AU610406B2 - Methods of chemiluminescence assay - Google Patents

Description

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AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

t610 0 6 Int. Class Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: .3 Applicant(s): Fujirebio Kabushiki Kaisha 6-7, Shimoochiai 4-chome, Shinjuku-ku, Tokyo, JAPAN Address for Service is: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Complete Specification for the invention entitled: METHODS OF CHEMILUMINENESCENCE ASSAY Our Ref 136436 POF Code: 1594/104462 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): -1- 6006

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1 i -14- Table 3 integral counts sec. from the maximum count) SDH I Relative Count ~1 i -1A- METHODS OF CHEMILUMINESCENCE ASSAY The present invention relates to two methods of I chemiluminescence assay. More particularly, the present invention relates to methods of chemiluminescence assay which comprise performing an enzymatic reaction at a pH of 4 10.5 using an acid or alkali phosphatase as an enzyme and a dioxetane derivative of the formula: 0--0

OR

Ar OP0 3 2-

(I)

rt.t t.'o (where R is a lower alkyl group, and Ar is an aromatic group) as a substrate, and carrying out a luminescent reaction under strong alkaline conditions and which produce a quantity of luminescence.

Luminescence assay is based on either a chemio0 luminescent or bioluminescent reaction and is utilized as .o a highly sensitive method of ultramicroanalysis. Known methods of chemiluminescence assay include: H 2 0 2 S...measurement with luminol/potassium ferricyanide in the S presence of an alkali [Bostick et al., Anal. Chem., 47, 447 452 (1975)]; glucose measurement with luminol-glucose oxidase [Bostick et al., Anal. Chem., 47, 447 452 (1975)]; and hemoglobin measurement with luminol/H 2 0 2 in the t presence of an alkali [Ewetz, L. et al., Anal. Biochem., 71, 564 570 (1976)]. Known methods of bioluminescence Sassay include: ATP measurement with Photinus pyralis -icount of the system amounts to that which is 50 times as high as the system m1, 4--4 luciferin-luciferase [Addanki et al., Anal. Biochem., 14, 261 264 (1966)]; measurement of intracellular free calcium ions with aequorin [Blinks et al., Pharmacol. Rev., 28, 1 93 (1976)]; and NADH measurement with bacterial luciferase [Hasting, J.W. et al., Annu. Rev. Microbiol., 31, 549 (1977)].

The conventional methods of chemiluminescence assay described above have several problems. Method is incapable of precise measurements since the sample is decomposed with an oxidizing agent used to produce luminescence O or because the oxidizing agent itself is decomposed. Method involves difficulty in measurements with an aqueous i system since the reagents used to produce luminescence are t low in water solubility. Method involves an intermit- 15 tent luminescent reaction, so it requires considerable skill in achieving timed measurements. Conventional methods of 0* bioluminescence assay and also have problems such as the need to use very expensive enzymes compared to t, those used in chemiluminescence assay and the inactivation of enzymes that occurs when these methods are applied to immunoassay.

In an effort to develop a convenient method of chemiluminescence assay that is capable of high sensitivity and high precision measurement, the present inventors made intensive studies and accomplished the present invention which includes a method of chemiluminescence assay comprising performing an enzymatic reaction at a pH of 4 10.5 using an acid or alkaline phosphatase as an enzyme and a 4, -16inches) coated with anti-TSH mouse IgG was added and left to stand at room temperature for 2 hours. After washing the bead with distilled water three times, 200 pi of a substrate i u I 0i

I

1401 0l 0 dioxetane derivative of the general formula as a substrate, and thereafter carrying out a luminescent reaction under strong alkaline conditions; and a method of chemiluminescence assay which comprises performing an enzymatic reaction at a pH of 4 10.5 using an acid or alkali phosphatase as an enzyme and a dioxetane derivative of the formula as a substrate, and subjecting only the solid phase of the enzymatic reaction system to a luminescent reaction under alkaline conditions.

