DK175273B1 - Determining antigen in liq. - using solid support coated with inert protein and tracer including label - Google Patents

Abstract

A method of determining an antigen in a liquid is claimed comprising; (a) combining a liquid suspected of containing the antigen with a solid support coated with an inert protein and a tracer including a label whereby the antigen attaches to the coated support and binds to the tracer to give a bound fraction including the label on the support, (b) sepg. the support from the liquid and (c) detecting the label on the support and inferring from the detecting that the antigen is present in the liquid. The inert protein is pref. casein or albumin.

Description

in DK 175273 B1

The present invention relates to a determination of a ligand and materials therein, and more particularly, to a determination which includes the capture of ligand directly onto a solid support previously coated with a nonspecific agent.

Many different measurement systems have been developed that are both rapid and sensitive to detect or determine the concentration of a ligand in a liquid. Conventional immune determinations are dependent on the binding of the ligand to a specific antibody and have been particularly useful because they provide high 10 degrees of specificity and sensitivity. These provisions generally use one of the above-mentioned reagents in labeled form, the labeled reagent being often referred to as the tracer.

Various means of labeling have been developed. Radioimraun Determination Methods (RIA) use radioisotopes as labels, 15 provide high degrees of sensitivity and reproducibility, and are suitable for automation for rapid processing of a large number of samples. Fluorine Immunoassay (FIA) uses fluorochromes as labels and provides direct detection of the label by estimating the dye with stimulating radiation of appropriate wavelength 20 and detecting its fluorescence.

Enzymes have also been used as labels by immunoassay. By enzyme immunoassay (EIA), the enzyme labeled reagents are inexpensive to produce and are very stable, thus providing a long shelf life and yet providing assays approaching the sensitivity of radioimmunoassay and providing objective results that can be determined either visually or fairly simply equipment such as a spectrophotometer.

At usual EIA, an enzyme is covalently conjugated with one component of a specific binding antigen-antibody pair, and the resulting enzyme conjugate is reacted with a substrate to produce a dye which is measured. Often, an unconjugated component, such as an antibody, becomes immobilized

I DK 175273 B1 I

i 2 I

In a solid support and serves to capture antigen in a specific binding reaction. Representative of such customary IJA is US Patent No. 3,654,090. IN

In U.S. Patent No. 4,588,680, determination of M protein in I I describes 5 different viruses, including Influenza A, B and C viruses. The I-I I ice cream includes blasting the virus to release the Μ-I I protein, which is directly absorbed onto a polymer carrier. IN

In PCT published application WO 86/02733, assay II describes Herpes simplex virus (HSV) in which antigenic glycoproteins gA / B, gc and gD are directly absorbed onto a polymer carrier II or captured by specific monoclonal antibodies on the carrier II. IN

In Sankolli et al. in Journal of Immunological Methods 104, I I 191 (1987) discloses an immunoassay for estradiol, in which a solid carrier is pretreated with a specific anti-IgG antibody. I This antibody specifically captures anti-estradiol antibody, which I 'in turn captures estradiol and provides a provision with I I improved reproducibility. IN

U.S. Patent No. 4,497,899 discloses a determination of the Chlamydia I antigen by which the antigen is directly absorbed onto a solid support such as a bead, glass, strip, disc or microtiter plate. The absorbed antigen is then determined by one of the usual EIA, RIA or FIA techniques. IN

In solid phase immunoassay, especially of high molecular weight molecules, there is often a tendency for materials in the sample to be determined to adhere to the solid carrier in a non-specific manner. A particular cause of loss of measurement sensitivity or irreversibility is nonspecific absorption of labeled antibody-I conjugate (tracer) directly at binding sites on the solid carrier which is not loaded with antibody. This problem has traditionally been solved by blocking non-uptake binding sites after application of anti-ligand with a protein which is not

3 DK 175273 B1 reacts with the labeled conjugate. U.S. Patent No. 4,407,943 discloses coating a porous membrane with a water-insoluble protein, such as a zein, attaching an antigen or antibody to the zein layer, and immobilizing an immunochemical neurite protein on the antigen to prevent nonspecific binding of foreign protein.

This traditional post-blocking has improved measurement sensitivity, but applying in the order of antiligand and blocking protein to a solid phase prior to ligand binding is cumbersome, time consuming and results in the loss of costly antiligand. Accordingly, there is a need for further improvement in solid-phase immunoassay technology, especially with regard to avoiding nonspecific binding.

