GB2175906A - Assay methods and reagents - Google Patents

Abstract

Improved assay methods and reagents are disclosed. Assays in accordance with the invention are undertaken at elevated temperatures but this had led to a need to increase stability of reagents used at these temperatures. The labelled conjugate is stabilised with a polyhydroxy compound such as glycerol. The support-ligand complex is stabilised by contacting with a polyalkylene glycol. Furthermore the assays are accelerated in the presence of a relatively hydrophilic, substantially neutral pH detergent.

Description

SPECIFICATION Assay methods and reagents The present invention relates to improved reagents and techniques useful in immunoassays, where, inter alia, enzymes are used as labels.

DESCRIPTION OF THE PRIOR ART Immunoassays used for the detection of antibodies found in human or animal sera, or for the detection of antigens, generally utilize radio immunoassay (RIA) or enzyme-linked immunosorbent (ELISA) techniques.

Generally these techniques utilise a predetermined antigen, known as a ligand, such as a nucleoprotein (viral protein) or pure protein, linked to a support such as a polymer matrix or glass beads. Commonly 96-well microtitre polystyrene trays are used. The antigen is adsorbed to the surface of the support and the antigen-support complex is contacted with a sample of sera or other sample to be tested. Alternatively, an antibody may be linked to the support.

The support is then washed to remove excess sera and then contacted with an enzymelabelled conjugate (ELISA) or radio-labelled conjugate (RIA) which forms a complex with any antibody (or antigen) which has bound to the antigen (body) support complex. After a suitable incubation time, the system is assayed for the presence of the labelled conjugate which has been bound to the support. The ELISA methodology is generally described in US Patent No.

3,654,090.

Although the above immunoassay techniques have been relatively well developed in the preceding several years, in many ways these immunoassays have not been optimised.

Assays for many different antigens are undertaken at reduced temperature or at room temperature. Clearly, it is inconvenient to undertake the assays at reduced temperature due to the requirement of having all reagents at the same temperature and the space having to be provided in cold-rooms etc. Although undertaking the assays at room temperatures is relatively convenient, it may be a source of error as room temperature will vary. However, the present inventors believe that the assay should be conducted not only at a uniform temperature but also at a temperature which is elevated so that the assay proceeds at an optimum rate.

DESCRIPTION OF THE INVENTION Therefore, in one form of the present invention, there is provided assay methods characterized in that they are conducted at temperatures of between about 35"C and 48"C, more preferably 40"-45"C. At temperatures in these ranges, the assay procedures are generally optimized and incubation times are significantly reduced thus leading to more economical assay procedures.

The use of elevated temperatures for the assay procedures does require certain modifications to reagents used in the assay. For example, many different antigens are available for the preparation of the antigen-support complex. However, it has always been considered that these complexes require refrigeration to retain activity for any significant period of time. There is also the risk that at reasonably high temperatures, the complex becomes unstable.

Also, the labelled-enzyme conjugate is often purchased commercially but these conjugates are generally considered to be unstable at room temperature and to require storage at temperatures of about 4"C to retain their activity for any significant period of time, particularly the final assay dilutions for routine procedure.

Therefore, it is one object of the present invention to provide stabilized reagents useful in immunoassay techniques which have increased stability at higher temperatures.

In one form of the present invention the labelled enzyme conjugate is stabilized in the presence of at least 10% v/v of a polyhydroxy compound having up to about 10 carbon atoms.

Preferably the stabilizing compound is glycerol. The labelled conjugate may comprise an enzyme conjugated with an antibody or an antigen. The conjugate may be diluted in solution with the requisite amount of polyhydroxy compound. Alternatively a solution containing the polyhydroxy compound may be used to reconstitute the lyophilized or freeze dried conjugate.

In a preferred embodiment of the invention, up to about 4% w/v polyethylene glycol or other polyalkylene glycol is added to the conjugate solution.

In another form of the present invention, there is provided a process for preparing a ligand conjugated to a solid support such as polymeric material or glass material, useful in enzymeimmunoassay methods, which process comprises the steps of: (i) incubating the ligand in the presence of a solid material for a period sufficient for significant binding between said ligand and support to occur, and (ii) washing the product of step (i) with a solution containing a polyalkylene glycol in a concentration of between 0.01 to 5% w/v.

The support-ligand complex prepared by this process may be provided in the form of a coating on cuvettes, slides, microtitre plates or test tubes, or it may be in the form of beads such as polystyrene or glass. Prepared by the above process it may be stored under prolonged storage conditions at room temperature without a significant loss of activity. When it is to be used, the complex, if in dry form, is hydrated, in a substantially neutral pH buffer and then contacted with the serum to be tested. Preferably the ligand is an antigen.