Fig. 1 is a graph showing the dependency of luminescence on the alkaline condition; Fig. 2 is a graph showing the profile of luminescence that is obtained when a luminescent reaction is performed under alkaline conditions after an enzymatic reaction is quenched with an enzyme inhibitor; Fig. 3 is a graph showing the profile of luminescence that is obtained when a luminescent reaction is quenched by addition of an acid and then resumed under alkaline conditions.

Fig. 4 is a graph showing the profile of luminescence and time that is obtained when 3-(2'-spiroadamantan)-4methoxy-4-(3"-phosphoryloxy)-phenyl-l,2-dioxetane disodium salt (hereinafter abbreviated as AMPPD) is totally decomposed with alkaline phosphatase and the pH is shifted; Fig. 5 is a graph showing the results obtained from chemical luminescence EIA of TSH, in this figure, the signal indicates the results obtained when the pH is not changed and the signal indicates the results obtained when the pH is changed from 9.5 to 13.5; t 1 'Ii

I.

x i 0000 0 0r 0 1 0 0004 *0 t 00 0 0 0C'i rr.

-17using a luminometer (Berthold, Inc.) for 10 seconds as an integral count.

I-

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-4- Fig. 6 is a graph showing the pH dependency of luminescence; Fig. 7 is a graph showing the effect of the frequency of washing on luminescence; and Fig. 8 is a photograph showing the spots obtained by exposing an X-ray film to the blots of HBVs (human hepatitis B virus surface antigen) DNA (100 pg, 10 pg, 1 pg, 0.1 pg) hybridized with alkaline phosphatase labelled oligonucleotide DNA and reacted with AMPPD and dipped into 1 N NaOH solution. (The control was dipped into 0.1 M EDTA solution.) In this figure, A is a film bearing spots due to the above sil* oo"* alkaline treatment (1 N NaOH) and B is the control. It can be clearly seen that the spots treated with the alkaline solution produce remarkably sharp and strong luminescence 15 in comparison with the control.

In the methods of chemiluminescence assay, an acid or a*t alkaline phosphatase is used as an enzyme. These phospha- °0 tases can be isolated and purified from animals or plants but commercial products may of course be used.

'$44 The substrate used in the method of the present invention is a dioxetane derivative represented by the general formula set forth hereinabove. Such dioxetane derivatives can be prepared in accordance with known methods Ssuch as those described in European Patent Publication 254,051, PCT Publication WO 8,800,695, and Tetrahedron Lett., 28, 1155 1158 (1987). In the general formula R may be illustrated by a lower alkyl group such as methyl, ethyl, propyl or butyl, and Ar may be illustrated by an aromatic group such as phenylene, naphthyl or anthranyl.

i -18control, a 100-second integral was taken for the sample to which only distilled water was added without 4 N NaOH.

The amplification rate for each type of polymer bead was The cation in the dioxetane derivative represented by the general formula may be illustrated by an alkali metal such as sodium or potassium, ammonium, or a quaternary ammonium represented by N(R2) 4 (where R 2 is an alkyl group such as methyl or ethyl, or an aralkyl group such as benzyl), In the present invention, it is essential that an enzymatic reaction be performed at a pH of 4 10.5. If an acid phosphatase is used as an enzyme, the enzymatic reaction is preferably performed at a pH of 4 7. If an alkaline phosphatase is used as an enzyme, the enzymatic reaction is preferably performed at a pH of 7 10.5. It is, therefore, necessary that the enzymatic reaction be performed in an optimal pH range for the phosphatase used.

o S" o In the present invention, a luminescent reaction is «99 S° 15 performed after the enzymatic reaction under strong alkaline conditions to induce chemiluminescence. The completion of the enzymatic reaction is related to the degree of reaction of the substrate and may be properly determined by taking into account such factors as the interrelationship between the amount in which an enzyme is present and the amount of substrate used.

The enzymatic reaction may be brought to an end by adding a stopper reagent. Useful stopper agents include enzyme inhibitors such as chelatans EDTA and EGTA), organic phosphoric acid esters phenylphosphates and naphthlphosphates) and inorganic acids orthophosphoric acid). In consideration of the Ki of enzyme inhibitors, stopper reagents are preferably used in amounts at least ten

I

-19- The membranes bearing immobilized HBV DNA were incubated in a prehybridization buffer (5 x ssc, 5 x Denhart's solution, 0.1% SDS) for 15 m.innute a- "n -6times the Ki value, for example, EDTA is used in an amount of at least 1 mM, and phenylphosphates are used in amounts of at least 30 mM.

