AU624650B2 - One-step immunoassay for determining antigen-specific antibodies of all immunoglobulin classes, and an agent therefor - Google Patents

Abstract

A process for preparing an agent with which a one-step immunoassay can be carried out for determining antigen-specific antibodies of all immunoglobulin classes is described. In the one-step immunoassay, carrier-bound antigen (solid phase) and dissolved labelled antigen of the same specificity are incubated, in a ratio determined in the preparation process, at the same time with the antibody-containing liquid sample. The liquid phase is separated from the solid phase and then the labelling on the solid phase is determined.

Description

Form COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952,69 COMPLETE SPECIFICATION (O RIG INAL) Class hInt. Class Application Number: Lodged: Complete Specification Lodged: Accepted., Published: Priority: Related Art:

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Wowii of Applicant: BEHRINGWERlE AKTIENGESELLSCHAVI' Ac~dress of Applicant: D-3550 Marburg, Federal Republic of Germny Actul Ivenor: UDO KRUPKA and H-ANS ERWIN PAUJLY Address fo Service atermark Patent Trademark Attorneys QUEEN STREEIT, MELABOURNE, AUSTRALIA, 3000.

Complete Specification for the invention entitled: ONE-STEP IMMUJNOASSAY FOR DETERMINING ANTIGEN-SPECIFIC ANTIBODIES OF ALL IMMUNOGLOBULIN CLASSES, AND AN AGENT THEREFOR The following statement Is a full description of this Invention, Including the best method of performing it known to :~us BEHRINGWERKE AKTIENGESELLSCHAFT 88/B 032 Ma 687 Dr.Ha/Sd One-step immunoassay for determining antigen-specific antibodies of all immunoglobulin classes, and an agent suitable therefor The invention relates to an immunochemical method for detecting and for determining antibodies which are specific for a particular antigen and which embrace all immunoglobulin classes.

Immunoglobulins are antibodies which are produced by the immune system of the body against foreign substances (antigens, for example proteins of pathogens, bacterial polysaccharides) but also against endogenous serum proteins and tissue proteins.

The determination of antigen-specific immunoglobulins is of particular importance for detecting particular diseases due to parasites, bacteria or viruses. A large number of immunological methods is known for determining immunoglobulins. Methods for the physical separation of immunoglobulins according to classes, for example immunodiffusion, immunoelectrophoresis or density-gradient centrifugation, are elaborate, inaccurate and susceptible to interference.

It is possible, in the so-called direct method, to determine antigen-specific immunoglobulins using immunoassay techniques; this entails the antigen being coupled to a solid carrier, resulting in a reactive solid phase.

The body fluid which contains the antigen-specific immunoglobulin is contacted with the solid phase, whereupon the antigen-specific immunoglobulin is bound to the solid phase, the solid phase and liquid phase are separated, the solid phase is subsequently contacted with a labeled antigen which is present in solution and which has the same specificity as the antigen on the solid 4 1\ 2phase, the solid phase and liquid phase are again separated, and the labeling 4s determined either on the solid phase or in the liquid phase. A method of this type is described in Example III of USP 4,016,043.

Used for the labeling are, for example, fluorescent and Schromophoric substances or radioactive isotopes or enzymes.

It is essential in the said method that, after the reaction (incubation) of the sample containing the antibodies to be detected with the solid phase and before reaction with the labeled antigen, unbound material is removed by washing. These methods are therefore called "two-step methods".

This means that an assay with a previously prepared solid phase requires at least three reaction steps, namely addition 1. of sample, 2. of labeled antigen and 3. of the reagents for the detection reaction, and each reaction step makes at least two washing steps necessary.

The object now was to make the assay shorter. It has now been found, surprisingly, that this is possible by the antigen-containing solid phase, the antibody-containing sample and labeled antigen being contacted without washing between the addition of the sample and the addition of the labeled antigen.

The preparation of an agent which allows a "one-step method" of this type to be carried out became possible after the amount of the antigen bound to the carrier, and the concentration of the labeled antigen present in the liquid phase, had successfully been suited to a particular analyte concentration range, and to the selected carriers for the antigen, for example microassay plate or r 3 beads in a tube, the assay volume and to the incubation conditions, such as the incubation time.