Furthermore, apart from the ability of a polyalkylene glycol to stabilize the ligand-support complex, it has also been found that such a compound will tend to stabilize protein-protein binding in the assay at elevated temperatures. Accordingly in a further form of the present invention assay methods are provided which are characterized in that they are conducted in the presence of a polyalkylene glycol, such as polyethylene glycol. Preferably, the concentration of the polyethylene glycol is between 0.01 to 5% w/v.

In particular, the first incubation step in which the sample to be tested is incubated with the support-ligand complex and the second incubation step, in which the enzyme labelled conjugate is incubated with the bound sample, are conducted in the presence of at least 0.01% w/v of a polyalkylene glycol such as polyethylene glycol having a molecular weight of between 6,000 to 8,000.

The present inventors have also found that not only can the incubation times for the assay be shortened by conducting the assay at elevated temperatures, but the immuno chemical reactions can be accelerated in the presence of a relatively hydrophilic, substantially neutral pH detergent having an HLB ratio of at least about 20. The preferred detergents are alkylbenzene sulphonates, preferably ammonium or sodium, lauryl or dodecyl benzene sulfonates.

Therefore, in a further form of the present invention there is provided an assay method based on immuno chemical reactions characterized in that the immuno chemical reactions are conducted in the presence of a relatively hydrophilic, substantially neutral pH detergent having an HLB ration of at least 20. Preferably the detergent is present in an amount of between 0.005 and 1% v/v, more preferably about 0.04% v/v.

In a preferred form of the present invention, there is provided an improved assay method for the detection of the presence of a predetermined antibody species in a sample of fluid, said method comprising: 1) providing a support coated with an antigen which will form a complex with said antibody species; 2) incubating said coated support with a sample of body fluid so that a complex is formed between said antigen and said antibody; 3) washing said coated support without disrupting said complex; 4) incubating said antibody-antigen complex with a labelled immunoprotein (conjugate) so as to form an antigen-antibody-labelled immunoprotein complex; 5) washing said coated support so as to remove excess labelled immunoprotein; 6) determining quantitatively or qualitatively the presence of said antigen-antibody-labelled immunoprotein complex, characterised in that step 2 and/or 4, and optionally step 6, is conducted in the presence of from 0.1% to 5% w/v of a polyalkylene glycol, and at least 0.05% v/v of a relatively hydrophilic, substantially neutral pH detergent having an HLB ratio of at least 20. Preferably the polyalkylene glycol is polyethylene glycol having a molecular weight of from 6000 to 8000.

Preferably the detergent is a sodium or ammonium salt of an alkyl benzene sulphonate.

DESCRIPTION OF PREFERRED EMBODIMENTS In a preferred embodiment of the process for preparing the ligand-support complex, a support material such as glass beads, or a matrix of polystyrene, polyvinyl chloride or polycarbonate is incubated with the required antigen in a buffered solution, having a pH between 5 and 10.

Preferably, the buffer is a non-phosphate group containing buffer such as Tris HCI or bicarbonate buffer. When the antigen is a nucleoprotein a preferred pH is about 9.6 but when the antigen is a relatively pure protein, the pH will be slightly less and most preferably between 8 and 9.

Preferably the antigen is incubated with the support material for about 3 hours at about 37"C.

The support is then washed to remove unbound antigen. After washing, the antigen is fixed by contacting the support, to which antigen is bound, with a polyalkylene glycol, preferably polyethylene glycol Preferably a hydrophobic detergent is used also to wash the support. The polyethylene glycol is diluted in a buffer which does not contain amino groups, and has a pH of about 7, for example a phosphate buffer. The polyethylene glycol has a molecular weight of about 6,000 to 8,000. The concentration of polyethylene glycol is in the range of from 0.01 to 5% w/v and most preferably is at a concentration of about 0.15% w/v. The detergent is substantially neutral pH detergent such as Tween, Triton or detergents having similar HLB values.

The HLB value of Tween 20 is 16.7 and Triton X-100 is 13.5. The concentration of the detergent is from about 0.005 to 1% v/v. Preferably, when using Tween the concentration is 0.05% v/v. Preferably the solution also contains glycerol in an amount up to about 1% by weight but the glycerol component is not essential.