When an alkaline phosphatase is used as an enzyme, the enzymatic reaction can be stopped by rendering the reaction conditions to be acidic. In this case, the enzymatic reaction may be resumed by readjusting the pH conditions to the above-stated optimal range for the alkaline phosphatase.

1G After the enzymatic reaction, a luminescent reaction n is performed under strong alkaline conditions and the chemi- .luminescence produced is measured. The term "strong alkaline conditions" as used herein means pHs of 11 and above.

Such strong alkaline conditions can be attained by adding S 15 compounds having hydroxide ions to the reaction system; exemplary compounds having hydroxide ions include hydroxides of alkali metals and alkaline earth metals such as NaOH, SKOH and Mg(0H 2 ammonium hydroxide and ethanolamine.

A luminescent reaction is initiated as soon as the reaction conditions are adjusted to strong alkaline ones.

The amount of luminescence produced can be readily measured Swith a commercial luminometer.

The luminescent reaction is quenched by removing the preset reaction conditions and may be resumed by reestablishing them. In other words, the luminescent reaction can be "turned on and off" by controlling the reaction conditions.

The luminescent reaction may be performed using at least one of a mammalian se';.m albumin, a polyalkyl

I

o o ca o V a o 9 o o oo ao o o 0 0 0

ILI

quaternary amine, a fluorescein, dimethyl sulfoxide, etc. as an enhancer. Such enhancers are typically used in amounts of 0.0001 10 wt% of the luminescent reaction system.

After the enzymatic reaction, a luminescent reaction is performed with only the solid phase being adjusted to alkaline conditions, and the chemiluminescence produced is measured. The expression "only the solid phase" should be taken to refer to the solid that is left after removing any other part of the enzymatic reaction system. The term "alkaline conditions" as used herein means pHs of 8 and above, preferably 20 and above.

The term "solid phase" as used herein means a solid.

material that is incorporated in the assay system and may be exemplified by an antibody bound solid material that is used in an immunoassay to be described hereinafter. Any solid material may be used as a solid phase and its shape and other factors are not limited in any way. Materials that are preferably incorporated to provide a solid phase are polystyrene, polyfluoroethylene, nylon and polyacetal.

The method of the present invention can be employed to perform an enzyme immunoassay. Antigens that can be assayed include drugs, hormones and trace components related with various disease in sera and urine. Suitable antibodies can be prepared by known methods. For instance, antibodies may be formed in warm-blooded animals such as rabbit, goat, horse, guinea pigs and chicks by injecting ca. 0.3 2 mg of a ligand per kg of body weight one to several times under the dorsal skin, in foot pads, into the femoral muscle, etc.

WOA

XKXXMSX The claims defining the invention are as follows: 1. A method of chemiluminescence assay comprising performing an enzvmati rpnn~-inn n T.T -A n together with an adjuvant. The antibodies produced may be used after decomposition into F(ab') 2 Fab', Fab, etc. with a proteolytic enzyme such as pepsin or papain.

Antibodies may also be obtained as monoclonal antibodies. In this case, a ligand is injected intraperitoneally or by some other route into a mouse together with an adjuvant and the spleen cells recovered are fused with mouse myeloma cells using polyethylene glycol. Positive hybridoma cells are selected by cloning and the monoclonal cells thus selected are grown within the pritonaeal cavity of a mouse to obtain a desired monoclonal antibody.

Various methods of immunoassay are described in "Koso Meneki Sokuteiho (Methods of Enzyme Immonoassay)" published j "a by Igaku Shoin in 1987, and a typical example that can be 15 used comprises reacting an antigen with an immobilizing antibody, then reacting an enzyme-labelled antibody with the antigen, and measuring the reaction product.