A method for determining a ligand suspected of being present in a liquid includes contacting the liquid with a solid support to which is attached an inert protein and with a tracer for the ligand which includes a tag whereby the ligand is captured on the carrier and binds to the tracer. In the present specification, the term "inert protein" means a protein that is immunologically unresponsive to 20 for any other component of the assay, with the understanding that the inert protein may well be immunologically responsive to other materials which are not part of according to the invention. After binding, the carrier is separated from the liquid and the label on the carrier is detected to indicate the presence of ligand in the liquid. The label may be a radioactive atom, a fluorescent dye, or an enzyme conjugated with an ligand or encapsulated in a liposome conjugated with the antibody.

A preferred assay of the invention is a membrane flow EIA for a viral antigen, wherein the label is an enzyme conjugated with an antibody specific to the antigen and the enzyme label is detected by reaction with a colorless substrate which is converted to a colored product.

DK 175273 B1 I

An alternative embodiment of the assay according to the invention I

sen is a double enzyme assay for a viral antigen. One

hydrolase conjugated with a specific antibody bound to anti-I

gene on the carrier removes a blocking group from a blocked I

5 inhibitor to release an inhibitor. The inhibitor I

the hydrolysis of an ester substrate to a colored product with an I

esterase, whereby lack of color development indicates presence- I

view of the antigen in the fluid. IN

Another aspect of the invention is an equipment of materials useful for carrying out a determination by the method of the invention. H

Thus, the invention provides a determination for a viral anti-I

gene by which substantially all binding sites on a fixed I

carrier is loaded with an inert protein. Antigen Capture I

15 on the carrier containing inert protein is obtained without one species

specific capture antibody, thereby avoiding the time-consuming H

and labor-intensive steps to produce specific capture * I

antibody. The inert protein substantially inhibits all I

nonspecific binding of other protein, including trace element, such as electricity

20 lers would reduce the sensitivity of the provision. As it indiffe- I

Interest rate protein is readily available and cheap. the invention discloses a simplified provision with significant savings. IN

The invention is further illustrated in the drawing, in which:

Figures 1-3 illustrate the results of determinations carried out I

25 by the method of the invention for RSV, I, respectively

influenza Ά and HSV. IN

The invention is satisfied by embodiments in many embodiments

different forms, but in the present description will be I

Detailed description of preferred embodiments of the invention, in the understanding that the present disclosure is intended

are taken as examples of principles of the invention and are not intended to limit the invention to the embodiments described. Op- H

the extent of the fin ice is determined by the requirements and their equivalents. H

DK 175273 B1 5

One embodiment of the invention is a method of solid phase immunoassay of a ligand in a liquid sample in which substantially all binding sites on the support are preconditioned with a coating of an inert protein. According to the invention, it has been discovered that the ligand in the sample to be determined can be non-immunogenically captured on the coated baser in an amount proportional to its concentration in the sample without reducing the inert protein's ability to block all nonspecific absorption of other proteins. The immunoassay of the invention can be designed to detect the presence of ligand in the sample or to determine its concentration, and the term "assay" should mean either qualitative or quantitative ligand measurement.

The ligand may be from any source and may be an antigen, an antibody or a hapten. For example, the ligand may be an antigen found in a body fluid or it may be isolated by a body fluid and then introduced into another fluid. such as a cushion. In other cases, the ligand may be from a different source than a body fluid, such as e.g. a culture of micro-organisms or a cell extract thereof. Preferred ligands are antigens, most preferably viral antigens found in a body fluid such as Adenovirus, Parainfluenza 3 virus and, most preferably, Herpes simplex virus, HSV, respiratory syncytial virus, RSV, and Influenza. A, Flu.A .. The invention is described in the following generically by viral antigen.

As to a detailed description of the assay components, the solid support, as is well known, may be any support which does not significantly generate other components or steps in the assay. Examples of solid carriers which may be used are glass and polymeric materials such as polyethylene, polyvinylidene fluoride, polystyrene and the like. These carriers may be made in any suitable form, e.g. like sheets, glass, recesses

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In microtiter plates or, most preferably, mem-

In fires. Preferred membranes may be of nylon or nitro

In cellulose. A particularly preferred membrane is a modified I

In commercially available nylon membrane from Pali Corp., Glen Cove, I

I (o)

In 5 New York, under the trade names Immunodyne ^ and Biodyne ^ A, B

I and C. I

Any inert protein may be coated on the membrane which I

You do not generate the subsequent binding reaction between antigen I

I and tracer, and which do not significantly bind nonspecifically to others

In 10 proteins in the assay medium. Representative, non- I

In limiting examples of suitable inert proteins, I

In casein and albumin, though, others will be obvious to those skilled in the art. IN

