GB2215047A - Analytical method using biospecific reagents labelled with colored macromolecular structures - Google Patents

Description

1 1 1 -047 ANALITICAL METHOD USING BIOSPECIFIC REAGENTS LABELLED WITH

COLORED MACROMOLECULAR STRUCTURES The present invention relates to an analytical method of "protein binding" which uses specific reagents, labeled with colored macromolecular structures. Protein binding assays are a type of analytical techniques based on the molecular recognition between biological molecules, such as antibodies, antigens (immunoassays), or receptor and receptor-binding molecules (receptor binding assays).

These types of assay generally require the use of biospecific binding reagents in a labelled form. Various types of labels have been used so far, each one having advantages and disadvantages. (Ref. 1: T. Chard. An Introduction to radioimmunoassay and related techniques. T.S. Work, eds., Elsevier Biomedica Press, New York (1982)).

The radioisotopes were and are nowadays widely used to label the biospecific binding reagents because they thevIre not endow the assay of high sensibility and precision, and affected by physical or chemical factors during the analysis.

The main drawbacks of this type of label is related to health hazard of radiations, for the user. Moreover, trained personell and expensive instrumentation for radioactivity counting are needed.

on the contrary, the label of the biospecific binding reagents using enzymes, which was subsequently developed, is not health hazardous and particular C instruments and/or radioactivity measures are not required.

Moreover qualitative assays with visual reading of signal are made possible by colored enzyme-catalyzed reactions. This is a remarkable advantage for home or medical office diagnostic tests (e.g. pregnancy and ferti-lity tests, sexually transmitted diseases, diagnostic tests etc.) where simplicity and rapidity of signal detection is required.

The disadvantages of enzymatic labels include the susceptibility of enzymes to denaturation by physical or chemical factors, the inhibition by components of the analytical matrix, and the requirement of additional : enzymatic activity.

reactions for measurement of These disadvantages have been eliminated in part by new types of labels based on the use of fluorescent, chemiluminescent or bioluminescent compounds. However all these types of labels, though generally stable and safe, require complex equipment for measuring the signal and thus do not fulfill he need of simplicity and rapidity of signal detection required, e.g., by home and medical office diagnostic tests.

For these reasons, alternative labels allowing visual reading of results and devoid of problems encountered with enzyme labels have been investigated. Examples are the use of colored particles, such as latex (Ref. 1), red blood cells (Ref. 1), and stained bacteria (EP-B-0074520), linked to the analytical reagent (e.g. antibodies or antigens) in order to make visible the biological reaction with the analyte. However, the is 1 J 4 -Z 30 - 3 relative huge mass of this type of labelled analytical reagents may limitate the choice of the best analytical system for this direct reading qualitative tests specially when soluble reagents free to diffuse into porus materials (membranes, chromatographic matrixes, filters etc) are required.

-The present invention is directed to a new label for protein binding assays, characterized in that a hydrophylic colored macromolecular structure is linked to the biospecific binding reagent.

According to the present invention the term "biospecific binding reagent" represents a molecular structure able to bioselectively recognize the analyte. Examples of biospecific binding reagent include antibodies, antigens, haptens, hormons, receptors, drugs, enzymes, enzyme inhibitors, coenzymes, membrane and bacterial cell wall binding structures. The biospecific binding reagent can be linked as such to the colored macromolecular by using bifunctional cross-linking reagents (e.g. glutaraldehyde, carbodiimide, N-NI-phenylenedimaleimide, m-sodium periodate, N-NI-oxydimethyenedimaleimide, N-succinimidyl 3-(2pyridyldithio)propionate, D-benzoquinone, N-succinimidyliodoacetate, bis(maleimido)-methyl ether, 3-maleimidobenzoyl-n-hydroxysuccinimide ester, maleimidohexanoyl-N-hydroxy-succinimide ester) or through formation of non covalent links with other molecular structures. Example of non-covalent conjugation of biospecific binding reagents to colored macromolecular structures include complexes between biotinylated analytical reagents and avidin (or streptavidin) coupled - 4 to the colored macromolecular structure; sandwich complexes between biotinylated analytical reagent, avidin, and biotinylated colored macromolecular structures; complexes - between analytical reagent, specific monoclonal or polyclonal antibodies against the analytical reagent covalently linked to colored macromolecular structures or, in their turn, complexed with protein A or anti-Fc antibodies linked to colored macromolecular structures.

