WO2008099279A1

WO2008099279A1 - A method for performing immunologic assays and product thereof

Info

Publication number: WO2008099279A1
Application number: PCT/IB2008/000381
Authority: WO
Inventors: Carlo Ciaiolo
Original assignee: Carlo Ciaiolo
Priority date: 2007-02-14
Filing date: 2008-02-12
Publication date: 2008-08-21
Also published as: ITTO20070106A1

Abstract

A method for performing an immunologic assay for the determination of an analyte in a sample, including the following operations: - placing said sample in a semisolid gel, wherein said semisolid gel contains at least one specific reagent for said analyte so that said analyte enters in contact with said reactant, so that said specific reactant reacts with said analyte with formation of an analyte-reactant complex, - detecting said analyte-reactant complex, characterised by the fact that said semisolid gel has a viscosity included between 10 and 16,000 cP.

Description

"A method for performing immunologic assays and product thereof"

* * * *

Field of the invention The present description relates to a new method for performing immunologic assays on biologic and chemical samples for the detection of an analyte of interest. In particular, the present description concerns a semisolid gel to detect precipitation reactions between the analyte of interest and a reagent specific for such analyte.

Technological background

Currently, to detect precipitation reactions in particular immunologic reactions, different methods are in use, some of which though, can present some problems in their use, for example:

• Methods according to Ouchterlony (long reaction times and scarce sensitivity) ,

• Crossed Star Immunofixation (CSI), FAST GEL, LINE GEL (complexity of methodology: use of masks that imply a certain dexterity) ,

• Turbidimetric reactions in liquid phase (lack of autoregulation in reaching optimal reagent/analyte equilibrium) . In particular, the gel immunodiffusion method according to Ouchterlony, consist of performing an

Antigen/Antibody reaction using a gel provided with two or more opposing wells. In one of these the antigen

(Ag) is deposited and in the other one the antibody (Ab) is deposited, the reactants will diffuse and if the ratio Ag/Ab is equilibrated they will form an immunocomplex detectable in the form of a precipitation line or arch in the gel. The space between the two wells determines a dilution of the reagent during the diffusion, therefore a scarce sensitivity, furthermore, a long time is also required before the reagents meet. Not being able to have the wells too close together for reasons of overlapping of the reactions, the methods like Crossed Star Immunofixation, FAST GEL and LINE GEL have been developed using masks instead of holes/cavities/wells in the agarose, allowing a higher reaction velocity to be obtained since the positioning of the Ag and Ab through the masks can occur at closer positions without running into the problems due to the formation of the reaction haloes around the well, since the wells are only virtual. However, as already mentioned, these latter methodologies include the use of masks and therefore they are more complicated from the operational point of view. The liquid phase reactions, in addition to lacking auto-regulation in reaching the optimal reagent/analyte equilibrium, involve high difficulty in evaluating the reaction by eye once it occurs since sometimes it is a matter of discriminating between a slight turbidity and a clear reaction.

Object and summary of the invention

The object of the present invention is that of overcoming the inconveniences linked to the traditional techniques for the determination of an analyte of interest in liquid phase immunoprecipitation assays or in-gel immunodiffusion.

According to the present invention, such an object is achieved by means of the solution specifically recalled in the claims that follow. The claims are an integral part of the technical teaching provided herein relative to the invention.

In an embodiment the invention concerns a method for performing immunologic assays on biologic and chemical samples for the detection of an analyte of interest employing a semi-solid gel containing a reactant specific for the analyte and having a viscosity included between 10 and 16,000 cP.

In an embodiment, the present invention concerns a semisolid gel mixed with a reactant, for example an antiserum, and arranged in discreet reaction areas for performing biologic and chemical reactions, in which the semisolid gel has a viscosity included between 10 and 16, 000 cP.

With the present invention, a reduction of the reaction times with respect to precipitation reactions in solid gels, is obtained, while with respect to the liquid-liquid reactions the visualisation of the, analyte-reactant complex is notably improved.

The advantages concerning the control of the excess of antigen and of sensitivity that are present in other in-gel methods, are preserved by the new method object of the patent.