Hence the invention relates to a process for the preparation of an agent with which antigen-specific immunoglobulins are determined, obtainable by Sa) contacting a dissolved antigen in a dilution series i with a carrier, removing unbound antigen by washing i 2j with a buffer, and thus preparing a plurality of i solid phases containing amounts of the antigen which differ from one another, b) contacting a plurality of samples in various volumes or dilutions with a dilution series of the labeled antigen of tne same specificity, resulting in mixtures which are all c) contacted with the solid phases prepared as described under a), S d) separating the solid phases from the liquid phases, e) determining the labelings on the solid phase and f) selecting those reaction mixtures whose labeling generates signals, which are obtained when the solid phases are first contacted with the sample, subsequently solid phases and liquid phases are separated, the solid phases are then contacted with dilutions of the labeled antigen obtained from a dilution series as described in solid phases and liquid phases are again separated, and then the labeling on the solid phase is determined.

The invention furthermore relates to an agent containing a carrier loaded with antigen as solid phase and a labeled antigen with the same specificity as the antigen on the solid phase, the amounts and concentrations of these having been suited to one another in the preparation process described.

The invention also relates to the use of an agent of this type in a method for determining antigen-specific 1 antibodies, in which the sample and the labeled antigen are simultaneously contacted with the solid phase, subsequently the solid phase is separated from the liquid phase, and the labeling on the solid phase is determined.

Suitable carrier materials for the solid phase are plastics such as polystyrene, polyvinyl chloride, polyamide or other synthetic polymers, natural polymers such as cellulose acetate or nitrocellulose, as well as glass, in particular glass fibers.

The carriers can have the form of beads, rods, tubes and microassay plates. Sheet-like structures such as paper strips, small plates and membranes are likewise suitable.

The surface of the carriers can be both permeable and impermeable to aqueous solutions. Preferred carriers are microassay plates.

A solid phase suitable for the method according to the invention is prepared by binding an antigen preparation irreversibly to a carrier.

The term "solid phase" is used in the present text for the carrier with immunochemical reactants bound thereto.

An irreversible binding within the meaning of the invention is present, for example, in the case of S1) adsorptive binding which is not broken by the agents i used in the method, such as labeled immunological reagents, or diluting or buffer solutions, 2) bioaffinity binding mediated by a spacer which binds immunochemically (high-affinity antibodies) or nonimmunochemically, in which case the spacer can be composed of biotin and avidin or of other pairs of receptors and ligands, 3) direct covalent bonding or 4) covalent bonding mediated by a chemically bifunctional spacer.

Covalent bonding is preferred when water-permeable carriers are used, or adsorptive binding is preferred when water-permeable as well as water-impermeable carriers are used.

Direct adsorptive binding of antigen preparations to, as carrier, polystyrene treated with gamma rays is particularly preferred.

Suitable as antigens for the solid phase and for preparing labeled antigens are classically purified proteins, synthetic peptides or proteins prepared by genetic ij manipulation, whose preparation is described as state of the art.

In general, the antigens used for preparing the labeled antigens are the same as for binding to the carrier. The 15 labeling is effected by methods described as state of the I art for the said labels.

Used for the labeling are, for example, fluorescent and chromophoric substances or radioactive isotopes, enzymes or particles, such as erythrocytes or latex particles, loaded with immunological components.

An enzyme is preferably used.

The agent according to the invention is used to detect and determine antibodies directed against, for example, cytomegalovirus, rubella, herpes simplex virus, varicella zoster virus, measles and mumps, Toxoplasma gondii, treponemas, hepatitis A and B virus, human immunodeficiency virus, hepatitis B c and e antigen, chlamydia, Epstein-Barr virus, parvovirus, rotavirus and against pathogens which are of importance in veterinary diagnosis, for example the pathogens of Aujeszki disease, bovine leukosis or brucellosis.

The agent according to the invention is preferably used -6for determining antibodies which are directed against hepatitis B core protein, against antigens of hepatitis A, human immunodeficiency (HIV), rubella or cytomegalovirus or antigens of Treponema pallidum or Toxoplasma gondii.

