CA1243944A - Reagent and process for quantitatively measuring antibodies - Google Patents
Reagent and process for quantitatively measuring antibodiesInfo
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- CA1243944A CA1243944A CA000460585A CA460585A CA1243944A CA 1243944 A CA1243944 A CA 1243944A CA 000460585 A CA000460585 A CA 000460585A CA 460585 A CA460585 A CA 460585A CA 1243944 A CA1243944 A CA 1243944A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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Abstract
Abstract Antibodies against bacterial peptidoglycans Antibodies from the classes of immunoglobulins A, M and E against the peptide sub-unit pentapeptide L-Ala-D-Glu(L-Lys-D-Ala-D-Ala) of peptidoglycan in biological test material and against bacterial cell walls containing these peptidoglycans may be extracted from human plasma and are useful in the investigation of bacterial infections. Such antibodies may be characterized by their ability to bind to:
a) pentapeptides of formula I
R - A1 -D -Ala (I) (wherein R represents an amino acid component with 1 - 3 amino acid residues selected from the group comprising Gly, L-Ala, D-Glu, L-Lys; and A1 represents one of the amino acid residues D-Ala, D-Ser, D-Val or D-Leu); and/or b) bacterial cell walls or cell wall material containing pentapeptides of general formula I; and/or c) Gram-positive bacteria containing pentapeptides of formula I.
a) pentapeptides of formula I
R - A1 -D -Ala (I) (wherein R represents an amino acid component with 1 - 3 amino acid residues selected from the group comprising Gly, L-Ala, D-Glu, L-Lys; and A1 represents one of the amino acid residues D-Ala, D-Ser, D-Val or D-Leu); and/or b) bacterial cell walls or cell wall material containing pentapeptides of general formula I; and/or c) Gram-positive bacteria containing pentapeptides of formula I.
Description
- 3~2~3~
ANTIBODIES AGAINST BACTERIAL PEPTIDOGLYCANS
This invention relates to antibodies against bacterial peptidoglycans and to processes for their quantitative determination.
As described in DE-A-32 31 204 ~eqUiVa1ent to Canadian Patent Application 434968) filed Aug. 19,1983, antibodies against the peptide sub-unit pentapeptide L-Ala-D-Glu(L-Lys~D-Ala-D-Ala) of peptidoglycan are indicators Eor acute or chronic bacterial infections.
It has been found that, in addition to the specific antibodies of the immunoglobulin class IgG which can be detected in the later stage of an infection, specific antibodies of the immunoglobulin classes IgAI IgM and IgE also occur during infection.
Thus, antibodies of the class Ig~ are found in the initial stages of an infection and in some cases even before any clinical symptoms occur.
Antibodies of class IgA can be detected in infections which initially manifest themselves essentially in the mucous membranes. Antibodies of class IgE
are found in diseases which involve allergic reactions.
According to one aspect, the present invention thus provides antibodies against the peptide sub-unit pentapeptide L-Ala-D-Glu(L-Lys-D-Ala-D-Ala) of peptidoglycans, characterized in that said antibodies are from the class of immmunoglobulins A, M or E. The antibodies of the invention are preferably isolated from other immunoglobulins and/or are in sterile solution and are suitably those characteri2ed by a) special binding to pentapeptides of formula I
R - Al - D-Ala (I) (wherein , , .
3~
R represents an amino acid component with 1, 2 or 3, preferably 3, amino acid residues selected from the group comprising Gly, L-Ala~ D-Glu and L-Lys; and Al represents one of the amino acid residues D-Ala, D-Ser, D-Val or D-Leu); and/or b) the ability to bind to bacterial cell walls or cell wa~l material which contain pentapeptides of formula I; and/or c) the ability to bind to Gram-positive bacteria which contain pentapeptides of for~ula I.
The antibodies of the invention, which can be used as standards for or as positive controls in the quantitative de~ermination of antibodies of the same class, can be extracted from plasma e.g. by affinity chromatograph~- as described hereinafter.
According to a further aspect, the invention provides a process for the quantitative determina~ion o~ antibodies from the class of immunoglobulins A, M and E against the peptide sub-unit pentapeptide L-~la-D-Glu(L-Lys-D-Ala~D-Ala) of peptidoglycans in a biological test material which process comprises contacting said test material with an antigen of formula I which is optionally labelled and optionally substrate bound. The process of the invention is conveniently performed according to immunochemical procedures known to the art, e.g. the double antibody ra~ioimmunoassay, the double antibody enzyme-immunoassay, the nephelometric method t the solid-phase sandwich radioimmunoassay, the solid-phase enzyme immunoassay, the solid~phase fluorescence immunoassay and the latex agglutination test.
Thus ~or example a general ~est scheme might involve: (a) coupling a pentapeptide of formula I bonded to a carrier polymer (e.g. albumin) to a solid phase (such as polystyrene~; (b~ contacting the solid phase with the test sample (e.g~ patient's 3~
serum) or a standard; (c) washing to remove unbound immunoglobulins; (d) contacting the solid phase on which the immunoglobulin is bound with corres-ponding anti-antibodies (anti-IgA, an~i-IgM or anti-IgE) which are coupled to an indicator system;
and (e) washing again to remove unreacted anti-antibodies. The indicator moieties can then be measured on the solid carrierO The tests for determining the differen~ types of immunoglobulin require different conditions which are described in detail in the Examples.
According to a further aspect, the invention provides a method of extracting the antibodies of the invention from human plasma which method comprises: (a) coupling a pentapeptide of formula I to a solid phase; (b) contacting said solid phase with IgA, Ig~ or XgE containing human plasma whereby said antibodies selectively bind to said pentapeptide coupled to said solid phase; and (c) subsequently selectively eluting said antibodies from said solid phase.
Standardisation of the test .
l. Obtainina a standard from s~ecific IaM and IgM and IgA are isolated by conventional methods (e.g. FPLC - ~ast Performance Liquid Cbromato-graphy) from sera which contain antibodies with specificity against the peptide sub-unit pentapeptide of formula I and the specific antibody fraction is obtained by affinity chromatography (with matrix-bonded peptides of formula I). After the specific antibodies have bound to the affinity matrix, any non-bound serum protein is removed by washing with buffer solutions. The elution of the antibodies bound to the soIid matrix is effected by specific desorption of the bound immunoglobulins with peptides ..~., of formula I. Desorption may also be effected by methods which have been published in principle, such as the use of low pH values (such as for example l-molar acetic acid) or high salt concentrations (such as for example 6-molar guanidinium chloride, 8-molar urea or 3-molar NaCl). After dialysis with buffer solutions, the content of specific antibodies against the peptide sub-unit pentapeptide o~ peptidoglycan is quan~ified by means of the immunoglobulin content of the purified fraction.
-
ANTIBODIES AGAINST BACTERIAL PEPTIDOGLYCANS
This invention relates to antibodies against bacterial peptidoglycans and to processes for their quantitative determination.