In coating the inert protein on the membrane can be carried out

I by any suitable method, preferably by incubating membrane I

In a solution of the protein, thereby absorbing the protein

You reside physically in the polymeric matrix of the membrane surface. IN

In the membrane having a coating of inert protein, I

You are put on a trial suspected of containing it

In real antigen. Preferably, the coated membrane is incubated with I

In the 20 sample in a transient flow format for approx. 1 - 15, I

Preferably for approx. 5 minutes at a temperature of approx. 0 - 50 ° C, I

Preferably for approx. ambient temperature. By this progress I

In a way, antigen is absorbed into the sample on the coated membrane of I

Relative to its concentration in the sample. Furthermore, you have

It has been found that viral antigen is preferentially absorbed even when I

In the sample contains a large excess of foreign protein, which I

In the case, when the sample is a body fluid. IN

In the tracer, an antibody specific for antigen I comprises

In mesh and which have a mark conjugated thereto. The mark may be you

In any usual mark, as after binding of the tracer I

I to the antigen captured on the coated carrier gives rise to

I to a signal that can be detected. Accordingly, the mark may be an I

In radioactive atom or fluorescent dye conjugated I

7 DK 175273 B1 with the antibody. When the label is a radioactive element, the signal is radioactive counts. When the label is a fluorescent dye, the signal fluorescence emission is detected after application to the dye of appropriate wavelength stimulating light. A typical radiolabel is e.g. I, and a typical fluorescent dye is e.g. fluorescein isothiocyanate (FITC). Conjugation of radioactive and fluorescent labels with antibodies is conventional, and no specific details are required on the preparation and use of radioactive and fluorescent labels for immunoassayment in order for those skilled in the art to have a complete understanding of the invention.

The preferred tracer of the invention is an antibody having an enzyme conjugated thereto. Any enzyme that can be conjugated to the antibody can be used and for which a substrate exists which can be converted into a colored product. Suitable enzymes are e.g. cyclases, isomerases and peroxidases. A preferred peroxidase is horseradish peroxidase. Preferred enzymes are hydrolases such as peptidases, esterases, phosphatases and glycosidases. The most preferred trace elements 20 include alkaline phosphatase or carboxyesterase conjugated to the antibody. Conjugation of enzymes to antibodies is well known and fully understood by those skilled in the art.

Alternatively, the radioactive atom, fluorescent dye or enzyme may be encapsulated in a liposome. Encapsulation of 25 labels in liposomes and conjugation of liposomes to antibodies for use as trace elements in immune determinations are also quite traditional.

The choice of substrate, of course, depends on the enzyme component of the tracer, and many different substrates are well known for 30 classes of enzymes. Preferred substrates are substrates which form insoluble precipitates on membranes. When the enzyme is a peroxidase, a preferred substrate is diaminobenzidine. Preferred substrates for hydrolases are indolyl derivatives. When

DK 175273 B1 I

eg. the enzyme, alkaline phosphatase, is a preferred sub-I

strat 3-indolyl phosphate. When the enzyme is an esterase,

drawn substrates 3-indolyl acetate and butyrate. These substrates I

utes are well known. IN

5 According to the preferred method of determination according to the invention I

the membrane coated with inert protein becomes I

having viral antigen captured thereon, as described above, I

incubated with a solution of the trace element in a liquid to

induce immunological binding of the antigen and antibody component- I

10 ter in the tracer. The membrane having a bound antigen I

antibody fraction thereon can then be separated from the liquid phase

view of the determination medium by any suitable method, preferably I

by causing the fluid to pass through the membrane. Liquid I

flow through the diaphragm may be by gravity or may increase

15 is gradually enhanced by capillary action induced by absorbance

running material placed under the diaphragm. The membrane can then su- I

is spent in another liquid, e.g. water, saline or shock- I

pad in which the enzyme substrate is dissolved. Enzyme in the bound

fraction converts the substrate into a detectable product

20 by means of a signal associated with color. The ten

The signal detected may thus be the development or disappearance of I

a color or change from one color to another, or an I

change in the rate at which the substrate is converted to pro- I

product; eg. the color of a substrate can be observed to remain

25 unchanged for a certain time. It is preferred that the substrate be color I

dissolved until it is cleaved by the enzyme label to form a - I

colored product. The extent of color formation is proportional to I

antigen concentration which can be determined by measuring fluid I

samples having predetermined amounts of antigen therein, and co- I

30 different color intensities. Measurements can be made either in-

strumentally or, preferably, with the naked eye. IN

An alternative assay method according to the invention is a double

belt enzyme measurement performed on the coated carrier having antigen I

absorbed thereon. In this embodiment of the assay method, the label can be considered as a first enzyme which removes

derives a blocking group from a blocked inhibitor. Suitable vehicles

B1 enzymes are generally hydrolases such as phosphatases, peptidases, esterases, glycosidases and the like. Examples of, but not limited to, suitable first enzymes are trypsin, thrombin, mammalian liver esterase, acetylcholinesterase, β-galactosidase or, most preferably, alkaline phosphatase.

