GB2086042A - Process for Carrying Out Analytical Determinations by Means of Chemiluminescence, and the Use of the Process for Immunoassay - Google Patents

Abstract

In a method for carrying out analytical determinations using chemiluminescence a reagent is labelled with fluoresceinisothiocyanate (FITC) and the chemiluminescence reaction is triggered by adding an oxidant in the form of an aqueous solution of sodium hypochlorite, which may have a concentration of Cl per litre between 2 and 100 g. This method permits the use of a single reagent to trigger the chemiluminescence reaction. An example relates to the detection of HBsAg using Ab(HBs) coupled to polystyrene beads or a microtitre plate, and FITC-labelled Ab(HBs).

Description

SPECIFICATION Process for Carrying Out Analytical Determinations by Means of Chemiluminescence, and the Use of the Process for Immunoassay This invention relates to the use of chemiluminescence in analytical determinations.

Chemiluminescence is a phenomenon which is based on a chemical reaction and is therefore fundamentally different from fluorescence. In fluorescence a molecule is excited by visible light or UV light but remains chemically unchanged and merely reradiates (emits) the absorbed light.

The emission is either at the same wavelength as the absorbed light or a longer wavelength.

In contrast, the mechanism of chemiluminescence is based on the formation of at least one high-energy, unstable intermediate product which decomposes, with emission of light, to give an end product.

Analytical processes which utilise fluorescence are known per se, but background fluorescence has been found to affect seriously the results obtained, and has made it necessary to employ some expedient to eliminate this interfering fluorescence from the measurement.

Thus, according to German Offenlegungsschrift 2,628,158, the effect of background fluorescence is avoided by using as the labelling material only those fluorescent substances which, with appropriate excitation and at room temperature, have a narrow fluorescence spectrum coupled with a long decomposition time, and also by ensuring, by an appropriate scanning mechanism, that the measurement takes place only at a point in time when the interfering background fluorescence has already decayed, i.e. after about 100 nano-seconds, calculated from the radiation pulse. Chelates of rare earths and pyrene butyrates are particularly suitable labelling substances for this procedure.

The method disclosed in German Offenlegungsschrift 2,628,1 58 has, however, the disadvantages that the actual measurement, in each case, can only take place after the decay phase, and that relatively expensive equipment is required to couple the radiation pulse to the detection device so that the measurement is made at the correct time.

A summary of the difficulties which occur in fluorescence immunoassay processes may be found in "Clinical Chemistry" (1979), pages 353-361.

Proposals to utilise chemiluminescence for quantitative analytical processes have been based on the property of, in particular, the organic compound known by the name Luminol, i.e. that Luminol reacts with oxygen in alkaline solution and thereby forms an unstable intermediate product which then decomposes, with emission of light.

However, this method has not found acceptance in analytical practice, especially in immunoassay, mainly because when Luminol is subjected to the chemical modification necessary to enable it to be coupled to proteins of the antigen and antibody type, its molecular structure is altered such that the reaction required for chemiluminescence is impaired. This view is also supported by the observations made by Simpson and coworkers (see, for example "Nature", Volume 279 (1979), pages 646/647) that the radiation intensity of diazotised Luminol is only about 1% of that of unmodified Luminol.

A system in which fluorescein can be excited to luminescence by chemical processes is also described, in "Journal of Physical Chemistry", 78 (1974), pages -1681-1 683.

In this reference a system consisting of H202 and NaOCI is used as a reactant for the chemiluminescence reaction. However, it was reported that the concentration of the fluorescein required for detection is extremely high. The smallest amount detected was 2 x 10-4 mol, so that, compared with the requirements of immunoassay, the sensitivity is at least six orders of magnitude too low. It was also reported that the life of the luminescence in the system mentioned is only 2 Ms, that is to say the emission of photons takes place instantaneously. These very rapid kinetics are completely unsuitable for sensitive measurement. Photon counters are used for detecting the photons because they are the most sensitive measuring devices, but because of their typical dead time of 20 ns, only limited maximum pulse rates can be processed.

Quantitative and sensitive measurement of luminescence phenomena which takes place instantaneously is thus impossible.

The object of the invention was thus to make the phenomenon of chemiluminescence applicable to analysis methods, and in particular immunoassay, in a manner which can be utilised in practice.

In order to enable the chemiluminescence method to be applied to immunoassay, it would be desirable to achieve the following: 1. The sensitivity should be increased so that as little as 10-'0 mol of the labelling agent can be detected.