As described in DE-A-32 31 204 ~eqUiVa1ent to Canadian Patent Application 434968) filed Aug. 19,1983, antibodies against the peptide sub-unit pentapeptide L-Ala-D-Glu(L-Lys~D-Ala-D-Ala) of peptidoglycan are indicators Eor acute or chronic bacterial infections.
It has been found that, in addition to the specific antibodies of the immunoglobulin class IgG which can be detected in the later stage of an infection, specific antibodies of the immunoglobulin classes IgAI IgM and IgE also occur during infection.
Thus, antibodies of the class Ig~ are found in the initial stages of an infection and in some cases even before any clinical symptoms occur.
Antibodies of class IgA can be detected in infections which initially manifest themselves essentially in the mucous membranes. Antibodies of class IgE
are found in diseases which involve allergic reactions.
According to one aspect, the present invention thus provides antibodies against the peptide sub-unit pentapeptide L-Ala-D-Glu(L-Lys-D-Ala-D-Ala) of peptidoglycans, characterized in that said antibodies are from the class of immmunoglobulins A, M or E. The antibodies of the invention are preferably isolated from other immunoglobulins and/or are in sterile solution and are suitably those characteri2ed by a) special binding to pentapeptides of formula I
R - Al - D-Ala (I) (wherein , , .
3~
R represents an amino acid component with 1, 2 or 3, preferably 3, amino acid residues selected from the group comprising Gly, L-Ala~ D-Glu and L-Lys; and Al represents one of the amino acid residues D-Ala, D-Ser, D-Val or D-Leu); and/or b) the ability to bind to bacterial cell walls or cell wa~l material which contain pentapeptides of formula I; and/or c) the ability to bind to Gram-positive bacteria which contain pentapeptides of for~ula I.
The antibodies of the invention, which can be used as standards for or as positive controls in the quantitative de~ermination of antibodies of the same class, can be extracted from plasma e.g. by affinity chromatograph~- as described hereinafter.
According to a further aspect, the invention provides a process for the quantitative determina~ion o~ antibodies from the class of immunoglobulins A, M and E against the peptide sub-unit pentapeptide L-~la-D-Glu(L-Lys-D-Ala~D-Ala) of peptidoglycans in a biological test material which process comprises contacting said test material with an antigen of formula I which is optionally labelled and optionally substrate bound. The process of the invention is conveniently performed according to immunochemical procedures known to the art, e.g. the double antibody ra~ioimmunoassay, the double antibody enzyme-immunoassay, the nephelometric method t the solid-phase sandwich radioimmunoassay, the solid-phase enzyme immunoassay, the solid~phase fluorescence immunoassay and the latex agglutination test.
Thus ~or example a general ~est scheme might involve: (a) coupling a pentapeptide of formula I bonded to a carrier polymer (e.g. albumin) to a solid phase (such as polystyrene~; (b~ contacting the solid phase with the test sample (e.g~ patient's 3~
serum) or a standard; (c) washing to remove unbound immunoglobulins; (d) contacting the solid phase on which the immunoglobulin is bound with corres-ponding anti-antibodies (anti-IgA, an~i-IgM or anti-IgE) which are coupled to an indicator system;
and (e) washing again to remove unreacted anti-antibodies. The indicator moieties can then be measured on the solid carrierO The tests for determining the differen~ types of immunoglobulin require different conditions which are described in detail in the Examples.
According to a further aspect, the invention provides a method of extracting the antibodies of the invention from human plasma which method comprises: (a) coupling a pentapeptide of formula I to a solid phase; (b) contacting said solid phase with IgA, Ig~ or XgE containing human plasma whereby said antibodies selectively bind to said pentapeptide coupled to said solid phase; and (c) subsequently selectively eluting said antibodies from said solid phase.
Standardisation of the test .
l. Obtainina a standard from s~ecific IaM and IgM and IgA are isolated by conventional methods (e.g. FPLC - ~ast Performance Liquid Cbromato-graphy) from sera which contain antibodies with specificity against the peptide sub-unit pentapeptide of formula I and the specific antibody fraction is obtained by affinity chromatography (with matrix-bonded peptides of formula I). After the specific antibodies have bound to the affinity matrix, any non-bound serum protein is removed by washing with buffer solutions. The elution of the antibodies bound to the soIid matrix is effected by specific desorption of the bound immunoglobulins with peptides ..~., of formula I. Desorption may also be effected by methods which have been published in principle, such as the use of low pH values (such as for example l-molar acetic acid) or high salt concentrations (such as for example 6-molar guanidinium chloride, 8-molar urea or 3-molar NaCl). After dialysis with buffer solutions, the content of specific antibodies against the peptide sub-unit pentapeptide o~ peptidoglycan is quan~ified by means of the immunoglobulin content of the purified fraction.
-
2. Standardlsation of the determination of specific IgE
This is done by using a pooled serum from test subjects with a high specific IgE and subsequently comparing the sample measured with a standard curve (corresponding dilution).
This is done by using a pooled serum from test subjects with a high specific IgE and subsequently comparing the sample measured with a standard curve (corresponding dilution).
3. Design of test Quantitative determination of specific antibodies of the above immunoglobul}n classes against the peptide sub-unit pentapeptide of peptidoglycans can be effected by two fundamentally different methods:
a) quantitative determination in a soluble system tmeasurement in a homogeneous phase); and b) quantitative determination by measurement in a heterogeneous system (measurement in a heterogeneous phase; solid-phase immunoassay).
3.1. Measurement in the homo~en ous phase 3.1.1. Quantitative determination of antibodies against the peptide sub-unit pentapeptide of peptido-glycans in a double antibody radioimmunoassay.
Peptides of formula I are radioactively labelled and ~he test mixture is prepared according to the following scheme:
~0 *Pept. + Ab ~ ~~ *Pept.-Ab (wherein *Pept. represents the labelled peptide and Ab represents an antibody thereto.) The separation of the radioactive free antigen from the radioactive antibody-bound antigen is effected by the double antibody method by specific precipitation of the immunoglobulins using a second anti-antibody according to the following scheme:
*Pept.-Ab ~ Anti-Ab -~ *Pept.-Ab m~(Anti-Ab)m-complexes (wherein Anti-Ab represents the anti-antibody).
Radioactive labelling of the antigen may be efected, for example:
a) by coupling tyrosine to the N-terminal end of the peptide and subsequentl-y iodinating with 125I
by conventional methods b) by iodinating with ~N-succinimidyl-3-(4-hydroxy-5-(125I)iodophenyl)propionate c) by tritiation of the antigen with N-succinimidyl (2,3~ H)propionate.
Xt is advisable to purify the anti-antibodies used by affinity chromatography on matrix-bonded peptides ~f ~ormula I before use in order to remove any antibodies against peptidoglycans which they may contain, or preferably to use monoclonal anti-antibodies.
3.1.2. Quantitative determination of antibodies against the peptide sub-unit pentapeptide in a double antibody enzyme-immunoassay.
