JPS5840142B2 - Method for detecting immunoglobulin or antigen or hapten - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for qualitatively or quantitatively detecting the presence of an immunoglobulin, the Fab portion of which binds or is capable of binding to an antigen or hapten. The present invention relates to a method for detecting the presence of an antigen or hapten that is present or capable of binding.

In this case, the immunoglobulin has one or two molecules that can be analytically detected before or after binding to the antigen or hapten.
The label is labeled with one or more analytically detectable atoms or groups upon contact with the polypeptide labeled with one or more atoms or groups.

The term "polypeptide" as used herein also includes proteins.

Such methods are already known.

That is, it is already known to use, for example, an antibody labeled with radioactive iodine as a labeled polypeptide.

Preparation of antibodies is a complex process. Furthermore, antibodies are heterogeneous substances with varying binding capacities.

Preparing Antibodies to Immunoglobulins For example, if antibodies to rabbit immunoglobulins are prepared in goats, the antibodies bind only to immunoglobulins from rabbits and usually not to immunoglobulins from other animals.

In order to label individual immunoglobulins, it is necessary to prepare special antibodies that can react with the immunoglobulin in question.

In addition, labeling antibodies with displayable atoms or groups often results in loss of immunological activity.

One object of the present invention is to at least substantially eliminate such drawbacks.

The main feature of the present invention is that in the above-mentioned method, the immunoglobulin belongs to the IgG class, and the polypeptide is obtained from a microorganism and has the ability to bind to the Fc part of the above-mentioned immunoglobulin, which belongs to the IgG class. It is in being.

Polypeptides with the above properties from microorganisms are relatively easily obtained and can be labeled with analytically detectable atoms or groups in good yields.

An important advantage obtained when a labeling polypeptide having the above properties is used for labeling immunoglobulins according to the method of the invention is that it can bind to the Fc portion of immunoglobulins from a variety of animals; by F
The ab moiety is freed and becomes more susceptible to binding antigens and haptens.

Known methods for labeling antibodies against the Fc portion of immunoglobulins require the preparation of the Fc fragment of the immunoglobulin in a pure state, which is a difficult process, and require individual labeling for each type of animal. It requires immunizing the animal with an Fc fragment of an immunoglobulin.

Another advantage offered by labeling methods is that, unlike antibodies,
The polypeptide is a single substance that can bind to the immunoglobulin in a defined manner.

The displayable atoms or groups that label the polypeptide may be radioactive and/or colored and/or fluorescent and/or contain free radicals and/or have enzymatic activity. Either is fine.

Methods for labeling polypeptides according to the above are well described in the literature and can be applied to polypeptides from microorganisms in the present invention.

Specific examples of radioactive isotopes related to this include iodine isotopes (e.g., 125■) 14C
and tritium, which are introduced directly into the polypeptide or by substituting the polypeptide with a group containing the isotope.

A convenient way to label polypeptides is to introduce fluorescent groups, for example with the aid of fluorescein inthiocyanate.

Enzymes (eg, peroxidases or hydrolases, such as phosphatases) can be coupled to polypeptides by CNBr or geltaraldehyde.

The polypeptide is labeled with an analytically detectable atom or group, and thus the immunoglobulin is also labeled), and can be readily detected and measured by conventional methods.

The polypeptide from the microorganism used may be, for example, the so-called protein A from Staphylococcus aureus or a fragment of this protein.

Said fragment has polypeptide properties and is capable of binding at least one immunoglobulin in its 10 parts.

The above polypeptide, protein A cleaved from Staphylococcus aureus and its fragments, can bind to immunoglobulins belonging to the IgG class in the Fc portion.

Labeling of immunoglobulins with labeled polypeptides can be advantageously applied to qualitatively or quantitatively display immunoglobulins or antigens or haptens, wherein said antigens or haptens can be labeled using the method of the invention to label the Fab portion of the immunoglobulin. join or be joined to.

Labeling of immunoglobulins with labeled polypeptides is
In the presence of a liquid (preferably an aqueous liquid) in which the labeled polypeptide is soluble, the polypeptide binds to the Fc of the immunostimulating purine.
This is carried out as appropriate under conditions that bind the moieties.

Thus, the pH is appropriately selected, for example, in the vicinity of the neutral point, for example within the range of pH 5 to 9, for example within the range of pH 6 to 8.