2. The reaction kinetics should be changed so that the emission of light does not take place instantaneously but, for example, over a period of several seconds.

It would furthermore be desirable: 3. to initiate the luminescence not by the addition of two reagents (H202+NaOCI) but only by a single reagent. This would on the one hand simplify the measuring equipment (only one dispenser would be required, instead of two).

Also, however, when two reagents are mixed, even in the absence of the chemiluminescent substance to be detected, considerable background luminescence is produced. This background is pronounced in the case of the system H202/NaOCI, and furthermore is not constant, so that there are again the disadvantages known in the case of fluorescence.

The present invention employs a surprisingly effective novel system of labelling material and oxidising agents which permits a high yield of photons and yet also, when the labelling material is coupled with, for example, proteins, aminoacids or polysaccharides it does not suffer troublesome decrease in reactivity towards the oxidising agent which induces the formation of the unstable intermediate products. This system is also sufficiently stable and sensitive for it to be possible for even very low concentrations of the labelling agent to be measured with a high accuracy.

According to the present invention there is provided a process for carrying out analytical determinations by means of chemiluminescence, characterised by employing fluorescein isothiocyanate (FITC) as a labelling agent, triggering a chemiluminescence reation by adding an aqueous solution of sodium hypochlorite and measuring the emission of light.

The structural formula of fluorescein isothiocyanate is

It is commercially available, one supplier being Messrs. EGA-Chemie, 7924 Steinheim, Federal Republic of Germany.

In a preferred embodiment, the determination is carried out in an aqueous medium without the addition of other oxidising agents and/or reducing agents for the chemiluminescence reaction.

Commercially available NaOCI solutions contain about 1 50 to 1 55 g of Cl per litre.

Aqueous dilute NaOCI solutions which contain, for example, 2 to 100 g of Cl/litre are generally used for the process according to the invention.

For determinations using FITC which is not bonded to protein it is suitable, for example, to use hypochlorite solutions containing 2.5 g of Cl per litre. The hypochlorite solutions used for the chemiluminescence reaction can thus contain 0.01 to 3% and advantageously 0.15 to 0.6% (weight/volume) of NaOCI, depending on the chlorine content of the starting solution to be diluted.

For FITC bonded to protein, the chorine content of the hypochlorite solution must be higher by a factor of 10, so that dilute solutions containing about 25 g of Cl per litre are advantageously used.

The chemiluminescence induced by the oxidation reaction is recorded quantitatively by a suitable instrument for measuring photons, without a special radiation pulse being required, as is the case, for example, with fluorescence analysis.

The process according to the invention is suitable both for solid phase assay and for liquid phase assay.

The target substances in question which are to be labelled by the compound FITC, which can be excited to chemiluminescence, are first separated off from other substances in a known manner, for example by means of chromatography or by fixing to antibodies (in this context, see, for example, "Proc. Soc. Experimental Biology", Volume 113 (1963), pages 394--397).

For example, the antibody specific for a certain target substance may be applied to a solid matrix substance, such as cellulose acetate, and the target substance to be investigated and to be determined analytically is brought into contact, in the form of a solution, with the antibody on the matrix and is thereby fixed on the substrate. The fixed target substance is then coupled to the labelling agent i.e. the fluorescein isothiocyanate (FITC), and the latter is subjected to further chemical reaction by adding aqueous NaOCI solution.

The FITC may be coupled to the antibody or the antigen, depending on whether a direct, an indirect or an anti-complementary serological activity method is chosen, by known methods, such as have already successfully been used for the coupling of fluorescein isothiocyanate (see for example, "Amer. J. Pathol.", (1958), page 1081 and "Proc. Soc. Experimental Biol. Med." 98 (1958), page 898 et seq.).

In the direct immunoassay method, for example, for determining a certain antigen in blood serum, the serum in question is subjected to customary preparation techniques. Then the antigen is fixed onto a solid substrate, and subsequently combined with a specific antibody, coupled with the FITC. Finally, excess labelled antibody is separated off.

According to the known "Sandwich" technique, which is described in "Proc. Soc. Ex.

