A METHOD OF IMMUNOASSAY DETECTION BY REACTION-RATE POTENTIOMETRY USING FLUORIDE ION-SELECTIVE ELECTRODE

Abstract: A universally applicable immunoassay detection me­ thod for use in quantitative determination of an analyte in a sample, following immunological reaction of said analyte with an element which specifically binds to (or is bound by) this analyte. Attached to this element is a catalytic unit which generates signals detectable electrometrically. Inherent in this invention is the facility to monitor the signal in a continuous fashion thus allowing kinetic or reaction-rate monitoring which offers, in turn, the opportunity for rapid analytical systems.

Description: A METHOD OF IMMUNOASSAY DETECTION BY REACTION-RATE POTENTIOMETRY USING FLUORIDE ION-SELECTIVE ELECTRODE Field of the Invention This invention embodies a universally applicable immunoassay detection method for use in quantitative determination of an analyte in a sample, following immunological reaction of said analyte with an element which specifically binds to (or is bound by) this analyte. Attached to this element is a catalytic unit which generates signals detectable electro metricaily. Inherent in this invention is the facility to monitor the signal in a continuous fashion thus allowing kinetic or reaction-rate monitoring which offers, in turn, the opportunity for rapid analytical systems generally applicable to immunoassay. Background of the Invention Immunoassays are based on the specific and avid binding reaction of an antibody to an antigen. Following this reaction, the antibodybound fraction is separated from the non-antibody bound fraction and either fraction quantified by measurement of a signal generator, defined prior to the immunological reaction. Commonly immunoassays are of two major forms, quantitative excess reagent and limited reagent techniques as defined by Ekins (Ekins R.P., Radioimmunoassay and Related Procedures in Medicine, IAEA, 1977, Vienna pp. 241-268). Here reaction between the two members of the immunological binding pair can be carried out in solution with up to one of the reaction members immobilised either by adsorption or covalent attachment to a solid-phase matrix, essentially for ease of separation. The aim of these assays which rely on the identification of unique structural elements of the analyte by the reciprocal member of the immunological binding pair, is to estimate the amount (mass or molecular number) of particular substance of unique molecular structure in a sample. The present invention has particular application to these two forms of assay. Definition of some terms: Antibody: One member of the immunological binding pair, commonly of the Immunoglobulin G (lug) class and represented by the abbreviation Ab. Antigen: The second member of the immunological binding pair, abbreviated to Ag. Label: The signal generator attached to either Ab or Ag; repre sented thus: *. The reagent excess methods rely on the reagent (either Ab or Ag) present in the assay system being in excess concentration relative to the reciprocal member of the binding pair which is the analyte. The essential feature of such assays is that: the response parameter directly reflects the amount of analyte in the system and are of the following forms (1) (2): (1) excess Abk + Ag Ab*Ag + Ab* (2) excess Ag* + Ab Ag*Ab + Ag$. (2) excess Ag* + Ab Ag*Ab + Ag Here separation of antibody-bound from non-antibody bound fraction is accomplished by a range of physical, chemical or immunological techniques (e.g. Ratcliffe J.G. (1974) British Medical Bulletin 30: 32-37), well known to persons skilled in the art. Limited reagent assays however are systems where the reagent is present in a limited and carefully defined amount, usually less than the concentration of test substance in the system. Here the analytical measurement relies on the distribution of the reagent between sample analyte and a fixed quantity of exogenous labelled analyte and is of the following form (3): (3) limited Ab + Ag + Ag* AbAg + AbAg* + Ag* + Ag As above, separation of the bound from the free moieties is accomplished by well tried means and either moiety can be analysed for signal. The above represents the conventional and widely used approaches to measuring numerous exogenous and naturally endogenous substances in the clinical investigation and detection of normal and pathological function in man. Typically endogenous factors are human plasma proteins, myeloma proteins, acute-phase reactants, tumour markers, blood clotting factors, peptide and protein hormones, steroid hormones and thyroid hormones. Exogenous factors are drugs, drug metabolites, bacterial