Turning to the protein-protein or immunoprotein-protein complex, this is used in RIA or ELISA techniques to bind to the immunoglobulin molecules which have reacted with the antigen. The immunoproteins or antisera used are, for example, antihuman or antianimal immunoglobulins such as those of the IgG, IgM or IgA class or antibodies to biologically active compounds such as thyroxin, corticosterone and insulin. These are conjugated with a protein which acts as a label for assay purposes. For example, preferred protein labels are enzymes such as alkaline phosphatase, horseradish peroxidase, ssgalactosidase or fi-glucosidase. Alternatively, 1'25-labelled thyroxin or other radio-labelled proteins may be used.

The immunoprotein or protein is mixed with the protein label, such as an enzyme, in predetermined proportions and incubating them in a buffered solution at between pH 6 to 8. Preferably, the solution of antisera and protein label is a dilute solution containing about 0.2% glutaraldehyde v/v and the buffer used is 0.1M Tris HCI or 0.1M phosphate buffer containing 10mM magnesium chloride or other cofactors according to enzyme requirements. In one known method of conjugation, glutaraldehyde is added during or subsequent to incubation. Up to this stage this is one of the standard techniques for conjugation of the protein label with the antiserum.

The next stage of the process seeks to remove non-conjugated immunoprotein or protein label from the conjugate.

Preferably the separation is effected by dialysis in the presence of 0.1 M Tris HCI buffer containing 10mM MgCI2 having a pH of 7 to 9. Alternatively, column chromatography may be used.

To the conjugate, there is added at least about 10% and up to about 50% v/v of glycerol.

Preferably the final concentration of the glycerol in the conjugate solution is about 20% v/v.

Preferably, up to about 5% of polyethylene glycol is also incorporated into the conjugate solution.

The present inventors have found that the addition of glycerol to the conjugate leads to a significant increase in the temperature stability of the conjugate and allows it to be stored and used at room temperature and in particular, allows this conjugate to be used in an assay at elevated temperatures such as about 45"C.

In this way, the assay using this conjugate can be maximised particularly as the time required for incubation of the conjugate with the antigen-antibody complex is reduced.

Turning to the assay procedure, a detergent is used to accelerate the immunological reactions.

Preferably, the detergent is an alkylbenzene sulfonate salt, e.g. sodium or ammonium, such as that marketed under the trade mark of NANSA by Albright & Wilson. The detergent is used in an amount between 0.005 and 1% v/v, preferably at levels of about 0.04% v/v. Too much detergent will lead to nonspecific binding and washing the protein from the support whilst too little will have no effect. The amount may be varied in the range given to suit the particular assay.

The HLB ratio of the detergent is relatively high so that the detergent is considered a hydrophilic detergent. Preferably the HLB ratio is at least about 20.

The detergent is used in the first incubation between support-ligand and sample as well as in the second incubation between support-ligand-bound antibody and the enzyme labelled conjugate. Optionally it is also used in the third incubation step of the substrate with the enzyme label if the ELISA technique is used.

Furthermore, the assay procedure is stabilized by the presence of a polyalkylene glycol such as polyethylene glycol (PEG), in the assay steps where protein-protein binding occurs. Thus PEG is used in the first incubation step where the ligand support will bind to antibodies present in the sample as well as the second incubation step wherein the enzyme labelled conjugate binds to the bound antibody. Optionally, PEG is also present in the third incubation but has little effect at that stage of the assay.

Preferably, PEG is used at a concentration of 0.1 to 5% w/v, more preferably about 0.15% w/v. Preferably PEG has a molecular weight of between 1,000-20,000, more preferably 6,000 to 8,000.

Notwithstanding other forms which may fall within the scope of the present invention, a preferred form will now be described by reference to the following examples.

EXAMPLE 1 The following is a general procedure for the preparation of the antigen support complex, the preparation of the antiserum-enzyme conjugate and the assay procedure of the present invention.

Antigen: Toxoplasma gondii Antigen Buffer: 0.1 M Bicarbonate Buffer, pH 9.6 Antigen is dispersed and sheered through a glass syringe. Alternatively and preferably this may be done by using a sonicator. The antigen is diluted with bicarbonate buffer from about 40 to 200 times depending on the antigen and the purity of the specimen.

250ul of antigen in the coating solution is applied to a polymer matrix such as Guildford polystyrene cuvettes or microtitre plates.

For each test, there are 2 wells or cuvettes used. The control well is coated with a buffer alone whilst the test well is coated with the test antigen in the buffer solution.

The plate is incubated at 37"C for between 2 to 4 hours.

The coated plates are then fixed in the solution of phosphate buffered saline containing 0.05% Tween 20 and 1 0mM MgCl2. The plates are then incubated for two hours at 37"C. Upon completion of incubation, the plates are allowed drain dry at room temperature.