If desired, the labeled immunoglobulin can be purified, for example by gel filtration or specific adsorption, when in solution or when in insoluble form, for example when bound to an insoluble or insolubilized antigen or hapten; When it is directly or indirectly bound to an insoluble polymer, it can be purified by washing.

Excess free labeled polypeptide can be removed during purification of labeled immunoglobulin.

The present invention also provides an adjunct for labeling immunoglobulins in free or bound form with one or more analytically detectable atoms or groups, which are used in the method of the present invention. The present invention relates to an adjuvant consisting of or containing a polypeptide labeled with an atom or group capable of being labeled.

A feature of the adjuvant is that the polypeptide is obtained from microorganisms and is capable of binding itself to the Fc part of immunoglobulins belonging to the IgG class.

Auxiliary agents can be advantageously used in the labeling methods described above.

The adjuvant may be present in the form of a solution, preferably an aqueous solution.

It may also be present in solid form, such as lyophilized form, so that it can easily be brought into solution at the time of use.

The method of the invention may form an important step in the qualitative or quantitative determination of immunoglobulins, for example together with an immunochemical reaction in which immunoglobulins are or can be bound to antigens or haptens.

The above-described labeling methods can be utilized to prepare reagents for qualitatively or quantitatively detecting antigens or haptens in free or bound form for use in the methods of the present invention.

Such reagents may consist of or contain the reaction product between a polypeptide and an immunoglobulin labeled with one or more analytically detectable atoms or groups.

According to the present invention, the reagent is such that the polypeptide is obtained from a microorganism and is capable of binding to the Fc part of an immunoglobulin belonging to the IgG class, so that the Fab part of the immunoglobulin having antibody activity is activated by an antigen or It is unique in that it can bind haptens.

Such reagents may be present in the form of a solution, preferably an aqueous solution.

It may also be present in solid form, for example in lyophilized form, so that it can be easily brought into solution at the time of use.

The reagent is appropriately used in the presence of a liquid (preferably an aqueous solution) under conditions such that the Fab portion of the immunoglobulin having antibody activity can bind to the antigen or hapten.

The antigen or hapten may be in free or bound form, for example bound to the surface of a cell or to a polymer, such as an insoluble polymer.

The antigen may also be bound to another antibody.

Hereinafter, the present invention will be explained by examples.

Example I Preparation of Labeled Protein A from S. aureus A S
Preparation of crude extract of protein A from S. aureus, strain Cowan I, as described in the European Journal of Biochemistry (
The cells were cultured by the method described in European J. Biochem, Vol. 29 (1972), p. 572 (Squist et al.).

Protein A was separated from bacteria by the enzyme preparation Lysostaphin.

Insoluble materials were removed by centrifugation and the liquid phase was collected.

The p) was adjusted to 1,1':3.5 using HCA, insoluble material was removed by centrifugation, and the liquid was collected.

The pH was then adjusted to 7.0 using NaOH.

All operations were performed according to the methods described in the above literature.

The liquid thus obtained is a crude extract containing protein A in a mixture with impurities.

B Preparation of IgG-conjugated agarose For this purpose, the commercial product Sepharose '4B (Pharmacia 7'-'7 Huynh Chemicals
Agarose in the form of Fine Chemicals AB Uppsala Sweden) was used.

Agarose is a small particle (40-190μ) swollen in water.
? 7L).

The particle assembly contained just over 4 weight agarose.

The particle aggregate was first washed with water.

A solution of 10 g of cyanogen bromide (CNBr) in 50 TL of water was added with stirring at 20°C to 100 rfL of a mass of particle aggregates mixed with 50 N of Na water.
The pH was kept at lO~11 by adding OH.

After 10 minutes, the particle assembly was thoroughly washed with ice water and then with 0.2 M sodium carbonate-sodium bicarbonate buffer in water (pH 9, temperature 4°C).

Particle aggregates activated with cyanogen bromide were added to 120 ml of the above buffer at pH 9.0 containing 3 g of human IgG (Kabi AB, obtained from Stockholm Sweden).
1 was used to form a slurry at 4°C with stirring.

After 4 hours, the particle aggregate was separated from the furnace and washed with the above buffer solution at pH 9.0, and then the particle aggregate was soaked in an aqueous solution containing 0.05 M2 amino-ethanol and 0.2 M sodium carbonate (pH 9.0). ) 1.51 and stirred at 4° C. for 18 hours.