The test procedure o~ ~.1.1. above is modified , ~, ~ ~3~
by effecting the labelling of the anti-antibodies by means of an enzyme such as alkaline phospha~ase, peroxidase, glucose-oxidase-peroxidase, glucose-6-phosphate~dehydrogenase, malate dehydrogenase, etcO, rather than labelling with a radioactive isotope.
3.1.3. Quantitative determination of antibodies against the peptide sub-unit pentapeptide of peptidoglycans by nephelometric methods.
Peptides of formula I are bound-, via their N-terminal amino group, to a soluble matrix, P.g~
proteins, polyvinyl pyrrolidone, etc., and the test is carried out according to the following scheme:
Matrix--(Pept.)n ~ Ab ~ Matrix-(Pept.)n-Ab-complexes turbidity The coupling of the peptides to the carrier material may be effected, for exampleO
1. by binding the iodoacetylated peptides to the matrix;
2. by the carbodiimide method;
3. by means of bifunctional reagents, e.g. diisocyanates, glutardialdehyde, etc.
3 D 2'~ Measur~ment_ in the heterogeneous phase Peptides of formula I are bound via their N-terminal amino group to a solîd insoluble matrix such as paper, plastics, latex, etc. Coupling is effected by conventional methods. The peptides may be bound either directly to the matrix or via so called "spacers". These "spacers" may be aliphatic hydrocarbon chains, proteins such as albumin, RNase, etc.
.
,.
~ 7 --3.2.1. Quantitative determination of antibodies against the peptide sub-unit pentapeptide of peptido-glycans in a solid phase "sandwich" radioimmuno assayO
3.2.2. Quantitative determination of antibodies against the peptide sub-unit pentapeptide of peptido-glycans in a solid phase enzyme-immunoassay.
The test procedure of 3.2.1. above is modified by effecting the labelling of the anti-antibodies by means of an enzyme such as those ~entioned in 3.1.2. above rather than by labelling with a radioactive isotop~.
I
3.2.3. Quantitative determination of antibodies against the peptide sub-unit pentapeptide of peptidoglycan in a solid phase 1uorescence immunoassay.
The test procedure of 3.2.1 above is modified by labelling the antibody by means of a fluorescen-t dye (cf~ also FI~ ~ - Sti ~ system~.
3.2.4. Quantitative determination of antibodies against the peptide sub-unit pentapeptide o~ peptidoglycan by means of a latex agglutination test.
Peptides of formula I are coupled to latex particles either directly or via "spacers" such as proteins.
In the methods of quantitative determination discussed at 3.1~ and 3.2. above the quantification is effected by means of a calibration curve which can be plotted with the aid of the standard solutions of antibodies described in 1 above.
Eor the determination of specific IgE a relative quantification over the pooled serum standard described at 2 above i5 effected.
The follo~ing Examples are provided to illustrate the scope of the invention without serving to restrict the scope of protection sought therefor-~243~
Example 1Determining specific antibodies of class a) Pre~ration of the conju~ate (antigen) Equimolar quanti~ies of peptides of formula I and iodoacetylsuccinimide ester in dioxan/water (1:2 v/v), containing 100 mmol/l of sodium hydrogen carbonate, are incubated at 4C for 20 hours with gentle stirring. The dioxan is then eliminated _ vacuo and ~he reaction product formed is precipitated from the aqueous phase by acidification with HCl.
After washing in a hydrochloric acid medium, the product is lyophilised.
100 mg of iodoacetylated peptide and carrier protein, e.g. 250 mg of human serum albumin, are incubated for 72 hours at 37C in 8 molar urea, containing 0.1 mM sodium bicarbonate. After 3 days' dialysis with distilled water (the dialysi-~liquid beiny changed 6 times) the peptide coupled to the albumin is lyophilisedO With the process described, on average 10 moles of peptide per mole of carrier protein are covalently bound.
b) Adsorption_of the conjugate on plastics surfaces, e.~. polystyrene In this example, polystyrene tubes were used having a capacity of barely 2 ml, a height of 4 cm and a diameter of 1 cm. Microtitre plates, halls, dishes, etc. of similar plastics material are also ~ suitable for bindingO
100 ~1 of conjugate solution (corresponding to 100 ng of conjugate in 0.1 molar bicarbonate buffer, pH 9.6) are pipetted into the tubes and dried at 37C for 24 ~hours. The dry tubes are each washed once with 200 microlitres of 10 mmol/l phosphate buffer (pH 7.2) in isotonic common salt solution containing 0.1% Twee ~ 80 and after the wash solution has been removed by suction filtering the tubes are again rinsed with 200 microlitres 3~
g of distilled water and then suction filtered again.
The tubes coated with the conjugate are stored in a drying cupboard at 37C.
c) Test procedure lQ0 microlitres of specific standard solution (see the preparation of the standard) or serum with a known content of specific IgA are pipetted into the tubes coated as described above and incubated at 20C for 2 hours. ~n order to stay within the measuring range of the standard curve-, samples of biological material, e.g. patients' sera, are diluted in the ratio of 1:5 to 1:500, depending on the titre, with PBS ~phosphate buffered saline)/Tween~
80 buffer and are used in the same volume. The incubation mixture is suction filtered and then washed twice with 200 microlitres of PBS/'I'ween~
80 buffer and then suction filtered again. 100 ~1 (anti-human IgA) - immunoglobulin (e~g. from goats) labelled with peroxidase or alkaline phosphatase are pipetted in~o the tubes and incubated for 1 hour at a constant temperature of between 20C
and 37C. Then the tubes are washed twice more, each time wi~h 200 microlitres of PBS/Twee ~ buffer.
If conjugated peroxidase is used as the indicator system, o~pheny}enediamine and H~02 are added as su~strate in a citrate buffer (0.1 mol/l, p~ 5.0) and the tubes are incubated for 30 minutes at constant temperature.
To stop the reaction, 50 pl of 5N H~SO4 are added~ The extinction at 492 nm is proportional to the ~uantity of immunoglobulin used.
Example 2 Determinin~ specific antibodies of class I~M
a) Preparation of the coniugate tantigen) The conjugate is prepared as described in Example 1.
~2~3~
-- 1() --b) Adsorption of the conjugate on plastics surfaces, e g. polystyrene The adsorption of the conjugate on plastics surfaces is also carried out as described in Example 1, except that instead of lO0 ng of conjugate 50 ng of conjugate in 0.1 molar bicarbonate buffer (p~I
9.6) are used.
c) Carrying out the test Pre-treat~ent of the sera: in order to exclude interference by l'rheumatic factors" t~e sera are treated on latex-bound IgG (e.g. latex-RF reagen before being used in the test system.
lOO~ul of specific standard solution (see preparation of standard) or serum with a known conten~ of specific IgM are pipetted into the tubes which have been coated as described above and then incubated for 2 hours at 20~C. In order to stay within the measuring range of the standard curv~, sample-s of biological material, e.g. patients' serar are diluted with 0~3 mol/l of glycine-NaCl buffer, pH 8.2, depending on the titre, and used in the same volume. The incubation mixture is suction filtered and then washed twice with 20~ ~l of 3.3 mol/l glycine-NaCl buffer, pH 8.2, and then suction filtered again. lO0/ul ~anti-human IgM) immuno~lobulin ~e.g. from goats) labelled with peroxidase or alkaline phosphatase are pipetted into the tubes and incubated for l hour at a constant temperature of between 20C and 37C. Then the tubes are washed twice more each time with 200/ul of 0.3 mol/l of glycine-NaCl buffer, p~ 8.2. If conju~ated peroxidase is used as the indicator system, o~phenylenediamine and H~O2 are added as substrate in a citrate buffer (0~1 mol/l, pH 5.0), and the tubes are incubated for 30 minutes at constant temperature.