toxins, structural properties of invading bacteria, viruses, actinomycetes, mycoplasms, fungi and organisms which cause sexually transmitted diseases. This list is readily applicable to immunoassay based on potentiometric detection of fluoride ion by reaction-rate techniques or fixed time reaction methods. Historically, immunoassay systems have been based on radioisotopic detection techniques (e.g. Kirkham K.E. and Hunter W.M., Radioimmunoassay Methods, Churchill-Livingstone, Edinburgh, 1971) but although extremely sensitive, these systems have the unwelcome aspect of handling, storage and disposal of radioactive materials and the purchase of expensive dedicated counting apparatus. Non-isotopic techniques have also been used whereby an enzyme is linked to one member of the immunological binding pair and the presence of the enzyme detected following the immunoassay reaction sequence (see for instance Alexander et al., Anal. Chem. 54 (1982), p. 67-71 and the publication of "BATTELLE TODAY" (29), August 1982, p. 1). The enzyme based immunoassays are attractive due to the available and considerable knowledge in detection, characterisation and preparation of antibody-enzyme conjugates and the inherent sensitivity of a signal generator (enzyme) which produces a number of signal units per molecule of generator (i.e. acts as a catalyst). These enzyme immunoassay systems exist as both limited and excess reagent immunoassays. Engvall and Perlmann (Immunochemistry 8: 871-874, 1971) were the first to utilise and coin the acronym enzymelinked immunosorbent assay (ELISA) which is the major example of reagent excess enzyme immunoassay. Since then, numerous forms of enzyme immunoassays have been developed (see Ishikawa E. et al., Enzyme Immunoassay, Igaku-Shoin, Tokyo, 1981) and have been the subject of a number of patents in this area (e.g. U.S. Patents: 3,654,090; 3,791,932; 3,839,153; 3,850,752; 3,879,262) each describing methods where an enzyme is coupled to the analyte or reagent. For instance, an analyte is immunoreacted with an excess of a substrate-bound first partner member after which an excess of an enzyme labelled second partner is added, whereby following reaction one of the products is immobilised and either the soluble or insoluble fraction estimated by means of enzyme activity. Other types of labels have been used to monitor the bound or nonbound fraction in immunoassay. Fluorescence labels using quenching (U.S. Patent 3,996,345) and enhancement techniques (U.S. Patent 3,901,654), electron spin resonance (U.S. Patent 3,850,578) and chemiluminescence (GB Patent 1,548,741 and Canada Patent 1,116,079) have been used. However most current non-isotopic methods rely on the use of optically clean surfaces and optically pure solutions. Biological samples are generally not optically pure and can contain particulate matter of a proteinaceous or lipaemic nature which interferes optically. Fluorescent or fluorogenic assays have advantages over enzyme-chromogenic immunoassays (Murachi T., Enzyme Immunoassay, Igaku-Shoin, Tokyo 1981, pp. 5-13) being potentially more sensitive. However these assays are subject to errors due to fluorescence quenching, light scattering and high fluorescent background due to the nature of complex biological samples such as serum, plasma or urine. Other references pertaining to the invention are WO-A8 001 081; DE-A-3 010 462; DE-A-2 824 742 and Arzneimittel Forsch. Band 28, No 11A (1978) L. Schrstt "Enzymimmunoassay", p. 1934-40. Thus there still exists a need for a safe, rugged, readily applicable detection method for general use in immunoassay signal generation. Radioisotopic techniques have the best sensitivity but have inherent dangers in their use. Optical assay techniques are readily interfered with due to the diverse nature of biological samples. However we have developed a universally applicable detection method which is not inherently hazardous to the operator, requires no specialised or expensive equipment of reagent handling and storage devices, and the signal is not nonspecifically altered by normal components of the biological specimen under analysis. More precisely, in this method which comprises using an enzyme marker in a specific reactant partner for a bound analyte, the detection of the enzyme signal involves measuring catalytically generated specific ionic species the amount of which is quantitatively related to the presence of the marker and the analyte. This technique relies on the known phenomenon that some fluoro-compounds are oxidised by H2O2 in the presence of peroxidase with the stoichiometric disruption of a fluorinecarbon bond to give fluoride