The strong alkaline conditions are set at pH 11 14 ap-roximately 5 60 minutes after the enzymatic reaction is carried out by adding the phosphatase at its optimum pH 4 10.5, for example, pH about 9.5 for alkaline phosphatase, to the substrate to amplify the quantity of luminescence whereby the immunoassay can be efficiently conducted.

The method of the present invention can also be used in polynucleotide measurements. Various methods of polynucleotide measurements are described in "Molecular and Cellular Probe", vol. 1, p. 177 ff. (1987); in a typical method, the DNA of a specimen immobilized on a -21- 8. A method according to Claim 7 wherein the enhancer is a mammalian serum albumin, a polyalkyl quaternary amine, a fluorescein and/or dimethyl sulfoxide.

nitrocellulose filter is reacted with a hapten-labelled complementary probe DNA, and an anti-hapten antibody bound to alkaline phosphatase is allowed to act on the reaction product. The activity of alkaline phosphatase may be assayed by using a dioxetane derivative as a substrate.

The following Examples illustrate this invention but do not limit the scope thereof to them.

Example 1 TSH Measurement: A sample containing 15 pl of TSH 2 pU/ml) was mixed with 135 pl of an anti-TSH mouse IgG Fab' conjugated 0a0o with alkaline phosphatase (conjugate cone., 0.5 pg/ml; 0.1 M Tris-HCl; 2% BSA; 1 mM MgC12; 0.1 mM ZnC12; pH To the mixture, a polystyrene bead (dia. 1/8 inches) coated with anti-TSH mouse IgG was added and left to stand at room temperature for 2 hours. After washing the bead with "o distilled water three times 200 pl of a substrate solution (0.1 M Tris-HCl; 1 Mm MgC1 2 0.1 mM ZnCl2; pH 9.8) containo o ing 100 pg/ml of 3-(2'-spiroadamantan)-4-methoxy-4-(3"- 0006 phosphoryloxy)-phenyl-1,2-dioxetane disodium salt (hereinafter abbreviated as AMPPD) having the structural formula O noted below was added: O I OM e O 2- O PO 2 2Na

(AMPPD)

7 I r I ~nmaarcll*r~ B00 0 00 00 0 O 0 0 0 0 00 OU 0 o 00o 0O O 0u 3 1 0 and left to stand at room temperature for 20 minutes.

Immediately upon adding an alkaline solution to the reacted substrate solution, the luminescence produced was counted with a luminometer (Berthold, Inc.) for 10 seconds as an integral count.

The results are shown in Fig. 1.

Example 2 A sample containing 15 p1 of TSH 40 pU/ml) was mixed with 135 pl of an anti-TSH mouse IgG Fab' conjugated with alkaline phosphatase (conjugate cone., 0.5 pg/ml; 0.1 M Tris-HCl; 2% BSA; 1 mM MgC12; 0.1 mM ZnC12; pH To the mixture, a polystyrene bead (dia. 1/8 inches) coated with anti-TSH mouse IgG was added and left to stand at room temperature for 2 hours. After washing the bead with 15 distilled water three times, 200 pl of the substrate solution (0.1 M Tris-HC1; 1 mM MgCl 2 0.1 mM ZnCl 2 pH 9.8) containing 100 pg/ml of AMPPD was added and the reaction left to stand at room temperature.

After 15 minutes, a luminescence was measured using a luminometer (Berthold, Inc.); at some point of time of the measurement, 100 pl of a solution (0.1 M Tris-HCl; 0.2 M Na 2 HPO; 10 mM EDTA; pH 9.8) was added to stop the enzymatic reaction; 8 minutes after the addition of the stopper solution, 20 pl of 1 N NaOH was added to elevate the pH of the reaction solution up to 11.2. The luminescence produced was counted utilizing the luminometer for 10 seconds as an integral count.

The results are shown in Fig. 2.