PREPARATION OF ANTISERUM-ENZYME CONJUGATE Enzyme: Alkaline Phosphatase (Sigma Laboratories) Antiserum: Sheep Anti Human IgG (Silenus Laboratories) Crude Alkaline phosphatase powder is purified by chromatography. The pooled alkaline phosphatase fractions, about 30-40ml, are mixed with 6-10ml of anti human IgG and dialysed in Tris HCI (0.1M pH 8.6+10mM MgCl2+0.15M NaCI), or a phosphate buffer with 1OmM MgCI2 (pH 6-8) overnight.

The mixture is then concentrated in polyethylene glycol flakes in a dialysis bag or, if necessary, in an Amicon Concentrator. To the dialysed concentrated mixture, up to 0.4% glutaraldehyde (final concentration) is added. The mixture is stirred for about 30 minutes. The mixture is then dialysed in Tris HCI-(0.1M pH 8.6+10mM MgCl2+0.15M NaCI), in the first dialysis overnight. The conjugate, immunoprotein and alkaline phosphatase can now be separated on a Sephadex G-200 or G-150 or Sephacryl S.300 column, if removal of non-conjugated material is necessary.

Upon purification, the conjugate is stabilised in a solution to achieve a final concentration as follows: Tris HCI (0.1M pH 7.4) buffer containing 20% glycerol, 0.15% polyethylene glycol, 10mM MgCl2, and 0.15M NaCI.

ASSAY TOXOPLASMA GONDII SPECIFIC IgG ASSAY REAGENTS: 1) Cuvettes/plates coated with Toxoplasma antigen 2) Anti human IgG conjugated to alkaline phosphatase diluted in stabiliser solution conveniently to allow a further 30 fold dilution for the assay.

3) Paranitrophenyl phosphate at 3mM concentration dissolved in 2-amino,2-methyl, 1 -propanol buffer at pH 9.9, 0.1M stored frozen in 5ml aliquots.

4) Serum and conjugate diluent made up as follows: Phosphate buffered saline (20 times concentrated) 50ml Polyethylene glycol (15% solution) 10ml Magnesium chloride 0.5M 16ml NANSA (10% solution) 2.5 ml Water (distilled) 921.5ml 5) Wash solution: a solution of phosphate buffered saline containing 0.05% Tween 20 and 5mM magnesium chloride 6) Substrate diluent 2-amino,2-methyl,1-propanol (0.1M), NANSA 0.045%, Magnesium Chloride 5mM pH 10.25 ASSAY METHOD: The coated plates/cuvettes are hydrated by washing in the wash solution three times. The last wash is allowed to remain for three minutes before being allowed to drain for a further three minutes. 10ul serum is then applied to both control and test cuvettes for each sample.

The serum diluent is pipetted into all wells, and the plate/cuvette is incubated for 30 minutes at 45"C. At the end of the incubation, the plates/cuvettes are washed in phosphate buffered saline containing Tween 20. The plates or cuvettes are then filled again with the conjugate which is diluted thirty fold. The plates/cuvettes are covered and incubated at 45"C for 15 minutes.

The plates or cuvettes are washed four times in phosphate buffered saline.

Frozen aliquots of paranitro phenyl phosphate is diluted 1:4 with the substrate diluent. The plate/cuvette is covered and incubated at 45"C for 20 to 30 minutes. All solutions are preincubated at 45"C before being dispensed into the cuvettes/plates.

Alkaline phosphatase activity is detected spectrophotometrically by the determination of paranitrophenol at 405nm. The results given are the average of duplicates. Significant deviations between duplicates led to repeats of the assay.

EXAMPLE 2 The example below shows that non-specific binding can be reduced if after the preparation of the antigen support complex the complex is washed with a protein solution such as bovine serum albumin at pH 9.6.

The test procedure used was the toxoplasma gondii assay described in Example 1. Toxoplasma complement fixing antigen was incubated for two hours with polystyrene microtitre trays.

After two hours incubation both control and test wells were drained and filled with 250ul aliquots of: (a) 2.5% bovine serum albumin in bicarbonate buffer: 0.1M at pH 9.6.

(b) 2.5% bovine serum albumin in Tris-HCI buffer: 0.1m at pH 7.4, and (c) 2.5% phosphate buffered saline at pH 9.6.

After thirty minutes the plates were washed with phosphate buffered saline containing 5mM magnesium chloride and 0.05% Tween 20.

The results are given in table 1.