Biol." 113(1963), page 349, a solid substrate with a deposit of antibody liquid is brought into contact with the sample to be investigated, the amount of antibody being greater than that required for bonding all the antigenic material in the sample. The substrate charged in this manner is then brought into contact with a solution containing the antibodies modified by the labelling agent, so that the antigens on the substrate become attached to these labelled antibodies. The substrate is then freed from excess labelled antibody and the solid substrate is subsequently treated with aqueous NaOCI solution in order to induce chemiluminescence which is then measured. This "Sandwich" process is always particularly suitable if the antigen in question has more than one bonding point for an antibody, that is to say in general for antigens in the form of large molecules.

If the antigens in question are relatively small molecules which contain only a single antibody specific bonding point per molecule, the indirect investigation process may be used. In this process, antigens which are identical to the material to be investigated are bonded to a solid substrate which forms a chemical bond with an antigen such that an antibody-specific bonding point or bond remains free. This process requires the formation of a chemical compound of the antigen in question and a protein, the bonding point on the antigen then remaining accessible to the animal immunological system, so that suitable antibodies can be produced (in this context, compare "Structural Basis of Antibody Specificity", Verlag Pressman a Goldberg (1968), especially pages 9 and 10).A suitable solid substrate which can form a chemical combination with an antigen is, for example, a styrene polymer with side chains which contain functional groups of a type such that the side chains match a group on the antigen to be investigated.

The solid substrate is then immersed in a solution containing an unknown amount of the antigen in question and the antibody specific to the antigen and modified by the labelling agent is subsequently added. The antigen contained in the solution and the antigen on the solid substrate compete for the labelled antibody, and the amount of the antibody which forms a combination with the solid substrate depends on the unknown amount of antigen in the solution.

The amount of labelled antibody on the solid substrate is then determined by adding NaOCI solution and inducing chemiluminescence, and the amount of antigen in the solution can thus be determined.

The process according to the invention is of particular interest in virological-serological diagnostics, for example in hepatitis A and hepatitis B diagnostics and rubella, CMVand EBV-serology. In addition, non radioactive sensitive and specific test systems are of extreme interest for clinical chemistry, for example for thyroid gland diagnostics (T3 and T4), for diabetes diagnostics (insulin) and the like.

The process of the invention will now be illustrated in more detail with the aid of the following examples, which refer to the accompanying drawings, in which: Fig. 1 is a graph of the kinetics of the luminescence reaction; and Fig. 2 is a graph showing the effect of FITC concentration on pulse rate; Fig. 3 is a graph showing detection of a HBs Ag-positive serum.

Example I Measurements were carried out with nonbonded FITC to determine the kinetics of the reaction. As shown in Fig. 1, it was found that the progress of the emission of light with respect to time, that is to say the kinetics, can be selected by choice of the NaOCI dilution. The known emission of light taking place instantaneously is still obtained above a concentration of about 8 g of Cl/litre (curve 1). It can be seen that optimum kinetics are obtained in the region of about 2.5 g of Cl/litre (curve 2), whilst with 0.8 g of Cl/litre (curve 3) the kinetics are still slower, but the sensitivity has decreased considerably.

If FITC bonded to protein is used in place of unbound FITC, then different kinetics are obtained as a function of chlorine concentration. It is necessary to raise the chlorine concentration by a factor of 10 in each case in order to obtain the same kinetics as with unbound FITC.

The measurements were carried out as follows: 200 ng of FITC, dissolved in 20 yl of distilled water, were first pipetted into a test tube. The sample was then placed in the darkened measuring position in front of the photomultiplier (measuring instrument: BIOLUMAT LD 9500 from Messurs Laboratorium Prof. Dr. Berthold, Wildbad, Federal Republic of Germany). 100 ul of Na0Cl of the given dilution were then injected onto the FITC through the dispenser system.

The kinetics were followed by recording via a rate meter connected to a pen recorder. The rate meter has two time constants of 20 ms and 1 s, the short time being automatically switched in for rapid changes and the long time being switched in for slow changes. At the same time, the number of pulses is integrated over a period of, preferably 1 Os.

Example II: In a second series of experiments, solutions of varying FITC concentration were prepared. For this, various amounts of FITC in the range from 1.25 ng to 200 ng, were in each case dissolved in 20 Ml of distilled water, in each case 100 jul of dilute NaOCI (2.5 g of chlorine/litre) were then added, and the measurments were taken.

Figure 2 shows the result. Perfect linearity over the entire range is obtained. The limits were determined only by the measuring conditions available at the time. Towards higher concentrations, problems appear as it becomes increasingly difficult to dissolve the required amount of FITC in water, whilst towards lower concentrations, a further factor of 10 may easily be achieved by lowering the background effect due to the measuring instrument, for example by cooling the photomultiplier.