The blocked inhibitor may be any material which can be converted by the first enzyme to an inhibitor of the second enzyme. The preferred blocked inhibitor has two components, the inhibitor and the blocking group, and is unresponsive to the second enzyme until its blocking group is removed by the first enzyme and the inhibitor is released into the measuring medium. Thus, the choice of components of the blocked inhibitor depends on the first and second enzymes to be used.

The blocking group must be one which can be covalently conjugated to the inhibitor by a bond which can be substantially selectively cleaved by the first enzyme, and the inhibitor component must inhibit the activity of the second enzyme while having essentially no effect. on the first enzyme. Therefore, the nature of the second enzyme and its substrate will be debated before further description of the blocked inhibitor and inhibitor.

The second enzyme in the double enzyme assay is usually a hydrolase which converts the substrate into a product which can be detected by a signal associated with color. It is preferred that the second enzyme be substantially unreactive to the blocked inhibitor. Suitable hydrolases are e.g. phosphatases, peptidases such as trypsin, chymotrypsin and pepsin, or preferably esterases such as acetylcholinesterase (AChE) and butylcholinesterase. The most preferred second enzyme is a carboxyesterase, e.g. swine or rabbit liver esterase (RLE), wherein the preferred substrate is an indolyl ester.

DK 175273 B1 I

io I

As mentioned above, the first enzyme component cleaves into trace I

drug the blocking group from the blocked inhibitor I

and provides the inhibitor of the second enzyme. Suitable inhibitors I

and blocked enzyme inhibitors are illustrated by the one below

5 indicated general formula I wherein the character of group B as I

described later determines whether the compound is an inhibitor I

or a blocked inhibitor: I

ACF2 - C - (CH2) n - B (I) I

In the formula I, R 1 may be H, lower alkyl of 1-6 carbon atoms, I

branched or unbranched, nitro, alkoxy, halogen and lignin I

10, X may be 0, S or NR 2, where R 2 may be H or low H

is alkyl of 1-6 carbon atoms, n can be 1-6, A can be H

And B may be H, a phosphoric acid or a salt, an I

glycosyl group, an amino acid residue such as a lysine or arginine I

residue covalently conjugated to X through the carboxylic H amino acid

15 group, an acyl group having 2-4 carbon atoms such as an acetyl H

or butyryl group, or a peptide of formula IIH

- C - CH - NH - C - CH - NH (C - CH - NH) C - Rc, ττν I

n t n i n t 5 »S H

0 R3 o r2 0 R4 o

^ nh I

where R 1 is {CH 2) NH 5, (CH 2) * NS - C or benzyl,

R 4 may be H, lower alkyl or hydroxy lower alkyl of 1-4 I carbon atoms, branched or unbranched, (jH2C00H or I

20 (CH2> 2COOH, Rg may be lower alkyl or lower alkoxy with

1-4 carbon atoms, branched or unbranched, phenyl or I

benzyloxy, and g may be 0-10. IN

When B is H, the formula I represents enzyme inhibitors. When B I

is any group other than H, the formula I represents block I

The enzyme inhibitors. When B is a phosphoric acid or a salt I

thereof, it is intended that B has the formula III I

11 where 175 is bonded to X / and n can be as described above.

The inhibitor and the blocked inhibitor of formula I can be synthesized by any sequence of usual chemical reactions imaginable by those skilled in the art. Suitable and convenient methods are set forth in the examples given below. The following list of effective enzyme inhibitors is provided by way of example only.