ions which are measured electrometrically with a specific fluoride sensitive electrode. This is described in Clinical Chemistry, 1982, 28, 1962, and has been applied to the detection of glucose and cholesterol in a previous patent (U.S. Patent 4,353,983). The present invention is applicable both to excess reagent immunoassay and limited reagent immunoassays. According to one embodiment of the invention for measuring a polyvalent analyte antigen (Ag) involving peroxidase linked to a substrate (I) in a "Sandwich-immunobinding" of the type "Peroxidase-Antibody (Ab2) / Antigen (Ag) / Antibody (Ab1)-Substrate (1), the determination is effected as follows: a) adding a liquid sample of analyte (Ag) to a first reaction container provided with a substrate (1) carrying an antibody (Ab1) and performing a first incubation (I) in which a selective coupling of the antigen (Ag) with the antibody (Ab-l) occurs; b) removing the remaining liquid sample from the substrate (1) and container; c) adding thereto a peroxidase solution which comprises a known excess of a peroxidase (POD)-antibody (Ab2) and effecting a second incubation (I I); d) transferring either the substrate (1) with the bonded peroxidase (POD) or the remaining peroxidase solution with the unbound peroxidase (POD) in a second container (2) containing a fluoride selective electrode (3) and adding thereto hydrogen peroxide (H202) and a fluoro-compound (RF) solution whereby a peroxidase catalyzed splitting of the fluorine into F ion occurs; e) reading the voltage from the electrode, this being the measuring signal of the marker relating to the amount of antigen (Ag) to be measured in the sample. In a modification of the above procedure, step a) can be performed differently as follows: instead of transferring either substrate or solution into a second container equipped with an F electrode, the solution can be removed from the first container, the substrate being left therein. Then the electrode can be introduced in the container and hydrogen peroxide and fluorophenol added thereto whereby the same reaction takes place and step e) can be performed as above. The method of the invention is illustrated by the formula below: EMI6.1 in which the vertical hatched line indicates the substrate, Abl is the first antibody, Ag is the analyte, Ab2 is the second antibody, POD is the enzyme marker and RF is the fluorocompound, preferably a fluorophenol or fluoroaniline as in US P 4,353,983. Brief Description of the Drawings The following examples will better illustrate the practical aspects of the invention with reference to the annexed drawing in which: Fig. 1 illustrates the experimental reaction-rate curves taken from example 1 which shows F variation calculated from the corresponding electrode potential and plotted in ng/l against time for samples of different human IgG concentrations. The rate curves 1-7 shown represent human IgG concentrations of 0, 30, 100, 300, 1000, 3000 and 10,000 ng/ml respectively. Fig. 2 is a graph obtained by plotting the mean dF /dt values of the rate curves of Fig. 1 versus the Log of the corresponding human IgG concentrations. Fig. 3 illustrates the experimental reaction-rate curves taken from example 2 which shows F variation calculated from the corresponding electrode potential and plotted against time for samples of different human alphafetoprotein (AFP) concentrations. The rate curves 1-7 shown represent human AFP concentrations of 0, 5, 12.8, 32, 80, 200 and 500 ng/ml respectively. Fig. 4 is a graph obtained by plotting the do /dot values of the rate curves of Fig. 3 versus the Log of the corresponding human AFP concentrations. Fig. 5 illustrates the experimental reaction-rate curves taken from example 3 which shows F variation calculated from the corresponding electrode potential and plotted against time for samples of different human placental lactogen (HPL) concentrations. The rate curves 1-8 represent HPL concentrations of 0, 0.24, 1, 3.9, 15.6, 62.5, 250 and 1000 ng/ml respectively. Fig. 6 is a graph obtained by plotting the dF /dt values of the rate curves of Fig. 5 versus the Log of the corresponding HPL concentrations. In order to demonstrate the subject invention, the following assays were carried out. These assays are offered by way of illustration and are not to be constituted as limiting. Example 1. Determination of human IgG: Reagents: 1.a) Phosphate buffered saline (PBS) pH 7.4 was prepared by dis solving potassium hydrogen orthophosphate 7 mM (1.22 g), potassium dihydrogen orthophosphate 3 mM (0.408 g) and sodium chloride 150 mM (8.77 g) in double distilled water. 