by adding sodium hydroxide NaOH and stirring for 1 minute.

Then poured 100 cm3 of aluminum filings previously mixed with 100 cm3 of magnetite filings, stirring continuously to mix the components well. Let cool. Cold water is used to remove all impurities. The plate is removed and placed in an electric crusher. Sieve at 100 microns. This powder is washed and placed in a glass vase placed on a strong magnet. All incomplete beads, aluminum scrap and impurities float while the quality microbeads stick to the bottom. The microbeads are separated and rinsed with the following solution: Buffer 0.01 M ammonium acetate pH 4
1M sodium chloride pH 7
0.01 M TRIS-HC1 buffer pH 8.5
1.2 Activation coating placement
The obtained matrix beads are placed in 100 mM phosphate buffer pH 6.8 to eliminate all interference. Buffers should be prepared with distilled, sterile 3 times water. 100 cm3 of 25% glutaraldehyde are added and the mixture is incubated for 24 hours at 37 ° C., rinsed with a pH 7.6, 0.5 M phosphate buffer and maintained at 4 ° C. in the presence of a bacteriostatic agent and 0.015% Merthiolate type. or sodium azide.

1.3 Ligand fixation
1.3.1 antigen binding
The activated microbeads are placed in a solution of 100 ml of 0.5 M phosphate buffer, pH 7.6 + 5 mg of antigen. The mixture is stirred for 18 hours at +4 ° C. and then washed with 10 volumes of the above-mentioned phosphate buffer.

 1.3.2. Fixing an antibody.

 To fix 1 mg of antibody, 0.25 ml of activated microbeads is used, which is rinsed with 10 volumes of sterile deionized water, after which 3 volumes of 0.5 M phosphate buffer, pH 7, are added. , In which 1 mg of freeze-dried or dissolved immunoglobulins was added.

 After stirring overnight, keeping at +4 ° C., the above-mentioned buffer is rinsed to remove non-fixed Ig, and then treated with the same volume of a pH 7.5-8.5 buffer, + 4 C, rinsed, added a bacteriostatic and maintained at +4 C. The microbeads obtained are ready for use; their diameter is advantageously 100 microns, their area of 0.0314 mm 2 and their volume of 0.0005236 mm 3.

 This solution seems to be the most efficient: we first fix an Ig x taking advantage of strong Ig binding capacity. The antigen to be assayed will bind to the Fab sites and will be revealed by an Ig against the epitope still free on the antigen.

 To assay an Ig, it will suffice to fix an antigen on IgO fixed. The Ig sought is fixed by its Fab sites on the antigen. It will be revealed by a labeled anti-Fragment Ig Fc.

 The length and the great mass of this convoy pose no problem. The detectability is multiplied by 10, while all the disadvantages of the direct fixation of antigens (background, steric gene, ...) are considerably diminished, the fixed Ig behaving like excellent spacing arms.

 Figures 10 and 11 illustrate a ligand for antigen detection and a ligand for antibody screening, respectively.

Example 2 Preparation of Overactivated Microbeads
The activated microbeads obtained according to Example 1.2 are placed in a solution of 100 ml of 0.5 M phosphate buffer, pH 7.6, containing 5 mg of freeze-dried protein A. The mixture is stirred (on Titertek force 4, for example) for 18 hours at +4 ° C., and washed with 10 volumes of the buffer above.

 To block the remaining free aldehyde groups of glutaraldehyde, treated with a buffer of ethanolamine or 0.1M Tris pH 7.5-8.5 for 3 hours at +4 C, then rinsed with 10 volumes of 0.5 M phosphate buffer, pH 7.6, containing 0.15 M NaCl; then equilibrated with 1 volume of 0.5M phosphate buffer, pH 7.6 containing a suitable bacteriostatic.

 On these hyperactivated microbeads, the ligand is fixed as described in Example 1.3 and microbeads are obtained ready for use, as shown in Figure 8 of the accompanying drawings.

Example 3: Dssaoe of antiaenes
0.25 ml of ready-to-use microspheres, i.e. coated with protein A and antigen-specific antibody, are placed in a NUNC minisorb tube (which absorbs only very little material). immunological) or in a glass tube already prepared by surface blocking agents. 0.75 ml of 0.5 M phosphate buffer, pH 7.6 and 1 ml of the test serum are added thereto, the tube is closed and the mixture is stirred horizontally for 3 minutes by hand or on a stirrer in case of series. A magnet is placed against the walls of the tube and all the liquids are thrown away.

 1 ml of the labeled antigen-specific Ig is added and stirred for 3 minutes. The magnet is placed against the tube and all liquids are thrown away. Rinse with the same buffer. If the marking is radioactive, the counts are counted. If it is enzymatic, 1 ml of substrate + chromogen is added and the color reaction is measured in D.O.

EXAMPLE 4 Antibody Dosaae
0.25 ml of overactivated microbeads are placed as described in Example 2 and prepared as described in Example 1, with an antigen corresponding to the desired antibody. 1 ml of serum is added to analyze, the desired antibody is fixed on the antigen. The liquids are discarded, rinsed and 1 ml to 1/2000 of a labeled Ig specific for the Fc segment of the desired antibody is added. Rinsed several times and read the reaction.