An agent of this type which is composed of a single element in which all the reagents necessary for the method are present in dry form is also preferred.

The possible uses of the described invention of a one- 110 step immunoassay are, in principle, identical to the uses of the direct and indirect multistage assays which have already been described previously. The present new method differs advantageously from the latter in three ways: The simultaneous incubation of analyte-containing sample and labeled immunological reagents dispenses with an rlr incubation step and a washing process, which results in a considerable simplification of the assay procedure.

As is evident from the examples, the one-step method allows the total duration of the assay to be considerably reduced, which, besides the principal advantage that the method is practical in operation, also has great importance for the rapid clinical detection of acute infections.

The examples which follow represent embodiments of the invention, without intending to restrict it to them.

Example 1 Determination of immunoglobulins specific for hepatitis B surface antigen in human sera or plasmas.

A. Preparation of hepatitis B surface antigen (HBs) HBs protein was prepared from human sera which had high concentrations of hepatitis B antigens of the subtype adw or ady. Methods of chromatography and L 7 centrifugation which complied with the state of the art were used: Gerlich et al., 1975, Developments in Biological Standardization 30, 78-87; Gerin et al., 1975 J.

Immunol. 115, 100-105. The concentration of the preparations of hepatitis B surface antigen prepared in this wey was quantified by the method described by Stamm et al., 1980, J. of Biological Standardization.

f B. Labeling of the hepatitis B surface antigen (HBs) with horseradish peroxidase (POD) The HBs preparation from human serum was labeled with POD after reconstitution of the protein solution to 4 mg/ml in phosphate-buffered saline, pH 7.2, using the periodate technique of Nakane et al., 1974, J. Histochem. Cytochem. 22, 1084-1090. This entails 20 mg of POD being dissolved in 1 ml of phosphate-buffered saline, pH 7.4, and, after addition of 2,4-dinitrofluorobenzene, being treated with sodium metaperiodate. Gel filtration was subsequently carried out on RBiogel P 6 equilibrated with 0.2 mol/l sodium carbonate/bicarbonate, pH and the fraction of the POD which contained aldehyde I groups was reacted with 4 mg/ml HBs. Addition of sodium borohydride was followed by renewed gel filtration, and the POD- and HBs-containing fraction I was obtained as POD-HBs conjugate with a protein A content of 1 mg/ml and a POD:HBs ratio of 1.5:1 to 2.0:1.

To this fraction were added 20 mg/ml bovine serum albumin and 1 mg/ml phenol. Used for further dilution was a solution, called conjugate buffer, of g/l hydrolyzed gelatin crosslinked with hexamethylene diisocyanate, a Behringwerke AG commercial product called polygeline, and 50 mmol/l tris, adjusted to pH 7.4 with HC1.

8 To establish the conjugate dilutions both for the method in Example D, the state of the art, and for the one-step method according to the invention in Example E, both two- and five-fold serial dilutions were prepared, contacting all dilutions with the plasma specified in the table and with the negative control and, in each case, all with various amounts of HBs-coated wells of the microassay plates in Saccordance with the two methods.

It was found that conjugate dilutions of 1:50 and wells coated with solutions of 0.4 pg/ml HBs (see Examples C and D) produced results in a two-step method comparable with conjugate dilutions of 1:20 and wells coated with solutions of 1.0 pg/ml HBs in the one-step method (see Examples C and E).

C. Coating of microassay plates with HBs Microassay plates, i.e. immunoplates II 96 F with a round bottom (from Nunc, Roskilde, Denmark, Cat. No.

262162) were incubated with 125 1 per well of solutions containing 1.6, 1.0, 0.8, 0.4, 0.2 and 0.1 pg/ml HBs antigen in phosphate-buffered saline, pH 7.5, at room temperature for several hours. The plates were subsequently sucked empty, washed with phosphate-buffered saline, dried with silica gel and placed in air- and moisture-tight packs.

