CA1218299A - Antibodies against bacterial peptidoglycans, processes for preparing them and methods of quantitively measuring them - Google Patents
Antibodies against bacterial peptidoglycans, processes for preparing them and methods of quantitively measuring themInfo
- Publication number
- CA1218299A CA1218299A CA000434968A CA434968A CA1218299A CA 1218299 A CA1218299 A CA 1218299A CA 000434968 A CA000434968 A CA 000434968A CA 434968 A CA434968 A CA 434968A CA 1218299 A CA1218299 A CA 1218299A
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- Prior art keywords
- ala
- antibodies
- formula
- amino acid
- pentapeptide
- Prior art date
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Classifications
-
- 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
- G01N33/531—Production of immunochemical test materials
-
- 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
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
-
- 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
Abstract
Abstract Antibodies against bacterial peptidoglycans Antibodies from the class of immunoglobulins G against the peptide sub-unit pentapeptide L-Ala-D-Glu(L-Lys-D-Ala-D-Ala) of peptidoglycans 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 collagen diseases. Such antibodies may possess the ability to bind to (a) pentapeptides of formula I
R - A1 - D - Ala (I) (wherein R represents an amino acid component with 1, 2 or 3, preferably 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); or (b) bacterial cell walls or cell wall material which contain pentapeptides of formula I; or (c) Gram-positive bacteria which contain pentapeptides of formula I.
R - A1 - D - Ala (I) (wherein R represents an amino acid component with 1, 2 or 3, preferably 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); or (b) bacterial cell walls or cell wall material which contain pentapeptides of formula I; or (c) Gram-positive bacteria which contain pentapeptides of formula I.
Description
1;~18;~9 Antibodies a~ainst bacterial peptidogLycans This invention relates to antibodies against bacterial peptidoglycans, to processes for the extraction of such antibodies, to a method of quanti-tatively measuring them in biological test material,and to their use as stardards in such methods.
The term "collagenoses7' (collagen diseases) is used nowadays to denote a group of diseases which occur in generalised form in the connective tissue, such as rheumatic fever, chronic polyarthritis, lupus erythematosus, arteritis temporalis Horton and many others. Some of these diseases, which can also be termed autoimmune diseases, are often difficult to distinguish from one another on the basis of their clinical symptoms. Moreover, the various manifestations of a collagen disease may take different forms in one and the same patient.
Of the diseases mentioned above, rheumatic fever is a special case since the etiological factor which causes it is a previous infection with haemolysing streptococci of group A. In these patients, the so-called "rheuma tests" in the serum produce a positive reaction to a high degree (80 to 90%).
ThiS iS caused by antibodies against intracellular components of streptococci, such as streptolysin O, desoxyribonuclease, hyaluronidase, etc.
Chronic polyarthritis (rheumatoid arthritis) on the other hand is the most common collagen disease and causes a significant deterioration in health.
Currently, it is accepted that at least 10~ of the total population are affected. However, the results of the "rheuma tests" are positive in only about half the patients. This is particularly true in children.
Hitherto, the emphasis in investigations into the pathogenesis of rheumatoid arthritis and also the diagnosis of this disease has been placed essentially on the idea that intracellular components .lZ~82~
of bacteria are effective as antigens, stimulate the production of antibodies and trigger chronic inflammatory reactions whilst forming immune complexes in the connective tissue.
However, recent investigations have shown that the bacterial cell wall and, in particular, the peptidoglycanes are of far greater significance in the persisting immune response on the induction of chronic inflammatory processes with or without any initial (bacteriologically) detectable infection.
Peptidoglycans (also known as mureines or mucopeptides) are the most important component of the bacterial cell wall and ~are present in all bacteria. Fungi, rickettsiae and viruses, on the other hand, have no corresponding components.
Animal experiments have shown that peptidoglycans can cause markedly chronic inflammations in mammals, as well as inducing acute inflammatory processes.
Moreover, tests have shown that, on account of their considerable resistance to all known animal enzymes, bacterial cell walls may remain localised in the tissues for periods ranging from months to years. It has therefore been suspected that pathogen components of this kind may possibly be implicated in the formation of primarily chronic inflammations of hitherto unknown etiology (e.g.
chronic polyarthritis).
Thus, peptidoglycans are highly potent antigens which occur practically ubiquitously and against which antibodies are frequently formed in-animals.