TABLE 1 Elimination of non specific binding sites on polystyrene-antigen 2.5S 2.51 2.51 Normal No. . 2.51 2.52 2.51 Normal Bicarb Tris PBS Without Bicarb Tris HC1 PBS Without BSA HC1 BSA BSA BSA BSA BSA BSA BSA 1 .08 0.16 0.11 0.21 9 0.24 0.24 0.31 0.32 2 0.09 0.27 0.21 0.34 10 0.21 0.21 0.29 0.31 0.17 0.25 0.18 0.30 11 0.17 p.20 0.27 0.29 4 0.11 0.21 0.17 0.31 12 0.20 0.26 0.29 0.26 5 0.12 0.23 0.16 0.24 13 0.17 0.18 0.27 0.27 6 0.12 0.22 0.17 0.28 14 0.29 0.23 0.29 0.30 7 0.11 0.24 0.24 0.28 15 0.25 0.18 0.34 0.34 8 0.21 0.30 0.26 0.30 16 0.30 0.30 0.33 0.32 The best post coating solution to eliminate non-specific binding is a phosphate buffered saline solution of bovine serum albumin. The sera specimens which have true positive specific IgG's will not be dampened by this procedure.

EXAMPLE 3 Several aspects of the assay described in Example 1 were tested so as to optimize variables in the assay. At temperatures of 45"C, incubation times were optimized.

The first incubation period i.e. the incubation period of the serum with the antigen-support complex was optimized at 30 minutes. The second incubation period i.e. the incubation of the anti IgG conjugate/alkalinephosphatase with the support was optimized at 15 minutes. The third incubation period being the incubation of the substrate with the bound labelled enzyme conjugate was optimized at 30 minutes.

The optimization procedure also involved testing for a temperature optimum. The assay procedure used was that described in Example 1 and incubation times were as above. The assay and all incubations were undertaken at the following temperatures: 21"C, 30 C, 37"C, 40"C, 45"C, 50"C and 55"C.

Table 2 below tabulates optical densities of eight separate samples of sera at the above temperatures. It can be seen that the optimum temperature for the assay and incubation is about 40-45"C.

TABLE 2 21 C 300C 370C 400C 450C 500C 550C l 0.09 0.12 0.14 0.10 0.18 0.12 0.06 2 0.13 0.23 0.22 0.33 0.31 0.25 0.03 3 0.17 0.20 0.24 0.32 0.28 0.18 0.08 4 0.16 0.26 0.28 0.33 0.36 0.19 0.07 5 0.14 0.24 0.26 0.31 0.33 0.23 0.06 6 0.16 0.25 0.27 0.34 0.33 0.19 -0.04 7 0.17 0.26 0.26 0.30 0.34 0.14 0.05 8 0.13 t.25 0.26 0.24 0.37 0.22 0.01 EXAMPLE 4 The long term storage of the enzyme labelled conjugate at room temperature in the presence of 20% v/v glycerol was checked. Assays using the conjugate were conducted at periods of 0, 6 days, 9 days, 12 days, 15 days and 140 days.The conjugate was compared to three commercial products A, B and C and the results given as optical densities in Table 3.

TABLE 3 Long Term Storage of Conjugate at Room Temperature With Glycerol DAYS DILUTION 1 6 9 12 15 140 XLNLsB IgG 1 1:20 0.68 0.79 0.77 0.80 0.78 0.84 + 20% glycerol MANLAB IgG 1 1:20 0.78 0.87 0.87 0.85 0.83 0.44 (no glycerol) MANLAB IgG 2 1:20 0.38 0.49 0.42 0.43 0.46 0.42 + 20% glycerol MANLAB IgG 2 1:20 0.31 0.36 0.33 0.32 0.31 0.19 (no glycerol) A 1:10 0.41 0.82 0.67 0.79 0.64 1.16 B 1.15 0.81 - 0.95 0.85 0.86 0.83 1.18 C 1:120 0.52 0.90 0.98 1.11 0.91 1.21 NOTE: All conjugates were diluted to give an optional reading on Day 1 from previous sample trial runs.

The above results show a marked variation at room temperatures after prolonged storage at room temperature, when the products are stored in the absence of glycerol. There is no significant variation in results after prolonged storage at room temperature in the presence of glycerol.

EXAMPLE 5 Human IgG-Fc fragment was coated onto Gilford cuvettes at 200ug/ml in 0.1 sodium bicarbonate-carbonate buffer at 37"C for two hours. The cuvettes were washed with PBS Tween 0.05% with 5mM magnesium chloride.

Conjugates of antihuman-lgG were diluted with: Tris-HCI 0.1M, pH 7.4 containing 0.15M sodium chloride and 10mM magnesium chloride.

Varying concentrations of glycerol were added to the diluent to get a final concentration of glycerol between 0% and 40% v/v. In one aspect, the conjugate solution further contained Nansa in an amount of about 0.04% w/v.