Kim)

Name NMR data (esterase)

1. 1,1,1-Trifluoro-3- (4- (CDCl3): 3.91 (s, 2H), 5.21 (bs, 1K), 2.0 x 10-6, RLE

hydroxyphenyl) - 6.90 (d, 2H), 7.10 (d, 2H) propanane

2. l, l, l-tri l Luoro-3- (3- (CDCl3): 4.00 (s, 2H), 4.80 (bs, 1H),> 10-4, FLE

hydroxyphenyl) -2- 6.80 (m, 3H), 7.30 (ro, 1H) propanane

3. 1,1,1-Trifluoro-4- (4- (CDCl3): 2.95 (m, 4H), 4.90 (bs, 1H), 2.0 x 10-8, RLE

hydroxyphenyl) -2- 6.92 (dd, 4H), J = 4.60 Hz butanane

4. 1.1, 1-Trifluoro-4- (3- (CDCl3): 2.94 (t, 2H), 3.05 (t, 2H), 1.0 x 10-7, RIE)

hydroxyphenyl) -2- 5.70 (bs, 1H), 6.80 (m, 3H), butanone 7.15 (m, 1H)

5. 1,1,1-Trifluoro-5- (4- (CDCl3): 1.19 (t, 2H), 2.59 (t, 2H), 1.0 x 10-8, RIE

hydroxyphenyl) -2-2.68 (t, 2H), 5.23 (bs, JH), 6.95 (d, 2H), pentanone 7.10 (d, 2H)

6. 1.1, 1-Trifluoro-5- (3- (CDCl3): 1.95 (p, 2H), 2.70 (t, 2H), 1.7 x 10-7, RIE)

Hydroxyphenyl) -2- 2.95 (t, 2H), 5.40 lb (1H), pentanone 6.70 (m, 3H), 7.30 (m, 1H)

7. 1,1,1-Trifluoro-6- (4- (CDCl3): 1.63 (m, 4H), 2.59 (q, 2H), 2.0 x 10-8, RIE

hydroxyphenyl) -2- 2.70 (g, 2H), 6.55 (bs, 1H), 6.77 (d, 2H), hexanone (O2) (d, 2H)

8. 1,1,1,2,2-pentafluoro- (CDCl3): 2.94 (m, 2H), 3.04 (m, 2H), 8.0 x 10-7, RLE

5- (4-hydroxyphenyl) -3- 4.75 (bs, 1H), 6.90 (d, 2H), pentanone 7.10 (m, 2H) PLE + pig liver esterase (EC3.1.1.1) RLE = rabbit liver esterase (EC3.1.1.1)

I DK 175273 B1 I

I 12 I

In another aspect of the invention is a reagent kit or one

In package of materials for carrying out a determination of an I

In ligand according to the method of invention I

In the late. The equipment may include a solid support, preferably an I

In 5 membrane coated with an inert protein, an antiligand, an I

In enzyme conjugated with the antiligand and a substrate for the enzyme. IN

The Equipment may also include another enzyme and a blocked in-

In the hibitor of the second enzyme, standards for the ligand such as I

In e.g. one or more ligand samples of known concentration, I

I or it may include other reagents, enzyme substrates I

I or other labeled or unlabeled specific ligands, ahti- I

In ligands or complexes thereof useful for embodiment I

I of the provision. It may include solutions such as salt I

In water or cushions. The components of the equipment may be the same

Lightly in a box, preferably of plastic, containing a material I

Placed underneath the membrane, such as absorbent paper, to facilitate

In flow of measuring fluids through the membranes by capillary action. IN

In Experimental: I

In Routine Analytical Techniques · Lightning Silica Gel Chromatography I

I 20 was performed on ICN silica gel 32-53 mesh at 3-7 psi. Analy- I

In tical TLC was performed on 0.25 mm 5 x 20 cm aluminum clad I

In silica gel plates from EM Scientific. Prasparative TLC was performed

I led on 2.0 mm 20 x 20 cm glass coated silica gel sheets from I

In EM Scientific. Melting points were determined on a Thomas Hoover I

In 25 capillary melting point apparatus and are uncorrected. NMR spectra I

You were recorded on an IBM WP-200SY spectrophotometer and keel I

In misaligned shifts are given in ppm relative to trimethyl-I

In the silane. HPLC was performed on a Waters 510 two-pump system with I

In UV detection using one of two solvents on I

In 30 a Brownlee AX-300 7 x 250 mm column. System A used an I

In the first hold for 5 minutes at 30 mM NR 2 OAc, pH 5.5, post-I

Followed by a linear gradient to 2.0 M NH 2 OAc over a period of time

For 30 minutes, followed by a hold at 1.0 M NH 2 OAc I

13 DK 175273 B1 for 5 minutes. System B used an isocratic buffer system of 30 mM NH 2 OAc, pH 6.5, for 40 minutes. Flow rates were 1.0 ml per ml. minute. Gas chromatography was performed on an H.P. 5840A gas chromatograph equipped with an FID 5 and an automatic injector using a 30 M DB-1

Megabore column, purchased by J&W Scientific, Inc. GC conditions were as follows: 3 minute holding at 100 ° C, followed by a 10 ° C / minute gradient to 250 ° C, followed by a 3.0 minute holding at 250 ° C at a flow rate 10 of 16.0 ml per minute.