The particle aggregate was then washed with 0.1 M sodium phosphate buffer (pH 6.0) containing 4 M urea, and further washed with 0.1 M sodium phosphate buffer (pH 7.0) containing 4 M urea.
), and the 0D280 nanometre (nm) of the cleaning solution is 0.
．． It was washed until it became 01 or less.

The gel was then washed with 0.1 M glycine-HC1 buffer in water (pH 3,0) and then washed with the above 0.1 M IJ.
The cells were washed again using sodium chloride buffer (pH 7.0).

The product thus obtained contained approximately 30 μg of bound IgG per 11 rLl of particle mass.

CS, Separation of Protein A from Crude Extract of C. aureus A mass of particle aggregates bound to IgG at pH 7.0 obtained by B above 1007717! was packed into a chromatography column.

pH 7.0 containing protein A obtained by A above
500 ml of the crude extract was slowly passed through a column packed with particle aggregates at 20°C.

The flow rate at this time was adjusted to 50 TLl/hour.

The particle aggregate in the column was dissolved in 0.1 M water at pH 7.0.
From the IJ sodium phosphate buffer, the OD28 of the washing solution
It was carefully washed until it became 0 nm or 0.02 or less.

The product thus obtained contained approximately 3 μ of binding protein A per liter of mass ITL of the particle assembly.

D. Separation of Protein A from Particle Aggregates Protein A bound to the particles obtained in step C above is separated from
．． 1 M glycine-H (1 buffer (pH 3,0) 10
0- was used to elute the column at acidic pH to separate it from the particle aggregates.

Collected protein A containing glycine-H (l buffer)
was dialyzed against distilled water.

Protein A was then obtained in solid form by lyophilization.

Approximately 250 μ of protein A was obtained in pure form.

(In an alternative method, desalting was performed by gel filtration instead of dialysis.

) It was confirmed by an immunological test that Protein A did not contain any impurities.

E. Preparation of Labeled Protein A Protein A obtained by the above method was labeled with 125■ by a method using -bromine chloride as a reagent.

Labeling can be performed in the following manner.

Radioactive iodine (12J) Iodine-125 is 0.1M N as NaI without carrier
A solution without reducing agent dissolved in aOH was used.

Iodine monochloride Iodine monochloride J, L, Izzo, W, F, Ba1e,
M.J.

Journal by Izzo, A. Roncone
of Biological Chemistry (J, Biol,
Chem,) Volume 239, No. 11 (1964), 37
It was prepared by the method described on page 42.

Iodine content was determined by reducing total iodine to iodide with arsenic trioxide followed by titration with 0.1M AgNO3.

The storage solution for I (J' is 0.02M KCl? and 2.OM N
It contains 2.54 iodine in one column of aCl, t, and OMHcl, and is stable for at least 18 months at room temperature.

Before use, the basic solution of ■Cl was diluted to the required concentration.

The diluent used is a hydrochloric acid-salt solution, the concentration of which is adjusted individually for each experiment, with I (l being above the lowest concentration of HCl and NaCl known to be stable). is adjusted to

(R, W, Helmkamp, M, A, Contre
ras and W.

F, Ba1e: Int, J, Appl, Radia
t, l5otopes.

18 (1967) 737) Labeling method Iodination is performed using McFairlane (McFairlane).
) - Iodine chloride method (A, S, McFair
1ane, Nature.

182 (1958) 53) with additional modifications.

(J, L, Izzo, W, F, Ba1e, M, J, Iz
zoand A, Roncone, J., Biol, C.
hem, 239, A 11 (1964) 374
3, W,F,Ba1e,R,W.

Helmkamp, T.P., Davis, M.J.
Izzo, R.L.

Cooland, M.A., Contreras an.
d J, L, 5par.

Proc, Soc, Exp, Med,, 122 (1
966) 407°R, W, He 1mkamp, M
, A. Contreras and W.F.

Ba1e, Int, J, Appl, Radiat, l5
otopes, 1s (1967) 737).

100 μg of protein A was labeled at a molar ratio of C4 to protein A of 1.875:1 and a molar ratio of Cl to 125 of 2:1.

The following solution was used.

Solution I: Protein A was dissolved in 0.05 M sodium phosphate buffer, pH 7.4, to a concentration of 51 v/1 rll.