The conjugate as diluted above was incubated with the IgG coated cuvettes at 45"C for 15 minutes, the cuvettes were washed, substrate added and incubated at 45"C for 30 minutes.

Table 4 below provides the results.

TABLE 4 Stabilization of Conjugate with Glycerol Glycerol With Nansa Without Concentration SSAL Nansa 1 Ot 0.18 0.31 2 5t 0.25 0.31 3 10% 0.26 0.30 4 15% 0.25 0.34 5 20% 0.29 0.32 6 30% 0.32 0.35 7 40% The addition of glycerol to the conjugate did not enhance or appear to significantly effect the immunochemical reaction of the conjugate. Glycerol acts only as a stabilizer for storage of the product at room temperature. The conjugate soon after preparation can be lyophilised and glycerol can be added, after hydration of the lyophilisate.

The addition of Nansa does not appear to effect conjugate activity over the short term.

EXAMPLE 6 The testing procedure used with the preceding example was used to study the effect of the incorporation of polyethylene glycol having a molecular weight of between 6,000 and 8,000 in varying amounts. The assay procedure employed the same samples but with concentrations of polyethylene glycol varying from 0% to 6% w/v in the dilution of the conjugate. The results obtained are shown in Table 5 below. Therefore, it was concluded that polyethylene glycol has no significant enhancing effect on the antigen-antibody complex formation.

TABLE 5 No. Conc. of PEG Optical Density 1 0% 0.18 2 0.2 0.19 3 0.4 0.19 4 < 0.6 0.16 5 1.0 0.18 6 2.0 0.18 7 4.0 0.16 8 6.0 0.12 EXAMPLE 7 The use of polyethylene glycol to stabilize the ligand-support complex is shown in this Example.

Polystyrene plates A and B were incubated with toxoplasma antigen for two hours, washed in PBS-Tween 20 (0.05%) and MgCI2 (5mM) which contained 0.15% w/v PEG.

Assays were performed in accordance with Example 2 after storage of the plates at room temperature for 0 days, 2, 10, 17 and 33 days and the results are given at Table 6 below.

No substantial loss of activity over a 33 day period of the coated plates was noted.

TABLE 6 TInE O DAYS 2 DAYS 10 DAYS 17 DAYS 33 DAYS NORMAL No. A B A B A B s B A B VALUE 1 .19 .15 .21 .15 .15 .10 .09 .15 .20 .22 .18 2 .18 .15 .13 .11 .10 .12 .09 .15 .16 .19 .16 3 .15 .14 .10 .11 .19 .08 .17 .13 .16 .19 .15 4 .14 .12 .14 .13 .12 .11 .13 .12 .18 .19 .14 5 .20 .16 .15 .13 .11 .08 .12 .20 .17 .16 .18 6 .17 .14 .18 .25 .19 .24 .21 .22 .21 .24 .25 7 .16 .13 .15 .26 .24 .26 .22 .28 .17 .22 .29 8 .44 .46 .40 .42 .31 .44 .32 .40 .36 .47 .50 effect on the antigen-antibody reaction with increasing concentration.

In this Example, the assay procedure of Example 1 is used on eight different samples. The concentration of polyethylene glycol is varied in the solution of antigen-enzyme label between 0 and 0.2% w/v. The results obtained are given in Table 7 below.

TABLE 7 CONCENTRATIOUS w/VL OF POLYETHYLENE GLYCOL NW 8000 ROUTINE VALUE WITH 0 0.025 0.05 0.075 0.10 0.125 0.150 0.175 0.20 0.155 1 0.10 0.09 0.14 0.16 0.10 0.12 0.15 0.16 0.14 0.20 2 0.25 0.26 0.26 0.28 0.22 0.25 0.22 0.25 0.24 0.22 3 0.24 0.29 0.28 0.28 0.25 0.27 0.23 0.27 0.28 0.26 4 0.16 0.15 0.15 0.19 0.14 0.13 0.12 0.12 0.13 0.13 5 0.25 0.27 0.32 0.26 0.25 0.29 0.26 0.27 0.29 0.28 6 0.19 0.24 0.26 0.25 0.22 0.24 0.20 0.20 0.23 0.22 7 0.22 0.24 0.26 0.26 0.26 0.26 0.24 0.23 0.27 0.23 8 0.25 0.27 0.31 0.26 0.27 0.29 0.24 0.22 0.27 0.27 It was concluded that the presence of PEG did not accelerate or enhance the formation of the antigen-antibody complex.