Detailed description of the invention

The invention will now be described, by way of non-limiting example, with reference to the enclosed figures, wherein:

- figure 1 illustrates three possible results of the reaction between a drop of reactive semisolid gel (which we will indicate as RSG) identifiable with the circular area with a dark background and the sample to analyse. The situation A is obtained in the presence of a negative sample, that is, no precipitation can be detected between the reactant dissolved in the semisolid gel and the content of the sample. Instead, situations B and C represent two examples of formation of precipitation areas following the reaction between the analyte contained in the sample (positive) and the reactant dissolved in the RSG. Such areas are characterised by white colouration and can occur, in a circular (B) or ring-like (C) shape; - figure 2 represents a simplified schematic of the performing of the procedure described in the present patent. Initially, a known quantity of RSG is placed on a circular reaction area (A) . Successively, the sample to be analysed in liquid form is introduced inside such drop (B) ;

- figure 3 shows a simplified representation of the instrumentation used for the visualisation of the precipitate inside the RSG (indicated as S) : a lamp L is located on a plane inclined with respect to the gel S so that the light incident on the gel has an optimal angle for the visualisation of the precipitate. The gel S can be observed in a perpendicular direction with the naked eye (E) on a black background B; - figure 4, left column, illustrates examples __αf reaction areas A on which the RSG is located (from top to bottom: circular, elliptical and petal-like shaped areas); in the central column, instead, the corresponding sample dispensing spots in the above-said areas are indicated with the letter P and in the right column, the corresponding precipitation . areas Z (visible as white areas on a black background) .

The present invention refers to a method for performing immunologic assays on biologic and chemical samples in liquid phase for the detection of an analyte of interest, in which a semisolid gel containing a reactant that can specifically precipitate the analyte of interest is used.

A drop of reactive semisolid gel is placed on a reaction area and the sample to be analysed, for example urine, is dispensed inside the drop; thusly a liquid drop inside a semisolid drop is obtained. Around the contact point between the two drops a gradient with different concentrations of Analyte (antigen) and of Reactant (antibody) will form and therefore also the one ideal for the precipitation reaction.

For semisolid gel it is intended a gel constituted of materials determining a viscosity not high enough to allow the formation of mechanically stable wells, holes or cavities inside of it, opposite to what occurs using an agarose gel commonly used in immunodiffusion assays according to the prior art. In other words, the semisolid gel of the present invention has an amorphous structure which impedes the creation of mechanically stable well, holes or cavities inside of it.

In a preferred embodiment, the semisolid gel according to the present invention is constituted by methylcellulose or by materials with similar physicochemical properties (and in particular viscosity). In an embodiment, the methycellulose is mixed in an aqueous solution made of a buffer solution + antiserum so that the final methylcellulose concentration was 2% resulting in a semisolid gel with a viscosity higher than 10 centi Poise (cP) and less than 16,000 cP. Preferably a gel with viscosity equal to 40 and 4,000 cP, preferably between 80 and 2,000 cP and even more preferably between 200 and 800 cP, even more preferably 400 cP. Such range of values is naturally to be intended with reference to the tolerances inherent in its determinations and/or measurements. The claimed range corresponds essentially to the range of viscosity values of a semisolid gel that do not allow mechanically stable openings to be obtained_^ inside of it.

Because of the viscosity of the RSG, the introduction of the liquid analyte inside the RSG can be performed, for example, through tips, capillaries, etc. In the zone of contact between the semisolid gel and the liquid phase, according to the present invention, an immediate mixing of the two phases does not occur, but there is a slow and gradual co- penetration or reciprocal diffusion of the phases themselves with the formation of a gradient at different concentrations of Reactant and Analyte. Therefore, in a point of the formed gradient the optimal concentration between analyte and reactant will be obtained. In this way, the reactant-analyte reaction will be optimised. The evaluation of the non-immediate mixing between the two phases has been realised by colouring the liquid phase, in particular bromophenol blue at the concentration of 0.2 gr/L weight/volume was used. After placing the liquid phase containing the coloured sample in the gel, no appreciable mixing of the two phases was noted for at lease 120 minutes.

The precipitation of the analyte-reactant complex inside the semisolid gel occurs in function of the diffusion of the analyte inside of^"" the RSG. Normally the precipitate has a white colour and can be observed in transparency on a dark background; in case of coloured reactions, they can be observed on light or coloured backgrounds. In Fig. 1 we observe an example of the result of a reaction between the RSG and a negative (A) or two positive (B, C) samples.