Biotinylated analytical binding reagents are, for instance, antibodies or antigens covalently linked to one or more biotin molecule! . Several types of "activated biotin". able to directly react with proteinaceous molecules, are commercially available (e.g. D-biotin-N-hydroxysuccinimide ester, D-biotinyl-f--aminocaproyl-Nhydroxysuccinimide ester, biotin-hydrazide, sulfosuccinimide biotine, sulfosuccinimidyl 6-(biotinamido)hexanoate, N- iodoacetyl-NIbiotinylhexilene-diamine).

The term "colored macromolecular structure" represents any water soluble colored polymer. Examples of water-soltible colored polymers are polysaccharides (e.g. blue-dextrans), commercially available colored proteins (natural or stained by synthesis, e.g. synthetically prestained phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, lysozyme), or newly synthetized colored proteins otained by conjugation with reactive- dyes.

Examples of reactive dyes are triazine dyes (Cibacron Blue F3GA, Reactive Red 120, Procion Blue MX-R, Texas Red).

1 iJ 1 Preferred embodiments of the invention are represented by those colored macromolecular structures where the reactive dye is covalently conjugated to a polymer of globular structure (e.g. a globular protein) in a 10-60 molar ratio. A further preferred embodiment of the invention is a copolymer of the colored macromolecular structure obtained by using crosslinking reagents (e.g. glutaraldehyde, carbodiiMides, N-NI-ophenylenedimaleimide, periodate, N-maleimidobenzoyl-N-hydroxysuccinimmide, mixed anhydrides, bifunctional imidates) or by non covalent bridges relying on biotin/avidin or antibodies/ protein interactions as described above.

EXAMPLE I

LABELLING OF AN ANTI-CHORIONIC GONADOTROPIN ANTIBODY WITH COLORED MACROMOLECULAR STRUCTURES, AND ITS USE IN A QUALITATIVE IMMUNOCOLORIMETRIC ASSAY FOR CHORIONIC GONADOTROPIN.

A) Conjugation of Cibacron Blue F3GA to bovine serum albumin Bovine serum albumin (BSA), 1 g, and Cibacron Blue F3GA (R) (CB) (Sigma Chemical Co., St Louis, MO 63178), 2 g, were dissolved in 100 ml of 0.1 M sodium bicarbonate and pH was brought to 10.5 by adding 5 M sodium hydroxyde.

The mixture was left to react for 24 h at room temperature and then gelfiltered through a Sephadex G-25 (R) column (Pharmacia Fine Chemicals, Uppsala, Sweden) (5 cm x 60 cm) preequilibrated with 0.1 M sodium bicarbonate. Fractions eluted with the "void volume" were pooled (this product was named CB-BSA) and kept at -200C 6 until use.

B) Characterization of CB-BSA The amount of CB in the CB-BSA conjugate was determined by spectrophotometric measures at 600 nm (A 100); BSA was determined indirectely by analysis of isoleucine (HPLC) after hydrolysis of conjugate in 6 N hydrochloric acid for 72 h at 110C. The molar ratios CB/BS in CB-BSA conjugates, turned out to be 54 moles of BSA CB/mole.

C) Copolymerization of CB-BSA CB-BSA, 10 ml, in 0.1 M sodium bicarbonate buffer pH 8.9 was mixed with 1 ml of 25% v/v glutaraldehyde solution and left to react at room temperature for 72 hours.Then 1 ml of 1 M lysine solution was added, is thorougly mixed and left to react at room temperature for 20 min. After reaction, the mixture was immediately gel-filtered to a Sephadex G- 25 (R) column (2.5 cm x 30 cm) preequilibrated with 0.1 M sodium bicarbonate. The blue colored peak, (50 ml) eluted with the "void volume" of the column, was recovered and kept at 4C for 24 h until the subsequent use. Throughout this work this product is indicated with the abbreviation (CB-BSA) n.

CB-CSA and (CB-BSA)n analysis by electrophoresis on revealed a remarkable reduction of (CB-BSA)n electrophoretic mobility in comparison with GB-BSA.