1.b) Coating buffer pH 9.6 was prepared by dissolving sodium carbonate 15 mM (2.94 g) and sodium bicarbonate 35 mM (1.59 g) in double distilled water. l.c) Assay buffer: 18 vol./vol. normal goat serum (Scottish Anti body production Unit, Lanarkshire, Scotland) in PBS. 1 .d) Wash buffer: 0.3% wt/vol. bovine serum albumin (Sigma Chemical Co., St. Louis, MO, USA) and 0.05% vol./vol. Tween 26 (Merck-Darmstadt, W. Germany) in PBS. 1.e) Blocking solution: 1% wt/vol. bovine serum albumin in PBS. 1.f) Goat immunoglobulin to human lgG (Scottish Antibody Pro duction Unit, Lanarkshire, Scotland) was diluted 1/100 with counting buffer for use. 1 .g) Human y-globulin, Cohn-fraction II (Serva Feinbiochemica, Heidelberg, W. Germany) was used as standard. Working standards of 0.03, 0.1, 0.3, 1.0, 3.0 and 10 Crg per ml were prepared by appropriate dilution in assay buffer. 1 .h) Peroxidase conjugated rabbit immunoglobulins to human IgG-y chains (Dakopatts, Copenhagen, Denmark) were diluted 1/40 in the assay buffer for use. 1.i) Organo-fluoro compound solution: 4-Fluorophenol (Aldrich Europe, Beerse, Belgium) 0.25% wt/vol. in 240 mM acetate buffer and 87 mM sodium chloride pH 5.5. 1.j) Colorimetry solution: Orthophenylenediamine hydrochloride (Fluka AG, Buchs, Switzerland) 20 mg was dissolved in 10 ml of 0.1 M citrate buffer pH 4.5. 1. k) Hydrogen peroxide: 30% commercial hydrogen peroxide solution (Merck, Darmstadt, W. Germany) was diluted to 50 mM in distilled water. Method: A 24 wells Linbro tissue culture plate (Flow Laboratories, Ayrshire, Scotland) was used. The wells were coated by pipetting 1 ml of diluted goat antiserum (solution 1.f) and left overnight at 40C. the plate was emptied by aspiration and washed three times with 1.5 ml of wash buffer per well. The wash buffer was left 5 minutes between successive washes in order to reduce non-specific adsorption. The wells were then filled with 1.5 ml of blocking solution and left 10 minutes at room temperature. The wells were then emptied by aspiration and small adhering droplets were released by shaking the plate vigorously. Two series of wells were then filled with 1 ml of IgG working standards (solution 1.g) and a blank solution. The plate was then incubated for 90 minutes at room temperature. The wells were emptied by aspiration and washed three times with 1.5 ml of wash buffer. The wells were then filled with peroxidase conjugated rabbit antiserum (solution 1.h) and incubated for 90 minutes at room temperature. The wells were emptied and washed three times with 1.5 ml of wash buffer as before. In one series, the wells were filled with 1 ml of fluoro-phenol solution (solution 1. i). Miniature teflon coated magnetic bars were then gently dropped in each well. The plate was then placed on a magnetic stirrer and the stirring-was started in a controlled manner to avoid spilling the solutions from the wells. The final analytical reactions involving F ions generation were then carried out in each well successively in the following manner: 20 ,ul of 50 mM hydrogen peroxide solution (solution 1. k) was injected in the well and the fluoride ion-selective electrode (type 96-09 Orion research) was then immediately inserted in the solution. This electrode had a reference electrode combined therewith but any other fluoride ion-selective electrode with separate reference electrode can also be used. The electrode can be connected to a suitabie voltmeter and in the present invention, a Corning EEL 112 Digital Research pH-meter from Corning Scientific Inc., Medfield, Mass. USA, was used. After-exactly 3 minutes, the electrode potential in mV (relative) was measured every successive 15 second for 180 seconds. The concentration of F for each measurement intervals was calculated from the measured potential from the Nernst equation which in this case has the-form: E = E' - S log [ F ] where E1 is a constant inherent to the system which is determined experimentally and which involves activity factors and the liquid junction potentials. S is the "Nernst slope" which is also a constant and is approximately 59 mV for a change of 10 units in the concen traction of F where the latter is expressed in moles/l. Graphical curves thus obfained are shown in Figure 1. The rate of F generation was calculated by linear regression of the dF /dt in the ascending portion of each curve. The relationship between the concentrations of IgG and rates of F generation is shown ia Figure 2, curve 2b, and the curve is thereafter used as a comparative standard template for the determination of unknown samples of IgG the analytical conditions being the same as for the determination of standards. In order to compare the present invention with the conventional photometric measurements, the second series of wells in the plate were treated as follows: A chromogen developing solution was prepared by mixing 3.5 jil of 30% H202 with 10 ml of orthophenylenediamine solution (solution 1 .j). This solution must be prepared fresh. 