D. Determination of antibodies specific for HBs antigen in a two-step method according to the state of the art a) 200 ip of the undiluted serum sample to be investigated were introduced into each well of a microassay plate coated with a solution of 0.4 pg/ml HBs. As reference, 200 Al of undiluted human serum free of HBs antigen antibodies .,ere 9 introduced (negative control) as well as serial dilutions of a sample containing antibodies against HBs antigen; b) the assay plate was covered and incubated at 18-25 0 C for 16 to 24 hours; c) subsequently the contents were sucked out and Swashed 3 times with phosphate-buffered saline containing 0.1 ml of Tween d) now 100 pl of 1:50 conjugate dilution were added to each well; e) the assay plate was covered and incubated at 40 0

C

for 1 h; f) renewed washing three times with phosphatebuffered saline, pH 7.5, was followed by addition S 15 of 100 pl of chromogen solution (tetramethylbenzidine (TMB) substrate formulation as in Example 1 of DE 3,541,979) to each well and incubation at room temperature for 30 min; e) 100 pl of 1 normal sulfuric acid were added to each well to stop the enzymatic conversion of substrate, and the solutions were measured at 450 nm; E. Determination of antibodies specific for HBs antigen by the one-step method according to the invention a) 25 pl of the 1:20 conjugate dilution are introduced uniformly into each well of a microtiter plate coated with a solution of 1 pg/ml HBs; J b) 100 pl of the undiluted serum sample to be determined were added. As reference, 100 pl of undiluted human plasma free of HBs antigen antibodies were introduced (negative control) as well as serial dilutions of a sample with a V~ wn content of antibodies against HBs antigen; c) the assay plate was covered and incubate for 1 h; d) the contents were subsequently sucked out, washed three times with phosphate-buffered saline L I I-.

10 containing 1 ml of TweenR 20, and 100 pA of chromogen solution (TMB substrate formulation) were added and incubated at room temperature for min; e) then 100 Al of 1 normal sulfuric acid were added to each well to stop the enzymatic conversion of I substrate, and the solutions were measured at 450 nm; f) Examples of results obtained: The samples were also assayed by the two-step methods described in sections D a) to D The results of the two methods are compiled in Table 1.

Table Extinctions i 1-step method 2-step method i Assay dura- Assay duration 90 min tion 18-25 h Strongly pos. plasma 2.000 2.000 Weakly pos. plasma 4 0.210 0.295 Negative control 0.021 0.035 plasma 0.029 0.041 i A sample was assessed as positive when the extinction was i 25 higher than a figure derived from the extinction of the j negative control plus 0.050 E.

It is evident from the results for the extinction, which represents a measure of the conteit of antibodies specific for HBs antigen in the human samples, that the onestep method produces, in the quantification via the standards of known antibody content, results equivalent to the two-step method.

In addition to the human samples listed in Table 1, 500 negative sera and plasmas and 150 sera and plasmas containing HBs antibodies were found to be negative and L i

-I

11 positive, respectively, in the two assays consistently.

The agreement of the antibody content in IU/ml determined with the two methods was excellent.

Accordingly, detection of antibodies specific for HBs antigen by the one-step method described is just as sensitive as the two-step assay. However, in contrast to the multistage assay method, the detection of the said and other antibodies is more rapid and straightforward, as is shown by comparison of the work involved in the two methods which is evident from sections D and E.

Example 2 Determination of immunoglobulins specific for hepatitis B core antigen A. Preparation of hepatitis B core antigen (HBc) The hepatitis B core antigen was prepared using DNA technology in Escherichia coli as described by Stahl et al., 1982, Proc. Nat. Acad. Sci. USA 79, 1606-1610.

B. Enzyme-labeling of the HBc antigen The labeling of HBc antigen was carried out as described for HBs antigen in Example 1 under B. The optimal dilution of the HBc-POD conjugate for use in the assay was correspondingly determined by checkerboard titration.

C. Coating of microassay plates with HBc antigen The coating was carried out as described for the HBs protein in Example 1 under C. However, the coating solution also contained 1 pg/ml gelatin in addition to the HBc antigen.