Inhibition constants were measured in 50 mM Tris, pH = 8.0. Enzyme and inhibitor were incubated at ambient temperature for 20 minutes. Substrate for the enzyme was then added and the rate of hydrolysis followed spectrophotometrically. The substrate for 15 PLE and RLE. was o-nitrophenyl butyrate and for AChE it was aoethyl thiocholine and Ellman's reagent.

The following examples are given to further describe the invention, but are not to be construed as limiting the invention.

EXAMPLE I.

Diammonium [4- (3-oxo-4,4,4-trifluorobutyl) phenyl) phosphate.

A. Preparation of Ethyl 2- (4-methoxybenzyl) -3-oxo-4,4,4-trifluorobutanoate.

A 1 liter four-necked round bottom flask, equipped with reflux, separating funnel, argon input tube and magnetic stirrer, was charged with 7.17 g (0.149 mol) of a 50% (w / v) oil dispersion. sodium hydride and 300 ml of dry ethyl ether. Absolute ethanol (9.0 ml) was slowly added to the stirred solution. After the evolution of hydrogen was stopped, a mixture of 25 g (0.136 mol) of ethyl 4,4,4-trifluoroacetoacetate and 21.3 g

I DK 175273 B1 I

I 14 I

In (0.136 mol) of 4-methoxybenzyl chloride added over one hour. IN

The resulting mixture was refluxed overnight

In chilled, extracted with water, 1 N hydrochloric acid, dried over anhydrous I

In magnesium sulfate and rotary evaporated under reduced pressure. The I

In 5 crude reaction mixture (33.5 g) was chromatographed on a 60 mm I

In x 300 mm silica gel column with ethyl acetate / hexane (25/75). A sieve

Fractions were combined to give 9.4 g (23%) of it (spectroscopy)

In whip complex) product like an oil. NMR (CDCl3): 1 * 26 (m, 3H), 1

I 3.77 (s, 3H), 4.12 (m, 2H), 7.08 (m, 2H). IN

Preparation of 1,1,1-trifluoro-4- (4-hydroxyphenyl) -butane-1

I 2-on. IN

I A 100 ml round bottom flask, equipped with a reflux condenser, I

In tical stirrer and argon inlet tube, was loaded with 2.05 g (6.7 l

Ethyl 2- (4-methoxybenzyl) -3-oxo-4,4,4-trifluorobutanoate I

In 15 (I), 20 ml of 31% (w / v) hydrogen bromide in acetic acid and 10 ml of water. IN

This mixture was heated overnight to 120 ° C, cooled, I

I concentrated under reduced pressure and separated between dichloromethane I

In and water. The organic layer was extracted in sequence with I

In aqueous bisulfite and saturated sodium bicarbonate, and then I

In 20 dried over anhydrous magnesium sulfate. The solvent was I

In removed, reduced pressure gains. The crude reaction mixture became I

I chromatographed on a 50 mm x 300 mm silica gel column with ethyl I

In acetate / hexane (50/50). Equal fractions were combined and dissolved

The solvent was removed under reduced pressure to give I

In 25 600 mg (41%) as a clear oil. NMR (CDCl3): 2.95 (m, 4H), 1

I 5.40 (bs, 1H), 6.93 (dd, 4H), J = 4.60.Hz. IN

In C. Preparation of diethyl- [4- (3-oxo-4,4,4-trifluorobutyl) - I

In phenyl phosphate. IN

I A 10 ml round bottom flask, equipped with argon-inlet and mag- I

Into 30 agitator agitator, charged with 400 mg (1.8 mmol) of 1,1,1

In 4- (4-hydroxyphenyl) butan-2-one, 400 mg (2.3 mmol) of diethyl chloro-I

In phosphate, 0.15 ml dry pyridine and 5 ml dichloromethane. Reaction I

The mixture was stirred overnight at ambient temperature, filtered to remove pyridinium hydrochloride, extracted with 0.2 N hydrochloric acid, extracted with water and dried over anhydrous magnesium sulfate. Removal of solvent under reduced pressure gave a crude yield of 600 mg of a brown oil.