To stop the reaction, SO~ul of 5N H2SO4 are ~3~
added. The extinction at 492 nm is proportional to the quantity of immunoglobulin used.
~ le 3 Determining specific antibodies of class I~E
a) Preparation of the conju~ate (antigenJ
The conjugate is prepared as described in Example 1.
b~ Adsorption of the conjugate on plastics surfaces, e.~. polystyrene The adsorption of the conjugate on plastics surfaces is also carried out as described in Example 1, except that instead of 100 ng of conjugate 200 ng of conjugate in 0.1 molar bicarbonate buffer (p~
9.6) are used.
c) Carrying out the test In order to arrive in the measuring range of the po~led -serum standard, patients' sera are used undiluted or in a dilution of up to 1:20.
100 ~1 of serum are pipetted with 100 ~1 of PBS/Tween~
80 buffer into the tubes coated as described above and then incubated for 3 hours at 20C. The incubation mixture is suction filtered and subsequently washed twice with 200 ~1 of PBS/Twee ~ 80 buffer and suction filtered again. 100 pl (anti-human IgE) immunoglobulin (e .~ . f rom horses), labelled with peroxidase or alkaline phosphatase, are pipetted into the tubes and incubated for 2 hours at constant temperature of between 20C and 37C. Then the tubes are washed twice more each time with 200 ~1 of PBS/Twee ~
80 buffer. If conjugated peroxidase i5 used as the indicator system, o-phenylenediamine and H2O2 are added as substrate in a ci~trate buffer of 0.1 mol/l, pH 5.0, and the tubes are incubated for 30 minutes at constant temperature.
To stop the reaction, 50 ~1 of 5W H2SO4 are , , , .
~3~
added. The extinction at 492 nm is proportional to the quantity of immunoglobulins used.
Example 4 Obtaining a specific human immuno~lobulin-A standard _ _ ~i~'' J A. ~eling the albumin-(L-Ala-D-G]
~` Ala-D-Ala)) con~ugate to Sepharose 4B
Synthesis of the conjugate is carried out~
as described for the test. 10 ml of ~ ~ es4~
gel are washed 5 times with a 4- to 5-fold excess of distilled water and then suspended in 10 ml of water. 430 mg of bromocyanogen, dissolved in 8 ml of water, are added thereto. The reaction takes place at ambient temperature and the pH i5 kept constant at 11.0 by the addition o~ 2 mol~l of sodium hydroxide solution. After 15 minutes' reaction with gentle stirring, the gel is suction filtered on to a BUchner funnel and washed with 60 ml of 0.1 mol/l of sodium-hydrogen carbonate solution. The sepharose is stirred into 7O5 ml of 0.1 mol/l of sodium hydrogen carbona~e containing 50 mg of albumin-peptide conjugate. Coupling is effected overnight a~ 4C with gentle stirring.
After the reaction time, the substance is suction filtered again and the coupled gel is wasbed twice with each of the following: 30 to 40 ml of water, then 0.1 mol/l of sodium hydrogen carbonate, 0.2 mol/l of sodium acetate buffer, pH 4.5, 0.2 mol/l of sodium phosphate buffer, p~ 7.2, and 0.01 mol/l of PBS, pH 7.~. The affinity gel is taken up in PBS buffer, pH 7.2, at 4C.
B. Procedure for affinity chromatography The gel is placed in a suitable column and serum containing antibodies or an isolated immuno-globulin-A fraction is added. The charged column is washed with PBS buffer ~without Tween) until 3g~
the extinction at 28~ nm in the eluate falls to below 0.03 (consumption of about twice as much buffer as serum).
~lution is carried out with acetic acid (0.1 to 1 mol/l)~ The antibody is eluted with the first fractions vf the acetic acid, the fractions containing antibodies are combined and neutralised, then dialysed and concentrated.
Alternatively, standardisation may be carried out as follows:
The concentration of specific I~A in a pooled serum is determined by affinity chromatographyO
For this, the isolated IgA fraction from the poolecl serum is added to the column and washed as described above. Total elution o~ the specifically bonded Ig~ is carried out with 6 mol/l of guanidinium chloride. After dialysis the protein content of the eluate is determined.
~0 Example 5 Determinin~ the content of specific IgM in a pooled serum standard A. Couplin~ the albumin-(L-Ala-D-Glu(L-Lys-D-Ala-D-Ala)) conjugate to Sepharose 4 B
Synthesis is carried out as described for the tes~. 10 ml o~ sepharose gel are wa~hed 5 times with a 4- to 5-fold excess of dis illed water and then su~spended in 10 ml of water. 430 mg of bromocyanogenl dissolved in 8 ml of water, are added thereto. The reaction takes place at ambient temperature and the pH is kept constant at 11.0 by the addition of~2 moI/l of sodium hydroxide solution~ After 15 minutes' reaction with gentle stirring, the gel is suction filtered on to a B~chner funnel and washed with 60 ml o 0.1 mol/l of sodium hydrogen carbonate solution. The sepharose is stirred into 7.5 ml of 0.1 moljl of sodium hydrogen ,, -~ ~43~
carbonate containing 50 mg of albumin-peptide conjugate.
Coupling is effected overnlght at 4C with gentle stirring. After the reaction time, the substance is suction filtered again an~ the coupled gel is washed twice with each of the following: 30 to 40 ml of water, then 0.1 mol/l of sodium hydrogen carbonate, 0.2 mol/1 of sodium acetate buffer, pH 4.5, 0.2 mol/l of sodium phosphate buffer, pH
7~2, and 0.01 mol/1 of PBS, pH 7.2. The affinity gel is taken up in PBS buffer, p~l 7.2, at 4C.
B Procedure for affinity chromatography The gel is placed in a suitable column and isolated IgM is added. The charged column is washed with PBS buffer (without Tween) until the extinction at 280 nm in the eluate falls to below 0.03 (consumption of about twice as much buffer as serum).
Total elution of the specifically bound IgM
is carried out with 6 mol/l guanidinium chloride.
After dialysis, the protein content of the eluate is determined.
a) quantitative determination in a soluble system tmeasurement in a homogeneous phase); and b) quantitative determination by measurement in a heterogeneous system (measurement in a heterogeneous phase; solid-phase immunoassay).