The semisolid gel can be pre-positioned in one or more distinct masses each susceptible of constituting a corresponding reaction site, and the sample can be advantageously placed in an eccentric position with respect to said gel mass.

Examples of analytes advantageously determinable with the method object of the present disclosure: free kappa light chains, free lambda light chains, kappa light chains, lambda light chains, IgG, IgA, IgM, IgD, IgE, C3, C4, acidic alpha 1-glycoprotein, antithrombin III, haptoglobin, alpha2-macroglobulin, alphal- antitripsin, ceruloplasmin, prealbumin, APO Al, APO B, lipoprotein (a) , myoglobin, ferritin, transferrin, albumin, alpha 1-microglobulin, retinol-binding protein, rheumatoid factor, C-reactive protein, anti streptolysin 0, salmonella, brucella, proteus, IL-lα, IL-lβ, IL-2, IL-4, IL-5, IL-6, IL-8, IL-IO, IL-12, IL- 12p70, IL-13, IFNa, IFNγ , ENA-78, Eotaxin, GROα, IP-IO, MCP-I, MDC, MIG, MIP-Ia, MIP-lβ, MPIF-I , RANTES, TARC,

TIMP-I, TIMP-2, ANG-2, PDGF-BB, Tpo, EGF, KGF, HGF, FGF Basic, VEGF, HB-EGF, MMP-I, MMP-2, MMP-3, MMP-8, MMP-9, MMP-IO, MMP-13, TH1/TH2, TNF, sCD40L, sP-selectin, t- PA, sVCAM-I. In addition to the above-described advantages, another advantage of the use of semisolid gel according to the present invention consists of the fact that the reaction occurs without the use of masks or other accessories, but by direct placement of the biologic sample. The advantages concerning the control of the excess of antigen and of sensitivity that are present on other gel methods, are maintained by the new method object of the patent.

With the method object of the patent a net improvement in terms of reaction velocity and sensitivity with respect to the methods according to Ouchterlony is obtained and in terms of operative simplicity with respect to Cross Star Immunofixation, FAST GEL, LINE GEL methods. Furthermore, the capacity to discriminate between a positive and a negative reaction with respect to liquid-phase reactions is notably improved.

An example of a preferred form of embodiment of the present invention Materials: • A Petri dish was used in which circular shaped reaction areas of 1 - 2 cm diameter and delimited by an adhesive border were provided.

• Chemical compound used to obtain the semisolid gel: Methylcellulose

• Buffer solution: 0.015 M phosphate buffer pH 7.8

• Antisera used: Anti free kappa light chains (DAKO), Anti free lambda light chains (DAKO), Anti Albumin

(DAKO) , Anti Retinol Binding Protein (DAKO) , Anti alpha-1-Microglobulin (DAKO) , Anti Transferrin, Anti IgG (DAKO) Preparation of the reactants:

• A mixture of 0.015 M phosphate buffer pH 7.8 and methylcellulose at 2.5% in weight/volume was brought to boiling, maintaining it under agitation, once gelled and cooled to ambient temperature, 4 parts of mixture and 1 part of specific antiserum were taken. All of this was agitated by inversion for 5 minutes. The RSG with methylcellulose at the concentration of 2% in weight volume was thusly obtained. Method:

• Position a drop of RSG of approximately 120 μl in the reaction area present on the Petri dish, with a micropipette take 10 μl of biologic sample and penetrating the RSG with the tip unload it inside (Fig. 2) . After a short while, if the case is positive, we will observe a precipitation button or circle.

• To improve the visualisation the gel is illuminated (Fig. 3/S) with a lamp L (Fig. 3/L) inclined so to provide a light incident on the gel with a certain angle. The gel is observed (Fig 3/E) perpendicularly on a black background (Fig. 3/B) . The angle of the light with respect to the gel is chosen so to obtain an optimal visualisation of the immunoprecipitate . • Based on the comparisons made with the other methods of the prior art a better result in terms of sensitivity and better visualisation of the result was always obtained.

In the following, we describe the characteristics of the instruments and the realisable reactants and of the consequent procedures for realising a reactant - analyte reaction.