The performances observed are as follows
- 1 ml of microbeads, having a reactive surface of 600 cm 2 fixed 3.3 mg of protein A
- As 1 mg of fixed protein A 12 mg of IgG, 600 cm 2 of microbeads set 40 mg of IgG, or 0.066667 mg or 66667 nanoaramme ~ pv 33
by comparison: The same microbeads without protein A fix only 4333 ng / cm2.

 A polystyrene surface, even when activated, sets only 400 ng / cm2 and only 100 ng / cm after 6 wipes.

The detection performance of this method is further increased, in particular by heating the IgG to be fixed at 135 ° C. for a few minutes. The Pc segment is denatured at this temperature and binds even more firmly to protein A. The Fab segment remains intact at this temperature
- It is possible to perform an assay by this method with only a drop of blood of 50 til, the thresholds of detectability very low recorded, allowing the microdosing on whole blood
- A problem can arise for very low concentrations: cf steric gene diagram. Mar-Ig is bivalent (binding to 2 antigens) but can be fixed by a single antigen.

 The measurement will then show twice the actual concentration. To remedy this, the best solution is to use labeled Fab fragments, as shown in Figure 12 of the accompanying drawings.

 Advantages: A Fab segment for AN antigen.

 Disadvantages: Fab segments have lower detectability than full Ig.

To remedy this: choose very high affinity Fabs and, if necessary, amplify the response with a second one.
Marked Fab directed against the previous.

 - It is also possible to place in the tube activated microbeads against different conditions for example: 0.25 ml of beads for HIV1, 0.25 for HIV2, 0.25 hepatitis. A positive reaction will indicate that one of the three infections is present, making it possible to guide the following examinations.

 It will be interesting to set a group of tumoral markers of the digestive or gynecological or respiratory sphere. In the case of a positive reaction, organ-by-organ exploration is carried out with identical tests but supporting only the markers of the organ explored.

 A positive reaction will guide the specialized examinations (Fibroscopy, Biopsies, ...). The use of the microbeads in accordance with the invention has made it possible, during the tests, to cross new detectability thresholds.

It provides new insights into hormonology, tumor markers, and HIV antigen that is virtually undetectable, thus completing antibody screening, of which negativity is frequent and late positivity in a growing number of patients. infected people. If the threshold of detectability of the HIV antigen is reached by this method, it will interest the whole virology.

 It is also intended for the early and rapid detection of many conditions (Syphilis, Hepatitis, ...) and to obtain rapid biological indications such as blood groupings, the evaluation of circulating enzymes and finally the diagnosis of difficult affections. to appreciate (Rabies, Malaria, ...).

The microbeads according to the present invention are capable of being used as non-limiting examples for
The diagnosis of viral diseases,
Diagnosis of microbial diseases,
Enzymatic assays,
Autoimmune diseases and Alzheimer's disease,
Rapid blood grouping,
Cancer screening,
The dosage of circulating drugs,
The detection of allergens. Allergy diagnosis,
The membrane receptor assays,
Hormone dosages,
Various assays (Anions Cations of the ionogram, metals, and generally all elements of the Mendeleieff Classification).

 As is apparent from the above, the invention is not limited to those of its modes of implementation, implementation and application which have just been described more explicitly; on the contrary, it embraces all the variants that may come to the mind of the technician in the field, without departing from the scope or scope of the present invention.

Claims (8)

 1. Solid phase useful for carrying out rapid biological tests consisting of microbeads, characterized in this Street each of these microbeads comprises  a matrix having, at its surface, chemical functions capable of securely fixing an activation coating  an activation coating capable of fixing a ligand  a ligand capable of capturing the molecule to be assayed in a liquid medium.  2. Solid phase according to claim 1, characterized in that the matrix of each of the microbeads is constituted by a combination of aluminum filings, magnetite, acrylic float and hydroxyapatite in substantially equal amounts.  3. Microbeads according to any one of Claims 1 and 2, characterized in that the activation coating which covers the surface of their matrix and is suitable for fixing a suitable ligand is constituted by an activator whose molecule comprises a -CH = C-reactive group to which a protein is attached.  4. Microbeads according to any one of claims 1 to 3, characterized in that they comprise an agent for superactivation of their surface bound to the activating coating agent.  5. Microbeads according to claim 4, characterized in that said overactivation agent is constituted by protein A for the binding of IgG.  6. Microbeads according to claim 4, characterized in that said overactivation agent is constituted by the G protein for the binding of IgM.  7. Microbeads according to claim 4, characterized in that said overactivation agent is constituted by protein A and protein G attached to the activation coating of the microbead matrix.  8. Microbeads according to any one of claims 1 to 7, characterized in that the ligand is taken from the group which comprises antibodies, antigens, immunoglobulins, antigens attached to appropriate sites of immunoglobulins, themselves depending on the case, the activation coating or the overactivation agent carried by the matrix of each of the microbeads.