3.1. Measurement in the homo~en ous phase 3.1.1. Quantitative determination of antibodies against the peptide sub-unit pentapeptide of peptido-glycans in a double antibody radioimmunoassay.
Peptides of formula I are radioactively labelled and ~he test mixture is prepared according to the following scheme:
~0 *Pept. + Ab ~ ~~ *Pept.-Ab (wherein *Pept. represents the labelled peptide and Ab represents an antibody thereto.) The separation of the radioactive free antigen from the radioactive antibody-bound antigen is effected by the double antibody method by specific precipitation of the immunoglobulins using a second anti-antibody according to the following scheme:
*Pept.-Ab ~ Anti-Ab -~ *Pept.-Ab m~(Anti-Ab)m-complexes (wherein Anti-Ab represents the anti-antibody).
Radioactive labelling of the antigen may be efected, for example:
a) by coupling tyrosine to the N-terminal end of the peptide and subsequentl-y iodinating with 125I
by conventional methods b) by iodinating with ~N-succinimidyl-3-(4-hydroxy-5-(125I)iodophenyl)propionate c) by tritiation of the antigen with N-succinimidyl (2,3~ H)propionate.
Xt is advisable to purify the anti-antibodies used by affinity chromatography on matrix-bonded peptides ~f ~ormula I before use in order to remove any antibodies against peptidoglycans which they may contain, or preferably to use monoclonal anti-antibodies.
3.1.2. Quantitative determination of antibodies against the peptide sub-unit pentapeptide in a double antibody enzyme-immunoassay.
The test procedure o~ ~.1.1. above is modified , ~, ~ ~3~
by effecting the labelling of the anti-antibodies by means of an enzyme such as alkaline phospha~ase, peroxidase, glucose-oxidase-peroxidase, glucose-6-phosphate~dehydrogenase, malate dehydrogenase, etcO, rather than labelling with a radioactive isotope.
3.1.3. Quantitative determination of antibodies against the peptide sub-unit pentapeptide of peptidoglycans by nephelometric methods.
Peptides of formula I are bound-, via their N-terminal amino group, to a soluble matrix, P.g~
proteins, polyvinyl pyrrolidone, etc., and the test is carried out according to the following scheme:
Matrix--(Pept.)n ~ Ab ~ Matrix-(Pept.)n-Ab-complexes turbidity The coupling of the peptides to the carrier material may be effected, for exampleO
1. by binding the iodoacetylated peptides to the matrix;
2. by the carbodiimide method;
3. by means of bifunctional reagents, e.g. diisocyanates, glutardialdehyde, etc.
3 D 2'~ Measur~ment_ in the heterogeneous phase Peptides of formula I are bound via their N-terminal amino group to a solîd insoluble matrix such as paper, plastics, latex, etc. Coupling is effected by conventional methods. The peptides may be bound either directly to the matrix or via so called "spacers". These "spacers" may be aliphatic hydrocarbon chains, proteins such as albumin, RNase, etc.
.
,.
~ 7 --3.2.1. Quantitative determination of antibodies against the peptide sub-unit pentapeptide of peptido-glycans in a solid phase "sandwich" radioimmuno assayO
3.2.2. Quantitative determination of antibodies against the peptide sub-unit pentapeptide of peptido-glycans in a solid phase enzyme-immunoassay.
The test procedure of 3.2.1. above is modified by effecting the labelling of the anti-antibodies by means of an enzyme such as those ~entioned in 3.1.2. above rather than by labelling with a radioactive isotop~.
I
3.2.3. Quantitative determination of antibodies against the peptide sub-unit pentapeptide of peptidoglycan in a solid phase 1uorescence immunoassay.
The test procedure of 3.2.1 above is modified by labelling the antibody by means of a fluorescen-t dye (cf~ also FI~ ~ - Sti ~ system~.
3.2.4. Quantitative determination of antibodies against the peptide sub-unit pentapeptide o~ peptidoglycan by means of a latex agglutination test.
Peptides of formula I are coupled to latex particles either directly or via "spacers" such as proteins.
In the methods of quantitative determination discussed at 3.1~ and 3.2. above the quantification is effected by means of a calibration curve which can be plotted with the aid of the standard solutions of antibodies described in 1 above.
Eor the determination of specific IgE a relative quantification over the pooled serum standard described at 2 above i5 effected.
The follo~ing Examples are provided to illustrate the scope of the invention without serving to restrict the scope of protection sought therefor-~243~
Example 1Determining specific antibodies of class a) Pre~ration of the conju~ate (antigen) Equimolar quanti~ies of peptides of formula I and iodoacetylsuccinimide ester in dioxan/water (1:2 v/v), containing 100 mmol/l of sodium hydrogen carbonate, are incubated at 4C for 20 hours with gentle stirring. The dioxan is then eliminated _ vacuo and ~he reaction product formed is precipitated from the aqueous phase by acidification with HCl.
After washing in a hydrochloric acid medium, the product is lyophilised.
100 mg of iodoacetylated peptide and carrier protein, e.g. 250 mg of human serum albumin, are incubated for 72 hours at 37C in 8 molar urea, containing 0.1 mM sodium bicarbonate. After 3 days' dialysis with distilled water (the dialysi-~liquid beiny changed 6 times) the peptide coupled to the albumin is lyophilisedO With the process described, on average 10 moles of peptide per mole of carrier protein are covalently bound.
b) Adsorption_of the conjugate on plastics surfaces, e.~. polystyrene In this example, polystyrene tubes were used having a capacity of barely 2 ml, a height of 4 cm and a diameter of 1 cm. Microtitre plates, halls, dishes, etc. of similar plastics material are also ~ suitable for bindingO
100 ~1 of conjugate solution (corresponding to 100 ng of conjugate in 0.1 molar bicarbonate buffer, pH 9.6) are pipetted into the tubes and dried at 37C for 24 ~hours. The dry tubes are each washed once with 200 microlitres of 10 mmol/l phosphate buffer (pH 7.2) in isotonic common salt solution containing 0.1% Twee ~ 80 and after the wash solution has been removed by suction filtering the tubes are again rinsed with 200 microlitres 3~
g of distilled water and then suction filtered again.
The tubes coated with the conjugate are stored in a drying cupboard at 37C.
c) Test procedure lQ0 microlitres of specific standard solution (see the preparation of the standard) or serum with a known content of specific IgA are pipetted into the tubes coated as described above and incubated at 20C for 2 hours. ~n order to stay within the measuring range of the standard curve-, samples of biological material, e.g. patients' sera, are diluted in the ratio of 1:5 to 1:500, depending on the titre, with PBS ~phosphate buffered saline)/Tween~
80 buffer and are used in the same volume. The incubation mixture is suction filtered and then washed twice with 200 microlitres of PBS/'I'ween~
80 buffer and then suction filtered again. 100 ~1 (anti-human IgA) - immunoglobulin (e~g. from goats) labelled with peroxidase or alkaline phosphatase are pipetted in~o the tubes and incubated for 1 hour at a constant temperature of between 20C
and 37C. Then the tubes are washed twice more, each time wi~h 200 microlitres of PBS/Twee ~ buffer.