D) Conjugation of (CB-BSA)n with the anti human chorionic gonadotropine monoclonal antibody INN22 The (CB-BSA)n solution (50 ml) was directely mixed with the anti-human chorionic gonadotropin monoclonal a 8-25% acrylamide gel in SDS antibody INN22 (Boehringer Mannheim GmbH, Federal Republic of Germany) and incubated for 24 h at room temperature. Then 5 ml of 1 M TRIS-HCI, pH 8.0, was added and incuba ted for 24 h to block any remaining active groups.

Throughout this work this product is indicated with the abbreviation (CBBSA)n-INN22.

E) Purification of (CB-BSA)n-INN22.

The dye-labelled antibody (CB-BSA)n-INN22 was separated from unconjugated (CB-BSA)n by affinity chromatography on hCG- CH-Sepharose prepared by coupling 20,000 IU of gonadotropin to Activated CH-Sepharose (Pharmacia Fine Chemicals, Sweden) according- to the manufacturer's instructions.

An aliquote of (CB-BSA)n-INN22, 3 ml, was adsorbed onto a 0.8 cm x 3 cm hCG-CH-Sepharose column preequilibrated with 1.15 M sodium chloride, 0.05 M sodium phosphate buffer, pH 7.3, (PBS) containing 1 mg/ml BSA (PBS-BSA). The column was then washed with the equilibrating buffer (20 ml), and eluted with 3 M potassium thiocyanate. The recovered product (10 ml) was immediately dialyzed against PBS, mixed with 1 ml of 1% W/v sodium azide solution and stored at 40C until use.

F) Qualitative immunocolorimetric assay Of hCG Aliquotes of (CB-BSA)n-INN22 (0.5 ml) containing 0.2 Optical Density units (600 nm) were mixed with 0.5 ml of hCG standard solutions at various concentrations (from 0 to 12.5 IU/ml) in PBS-BSA and incubated for 30 min at room temperature; then 0.5 ml of each solution were loaded on a battery of hCG-CH-Sepharose columns (0.5 x 0.7 cm) preequilibrated with PBS-BSA. After 5 minutes of incubation at room temperature, the columns were washed with 3 ml of PBS-BSA and the color bound was visually evaluated. Control experiments were carried out in parallel using batteries of columns containing CH-Sepharose (R) (Pharmacia Fine Chemicals, Sweden) without immobilized hCG, and columns containing BSA-CH-Sepharose prepared as described above for hCG-CH- Sepharose by using BSA instead of hCG in equal molar amount. The results, summarized in table 1, clearly indicate a (CBBSA)n-INN22n binding, which is visually detectable only in hCG-CHsepharose columns. This binding is completely inhibited when (CB-BSA)nINN22 is preincubated with hCG at concentrations which are higher than 0.5 IU/ml.

Finally, the colored antibody (CB-BSA)n-INN22 can be successfully and with good sensitivity used in the aualitative detection of hCG.

Table 1. Immunocolorimetric assay of hCG (see text).

I --------------------------------------------------------- Color bound to columns 1 Sample hCG M/m1) 0 0.1 0.5 2.5 12.5 1) As visually evaluated A 2 B c 2) hCG-CH-Sepharose 3) BSA-CH-Sepharose 4) CH-Sepharose EXAMPLE 2

Labelling of anti hCG monoclonal antibody M34111 with colored macromolecules and its use in a qualitative immunocolorimetric membrane assay.

A) Conjugation of CB-BSA with Mab M34111.

A 2 ml portion of CB-BSA, prepared as described in example 1, was incubated in 0.1 M sodium carbonate buffer, pH 8.7, containing 2.5% glutaraldehyde, for 16 h at room temperature. The product was gel-filtered through a Sephadex F-25 PD1010 (Pharmacia) preequilibrated with 0.1 M sodium carbonate buffer, pH 9,5. The peak eluted with the void volume of the column was recovered and mixed with a solution of MabM34111 (Boehringer Mannheim GmbH) (1 mg/ml in 0.1 M sodium carbonate buffer, pH 9.5, 0.27 ml). After incubation for 3 h at room temperature the solution was mixed with 1 ml of 1M lysine and further incubated overnight at 40C. The product was gel filtered on a Sephacry S-300 HR column (2.6 cm x 60 cm), preequilibrated with PBS (flow rate, 50 ml/h). The peak (Do at 280 nm) corresponding to the void volume of the column was recovered and kept at 40C until use. This product is hereinafter called CB-BSA-M34111.