-11- Example 3 Fifteen pl of a sample containing TSH 40 pU/ml) was mixed with 135 p1 of an anti-TSH mouse IgG Fab' conjugated with alkaline phosphatase (conjugate cone., 0.5 pg/ml; 0.1 M Tris-HCl; 2% BSA; 1 mM MgC12; 0.1 mM ZnC12; pH To the mixture, a p 'ystyrene bead (dia. 1/8 inches) coated with anti-TSH mouse IgG was added and left to stand at room temperature for 2 hours. After washing the bead with distilled water three times, 200 pl of the substrate solu- 0000 oo.* 10 tion (0.1 M Tris-HC1; 1 mM MgCl 2 0.1 mM ZnC12; pH 9.8) Q068 oo.0 containing 100 pg/ml of AMPPD was added and the reaction left to stand at room temperature.

8 00 After 40 minutes, a luminescence was measured utiliz- 00 00 0 0 0 ing a luminometer (Berthold, Inc.); at some point of time of the measurement, 200 pl of a 0.1 M phosphate acid-citric 0 0 Sacid solution (pH 3.0) was added to lower the pH of the reaction solution to 4.8; 3 minutes later, 50 pl of 1 N NaOH 0 08 .oo. was added to raise the pH of the reaction solution to 12.0.

The luminescence produced was counted employing the lumino- S 20 meter for 10 secjnds as an integral count.

o The results thus obtained are shown in Fig. 3.

Example 4 Measurement of TSH Using DMSO-Supplemented: Fifteen pl of a sample containing TSH 2 pU/ml) was mixed with 135 P1 of an anti-TSH mouse IgG Fab' conjugated with alkaline phosphatase (conjugate cone., 0.5 pg/ml; 0.1 M Tris-HCl; 2% BSA; 1 mM MgC12; 0.1 mM ZnCl 2 pH To the mixture, a. polystyrene bead (dia. 1/8 inches) coated -12with anti-TSH mouse IgG was added and left to stand at room temperature for 2 hours. After washing the bead with distilled water three times, 200 pl of the substrate solution (0.1 M Tris-IICl; 1 mM MgCl 2 0.1 mM ZnCl 2 pH 9.8) containing 100 pg/ml of AMPPD was added tD the bead and the mixture was reacted at room temperature.

Immediately after adding 200 pl of 2 N NaOH or a mixture of 2 N NaOH and DMSO (2 N NaOH to DMSO ratio, 2:1, 1:1, or 1:2) to the substrate solution, the luminescence produced was counted using a luminometer (Berthold, Inc.) for 10 seconds as an integral count.

The results are shown in Table 1 below.

Table 1 DMSO Counts/10 sec. Relative count I U t C t tl f t I t 1 t 0 (2 N NaOH 100%) 41,100 33 75,000 1.8 50 182,000 4.4 66 549,600 13.4 Example Measurement of AFP Using Quaternary Amine: Ten pl of a sample containing AFP 10, 50 ng/ml) was mixed with 150 pl of an anti-AFP mouse IgG Fab' conjugated with alkaline phosphatase (conjugate conc., 0.5 pg/ml; 0.1 M Tris-HCl; 2% BSA; 1 mM MgCI 2 0.1 mM ZnCl 2 pH To the mixture, a polystyrene bead (dia. 1/8 inches) coated with anti-AFP mouse IgG was added and left to stand at room temperature for 30 minutes. After washing the bead with TI ,I I' -13distilled water three times, 200 pl of the substrate solution (0.1 M Tris-HC1; 1 mM MgCI 2 0.1 mM ZnCl 2 pH 9.8) containing 100 pg/ml of AMPPD or the substrate solution further containing 0.05% polydiaryl dimethyl ammonium chloride (PDDAC) was added and subjected to reaction at room temperature for 20 minutes. The luminescence produced was counted using a luminometer (Berthold, Inc.) for 10 seconds as an integral count.

The results are shown in Table 2 below.

Table 2 Pooa 0000 0 0 0 .0 0 P)DAC Counts/10 sec. Relative count 0 12,390 1 0.05 96,050 7.8 j h Example 6 0 00 1 A solution of alkaline phosphatase obtained from bovine small intestine (0.2 mg/ml; 10 mM Tris-HCl; 1 mM 0 MgCl 2 0.1 mM ZnCl2; pH 8.0) was added to 100 pl of a substrate solution (10 mM Tris-HCl; 1 mM MgC1 2 0.1 mM 0 15 ZnCl 2 pH 8.0) containing 5 x 10- 2 pg/ml of AMPPD.

o 0 Immediately upon adding 1 M diethanol amine solution to the reaction mixture so as to make the pH of the reaction mixture to 8.6; 9.5; 10.3; 11.7; 12.6; 30 seconds after adding the amine solution, the luminescence produced was counted using a luminometer (Berthold, Inc.) for 10 seconds as an integral count.