EXAMPLE 9 A comparison of detergents was undertaken using Triton X-100, Tween 20, Tween 80, sodiumlaurylsulphate, sodium dodecyl benzene sulphonate and Nansa (sodium alkyl benzene sulphonate) at varying concentrations. The assay procedure incorporated specific amounts of the above selected detergents as part of the diluents for the serum and body anti-enzyme labelled conjugate.

The results are provided in table 8 below based on the assay procedure described in Example 1.

The use of Nansa which is a sodium alkyl benzene sulfonate detergent and/or the use of sodium dodecyl benzene sulfonate significantly enhances the sensitivity of the assay.

TABLE 8 TRITONX-100 TWEEN 20 TWEEN 80 SODIUM LAUREL SODIUM DODECYL NANSA SULPHATE BENZENE SULPHONATE 0.01% 0.05% 0.01% 0.05% 0.01% 0.05% 0.01% 0.05% 0.01% 0.05% 0.0461 1 0.17 0.19 0.10 0.10 0.03 0.08 0.01 0.03 .02 .21 0.18 2 0.06 0.06 0.09 0.06 0.04 0.05 0.08 0.09 .13 .25 0.29 3 0.16 0.01 0.17 0.24 0.17 0.11 0.19 0.10 .15 .20 0.29 4 0.01 0.04 0.05 0.05 0.04 0.03 0.08 0.04 0.06 0.20 0.21 5 0.09 0.04 0.04 0.09 0.03 0.11 0.08 0.06 0.12 0.16 0.24 6 0.03 0.06 0.03 0.04 0.03 0.03 0.04 0.05 0.05 0.20 0.26 7 0.05 0.05 0.05 0.05 0.04 0.02 0.06 0.05 0.11 0.17 0.21 8 0.07 0.07 0.06 0.05 0.05 0.07 0.01 0.10 0.10 0.17 0.20 EXAMPLE 10 The accelerating effect of a detergent was shown using Nansa at various concentrations using the standard assay of Example 2 and a standard incubation time for each of the first, second and third incubations. The Nansa was added at predetermined concentrations in the first and second incubations and results shown in Fig. 1.

It can be seen that increasing concentrations of detergent up to a maximum will give increased optical densities at set periods of incubation. However, it was determined that there is an optimum concentration after which the increase in optical density will lead to false positive results. Therefore it was concluded that the optimum concentration of NANSA was between 0.04 and 0.05% v/v.

EXAMPLE 11 SIMULTANEOUS IgG & IgM WITHOUT SEPARATION OF IgG Support matrix, polystyrene cuvettes by Guilford Standards: Behring plasma protein standard containing pre-assayed levels of IgG and IgM.

Pure standard: pure human IgG (Fc fragment) from Calbiochem-Behring.

Coating: The plasma protein and pure IgG standard were diluted in bicarbonate-carbonate buffer (0.1M) and then incubated with the solid matrix for two hours. The cuvettes were washed with PBS Tween (0.05%) MgCI2 (5mM).

The conjugate used was a routine anti IgG-alkaline phosphatase conjugate, at a final dilution of 1050 and anti IgM alkaline phosphatase at a final dilution of 900.

The cuvettes were incubated with 250ul of the conjugate for 15 minutes.

250ul of substrate solution were added after washing the plates after the above incubation and this incubation undertaken for 30 minutes.

All incubations were carried out at a temperature of 45"C.

The results are provided as Table 9.

TABLE 9 IgG IgM SUBSTANCE CONCENTRATION OPTICAL CONCENTRATION OPTICAL DENSITY DENSITY Behring Plasma-Protein 208ug/ml 0.63 23u6/ml 0.37 Standard 2075ug/ml 0.74 232ug/ml 0.33 4150ug/ml 0.71 464u6/ml 0.19 8300ug/ml - 930u6/ml 0.11 Pure Standard 200ug/ml 0.57 0 0.07 Conclusions: (1) Pure IgG standard gave comparable results to the plasma protein standard at similar concentrations. Pure IgG standard gave insignificant readings with IgM conjugate while a significant reading was obtained with 23ug/ml of IgM in plasma protein standard.

At the IgG concentration of 10 fold, it is possible to obtain a reasonable IgM measurement although significant inhibition of IgM measurement occurs at very high (4150ug/ml) concentrations of IgG. However, the concentration of specific IgG and IgM would not normally reach such concentrations.

Most assay methods using ELISA for the measurement of specific IgM antibodies suggest that to prevent swamping of antigenic sites by high levels of IgG, it is necessary to remove the IgG in serum before assaying for IgM. A common method of performing this removal is to use protein A which binds specifically to IgG. However, it is considered that sensitivity of the assay procedures used in the present invention are not reduced by measuring IgG and IgM without separation of IgG. It is considered that due to the shorter incubation times at higher temperatures the exclusion of IgM for the antigenic sites by IgG is not likely to occur and therefore a further advantage of the assay procedures of the present invention is that removal of IgG is not necessary in assaying for IgM antibodies.