A semisolid gel mixed with a reactant, normally an antiserum, but also an antigen, a chemical reactant, other is used. After the preparation, the gel is conveniently placed on a dedicated reaction area, not necessarily circular. It was noted experimentally that if the sample is placed non-centrally to the gel (that is, in an acentric position with respect to the gel mass) a reaction non symmetric to the gel will be obtained and therefore, more easily identifiable with respect to the part of gel in which the reaction did not occur. In Fig. 4 we observe some examples of reaction areas (A) on which are placed the gel, spots of acentrically placed samples (P) and relative zones in which the reaction occurred (Z). The instruments and reactant described allow the formation of a reaction front on the surface and inside of a semisolid gel containing a reactant and placed on an appropriate reaction area. The diameter of the reaction area used or the greater dimension (in case it was not circular), was never more than 100 mm, the diameter or the smaller dimension was never less than 0.1 mm, the volume of the gel-antiserum used for each well was never more than 100 ml and less than 0.1 μl .

The reaction occurring between a sample in liquid phase and a semisolid one notably improves the reaction times with respect to a liquid phase and a solid one since the semisolid phase is less rigid and accelerates notably the penetration of the sample, but with respect to a reaction between two liquid phases there is the notable advantage of maintaining a certain separation between the two phases (liquid and semisolid) and therefore benefiting from the advantages provided by the notable reduction of convective motion occurring between reagent and analyte.

These instruments and procedures guarantee an analytical efficiency such as simplicity of execution due to only two operative moments (preparation of the analysis and reading) , analytical reliability also for great disequilibria of analyte/reagent concentrations, easy interpretation (characteristic precipitate in the gel), high sensitivity, adequacy of the response times, method widely applicable to the different routine and/or research diagnostics.

The methods, the instruments and everything else described and/or represented in this patent are examples, and therefore are not binding or limiting without departing from the field of protection of the present invention, as defined in the annexed claims.

Claims

1. Method for performing an immunologic assay for the determination of an analyte in a sample in liquid phase, including the following operations: dispencing said sample in a semisolid gel, wherein said semisolid gel contains at least one specific reactant for said analyte so that said analyte enters in contact with said reactant, so that said specific reactant reacts with said analyte with the formation of an analyte-reactant complex,

- detecting said analyte-reactant complex, characterised in that said semisolid gel has a viscosity included between 10 and 16,000 cP.

2. Method for performing an immunologic assay for the determination of an analyte in a sample, including the following operations:

- mixing said sample in a semisolid gel, - dispensing a reactant in liquid phase specific for said analyte in said semisolid gel containing said sample so that said specific reactant enters in contact with said analyte with the formation of an analyte- reactant complex; - detecting said analyte-reactant complex, characterised in that said semisolid gel has a viscosity included between 10 and 16,000 cP.

3. Method according to claim 1 or claim 2, wherein said semisolid gel has a viscosity included between 40 and 4,000 cP, preferably between 80 and 2,000 cP.

4. Method according to claim 1 or claim 2, wherein said semisolid gel has a viscosity included between 200 and 800 cP, preferably 400 cP.

5. Method according to any of the previous claims, wherein said semisolid gel is a methylcellulose-based gel .

6. Method according to any of the previous claims, wherein after dispensing the sample in the semisolid gel there is no mixing between the semisolid gel and the liquid phase of the sample or of the reactant for at least 120 minutes.

7. Method according to any of the previous claims, wherein said specific reactant is an antibody to which said analyte binds specifically.

8. Method according to any of the previous claims, wherein said specific reactant is an antigen to which said analyte binds specifically.

9. Method according to any of the previous claims, wherein said semisolid gel is previously predisposed in a plurality of distinct masses, each susceptible of constituting a corresponding reaction site.

10. Method according to any of the previous claims, wherein said semisolid gel is previously predisposed in at least one distinct mass, said distinct mass having a circular, elliptical or pedal-like configuration.

11. Method according to any of the previous claims, wherein said semisolid gel is previously predisposed in at least one distinct mass, said distinct mass and in which said sample is placed in said at least one distinct mass in an acentric position with respect to said at least one mass.