The results are shown in Table 3 and Fig. 4.

ii 1 ,t i '~FkpYI l-~-~laarasraao*asnmra~san~ rr- ii i i-I -14- Table 3 integral counts sec. from the maximum count) pH Relative Count 12.6 21092 714% 11.7 19166 650% 10.3 16344 550% 9558 420% 8.6 2945 100% f t Example 7 Chemical Luminescence EIA of TSH Fifteen pl of a sample containing TSH (0 1 pU/ml) was mixed with 135 pl of an anti-TSH mouse IgG Fab' conju- S 5 gated with alkaline phosphatase (conjugate conc., 0.5 pg/ml; 0.1 M Tris-HCl; 2% BSA; 1 mM MgC12; 0.1 mM ZnCl 2 pH t I To the mixture, a polystyrene bead (dia. 1/8 inches) coated with anti-TSH mouse IgG was added and left to stand at room ,I o temperature for 2 hours. After washing the bead with distilled water three times, 200 p1 of the substrate solu- S tion (0.1 M Tris-HCl; 1 mM MgCl 2 0.1 mM ZnCl 2 pH 9.8) containing 100 pg/ml of AMPPD was added and left to stand for reaction for 80 minutes at room temperature.

Immediately after adding 4 N NaOH solution (100 pl) (pH 13.5) or 100 pl of distilled water to the reaction mixture, the luminescence produced was counted using the luminometer for 10 seconds as an integral count.

!i The results are shown in Fig. 5. It indicates that in I the system supplemented with NaOH (pH 13.5) counts at I 20 1 pU/ml TSH were over the measure. Even at 0.5 pU/ml TSH, i I D 1 4 1

*L

count of the system amounts to that which is 50 times as high as the system This indicates remarkable increase in production of luminescence due to addition of NaOH solution.

Example 8 TSH Measurement: Fifteen pl of a sample containing TSH (2 pU/ml) was mixed with 135 pl of an anti-TSH mouse IgG Fab' conjugated with alkaline phosphatase (conjugate cone., 0.5 pg/ml; 0.1 M Tris-HCl; 2% BSA; 1 mM MgCl 2 0.1 mM ZnC12; pH To the mixture, a polystyrene bead (dia. 1/8 inches) coated with I anti-TSH mouse IgG was added and left to stand at room temperature for 2 hours. After washing the bead with distilled water three times 200 pl of a substrate solution 15 (0.1 M Tris-HCl; 1 mM MgC12; 0.1 mM ZnCl2; pH 9.8) containtC ing 100 pg/ml of AMPPD and left to stand at room temperature for 20 minutes. Immediately after adding an alkaline soluttion to the reacted substrate solution, the luminescence produced was counted using a luminometer (Berthold, Inc.) I 20 for 10 seconds as an integral count.

S<tr The results are shown in Fig. 6.

Example 9 Effect of the Frequency of Washing on TSH Measurement: Twenty pl of a sample containing TSH 0.5, 2, 20 pU/ml) was mixed with 300 pl of an anti-TSH mouse IgG Fab' conjugated with alkaline phosphatase (conjugate cone., pg/ml; 0.1 M Tris-HCl; 2% BSA; 1 mM MgC1 2 0.1 mM ZnCl2; pH To the mixture, a polystyrene bead (dia. 1/4 i -16inches) coated with anti-TSH mouse IgG was added and left to stand at room temperature for 2 hours. After washing the bead with distilled water three times, 200 pl of a substrate solution (0.1 M Tris-HCI; 1 mM MgCl 2 0.1 mM ZnC1 2 pH 9.8) containing 100 pg/ml of AMPPD was added and the reaction left to stand at room temperature. After 20 minutes, the bead was washed 1 10 times with 2 ml of distilled water.