D. Determination of antibodies specific for HBc antigen 12 (anti-HBc) in a competitive assay according to the state of the art Al of sample and subsequently 100 pl of an anti- HBc-POD conjugate were pipetted into each well of the microassay plate. The samples were in the form of serial dilutions of the anti-HBc standard material from the Paul-Ehrlich Institute in the concentration range from 0 to 5 U/ml. In the subsequent S° incubation at 37 0 C in a water bath for one hour, the anti-HBc antibodies in the sample compete with the o'°o anti-HBc-POD conjugate for the binding sites on the O o solid phase.

-0 SAfter the wells had been washed, 100 pl of a solution of tetramethylbenzidine greater than the TMB substrate formulation in Example 1 of DE 3,541,979 were added and incubated at room temperature for S' 30 min.

The reaction was then stopped by addition of 100 pl of 1 N sulfuric acid, and the solution was measured at 450 nm.

The competitive principle means that samples which contain no anti-HBc produced high signals whereas samples with anti-HBc antibodies brought about an inhibition of the signal depending on the content.

Thus, the height of the signal was inversely propor- Stional to the anti-HBc concentration.

A sample was assessed as anti-HBc-positive when the reduction in the signal in comparison with the negative control was less than I 13 E. Determination of antibodies specific for HBc antigen by the one-step method according to the invention 100 pl of the HBc-POD conjugate were pipetted into each well of the microassay plate and subsequently 25 pl of sample were added. The samples were serial dilutions of the anti-HBc standard material from the Paul-Ehrlich Institute in the concentration range from 0 to 5 U/ml.

j! During the subsequent incubation at 37°C in a water bath for one hour, the sandwich complex between the solid-phase HBc, the anti-HBc and the HBc-POD conjugate was formed in the case of samples containing anti-HBc.

After the wells had been washed, 100 pl of TMB substrate formulation were added and incubated at room temperature for 30 min.

The reaction was then stopped by addition of 100 pl of 1 N sulfuric acid, and the solution was measured at 450 nm.

The sandwich principle means that samples which contain no anti-HBc produced no signal (or only the background signal), whereas samples containing anti- HBc antibodies bring about an increasing signal which depends on the content. Thus, the height of the signal was directly proportional to the anti-HBc concentration.

A sample was assessed as anti-HBc-positive when its extinction was more than 0.025 E above the signal for the negative control. The results obtained by the method according to the invention agreed with the results of the competitive method.

L i 1

Claims (8)

1. 2. The process as claimed in claim i, wherein hepatitis B surface (HBs), core (HBc) or envelope (HBe) antigen is used.
2. 3. The process as claimed in claim i, wherein one or more constituents or fragments of HIV 1 or HIV 2 or of both is used as antigen.
3. 4. The process as claimed in claim i, wherein one or I i' more constitutents of hepatitis A virus are used as antigen. The process as claimed in claim i, wherein one or more constituents of non-A, non-B hepatitis virus are used as antigen.
4. 6. An agent containing a carrier loaded with antigen as solid phase and a labeled antigen with the same specificity as the antigen on the solid phase, where the amount of the antigen on the solid phase and that of the labeled antigen are selected in a process as claimed in claim i.
5. 7. An agent as claimed in claim 6, wherein the antigen is HBs, HBc, HBe or a fragment thereof.
6. 8. An agent as claimed in claim 6, wherein the antigen contains one or more constituents or fragments of HIV 1 and/or of HIV 2.
7. 9. An agent as claimed in claim 6, wherein the antigen contains one or more constituents or fragments of hepatitis A virus. An agent as claimed in claim 6, wherein the antigen contains one or more constituents or fragments of Snon-A, non-B hepatitus virus.
8. 11. The use of an agent as claimed in claim 6 in a method for determining antigen-specific antibodies, in which the sample and the labeled antigen are simultaneously contacted with the solid phase, sub- sequently the solid phase is separated from the liquid phase, and the labeling on the solid phase is determined. DATED this 2nd day of November 1989. BEHRINGWERKE AKTIENGESELLSCHAFT WATERMARK PATENT TRADEMARK ATTORNEYS QUEEN STREET MELBOURNE. VIC. 3000.