Solution II: Dissolve 5.84 g (7) of NaCl in 12 ml of 1.0 MH (l) and 50 mA of distilled water, add distilled water to make a total volume of 100-1, and add diluent for Cl stock solution ( 0,12M HCl, 1, OM NaCl) was prepared.

Solution ■: The final Ice solution was prepared by diluting 100 μl of ■C4 stock solution with 15.58 g of solution ■.

Solution ■: Add 19.4 μl of radioactive iodine (5,9 mC1) to 40 μl of Solution ■ and keep on water bath until use.

All solutions were kept cool and the iodination reactions were carried out at 0°C.

Reagents were added in the following order into small glass tubes.

Mix 50 μl of solution 1 with 70 μl of sodium borate-sodium carbonate.

solution (pH 9.1 with 0.24 M sodium borate and 0.16 M sodium carbonate) and 20 μl of solution I (100
(containing μg of protein A) and immediately mixed well.

After 120 seconds, 2Jtlj of 0.01M Na2S2O3
, 50 μl of 2% KI and average molecular weight Mw = 20
200 μl of a 3% solution of dextran with 1,000 ml of dextran was added.

The 125-iodinated Protein A preparation was purified by gel filtration or dialysis using epichlorohydrin-crosslinked dextran (Sephadex- or polyacrylamide) in alkaline solution.

The fraction from the gelled percolumn was 0.05M phosphorus containing 0.05% Tween 20 (sorbitan monolaurate of polyethylene glycol 900) and 3% dextran with an average molecular weight Mw = 20,000. acid sodium buffer (pH 7
, 4) at approximately 4°C.

The fractions in which labeled protein A was detected were combined and stored frozen.

In this method, a yield of about 75% was obtained based on the theoretical amount of iodine that could be combined.

Labeled protein A has a molecular weight Mw = 42,000
125 ■ of about 0.5 atoms and 1 of about 160 atoms per )
271 and had a specific radioactivity of approximately 24 m, Ci/■.

Example 2 Protein A from S, Aureas for labeling purposes
Preparation of a polypeptide fragment of protein A from AS, S. aureus, strain Cowan 1, European Journal of Biochemistry Vol. 29 (1972); 572 pages (SJ''
The cells were cultured according to the guidelines described in 6quist et al.).

Bacteria were removed by centrifugation and 0.9% N in water.
Washed with aCl solution.

100g (wet weight) of bacteria in 1 part normal saline
50 to form a slurry.

To this was added 10 μl of trypsin (without chymotrypsin).

The pH was 7.2.

Enzyme treatment was carried out for 30 minutes at 30° C. and pH 7.2, after which trypsin inhibitor 201n9 obtained from soybean was added.

The suspension was then centrifuged.

Next, the supernatant liquid was collected, and Millipore (Millipore)
) Filtered and sterilized.

The sterilized liquid was a mixture of contaminants and contained fragments of polypeptide protein A.

In order to purify fragments that are capable of binding the Fc portion of IgG molecules, the above fragments are combined with IgG
was isolated using agarose binding.

B Preparation of IgG-conjugated agarose IgG-conjugated agarose was prepared as in Example 18.

The product obtained was about 301 v per particle mass ITrLl.
of bound IgG.

Preparation of Polypeptide Fragment of Protein A from CS, Aureus The IgG-conjugated particle aggregate 100- of pH 7.0 obtained in Section B above is loaded into a chromatography column.

100 - of the crude extract at pH 7.0 containing the polypeptide fragment of protein A from Section A above is passed slowly through the column containing the particle agglomerate at a flow rate of 50 vertical strokes per hour at 20°C.

Next, the particle aggregate in the column was thoroughly washed with 0.1 MIJ sodium phosphate buffer in water (pH 7.0).

The cleaning is continued until the OD280nm of the cleaning solution becomes 0.02 or less.

The product thus obtained is about 1 -
of the binding polypeptide.

D. Separation of polypeptide fragments from protein A from particle assemblies.
H3,0) was separated from the particle aggregates by eluting the column at acidic pH with 100 vtl.

The collected polypeptide containing the glycine-HC1 buffer, which is capable of binding the Fc portion of the IgG-molecule, is removed by gelation with Sephadex G25 (a particle assembly of dextran cross-linked with epichlorohydrin). I salted it.

Approximately 100 μm of polypeptide with a molecular weight of approximately 7000 was isolated by lyophilization.