Surprisingly, a further increase in sensitivity of around two orders of magnitude is obtained if the FITC is not free but is bonded to protein.

Example Ill The process according to the invention is used for detection of HBs antigen (abbreviated to HBsAg).

This detection is a solid phase assay. Anti-HBs from humans was coupled to the solid phase in the customary manner (solid phase: polystyrene beads or a microlitre plate). After this the solid phase was saturated with calf serum, this phase was then ready for use.

The solid phase was then incubated with the serum sample (0.1 or 0.2 ml) to be tested for HBsAg. For the rapid test, the incubation period was 2 hours at 37 0C, and for the standard test it was 1 6 hours at room temperature. The solid phase was then rinsed thoroughly with a customary phosphate buffer, which can have, for example, the following composition: NaCI 8,0 g KCI 0,2 g Na2HPO4 l 2H2O 2,89 g KH2PO4 0,2 g Doubly distilled water to 1000 ml HBs antigen present in the serum sample was now bonded to the solid phase.

The tracer was then added. An FlTC-labelled human anti-HBs which has been isolated by affinity chromatography was used as the tracer.

The solid phase was incubated with the tracer at room temperature for 4 hours. It was then rinsed thoroughly again with phosphate buffer. The HBsAg adhering to the solid phase was now bonded to the tracer.

The serum sample to be investigated was used in undiluted form an in various concentrations, having been diluted with human serum. However, phosphate buffer can also be used as the diluent, if appropriate with the addition of NaN3 (0.1%), which acts as a bactericide and inhibits bacteria growth in the serum samples.

After aqueous NaOCI solution (25 g of Cl/litre) has been added to the individual solid phases with tracer bonded thereto, the samples were measured and the number of pulses were determined as a function of the serum concentration.

The results obtained in this experiment are given in Figure 3.

The detection limit for the serum concentration of HBsAg is 0.1 ng/ml. This curve can be used as the calibration curve for determining unknown concentrations of HBsAg in serum samples.

Decisive advantages of the process according to the invention are: 1. Only a single reagent (instead of two in other cases) must be added in order to trigger off the chemiluminescence reaction; the measuring instrument can thus be simplified, because only one, not two, automatic injectors is required.

2. The zero value is now only determined by the instrument itself. In contrast, if two reagents are required, in addition to the FITC, these usually produce a considerable luminescence, as an interfering factor, in the absence of FlTC.

3. Only with this system could perfect linearity be established between the indication and the FITC amount over an initial concentration range 100 to 2x102 (Figure 2) which corresponds to a factor of 200.

4. A wide concentration range could also be recorded perfectly with protein-bonded FITC, the curve at very low concentrations being significantly flatter than that in the concentration range from 10 to 1,000 ng/ml.

Claims (9)

Claims

1. A process for carrying out analytical determinations by means of chemiluminescence, characterised by employing fluorescein- r isothiocyanate (FITC) as a labelling agent, triggering a chemiluminescence reaction by adding an aqueous solution of sodium t hypochlorite, and measuring the emission of light.

2. A process according to claim 1, wherein the determination is carried out in an aqueous medium, without the addition of other oxidising agents and/or reducing agents for the chemiluminescence reaction.

3. A process according to claim 1 or claim 2, wherein the light emitted is measured by means of an instrument in which the quanta of light are detected by a photomultiplier and the individual pulses triggered off by the photons are counted.

4. A process according to any one of claims 1 to 3, wherein the sodium hypochlorite solution contains 2 to 100 g of Cl/litre.

5. A process according to claim 4, wherein sodium hypochlorite solution containing about

2.5 g of Cl/litre is used for the determination of free FITC.

6. A process according to claim 4, wherein sodium hypochlorite solution containing about 25 g of Cl/litre is used for the determination of protein-bonded FITC.

7. A process according to any one of claims 1 to 6, wherein distilled water, phosphate buffer or a mixture of phosphate buffer and NaN3 solution is used as diluent for dilution of the sodium hypochlorite and the sample to be determined.

8. A process substantially as any one herein described.

9. Use of the process according to any one of claims 1 to 8 for carrying out immunoassay.

1 0. Process according to any one of claims 1 to 8 employed in an immunoassay procedure, to determine the produce of an immune reaction.