If conjugated peroxidase is used as the indicator system, o~pheny}enediamine and H~02 are added as su~strate in a citrate buffer (0.1 mol/l, p~ 5.0) and the tubes are incubated for 30 minutes at constant temperature.
To stop the reaction, 50 pl of 5N H~SO4 are added~ The extinction at 492 nm is proportional to the ~uantity of immunoglobulin used.
Example 2 Determinin~ specific antibodies of class I~M
a) Preparation of the coniugate tantigen) The conjugate is prepared as described in Example 1.
~2~3~
-- 1() --b) Adsorption of the conjugate on plastics surfaces, e g. polystyrene The adsorption of the conjugate on plastics surfaces is also carried out as described in Example 1, except that instead of lO0 ng of conjugate 50 ng of conjugate in 0.1 molar bicarbonate buffer (p~I
9.6) are used.
c) Carrying out the test Pre-treat~ent of the sera: in order to exclude interference by l'rheumatic factors" t~e sera are treated on latex-bound IgG (e.g. latex-RF reagen before being used in the test system.
lOO~ul of specific standard solution (see preparation of standard) or serum with a known conten~ of specific IgM are pipetted into the tubes which have been coated as described above and then incubated for 2 hours at 20~C. In order to stay within the measuring range of the standard curv~, sample-s of biological material, e.g. patients' serar are diluted with 0~3 mol/l of glycine-NaCl buffer, pH 8.2, depending on the titre, and used in the same volume. The incubation mixture is suction filtered and then washed twice with 20~ ~l of 3.3 mol/l glycine-NaCl buffer, pH 8.2, and then suction filtered again. lO0/ul ~anti-human IgM) immuno~lobulin ~e.g. from goats) labelled with peroxidase or alkaline phosphatase are pipetted into the tubes and incubated for l hour at a constant temperature of between 20C and 37C. Then the tubes are washed twice more each time with 200/ul of 0.3 mol/l of glycine-NaCl buffer, p~ 8.2. If conju~ated peroxidase is used as the indicator system, o~phenylenediamine and H~O2 are added as substrate in a citrate buffer (0~1 mol/l, pH 5.0), and the tubes are incubated for 30 minutes at constant temperature.
To stop the reaction, SO~ul of 5N H2SO4 are ~3~
added. The extinction at 492 nm is proportional to the quantity of immunoglobulin used.
~ le 3 Determining specific antibodies of class I~E
a) Preparation of the conju~ate (antigenJ
The conjugate is prepared as described in Example 1.
b~ Adsorption of the conjugate on plastics surfaces, e.~. polystyrene The adsorption of the conjugate on plastics surfaces is also carried out as described in Example 1, except that instead of 100 ng of conjugate 200 ng of conjugate in 0.1 molar bicarbonate buffer (p~
9.6) are used.
c) Carrying out the test In order to arrive in the measuring range of the po~led -serum standard, patients' sera are used undiluted or in a dilution of up to 1:20.
100 ~1 of serum are pipetted with 100 ~1 of PBS/Tween~
80 buffer into the tubes coated as described above and then incubated for 3 hours at 20C. The incubation mixture is suction filtered and subsequently washed twice with 200 ~1 of PBS/Twee ~ 80 buffer and suction filtered again. 100 pl (anti-human IgE) immunoglobulin (e .~ . f rom horses), labelled with peroxidase or alkaline phosphatase, are pipetted into the tubes and incubated for 2 hours at constant temperature of between 20C and 37C. Then the tubes are washed twice more each time with 200 ~1 of PBS/Twee ~
80 buffer. If conjugated peroxidase i5 used as the indicator system, o-phenylenediamine and H2O2 are added as substrate in a ci~trate buffer of 0.1 mol/l, pH 5.0, and the tubes are incubated for 30 minutes at constant temperature.
To stop the reaction, 50 ~1 of 5W H2SO4 are , , , .
~3~
added. The extinction at 492 nm is proportional to the quantity of immunoglobulins used.
Example 4 Obtaining a specific human immuno~lobulin-A standard _ _ ~i~'' J A. ~eling the albumin-(L-Ala-D-G]
~` Ala-D-Ala)) con~ugate to Sepharose 4B
Synthesis of the conjugate is carried out~
as described for the test. 10 ml of ~ ~ es4~
gel are washed 5 times with a 4- to 5-fold excess of distilled water and then suspended in 10 ml of water. 430 mg of bromocyanogen, dissolved in 8 ml of water, are added thereto. The reaction takes place at ambient temperature and the pH i5 kept constant at 11.0 by the addition o~ 2 mol~l of sodium hydroxide solution. After 15 minutes' reaction with gentle stirring, the gel is suction filtered on to a BUchner funnel and washed with 60 ml of 0.1 mol/l of sodium-hydrogen carbonate solution. The sepharose is stirred into 7O5 ml of 0.1 mol/l of sodium hydrogen carbona~e containing 50 mg of albumin-peptide conjugate. Coupling is effected overnight a~ 4C with gentle stirring.
After the reaction time, the substance is suction filtered again and the coupled gel is wasbed twice with each of the following: 30 to 40 ml of water, then 0.1 mol/l of sodium hydrogen carbonate, 0.2 mol/l of sodium acetate buffer, pH 4.5, 0.2 mol/l of sodium phosphate buffer, p~ 7.2, and 0.01 mol/l of PBS, pH 7.~. The affinity gel is taken up in PBS buffer, pH 7.2, at 4C.
B. Procedure for affinity chromatography The gel is placed in a suitable column and serum containing antibodies or an isolated immuno-globulin-A fraction is added. The charged column is washed with PBS buffer ~without Tween) until 3g~
the extinction at 28~ nm in the eluate falls to below 0.03 (consumption of about twice as much buffer as serum).
~lution is carried out with acetic acid (0.1 to 1 mol/l)~ The antibody is eluted with the first fractions vf the acetic acid, the fractions containing antibodies are combined and neutralised, then dialysed and concentrated.
Alternatively, standardisation may be carried out as follows:
The concentration of specific I~A in a pooled serum is determined by affinity chromatographyO
For this, the isolated IgA fraction from the poolecl serum is added to the column and washed as described above. Total elution o~ the specifically bonded Ig~ is carried out with 6 mol/l of guanidinium chloride. After dialysis the protein content of the eluate is determined.