It is known that the pentapeptide with the structure L-Ala-D-Glu(L-Lys-D-Ala-D-Ala), which occurs particularly in streptococci and staphylococci and does not participate in cross linking, is an essential antigenic determinant of peptidoglycan-s.
This peptide is hereinafter referred to as "peptide sub-unit pentapeptide". The C-terminal sequence -D-Ala-D-Ala oE the peptide sub-unit pentapeptide, 12~ 9 being an "immune-dominant group", is of critical importance in the binding of specific antibodies to the antigenic determinant L-Ala-D-Glu(L-Lys-D-Ala-D-Ala).
Specific antibodies against this peptide sub-unit pentapeptide are therefore of particular diaqnostic interest for the recognition and monitoring of the therapy of "autoimmune diseases" (such as chronic polyarthritis); their presence indicates streptococci or staphylococci as ètiological factors.
Up till now, the presence of antibodies against the peptidoglycan component of bacterial cell walls in animal sera has been described, using various techniques, such as immune precipitation. For detecting antibodies against the peptide sub-unit pentapeptide of peptidoglycans in animal sera, a hapten binding assay is described (J. Immunol.
114, 1191-1196 (1975)). However, the process pu~lished will permit only a rough quantitative estimate of the antibody content in human sera since the quantification is effected using hyperimmune sera - from rabbits.
This method has a number of disadvantages:
~5 1. It cannot be used to detect non-precipitatable antibodies. The authors themselves give a sharp `
deviation in the antibody content determined by various methods (e.g. specific antibody content determined by the hapten binding assay 4.6 mg/ml;
specific antibody content determined by the precipi-tation method 2.2 mg/ml).
The term "collagenoses7' (collagen diseases) is used nowadays to denote a group of diseases which occur in generalised form in the connective tissue, such as rheumatic fever, chronic polyarthritis, lupus erythematosus, arteritis temporalis Horton and many others. Some of these diseases, which can also be termed autoimmune diseases, are often difficult to distinguish from one another on the basis of their clinical symptoms. Moreover, the various manifestations of a collagen disease may take different forms in one and the same patient.
Of the diseases mentioned above, rheumatic fever is a special case since the etiological factor which causes it is a previous infection with haemolysing streptococci of group A. In these patients, the so-called "rheuma tests" in the serum produce a positive reaction to a high degree (80 to 90%).
ThiS iS caused by antibodies against intracellular components of streptococci, such as streptolysin O, desoxyribonuclease, hyaluronidase, etc.
Chronic polyarthritis (rheumatoid arthritis) on the other hand is the most common collagen disease and causes a significant deterioration in health.
Currently, it is accepted that at least 10~ of the total population are affected. However, the results of the "rheuma tests" are positive in only about half the patients. This is particularly true in children.
Hitherto, the emphasis in investigations into the pathogenesis of rheumatoid arthritis and also the diagnosis of this disease has been placed essentially on the idea that intracellular components .lZ~82~
of bacteria are effective as antigens, stimulate the production of antibodies and trigger chronic inflammatory reactions whilst forming immune complexes in the connective tissue.
However, recent investigations have shown that the bacterial cell wall and, in particular, the peptidoglycanes are of far greater significance in the persisting immune response on the induction of chronic inflammatory processes with or without any initial (bacteriologically) detectable infection.
Peptidoglycans (also known as mureines or mucopeptides) are the most important component of the bacterial cell wall and ~are present in all bacteria. Fungi, rickettsiae and viruses, on the other hand, have no corresponding components.
Animal experiments have shown that peptidoglycans can cause markedly chronic inflammations in mammals, as well as inducing acute inflammatory processes.
Moreover, tests have shown that, on account of their considerable resistance to all known animal enzymes, bacterial cell walls may remain localised in the tissues for periods ranging from months to years. It has therefore been suspected that pathogen components of this kind may possibly be implicated in the formation of primarily chronic inflammations of hitherto unknown etiology (e.g.
chronic polyarthritis).
Thus, peptidoglycans are highly potent antigens which occur practically ubiquitously and against which antibodies are frequently formed in-animals.
It is known that the pentapeptide with the structure L-Ala-D-Glu(L-Lys-D-Ala-D-Ala), which occurs particularly in streptococci and staphylococci and does not participate in cross linking, is an essential antigenic determinant of peptidoglycan-s.