1 ml of this solution was added to each well and the plate was then incubated in the dark, at room temperature for 30 minutes. Following this, 500 xul of the solution from each well was transferred to series of test tubes containing 2 ml of 0.2 M HCI. The absorbances of these solutions were then measured at 492 nm wavelength in a spectrophotometer. The photometric standard curve of lgG is shown as curve 2a in Fig. 2. A comparison of the two curves 2a and 2b clearly shows that the present technique is at least as sensitive as the conventional photometric technique. The present technique is however considerably faster. Example 2: Determination of human Alphafetoprotein (AFP) Reagents: 2.a) Assay buffer: Bovine serum albumin (Sigma) 0.3% wt/vol. in PBS (solution 1.a) 2.b) Rabbit immunoglobulin to human AFP (Dakopatts, Copenhagen, Denmark) was diluted 1/250 in the coating buffer (solution 1.b). 2.c) AFP standards: AFPL (Behringwerk, Marburg, W. Germany) was diluted in the assay buffer to obtain 10, 30, 100, 300 and 600 ng/ml AFP working standard solution. 2.d) Peroxidase conjugated rabbit immunoglobulin to human AFP (Dakopatts, Copenhagen, Denmark) was diluted 1/125 in PBS (solution 1.a). 2.e) Wash buffer. Surfactant Tween 20 (Merck Darmstadt, Germany) 0.1 ml was added to 1 litre of assay buffer. Method: The method was essentially similar to the one described in Example 1. The wells of the Linbro tissue culture plate were coated with 1 mf solution of rabbit antibody to AFP (solution 2.b). After the usual washing and blocking steps as described in Example 1, two series of wells were filled with 1 ml AFP working standards (solution 2.c) and a blank solution. The plate was incubated for 90 minutes at room temperature. The wells were then emptied and washed as described earlier. 1 ml of peroxidase conjugated rabbit antibody to AFP (solution 2.d) was then added and the plate incubated for 90 minutes at room temperature. Following this the wells were again washed, and finally filled with fluorophenol solution (solution i). Potentiometric reaction-rate measurements for F were then carried out in one series of wells as described earlier. Reaction-rate curves are shown in Fig. 3 and the calibration curves for AFP are shown in Fig. 4, curve 4b. In the second series of wells colorimetric reactions were carried as described earlier and the data are represented by curve 4a for comparison with the present technique. Example 3: Determination of human placental lactogen (HPL). Reagents: 3.a) Assay buffer. Bovine serum albumin (Sigma) 0.396 wt/vol. and normal horse serum (Scottish Antibody Production Unit, Scotland) 2% vol./vol. in PBS (solution 1.a). 3.b) Wash buffer. Bovine serum albumin (Sigma) 3 g and surfactant Tween 20 (Merck) 0.1 ml in 1 litre of PBS'(solution 1.a). 3.c) Rabbit immunoglobulin to HPL (Dakopatts, Copenhagen, Denmark) was diluted 1/125 in the coating buffer (solution 1.b). 3.d) Peroxidase conjugated rabbit immunoglobin to HPL (Dakopatts, Copenhagen, Denmark) was diluted 1/125 in the assay buffer. 3.e) HPL standard solutions. HPL standard (Biodata-Serono, Rome, Italy) was diluted in the assay buffer to give 0.060, 0.240, 0.980, 3.90, 15.60, 62.50, 250 and 1000 ng/ml HPL working standards. Method: The method was essentially the same as described in Example 1. Two series of wells of the Linbro tissue culture plate were coated with 1 ml solution of rabbit antibody to HPL (solution 3.c). The washing and blocking steps were performed as in Example 1. The wells were filled with HPL working standards (soiution 3.e) including a blank solution and the plate incubated for 90 minutes at room temperature. The wells were then emptied and washed as described earlier. 1 ml of peroxidase conjugated rabbit antibody to HPL (solution 3.d) was added and the plate incubated again for 90 minutes at room temperature. Following this the wells were washed. Finally one series of wells were filled with 1 ml of fluorophenol solution (Solution i). Potentiometric reaction-rate measurements were then carried out as described earlier (see Fig. 5). The reaction-rate curves and the calibration curve are shown in Figs 5 and 6 respectively (see curve 6b). Comparative colorimetric measurements were carried on a second series of wells in the usual manner and the data is shown in Fig. 6, curve 6a, for comparison with the present technique.