The polypeptide fraction was found by chromatography to consist of several closely related polypeptides, all of which are capable of binding the Fc portion of the IgG molecule.

In particular, the polypeptide fraction was found to be free of contaminants by immunoassay.

A polypeptide fragment capable of binding the Fc part of an IgG-molecule is described in Swedish Patent Application No. 14329/
72 by first isolating protein A and then treating protein A in solution (e.g. treatment with trypsin at pH 8.2 similar to that described above).
Polypeptides that can be isolated and then have the desired properties are separated by binding the fragments to agarose binding IgG in a manner corresponding to that described in Example 2 and then separating the polypeptide fragments. be done.

The resulting polypeptide product can be used to label with, for example, fluorescent or radioactive atoms or groups.

Similar to protein A, the resulting polypeptide product can be labeled in conventional manner, for example with fluorescein inthiocyanate.

Fluorefine inthiocyanate labeled products can advantageously be used to label immunoglobulins of the IgG-class.

This product can be used to prepare immunoglobulin Fa by the method of the present invention.
It can be used to qualitatively or quantitatively detect immunoglobulins or antigens or haptens bound or about to be bound to the b moiety.

Labeling with fluorescein inthiocyanate can be carried out, for example, as follows.

0.25M sodium carbonate-sodium bicarbonate buffer containing 0.45% Na C13 (pH 9.0)
To 10 m9 of polypeptide product dissolved in 1 TIL1 was added 1 m1j of fluorescein inthiocyanate.

The mixture was then stirred for a few minutes and left at 4°C for 12 hours.

The mixture was then centrifuged.

To clean the solution with labeled polypeptide from unreacted fluorescein isothiocyanate, the mixture was
% NaCl aqueous solution swollen with epichlorohydrin cross-linked dextran (Sephadex G25@)
and eluted with the same solution.

Next, the eluted fraction containing the labeled polypeptide was collected.

Adjust pH to 7.0. The solution may be stored frozen and the product lyophilized.

The product exhibits fluorescence under ultraviolet light. Labeling can be carried out in a similar manner, for example using tetramethylrhodamine inthiocyanate.

A labeled polypeptide can be attached to the Fc portion of an IgG molecule and used in the methods of the invention.

This means that cells with IgG-class antibodies, immunochemically bound to cell surface antigens, are exposed to phosphate buffer (p
This was demonstrated by treatment with a solution of protein A labeled with fluorescein isothiocyanate in H7,2).

After washing the cells with the same buffer, it was possible to detect and measure the fluorescence from labeled protein A that had bound to IgG on the cell surface.

Example 3 Detection of IgG-Class Antigen-Bound Immunoglobulins with Labeled Protein A For this test, 125■ labeled Protein A from Example 1 above was used to detect rabbit IgG antibodies against human IgE.

This IgG antibody is immunochemically conjugated to human IgE (antigen).
It was covalently bound by cyanogen bromide to small particles of dextran (Sephadex G25■, Ultrafine) which were bound by ab moieties and then cross-linked with epichlorohydrin in an alkaline solution.

During the test, 0.05 μ of the above particles conjugated with IgE were suspended in 1 ml of normal physiological saline.

The solution was then mixed with 75×10 9.9 rabbit anti-I
The cells were incubated for 3 hours with 100 μl of normal saline containing gE-antibody.

The particles were then centrifuged three times with 2.5 rnl of normal saline each time.

100 μl of normal saline containing 2×10 −9.9 of labeled protein A was added and the mixture was diluted with 1
The cells were incubated for 8 hours and then washed in a centrifuge five times with 2.5 ml of normal saline each time.

The gamma radiation of the particulate matter is then exposed to Walack.
lac) Measured using a gamma ray measuring device.

Approximately 25,000 counts were obtained every 5 minutes compared to a blank test.

This indicates that IgG-antibodies bound to IgE (antigen) are present on the particles.

Example 4 Detection of IgE (antigen) by labeled protein A bound to an antibody against IgE Fc of an antibody against IgE (belonging to the IgG-class)
The 125■ labeled protein A (obtained in Example 1) bound to the moiety was used for the test.

Reagents are 40X10-9 in 2TLl of normal saline.
g of 125 ■ Labeled protein A and 1100XIO-9
rabbit anti-IgE was cultured for 2 hours.

(Extra labeled protein A was present).

0.05 m9 of Sephadex 025 particles (Epichlorohydrin cross-linked dextran, Ultrafine) with human IgE (antigen) bound with cyanogen bromide were suspended in normal saline lTtl.