B) Membrane immunocolorimetric assay of hCG.

Nitrocellulose membranes (2.25 cm 2 each) (Biorad, 0.45 / um) were washed for 10 min with distilled water and left to dry for 15 min. Then 2 / um portions of a solution of Mab INN22 (Boehringer Mannheim GmbH) (1 mg/ml) inPBS - c were spotted in the middle of each piece of membrane. Then the membranes were left to dry for 15 min and incubated for 30 min in PBS containing 104 BSA at room temperature. Each membrane was further washed twice with PBS, left to dry for 15 min and tightly sealed into a plastic box (5 cm x 5 cm x 3 cm), previously filled with a few-sheets of absorbent soft cellulose paper. The cover of the box was made with a 1 cm 2 hole in the center, in correspondence of the membrane area bearing MabINN22, and with a funnel around the hole able to contain at least 5 ml of solution.

Various devices prepared as described above were used to test hCG solutions as follows:

ml aliquots of hCG solutions at various concentrations (from 0 to 10 IU/ml) in 0.14 M sodium chloride containing 0.104 BSA, were pipetted into the tunnel or each device and completely left to absorb through the membrane. Then 0.5 ml of CB-BSA-M3411 diluted 1:5 with 0.02 M TRIS-HCl buffer, pH 8.3, containing 0.15 M sodium chloride and 104 BSA, were let to absorb through each membrane. The color of the spots obtained was visually evaluated. The results are reported in table 2.

7 1 I; Table 2. Immunocolorimetric assay of hCG on nitrocellulose membranes (see text) Sample hCG W/m1 0 0,5 1 2 4 ------------------------------------------- Color 1) (bound to the membrane) A 2) B 3) 1) as visually evaluated 2) membrane area carrying blocked MabINN22 3) membrane area without MabINN22 (blocked with BSA, negative control).

Claims (9)

1. Protein binding analytical method including the interaction between the analyte and an analytical binding reagent labelled with visually detectable systems, characterized in that the label is obtained by hydrophylic colored macromolecular structures.
2. Method according to claim 1, wherein the hydrophilic colored macromolecular structures are chosen in a group consisting of dextran covalently bound to dyes, colored proteins commercially available, polymers obtained by conjugation of reactive dyes with proteins or hydrophylic polysaccharides.
3. 3. Method according to claims 1 or 2, wherein the hydrophylic colored macromolecular structures are obtained by conjugation, through covalent binding, of reactive dyes with polymers having globular structure.
4. 4. Method according to claims 1 or 2, wherein the hydrophylic colored polymers are conjugated by reaction with glutaraldehyde, carbodiimide, periodate, N-NI-0-phenylenedimaleimide, N-maleimidobenzoyl-N-hydroxysuccinimide, mixed anhydrides, bifunctional imidates.
5. 5. Method according to claims 1 or 2, wherein the hydrophylic colored polymers are linke d to avidine or streptoavidine, to which the binding reagent is bound by non covalent bridges.
6. 6. Method according to claims 1 or 2, wherein the hydrophilic colored polymers are subjected to biotinylation and to the formation of an avidine streptavidin - biotinylated binding reagent complex.

   n R or 1 k
7. 7. Method according to claims 1 or 2, for the qualitative immunocolorimetric determination of human chorionic gonadotropin (hCG), characterized in that an anti-hCG antibody labelled with bovine serum albumin conjugated with Cibacron Blu F3GA and copolymerized with glutaraldehyde and lysine is used.
8. 8. Colored analytical reagent for protein binding assays consisting of a biospecific analytical reagent conjugated to hydrophilic colored polymers.
9. 9. An analytical reagent according to claim 8, for the determination of chorionic gonadotropin costituted by an anti-hCG antibody, conjugated to bovine serum albumin, which is copolymerized and conjugated to a triazine dye.

   Published 1989 atThe Patent Office, State House, 6671 High HolbornLondonWC1R4TP. Further copies maybe obtained from The Patent Office. &Ale itwilLumuc,btaDeklouse,t>t),(i xugnxioicoiuijoiiuuji