This peptide is hereinafter referred to as "peptide sub-unit pentapeptide". The C-terminal sequence -D-Ala-D-Ala oE the peptide sub-unit pentapeptide, 12~ 9 being an "immune-dominant group", is of critical importance in the binding of specific antibodies to the antigenic determinant L-Ala-D-Glu(L-Lys-D-Ala-D-Ala).
Specific antibodies against this peptide sub-unit pentapeptide are therefore of particular diaqnostic interest for the recognition and monitoring of the therapy of "autoimmune diseases" (such as chronic polyarthritis); their presence indicates streptococci or staphylococci as ètiological factors.
Up till now, the presence of antibodies against the peptidoglycan component of bacterial cell walls in animal sera has been described, using various techniques, such as immune precipitation. For detecting antibodies against the peptide sub-unit pentapeptide of peptidoglycans in animal sera, a hapten binding assay is described (J. Immunol.
114, 1191-1196 (1975)). However, the process pu~lished will permit only a rough quantitative estimate of the antibody content in human sera since the quantification is effected using hyperimmune sera - from rabbits.
This method has a number of disadvantages:
~5 1. It cannot be used to detect non-precipitatable antibodies. The authors themselves give a sharp `
deviation in the antibody content determined by various methods (e.g. specific antibody content determined by the hapten binding assay 4.6 mg/ml;
specific antibody content determined by the precipi-tation method 2.2 mg/ml).
2. There is no evidence that the protein precipitated consists of immunoglobulins. Therefore, some co-precipitation of non-immunoglobulins cannot be ruled out.
i2~
i2~
3. It is known from a number of immunological studies that animal antibodies cannot be compared with human antibodies directed against the same antigen in terms of their affinity.
With regard to the hapten binding test itself, there is the objection that it is impossible to differ-entiate between the classes of immunoglobulin in the form in which the test is carried out.
Moreover, the hapten binding test in the form described can easily be disrupted by sùbstances occurring in the test material, e.g. any free antigen present or any antibiotics of similar structure.
This high degree of unreliability is a result of the fact that substances of this kind can very easily influence the state of equilibrium of the soluble antibody/hapten complexes.
However, the main objection to the use of the hapten binding test as a routine method of detecting antibodies against peptidoglycans is the fact that 22Na is used as an internal standard, in addition to 125I for labelling the antigen.
The use of a second isotope (22Na, a hard ~-emitter) used with some considerable radioactivity may be justified in exceptional cases for scientific investi~-- gation, but such a method cannot be recommended for routine tests.
According to one aspect of the present invention there are thus provided antibodies against the peptide sub-unit pentapeptide L-Ala-D-Glu-(L-Lys-D-Ala-D-Ala) of peptidoglycans, said antibodies being from the class of immunoglobulins G. The antibodies of the invention are preferably isolated from other immunoglobulins and/or are in sterile composition and are suitably those characterised a) by the ability to bind to pentapeptides of formula I
-3L2~
R - A - D - Ala (I) (wherein R represents an amino acid component with l, 2 or 3, preferably 3 amino acid residues selected from Gly, L-Ala, D-Glu and L-Lys, and Al represents one of the amino acid residues D-Ala, D-Ser, D-Val and D-Leu); and/or b) by the ability to bind to bacterial cell walls or cell wall material which contain pentapeptides of formula I; and~or c) by the ability to bind to Gram-`positive bacteria which contain pentapeptides of formula I.
According to a further aspect of t~he invention there is provided a method of extracting antibodies according to the invention from human plasma which method comprises: (a) coupling a pentapeptide of formula I to a solid phase, e.g. using coupling techniques known to the art; (b) contacti~g said solid phase with immunoglobulin G-containing human plasma, preferably an immunoglobulin fraction from human plasma which is enriched in immunoglobulin G by a precipitation reaction, whereby said antibodies selectively bind to said pentapeptide coupled to said soiid phase; and (c) subsequently eluting said antibodies selectively from said solid phase.
According to a yet further aspect of the invention there is provided a process for the quanti-tative determination of immunoglubulin G antibodiesagainst the peptide subunit pentapeptide L-Ala-D-Glu-(L-Lys-D-Ala-D-Ala) of peptidoglycans in a biological test material, in particular immunoglobulin containing human plasma, which process comprises contacting said test material with an antigen of formula I which is optionally labelled or substrate b~und. The process oE the invention, which is suitably perormed in vitro, is conveniently performed .