200 mg (31%) of a clear oil was isolated by a preparative TLC plate developed with ethyl acetate / hexane (50/50). NMR (CDCl3): 1.50 (m, 6H), 3.0 (m, 4H), 4.20 (m, 4H), 7.15 (s, 4H).

D. Preparation of diammonium [4- (3-oxo-4,4,4-trifluorobutyl) -phenyl] phosphate.

A 25 ml single neck round bottom flask equipped with argon input tubes and magnetic stirrer was charged with 5.0 ml of dichloromethane, 140 mg (0.40 mmol) of diethyl [4- (3-oxo-4,4,4-trifluorobutyl) phenylI-phosphate. (III) and 2.0 ml of bromotrimethylsilane. After stirring this mixture for 3 hours at ambient temperature, 10 ml of methanol was added and the volatiles removed under reduced pressure. The residue was dissolved in water and adjusted to pH 7.0 with 1.0 N sodium hydroxide. The aqueous solution was extracted with diethyl ether and lyophilized to give 20 190 mg of a white solid. This material was dissolved in 10 ml of water and purified by anion exchange HPLC. Gradient conditions: Initial retention for 5 minutes at 20 mM ammonium acetate, pH 6.5, followed by a linear increase to 1.0 M ammonium acetate over a period of 20 minutes, followed by 25 retention at 1.0 M ammonium acetate . At a flow rate of 2.5 ml / minute, the product eluted at ca. 32 minutes. The column capacity was 20 mg. Product fractions from several HPLC experiments were pooled and lyophilized to give 50 mg (37%). Melting point 235 - 240 ° C. NMR (D 2 O): 1.90 (m, 2H), 2.56 (m, 2H), 4.65 (s, DOH), 6.88 (dd, 4H), J = 6.82 Hz.

I DK 175273 B1 I

I 16 I

In Example II. IN

In Measurement of Respiratory Syncytial Virus (RSV). IN

A membrane filter stack was assembled with the following composition:

Top layer: 3 microns Immunodyne immunoaffinity membrane I

I 5 (Pall, East Hills, New York, t BIA0030HC5). IN

Pre-coated by immersion in saline with

In phosphate buffer containing 0.3% casein I

For 30 minutes at ambient temperature. IN

In Next Layer: Nonwoven Rayon Sheets (Schleicher & Schuell, I

10

Keene, New Hampshire, 5-S). IN

In Bottom layer: Absorbent cellulose pads (2) (Filtration I

In Sciences, Mount Holly Springs, Pennsylvania, I

In T ^ ED 320-200). IN

The 15 membrane layers were contained in a plastic holder containing I

In a receiving recess formed above the top layer. Inside this I

A recess for limiting the flow was placed in the recess

In the gene, which had an opening that was narrower than the received one

You see the recess and lying close to the top membrane. IN

I An antigenic stock was prepared with HEp-2 cells I

In infected with respiratory syncytial virus (RSV) (long strain) I

I in a buffer containing: 250 mM Tris (hydroxymethyl) amino-I

In methane hydrochloride (Tris-HCl), 150 mM sodium chloride (NaCl), I

In 10mM ethylene diamine tetraacetate (EDTA), 4% (v / v) polyoxyethylene-I

In sorbitan monolaurate (Tween 20), 1% n-acetylcysteine, 0.2% sodium I

In azide (NaN +), pH 8.5. Control antigen was prepared on equil

In no way from uninfected HEp-2 cells. IN

A portion of 150 µl of this antigen (or control) was applied

You operated the appliance and were allowed to run through the flow limiter

In 30 send efforts and down on the top membrane layer. (Liquid is withdrawn

In the easy top membrane by the capillary action of the supporting ab- I

17 DK 175273 B1 sorbing layer). The flow limiting insert was then removed and to the apparatus was added 150 ° ol of a wash solution consisting of 50 mM Tris-HCl, 150 mM NaCl, 0.2% NaN 2, pH 7.2 (saline with Tris buffer (TBS)). ) and further containing 5 1 mg / ml rabbit IgG.

A solution containing 27 µg / ml anti-RSV antibody conjugated with alkaline phosphatase was prepared in a buffer containing 50 mM Tris-HCl, 100 mM NaCl, 200 mM sodium phosphate, 1% casein, 1 mM magnesium chloride, 0.1 mM zinc chloride and 1 mM 10 2-mercaptoethanol, pH 7.5. A 150 ml portion of this mixture was added to the apparatus and allowed to absorb into the membrane stack. After a short (2 minutes) incubation, the apparatus was washed with 300 µl TBS (without IgG).