The examples provided above have used a specific toxoplasma gondii assay. However, the assay of the present invention is applicable to a wide range of assay procedures for the detection of a wide range of viral antibodies and/or antigens, such as rubella, CMV, herpes 1 and 2, toxoplasma, influenza A, B and C, parainfluenza 1, 2 and 3, mycoplasma, adenovirus, Q fever, measles, varicella zoster, Ross River virus, immune complexes, HCG and thyroxine. Animal antibodies or antigens can be detected as well as human antibodies or antigens by use of the appropriate antisera.

Claims (22)

1. A composition of a protein-protein conjugate which composition contains at least 10% v/v of a polyhydroxy compound having up to 10 carbon atoms.

2. The composition of claim 1 wherein said polyhydroxy compound is glycerol.

3. The composition of claim 1 or 2 which contains at least about 20% w/v of said polyhydroxy compound.

4. The composition of any one of claims 1 to 3 which further contains up to about 5% w/v of a polyalkylene glycol.

5. The composition of claim 4 wherein said polyalkylene glycol is polyethylene glycol.

6. The composition of any one of claims 1 to 5 wherein said polyhydroxy compound is in a concentration of between 20 and 50% w/v.

7. An assay method based on immunological reactions between a predetermined species in a sample and one or more immuno-proteins relatively specific for said species, characterised in the incubations between said species and said immuno-proteins in said assay are undertaken at temperatures of between 37"C-50"C for predetermined periods of time.

8. The method of claim 7 wherein said temperature is about 45"C.

9. An assay method for the qualitative and/or quantitative determination of a predetermined species in a sample, said method comprising: (1) providing a support to which is bound or adsorbed a protein or nucleoprotein capable of binding with said species by immunological reaction; (2) contacting said support with said sample so that said species binds to said protein.

(3) removing excess sample from said support; (4) contacting said support with a labelled protein capable of binding relatively specifically to said species when bound to said support, so that said labelled protein becomes bound to said species; (5) removing unbound labelled protein; (6) determining quantitatively or qualitatively the presence of said labelled protein; wherein step (2) and/or (4) are conducted in the presence of up to 5% w/v of a polyalkylene glycol.

10. The method of claim 9 wherein said polyalkylene glycol is polyethylene glycol.

11. The method of claim 9 or 10 wherein said polyalkylene glycol has a molecular weight of between 6,000 and 8,000.

12. The method of any one of claims 9 to 11 wherein said polyalkylene glycol is present in an amount of about 0.15% w/v.

13. The method of any one of claims 9 to 12 wherein steps (2) and (4) are conducted in the further presence of up to 1% v/v of a substantially hydrophilic detergent.

14. The method of claim 13 wherein said detergent is a salt of an alkyl benzene sulphonate.

15. An assay method for the qualitative or quantitative determination of a predetermined species in a sample, said method comprising: (1) providing a support to which is bound or adsorbed a protein capable of binding with said species by immunological reaction; (2) contacting said support with said sample so that said species binds to said protein; (3) removing excess sample from said support; (4) contacting said support with a labelled protein capable of binding relatively specifically to said species when bound to said support so that said labelled protein becomes bound to said species, and consequently said support; (5) removing unbound labelled protein;; (6) determining quantitatively or qualitatively the presence of said labelled protein, wherein steps (2) and (4) are conducted in the presence of up to about 1% v/v of a relatively hydrophilic detergent.

16. The method of claim 15 wherein said detergent is selected from the salts of a alkyl benzene sulphonate.

17. The method of claim 16 wherein said detergent is the sodium or ammonium salts of dodecyl and/or lauryl benzene sulphonate.

18. A method for the preparation of a storage stable support-ligand complex for immunological based assays said method comprising: (1) providing a solid or particulate support or matrix for said assay; (2) binding to said surface of said support a predetermined protein; (3) contacting said support to which said protein is bound with a solution of between 0.01 and 5% w/v of a polyalkylene glycol.

19. The method of claim 18 wherein said polyalkylene glycol is polyethylene glycol.

20. The method of claim 18 wherein said support is polystyrene, polyvinylchloride or glass

21. A storage stable support-ligand when produced by the method of claims 18 to 20.

22. A kit for an assay method characterised in that it contains the storage stable support of claim 21 and/or the composition of any one of claims 1 to 6.