Immediately after addition of 300 pl of 4 N Na0H (pH 13.5), the luminescence produced was counted using a luminometer 10 (Berthold, Inc.) for 10 seconds as an integral count.

t9 to: The results are shown in Fig. 7.

Example Measurement of TSH: 0a ao Twenty pl of a sample containing TSH 1, 10 pU/ml) was mixed with 300 pl of an anti-TSH mouse IgG Fab' conju- 900 gated with alkaline phosphatase (conjugate cone., 0.5 pg/ml; 0.1 M Tris-HC1; 2% BSA; 1 mM MgCl 2 0.1 mM ZnCl 2 pH 4 00 To the mixture, a polystyrene bead (dia. 1/4 inches) coated with anti-TSH mouse IgG was added and left to stand at 9 0 20 room temperature for 1 hour. After washing the bead with S distilled water three times, 300 pl of a substrate solution (0.1 M Tris-HCl; 1 mM MgC12; 0.1 mM ZnC12; pH 9.8) containing 100 pg/ml of AMPPD was added and the reaction left to stand at room temperature. After 2 minutes, the bead was washed with distilled water three times and dried. Immediately after adding 300 pl of a mixture of 2 N NaOH and DMSO (2 N NaOH to DMSO ratio, 2:1, 1:1 or 1:2) to the reacted substrate solution, the luminescence produced was counted COUNTS Fio e -17using a luminometer (Berthold, Inc.) for 10 seconds as an integral count.

The results are shown in Table 4 below.

Table 4 DMSO Counts/10 sec.

0 14,000 33 22,000 59,000 66 236,000 4 t It fl I 4' 4' 4 I I I I t 4 a 4 4 Example 11 5 AFP Measurement by ELA Using Various Polymer Beads: Twenty pl of each of samples containing AFP (1 ng/ml) was mixed with 300 pl of an anti-AFP mouse IgG Fab' conjugated with alkaline phosphatase (conjugate conc., 0.5 pg/ml; 0.1 M Tris-HCl; 2% BSA; 1 mM MgC12; 0.1 mM ZnCl; pH To each of the mixtures, a polymer bead (for the name of polymer and the bead diameter, see Table 2) coated with anti-AFP mouse IgG was added and left to stand at room temperature for 2 hours. After washing the bead with distilled water three times, 200 pl of a substrate solution (0.1 M Tris-HCl; 1 mM MgCl 2 0.1 mM ZnCl 2 pH 9.8) containing 100 pg/ml of AMPPD was added and reacted at room temperature for 20 minutes. The bead was again washed with distilled water three times. Immediately after adding 300 pl of 4 N NaOH and 100 pl of distilled water, the luminescence produced was counted using a luminometer (Berthold, Inc.) for 100 seconds as an integral count. As a

I

-18- 00 00 0o a 6 8 8 88 8 0 00 8 0 0 o a 0888 8 o S0 0 0 control, a 100-second integral was taken for the sample to which only distilled water was added without 4 N NaOH.

The amplification rate for each type of polymer bead was determined and the results are shown in Table Table Counts Counts Amplifica- Bead tion rate (A B) Polystyrene 1/4 inches Meiwa #80 950 12,830 13.5 #280 1,090 26,710 24.5 #0 890 5,990 6.7.

Sekisui #280 1,150 67,650 58.8 1/8 inches Meiwa #80 240 7,260 30.3 Polyfluoroethylene 1/4 inches #80 910 5,020 #0 380 860 2.3 Polyacrylonitrile 1/4 inches #0 790 1,130 1.4 Duracon polycetal 1/4 inches #0 880 2,290 2.6 Polymethylpentene 1/4 inches #0 880 2,470 2.8 5 Example 12 Detection of Human Hepatitis B Virus Surface Antigen

(HBV

s

DNA:

HBV DNA (100 pg, 10 pg, 1 pg, 0.1 pg) was denatured by the addition of an equal volume of 0.6 N NaOH. These samples were blotted onto nylon membranes (Hybond-N: Amersham) with a gentle vacuum. After washing the membranes with 2 M ammonium acetate and 5 x ssc, the DNA was immobilized on the membranes by UV irradiation.