A 150 ml solution containing 0.33 mg / ml nitro blue tetrazolium, 15% methanol and 0.2% NaN3 was added to the apparatus. This was followed by the addition of 150 µl of a solution containing 12 mM levamisole in 50 mM 2-amino-2-methyl-1-propanol acetate (AMP-HOAc), 0.2% NaN 2, 19 mM magnesium chloride at pH 9, 8th After 5 minutes incubation at ambient temperature, the 20 color-forming reaction was stopped by the addition of 150 μ en of a solution containing 200 mM potassium phosphate, 10 mM EDTA and 0.2% NaN 3, pH 7.2.

The color density of the resulting membrane was measured with a reflectance densitometer (Gretag, Seattle, Washington, model 183).

The results of an experiment with a variety of antigen dilutions are shown in Figure 1.

EXAMPLE III.

Influenza virus type A measurement

An influenza virus measurement was performed in a similar manner as in Example II with the following exceptions:

18 I

DK 175273 B1 I

1) The antigenic stock was made by Madin-Darby I

canine kidney (MDCK) cells infected with influenza A (strain I

WSN). IN

2) The top layer membrane was 3 microns of Biodyne C (Pall, East Hills, I

5 New York, ^ ÉBNPCH5) in place of Immunodyne. IN

3) The buffer for the antigenic stock was prepared with I

Tris acetate instead of Tris hydrochloride (contained no I

sodium chloride) and additionally contained 1 mM ethylene bis (oxy-I)

ethylene nitrilo) tetraacetic acid (EGTA). IN

4) The conjugate diluent contained 100 mM instead of I

50 mM Tris and 150 mM instead of 100 mM NaCl. IN

5) Levamisole concentration was 16 mM instead of 12 mM. H

The results of an experiment performed with a variety of antigen diluents

donors are shown in Figure 2. H

EXAMPLE IV. IN

Measurement of Herpes simplex virus (HSV). IN

A measurement of Herpes simplex virus (types I and II) was performed on I

similar to Example II with the following exception:

1) The antigenic stem material was made from vero cells infi- I

20 cated with HSV, type li. IN

The results of an experiment performed with a variety of antigen diluents

gears are shown in Figure 3.

EXAMPLE V.

Double enzyme cascade measurement of respiratory syncytial virus (RSV).

A double enzyme cascade measurement of RSV is performed in a similar manner to that of Example II with the following exceptions s 1) Rabbit liver carboxyesterase is coated on the surface of an Immuno-nodyne immunoaffinity membrane. The membrane is then blocked with casein in the manner previously described.

2) After applying the alkaline phosphatase and antibody conjugate and subsequent washing, a solution of 0.15 mM masked inhibitor [4- (3-oxo-4,4,4-trifluorobutyl) phenyl phosphate] is added to 50 mM diethanolamine, pH 9.0, to the apparatus.

This solution is allowed to incubate at ambient temperature for 20 minutes. A chromogenic solution containing 15 0.5 mM 3-indolyl butyrate (Research Organics, Cleveland, Ohio) in TBS is then added to the apparatus. The color development is stopped after an appropriate time by the addition of 1 mM 1,1,1-trifluoroacetophenone (Aldrich, Milwaukee, Wisconsin). The color density is measured with a reflectance densitometer as described in the preceding example.

The color density of the unknown sample is compared to an uninfected control. If the unknown is clearly brighter than the control (less than half the reflectance density of the control), it is judged to be a positive result, showing the presence of viral material.

Thus, the invention provides a determination for a viral antigen which does not contain a specific capture antibody for the antigen. Instead, a solid support is pre-coated with an inert protein and antigen is captured directly on the subject.

I DK 175273 B1 I

I 20 I

In laid bear. Although the inert protein is applied as I

In a pre-antigen capture coating, it also prevents I

In you essentially all the nonspecific binding of other proteins, so-4 · I

You as trace elements which would otherwise reduce the sensitivity of be- I

In the 5 ballot box. IN

Claims (2)

A method for determining an antigen in a liquid, characterized in that (a) associating a liquid suspected of containing a viral antigen with a solid carrier coated with an inert protein and a tracer; containing a tag whereby the viral antigen binds to the coated carrier and binds to the tracer to form a bound fraction containing the label on the carrier, 10 b) separating the carrier from the liquid and c) detecting the label on the carrier and ending from this detection; that the viral antigen is present in the fluid. 15 Method according to claim 1, characterized in that the label is selected by the group consisting of a radioactive atom detected by counts of radioactivity therefrom and a fluorescent dye detected by applying stimulating light from the dye and observing fluorescence emitted therefrom. 20