Next, 100 μl of the above reagent was added. After 18 hours, the particles were centrifuged five times with 2.5 TLl of normal saline each.

Next, gamma radiation of the particulate material was measured using a Wallac gamma ray measuring machine.

40.000 counts were obtained every 2 minutes compared to the blank, indicating that IgE (antigen) is present on the particles.

Example 5 Peroxidase-labeled protein A peroxidase was labeled with S in Example 1 by the following method.

It combined with protein A from Aureus.

15 μm of peroxidase (obtained from horseradish, manufactured by Sigma) was added to a 0.1 M aqueous sodium phosphate solution (p
0.25% solution of glutaraldehyde in H6,8).
It was dissolved in 2 ml.

This solution was left at 20°C for 18 hours.

Next, this solution was filled with swollen particles of epichlorohydrin-crosslinked dextran (Sephadex G-2'5@, Pharmacia Fine Chemicals AB, Uppsala Sweden) and a 0.1 M aqueous sodium phosphate solution (pH 6,8). Column (length 50CrrL1 diameter 1cIrL)
passed through.

The same phosphate buffer was also used to elute the column.

A colored fraction eluted in the void was collected during this gel chromatography.

The collected colored fractions were purified by ultrafiltration (PM-
10 membrane, those with a molecular weight of 10.000 or more will not pass through).

Five inches of protein A dissolved in the above phosphate buffer 1rILl was added to the geltaraldehyde activated solution of peroxidase (1TLl).

Thereafter, 0.2 TLl of 1M sodium carbonate-sodium hydrogen carbonate buffer (pH 9.5) was added.

The mixture was left at 4°C for 24 hours.

Next, a 0.2M aqueous lysine solution of 0.2a was added.

(Adjust the pH of the lysine solution to 7.0 in advance with a 5M NaOH aqueous solution).

After adding the lysine, the mixture was left at 20° C. for 2 hours.

Next, the solution was transferred to a column packed with swollen IgG agarose particles and 0.1 M sodium phosphate solution (pH 6,8).
The length was 10crfL1 and the diameter ICrrL).

(IgG agarose was prepared in the same manner as in Example 1).

Protein A with peroxidase attached was attached to IgG on the agarose particles.

A 0.1 M aqueous sodium phosphate solution (pH 6.8) was passed through the column to wash away unbound substances.

The column was then washed with 0.1 M glycine-HC at pH 3.0.
4 buffer.

The colored fractions were collected and combined. The solution was neutralized with 5M aqueous NaOH.

The solution was subjected to ultrafiltration (UM-2 membrane, molecular weight approximately 2.0
00 or higher (does not pass through) and concentrated to 1 ml.

The solution was dialyzed against pH 7.2 buffer for 12 hours. (This buffer contains 8 g NaCl/14 solution.

(prepared by dissolving 0.2 g of KCL, 1.15 g of Na2HPO4 and 0.2 g of KH2PO4 in water).

The peroxidase-labeled protein A solution was sterilized by passing through a Millipore filter.

The solution was appropriately stored at 4°C. Protein A conjugated with peroxidase can be used in the method of the present invention,
Peroxidase activity was measured by conventional methods.

In the same manner as in Example 2, it was confirmed by peroxidase detection that the protein A bound to peroxidase was bound to an IgG-antibody (against the antigen) that was immunochemically bound to the antigen on the cell surface.

In this way, the immunoglobulin IgG bound to the antigen
However, ■antigens bound to gG can also be detected indirectly by a simple enzymatic chemical assay.

Claims (1)

[Claims] 1 Qualitatively or quantitatively determining the presence of an immunoglobulin whose Fab portion of an immunoglobulin binds or can bind to an antigen or hapten, or the presence of an antigen or hapten that binds or can bind to a Fab portion of an immunoglobulin. A method of detecting, the method comprising: contacting the immunoglobulin with a polypeptide labeled with one or more analytically detectable atoms or groups, either before or after binding of the immunoglobulin to an antigen or hapten. and labeled with said atom or group, and said immunoglobulin belongs to the IgG class and said polypeptide is protein A from Staphylococcus aureus or a polypeptide-like fragment of protein A thereof and belongs to the IgG class. A detection method, characterized in that it is capable of binding itself to the Fc portion of the immunoglobulin.