'S t i -19- The membranes bearing immobilized HBV DNA were s incubated in a prehybridization buffer (5 x ssc, 5 x Denhart's solution, 0.1% SDS) for 15 minutes at 50 0

C.

Ten pl of a probe DNA (alkaline phosphatase labelledoligonucleotide DNA: Dupont) was added to 2 ml of the prehybridization buffer and hybridization was performed at 50 0

C

for 30 minutes. After the hybridization, the membranes were washed twice with the solution 1 (1 x ssc, 1% SDS) at room temperature for 5 minutes each time. Then the membranes were washed twice with solution 2 (1 x ssc, 1% Triton X-100) at 50*C for 5 minutes each time. Finally the membranes were washed under agitation twice for 5 minutes each time with 1 x ssc at room temperature.

The alkaline phosphatase was assayed in a solution containing AMPPD (100 pg/ml) and BDMQ for 5 minutes at room temperature. The membranes were dipped into an alkaline solution (1 N NaOH). Immediately thereafter, an SsX-ray film was exposed to the membranes for 3 minures. The control membrane was dipped into a 0.1 M EDTA solution s 20 (pH 5.2).

The results are shown in Fig. 8.

The present invention provides methods of chemiluminescence assay that combines an enzymatic reaction with a luminescent reaction under strong alkaline conditions.

These methods yield such a large count of luminescence that it is capable of high sensitivity and high precision measurements. The utility of these methods is further enhanced by its ability to control the timing of luminescence.

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Claims (13)

1. A method of chemiluminescence assay comprising performing an enzymatic reaction at a pH of 4 10.5 using an acid or alkaline phosphatase as an enzyme and a dioxetane derivative of the general formula: 0 0 OR Ar OP0 3

2- (I) (where R is a lower alkyl group, and Ar is an aromatic group) as a substrate, and thereafter carrying out a luminescent reaction under a strong alkaline condition. 2. A method according to Claim 1 which is used in an enzyme immunoassay.

3. A method according to Claim 1 wherein the luminescent reaction is carried out at a pH of 11 14.

4. A method according to Claim 1 which is used in a measurement of polynucleotide.

5. A method according to any one of Claims 1 4 wherein the luminescent reaction is performed in a strong alkali after the enzymatic reaction is stopped by addition of a stopper reagent.

6. A method according to Claim 5 wherein the stopper reagent is an enzyme inhibitor or an acid.

7. A method according to any one of Claims 1 4 wherein an enhancer is also present when the reaction is performed I in a strong alkali. -21-

8. A method according to Claim 7 wherein the enhancer is a mammalian serum albumin, a polyalkyl quaternary amine, a fluorescein and/or dimethyl sulfoxide.

9. A method of chemiluminescence assay using a solid phase comprising performing an enzymatic reaction at a pH of 4 10.5 using an acid or alkaline phosphatase as an enzyme and a dioxetane derivative of the general formula: 0 -0 OR Ar OP0 3 2- group) as a substrate, and thereafter subjecting only the solid phase of the enzymatic reaction system to a lumines- cent reaction under alkaline conditions.

10. A method according to Claim 9 which is used in an o 4 eso enzyme immunoassay. 15 measurement of polynucleotide. r 12. A method according to any one of Claims 9 11 where- in the luminescent reaction is performed in an alkali after the enzymatic reaction is stopped by addition of a stopper reagent.

13. A method according to Claim 12 wherein the stopper reagent is an enzyme inhibitor or an acid.

14. A method according to any one of Claims 9 11 wherein an enhancer is also present when the reaction is performed in an alkali. e g o -22- A method according to Claim 14 wherein the enhancer is a mammalian serum albumin, a polyalkyl quaternary amine, a fluorescein and/or dimethyl sulfoxide.

16. A method according to Claim 9 wherein the solid phase is polystyrene, polyfluoroethylene, nylon or polyacetal. DATED: 13 June, 1989 PHILLIPS ORMONDE FITZPATRICK Attorneys for: FUJIREBIO KABUSHIKI KAISHA i t I f f