~0 Example 5 Determinin~ the content of specific IgM in a pooled serum standard A. Couplin~ the albumin-(L-Ala-D-Glu(L-Lys-D-Ala-D-Ala)) conjugate to Sepharose 4 B
Synthesis is carried out as described for the tes~. 10 ml o~ sepharose gel are wa~hed 5 times with a 4- to 5-fold excess of dis illed water and then su~spended in 10 ml of water. 430 mg of bromocyanogenl dissolved in 8 ml of water, are added thereto. The reaction takes place at ambient temperature and the pH is kept constant at 11.0 by the addition of~2 moI/l of sodium hydroxide solution~ After 15 minutes' reaction with gentle stirring, the gel is suction filtered on to a B~chner funnel and washed with 60 ml o 0.1 mol/l of sodium hydrogen carbonate solution. The sepharose is stirred into 7.5 ml of 0.1 moljl of sodium hydrogen ,, -~ ~43~
carbonate containing 50 mg of albumin-peptide conjugate.
Coupling is effected overnlght at 4C with gentle stirring. After the reaction time, the substance is suction filtered again an~ the coupled gel is washed twice with each of the following: 30 to 40 ml of water, then 0.1 mol/l of sodium hydrogen carbonate, 0.2 mol/1 of sodium acetate buffer, pH 4.5, 0.2 mol/l of sodium phosphate buffer, pH
7~2, and 0.01 mol/1 of PBS, pH 7.2. The affinity gel is taken up in PBS buffer, p~l 7.2, at 4C.
B Procedure for affinity chromatography The gel is placed in a suitable column and isolated IgM is added. The charged column is washed with PBS buffer (without Tween) until the extinction at 280 nm in the eluate falls to below 0.03 (consumption of about twice as much buffer as serum).
Total elution of the specifically bound IgM
is carried out with 6 mol/l guanidinium chloride.
After dialysis, the protein content of the eluate is determined.
Claims (33)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. An antibody against the peptide sub-unit pentapeptide L-Ala-D-Glu(L-Lys-D-Ala-D-Ala) of a peptidoglycan characterized in that said antibody is selected from the class consisting of immunoglobulins A, M and E.
2. An antibody as claimed in claim 1, which possesses at least one of the following abilities:
(a) the ability to bind to a pentapeptide of formula I
R - Al - D-Ala (I) (wherein R represents an amino acid component with 1, 2 or 3 amino acid residues selected from Gly, L-Ala, D-Glu and L-Lys; and A1 represents one of the amino acid residues D-Ala,D-Ser, D-Val and D-Leu); and (b) the ability to bind to bacterial cell wall or cell wall material which contains the pentapeptide of formula I; and (c) the ability to bind to Gram-positive bacterium which contains the pentapeptide of formula I.
(a) the ability to bind to a pentapeptide of formula I
R - Al - D-Ala (I) (wherein R represents an amino acid component with 1, 2 or 3 amino acid residues selected from Gly, L-Ala, D-Glu and L-Lys; and A1 represents one of the amino acid residues D-Ala,D-Ser, D-Val and D-Leu); and (b) the ability to bind to bacterial cell wall or cell wall material which contains the pentapeptide of formula I; and (c) the ability to bind to Gram-positive bacterium which contains the pentapeptide of formula I.
3. An antibody as claimed in claim 2, which possesses the ability to bind to the pentapeptide of formula I wherein R represents an amino acid component with 3 amino acid residues selected from Gly, L-Ala, D-Glu and L-Lys and Al has the meanings as defined in claim 2.
4. An antibody as claimed in claim 1, 2 or 3, which is isolated from the other immunoglobulins.
5. An antibody as claimed in claim 1, 2 or 3, which is isolated from other immunoglobulins by affinity chromatography.
6. An antibody as claimed in claim 1, 2 or 3, which is in a sterile composition.
7. A process for the quantitative determination of anti-bodies selected from the class consisting of immunoglobulins A, M and E against the peptide sub-unit pentapeptide L-Ala-D-Glu(L-Lys-D-Ala-D-Ala) of a peptidoglycan in a biological test material which process comprises contacting said test material with an antigen of formula I
R - A1 - D-Ala (I) (wherein R represents an amino acid component with 1, 2 or 3 amino acid residues selected from Gly, L-Ala, D-Glu and L-Lys; and A1 represents one of the amino acid residues D-Ala, D-Ser, D-Val and D-Leu), which may be labelled and may be substrate bound.
R - A1 - D-Ala (I) (wherein R represents an amino acid component with 1, 2 or 3 amino acid residues selected from Gly, L-Ala, D-Glu and L-Lys; and A1 represents one of the amino acid residues D-Ala, D-Ser, D-Val and D-Leu), which may be labelled and may be substrate bound.
8. A process as claimed in claim 7, wherein in said antigen of formula I R represents an amino acid component with three amino acid residues selected from Gly, L-Ala, D-Glu and L-Lys.
9. A process as claimed in claim 7 or 8, wherein the quantitative determination of antibodies is (a) effected in a double antibody radioimmunoassay;
(b) carried out in a double antibody enzyme-immunoassay;
(c) carried out using a nephelometric method;
(d) carried out in a solid phase sandwich radioimmuno-assay;
(e) carried out in a solid phase enzyme-immunoassay;
(f) carried out in a solid phase fluorescence immuno-assay; or (g) carried out by means of a latex agglutination test.
(b) carried out in a double antibody enzyme-immunoassay;
(c) carried out using a nephelometric method;
(d) carried out in a solid phase sandwich radioimmuno-assay;
(e) carried out in a solid phase enzyme-immunoassay;
(f) carried out in a solid phase fluorescence immuno-assay; or (g) carried out by means of a latex agglutination test.
10. A process as claimed in claim 7, for the quantitative determination of an antibody of the class of immunoglobulin A
against the peptide sub-unit pentapeptide L-Ala-D-Glu(L-Lys-D-Ala-D-Ala) of a peptidoglycan in a biological test material wherein an IgA standard prepared by affinity chromatography on Sepharose?-(albumin-peptide of formu1a I) is used as a standard or as a positive control.
against the peptide sub-unit pentapeptide L-Ala-D-Glu(L-Lys-D-Ala-D-Ala) of a peptidoglycan in a biological test material wherein an IgA standard prepared by affinity chromatography on Sepharose?-(albumin-peptide of formu1a I) is used as a standard or as a positive control.
11. A process as claimed in claim 7, for the quantitative determination of an antibody of the class of immunoglobulin A
against the peptide sub-unit pentapeptide L-Ala D-Glu(L-Lys-D-Ala-D-Ala) of a peptidoglycan in a biological test material wherein the quantity of an isolated IgA fraction is measured by affinity chromatography on Sepharose ?- (albumin-peptide of formula I).
against the peptide sub-unit pentapeptide L-Ala D-Glu(L-Lys-D-Ala-D-Ala) of a peptidoglycan in a biological test material wherein the quantity of an isolated IgA fraction is measured by affinity chromatography on Sepharose ?- (albumin-peptide of formula I).
12. A process as claimed in claim 7, for the quantitative determination of an antibody of the class of immunoglobulin M
against the peptide sub-unit pentapeptide L-Ala-D-Glu(L-Lys-D-Ala-D-Ala) of a peptidoglycan in a biological test material wherein the quantity of an isolated IgM fraction is measured by affinity chromatography on Sepharose ?- (albumin-peptide of formula I).
against the peptide sub-unit pentapeptide L-Ala-D-Glu(L-Lys-D-Ala-D-Ala) of a peptidoglycan in a biological test material wherein the quantity of an isolated IgM fraction is measured by affinity chromatography on Sepharose ?- (albumin-peptide of formula I).