Claims: CLAIMS 1. A method for the immunochemical determination of an analyte (Ag) in a liquid sample in which this analyte is immuno-reacted with a reactant (ambi) attached to a solid substrate (1) and whereby measuring of the results involves an enzyme signal generator whose detection comprises measuring enzymatically generated ionic species, characterized in that the amount of the ions present or formed in the immuno-reaction relates to the quantity of analyte to be detected and in that the determination is effected by means of an electrode selective to such ions. 2. A method according to claim 1 for measuring one (Ag) of the immunochemical reaction member involved in said immunoreaction, said member being bound on one hand to a solid substrate (1) and on the other hand to peroxidase (POD), an excess of the latter in free form being also present, characterized in adding thereto hydrogen peroxide (H202) and a fluorocompound (RF) whereby a splitting of said fluorocompound (RF) catalyzed by the bonded or free peroxidase (POD) occurs and wherein one measures the resulting fluoride ion with an electrode selective thereto. 3. A method for measuring a polyvalent antigen (Ag) involving per oxidase linked to a substrate (1) in a "Sandwich-immunobinding" of the type "Peroxidase-Antibody (Ab2)/ Antigen (Ag)/Antibody (Ab1)-Substrate (1), characterized in performing the following steps: : a) adding a liquid sample of analyte (Ag) to a first reaction container provided with a substrate (1) carrying an antibody (ambi) and performing.- a first- incubation (I) in which a selective coupling of the antigen (Ag) with the antibody (Ab1) occurs; b) removing the remaining liquid sample from the substrate (1) and container; c) adding thereto a peroxidase solution which comprises a known excess of a peroxidase (POD)-antibody (Ab2) and effecting a second incubation (it); ; d) transferring either the substrate (1) with the bonded peroxidase (POD) or the remaining peroxidase solution with the unbound peroxidase (POD) in a second container (2) containing a fluoride selective electrode (3) and adding thereto hydrogen peroxide (H202) and a fluoro-compound (RF) solution whereby a per oxidase catalyzed splitting of the fluorine into F ion occurs; e) reading the voltage from the electrode, this being the measuring signal of the marker relating to the amount of antigen (Ag) to be measured in the sample. 4. A method for measuring a polyvalent antigen (Ag) involving peroxidase linked to a substrate (1) in a "Sandwich-immuno binding" of the type "Peroxidase-Antibody (Ab2)/Antigen (Ag)/ Antibody (Ab1)-Substrate (1), characterized in performing the following steps: a) adding a liquid sample of analyte (Ag) to a reaction container provided with a substrate (1) carrying an antibody (at1) and performing a first incubation (I) in which a selective coupling of the antigen (Ag) with the antibody (Ab1) occurs; b) removing the remaining sample liquid from the substrate (1) and container; c) adding thereto a peroxidase solution which comprises a known excess of a peroxidase (POD)-antibody (Ab2) labelled complex and effecting a second incubation (ill); ; d) removing the solution from the container but leaving the substrate with bound Ab1/Ag/Ab2-POD immunological pair, introducing into said containers a fluoride selectively sensitive electrode, adding thereto hydrogen peroxide and a fluoro compound solution and allowing the consecutive reaction to occur; e) reading the voltage from the electrode, this being the measuring signal of the marker relating to the amount of antigen (Ag) to be measured in the sample. 5. A method according to claims 3 or 4, characterized in that the fluorocompound is | fluorophenol.

Inventors: BROCHOT, JEAN, SIDDIQI, IQBAL
Applicants: BATTELLE MEMORIAL INSTITUTE
Application year: 1983
Main classification: G01N33
IPC codes: G01N33