12. Method according to any of the previous claims, wherein said analyte is selected from among: free kappa light chains, free lambda light chains, kappa light chains, lambda light chains, IgG, IgA, IgM, IgD, IgE, C3, C4, acidic alpha 1-glycoprotein, antithrombin III, haptoglobin, alpha2-macroglobulin, alphal-antitripsin, ceruloplasmin, prealbumin, APO Al, APO B, lipoprotein (a) , myoglobin, ferritin, transferrin, albumin, alpha 1-microglobulin, retinol-binding protein, rheumatoid factor, C-reactive protein, anti streptolysin 0, salmonella, brucella, proteus, IL-lα, IL-lβ, IL-2, IL-4, IL-5, IL-6, IL-8, IL-IO, IL-12, IL-12p70, IL-13, IFNa, IFNγ,ENA-78, Eotaxin, GROα, IP-IO, MCP-I, MDC, MIG, MIP- lα, MlP-lβ, MPIF-I, RANTES, TARC, TIMP-I, TIMP-2, ANG-2, PDGF-BB, Tpo, EGF, KGF, HGF, FGF Basic, VEGF, HB-EGF, MMP-I, MMP-2, MMP-3, MMP-8, MMP-9, MMP-IO, MMP-13, TH1/TH2, TNF, sCD40L, sP-selectin, t-PA, sVCAM-I.

13. Method according to any of the previous claims, wherein said sample is selected from urine, serum, plasma, blood, ascites, bacterial culture media, cell culture media, liquor.

14. Semisolid gel for the determination of an analyte in a sample, wherein said semisolid gel contains an antibody specific for said analyte and wherein said semisolid gel has a viscosity included between 10 and 16,000 cP.

15. Semisolid gel according to claim 14, wherein said semisolid gel has a viscosity included between 40 and 4,000 cP, preferably between 80 and 2,000 cP.

16. Semisolid gel according to claim 14 or claim 15, wherein said semisolid gel has a viscosity included between 200 and 800 cP, preferably 400 cP.

17. Semisolid gel according to any of the claims 14 to 16, wherein said semisolid gel is a methylcellulose- based gel.

18. Semisolid gel according to any of the claims 14 to 17, wherein said antibody is selected from: free kappa light chains, anti-free lambda light chains, anti-kappa light chains, anti-lambda light chains, anti-IgG, anti- IgA, anti-IgM, anti-IgD, anti-IgE, anti-C3, anti-C4, anti-acidic alpha 1-glycoprotein, anti-antithrombin III, anti-haptoglobin, anti-alpha2-macroglobulin, anti- alphal-antitripsin, anti-ceruloplasmin, anti- prealbυmin, anti-APO Al, anti-APO B, anti-lipoprotein

(a), anti-myoglobin, anti-ferritin, anti-transferrin, anti-albumin, anti-alpha 1-microglobulin, anti-retinol- binding protein, anti-rheumatoid factor, anti-C- reactive protein, anti-anti streptolysin O, anti- salmonella, anti-brucella, anti-proteus, anti-IL-lα, anti-IL-lβ, anti-IL-2, anti-IL-4, anti-IL-5, anti-IL-6, anti-IL-8, anti-IL-10, anti-IL-12, anti-IL-12p70, anti- IL-13, anti-IFNα, anti-IFNγ , anti-ENA-78, anti-Eotaxin, anti-GROα, anti-IP-10, anti-MCP-1, anti-MDC, anti-MIG, anti-MIP-lα, anti-MIP-lβ, anti-MPIF-1, anti-RANTES, anti- TARC, anti-TIMP-1, anti-TIMP-2, anti-ANG-2, anti-PDGF- BB, anti-Tpo, anti-EGF, anti-KGF, anti-HGF, anti-FGF Basic, anti-VEGF, anti-HB-EGF, anti-MMP-1, anti-MMP-2, anti-MMP-3, anti-MMP-8, anti-MMP-9, anti-MMP-10, anti- MMP-13, anti-THl/TH2, anti-TNF, anti-sCD40L, anti-sP- selectin, anti-t-PA, anti-sVCAM-1.

19. Realisation of a semisolid gel mixed with a reactant and corresponding circular or other shaped reaction areas apt to delimiting the placement of the gel and of a sample to be analysed.

20. Realisation according to claim 19 of a semisolid gel mixed with substances capable of providing detectable reactions for biologic and chemical analyses .

21. Realisation according to claims 19 or 20 of a semisolid gel mixed with antisera or antigens.

22. Realisation according to claims 19 or 20 of the use of methylcellulose for the preparation of the semisolid gel.