13. A process for the quantitative determination as claimed in claim 7, wherein the antigen of formula I is used as a standard or as a positive control.
14. A process as claimed in claim 7 or 8, wherein the antigen of formula I is conjugated with a carrier protein.
15. A process as claimed in claim 7 or 8, wherein the antigen of formula I is conjugated with a carrier protein bound to a solid phase.
16. A process as claimed in claim 7, which comprises (i) contacting a test sample or a standard with the anti-gen of formula I which is bound to a solid insoluble matrix, (ii) washing off unbound immunoglobulins, (iii) contacting the solid matrix to which the immunoglobulin to be determined is bound with a corresponding anti-antibody which is coupled with an indicator, (iv) washing off the unreacted anti-antibody, and (v) measuring the amount of the anti-antibody remaining on the solid phase by means of the indicator.
17. A process as claimed in claim 16, wherein the anti-body to be determined belongs to the class of immunoglobulin A and an (anti-human IgA) immunoglobulin labelled with an enzyme indicator is used as the anti-antibody.
18. A process as claimed in claim 17, wherein the enzyme is a peroxidase or an alkaline phosphatase.
19. A process as claimed in claim 17, wherein the antigen is bound to the solid matrix via a protein spacer.
20. A process as claimed in claim 16, wherein the anti-body to be determined belongs to the class of immunoglobulin M and an (anti-human IgM) immunoglobulin labelled with an enzyme indicator is used as the anti-antibody.
21. A process as claimed in claim 20, wherein the enzyme is a peroxidase or an alkaline phosphatase.
22. A process as claimed in claim 20, wherein the antigen is bound to the solid matrix via a protein spacer.
23. A process as claimed in claim 16, wherein the anti-body to be determined belongs to the class of immunoglobulin E and an (anti-human IgE) immunoglobulin labelled with an enzyme indicator is used as the anti-antibody.
24. A process as claimed in claim 23, wherein the enzyme is a peroxidase or an alkaline phosphatase.
25. A process as claimed in claim 23, wherein the antigen is bound to the solid matrix via a protein spacer.
26. A method of obtaining an antibody as claimed in claim 1 from human plasma which method comprises:
(a) contacting a pentapeptide of formula I
R - A1 - D-Ala (I) (wherein R represents an amino acid component with 1, 2 or 3 amino acid residues selected from Gly, L-Ala, D-Glu and L-Lys; and A1 represents one of the amino acid residues D-Ala, D-Ser, D-Val and D-Leu) coupled with a solid phase, with human plasma containing IgA, IgM
or IgE whereby said antibody is selectively bound to said pentapeptide coupled to said solid phase; and (b) subsequently selectively eluting said antibody from said solid phase.
(a) contacting a pentapeptide of formula I
R - A1 - D-Ala (I) (wherein R represents an amino acid component with 1, 2 or 3 amino acid residues selected from Gly, L-Ala, D-Glu and L-Lys; and A1 represents one of the amino acid residues D-Ala, D-Ser, D-Val and D-Leu) coupled with a solid phase, with human plasma containing IgA, IgM
or IgE whereby said antibody is selectively bound to said pentapeptide coupled to said solid phase; and (b) subsequently selectively eluting said antibody from said solid phase.
27. A method as claimed in claim 26, wherein the pentapeptide is coupled with the solid phase via a protein spacer.
28. A method as claimed in claim 27, wherein the protein is albumin.
29. A method as claimed in claim 28, wherein the pentapeptide is coupled with the solid phase via albumin using a iodoacetylated albumin.
30. A method as claimed in claim 27, wherein the pentapeptide is coupled with the solid phase via human serum albumin using a iodoacetylated albumin.
31. A method as claimed in claim 28, wherein the contact of the pentapeptide with the antibody is carried out using affinity chromatography of the solid phase with which the pentapeptide is coupled via the albumin spacer.
32. A method as claimed in claim 31, wherein an isolated IgA fraction is added to the column.
33. A method as claimed in claim 31, wherein an isolated IgM fraction is added to the column.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19833328832 DE3328832A1 (en) | 1983-08-10 | 1983-08-10 | REAGENT AND METHOD FOR THE QUANTITATIVE DETERMINATION OF ANTIBODY |
| DEP3328832.1 | 1983-08-10 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1243944A true CA1243944A (en) | 1988-11-01 |
Family
ID=6206209
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000460585A Expired CA1243944A (en) | 1983-08-10 | 1984-08-09 | Reagent and process for quantitatively measuring antibodies |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP0133553A3 (en) |
| JP (1) | JPS6070356A (en) |
| AU (1) | AU3182484A (en) |
| CA (1) | CA1243944A (en) |
| DE (1) | DE3328832A1 (en) |
| IL (1) | IL72632A0 (en) |
| ZA (1) | ZA846167B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1987001374A2 (en) * | 1985-08-28 | 1987-03-12 | George Pieczenik | Method and means for sorting and identifying biological information |
| DE3621570A1 (en) * | 1986-06-27 | 1988-01-07 | Hoechst Ag | DIAGNOSTIC FOR SCINTIGRAPHIC REPRESENTATION OF BOUGY TUMORS |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3966898A (en) * | 1973-06-28 | 1976-06-29 | Pharmacia Diagnostics Ab | Method and reagent for determining immunologic materials |
| DE3231204A1 (en) * | 1982-08-21 | 1984-03-01 | Boehringer Ingelheim Diagnostika GmbH, 8046 Garching | ANTIBODIES AGAINST BACTERIAL PEPTIDOGLYCANES, METHODS FOR THEIR PRODUCTION AND METHODS FOR THEIR QUANTITATIVE DETERMINATION |
-
1983
- 1983-08-10 DE DE19833328832 patent/DE3328832A1/en not_active Ceased
-
1984
- 1984-08-02 EP EP84109177A patent/EP0133553A3/en not_active Withdrawn
- 1984-08-09 IL IL72632A patent/IL72632A0/en unknown
- 1984-08-09 ZA ZA846167A patent/ZA846167B/en unknown
- 1984-08-09 CA CA000460585A patent/CA1243944A/en not_active Expired
- 1984-08-10 AU AU31824/84A patent/AU3182484A/en not_active Abandoned
- 1984-08-10 JP JP59167702A patent/JPS6070356A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| EP0133553A2 (en) | 1985-02-27 |
| AU3182484A (en) | 1985-02-14 |
| DE3328832A1 (en) | 1985-02-28 |
| EP0133553A3 (en) | 1986-07-30 |
| JPS6070356A (en) | 1985-04-22 |
| IL72632A0 (en) | 1984-11-30 |
| ZA846167B (en) | 1986-04-30 |
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