12~8~9 according to immunochemical techniques known to the art, such as the double antibody radioimmunoassay, the double antibody enzyme-immunoassay, the nephelo-metric method, the solid-phase sandwich radioimmuno-assay, the solid-phase enzyme immunoassay, the solid-phase fluorescence immunoassay and the latex agglutination test.
According to a still further aspect of the invention there is provided the use of antibodies of the invention, or of antibodies extracted according to the method of the invention, as standards for or as positive controls in the quantitative determination process of the invention.
Antibodies with a specificity against the peptide sub-unit pentapeptide ~-Ala~D-Glu(L-Lys-D-Ala-D-Ala), i.e. antibodies according to the invention, may be obtained by afEinity chromatography (with matrix-bonded peptides of formula I, e.g.
of the sequence R-D-Ala-D-Ala) from patients' sera in which such antibodies have been detected. 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 solid matrix may be effected by specific desorption of the bound immunoglobulins with peptides of formula I e.g.
of the sequence R-D-Ala-D-Ala. Desorption may also be effected by methods which have been published in principle, such as the use of low pH values or high salt concentrations. After dialysis with buffer solutions, the content of specific antibodies against the peptide sub-unit pentapeptide of peptido-glycans is quantified by means of the immunoglobulin content of the purified fraction.
- Quantitative determination of specific antibodies of the various immunoglobulin classes against the peptide sub-unit pentapeptide of peptidoglycans -~L8;~9 can be effected by two fundamentally different methods.
a) Quantitatlve determination in a soluble system (measurement in a homogeneous phase), bj Quantitative determination by measurement in a heterogeneous system (measurement in a heterogeneous phase; solid-phase immunoassay).
Examples of these two methods of quantitative determination are discussed below a) easurement in the homoqeneous'phase (a.l) quantitative determination of antibodies against the peptide sub-unit pentapeptide of peptidoglycans in a double antibody radioimmunoassay:
Peptides of formula I, e.g. of the R-D~Ala-D-Ala sequence, are radioactively labelled and , the test mixture is prepared according to the following scheme:
*Pept. + Ab ~' *Pept.-Ab twherein *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 3Q precipitation of the immunoglobulins using a second anti-antibody according to the following scheme:
*Pept.-Ab ~ Anti-~b ---~ *Pept.-Ab m~(Anti-Ab)m-complexes (where Anti-Ab represents the anti-antibody) Radioactive labelling of the antigen may be efEected, for example:
iz~
i) by coupling tyrosine to the N-terminal end of the peptide and subsequently iodinating with 125I
by conventional methods ii) by iodinating with (N-succinimidyl-3-(4-hydroxy 5-( I)iodophenyl)propionate iii~ by tritiation of the antigen with N-succinimidyl (2,3-3H)propionate.
By suitable selection o the specificity of the second antibody it is possible to determine separate different classes o immunoglobulin.
It is advisable to purify the anti-antibodi-es used by affinity chromatography on matrix-bonded peptides of formula I, e.g. of the sequence R-D-Ala-D-Ala, in order to remove any antibodies against peptidoglycans which they may contain.
(a.2) Quantitative determination of antibodies against the peptide sub-unit pentapeptide in a double antibody enzyme-immunoassay:
The test procedure of (a.lj above is modified by effecting the labelling of the anti-antibodies by means of an enzyme such as alkaline phosphatase, peroxidase, glucose-oxidase-peroxidase, glucose-6-phosphate-dehydrogenase, malate-dehydrogenase, etc., rather than labelling with a radioactive isotope.
~a.3) Quantitative determination of antibodies against the peptide sub-unit pentapeptide of peptido-glycans by nephelometric methods:
Peptides of formula I, e.g. of the R-D-Ala-D-Ala sequence, are bound, via their N-terminal amino group, to a soluble matrix, e.g. a protein, polyvinyl pyrrolidone, etc., and the test is carried out according to the following scheme:
~2~8~
g Matrix-(Pept.)n + Ab ---~DMatrix-(Pept.)n-Ab-complexes turbidity The coupling of the peptide!s to the carrier material may be effected, for example:
l. by binding iodoacetylated peptides-to the matrix 2. by the carbodiimide method 3. by means of bifunctional reagents, e.g. diiso-cyanates, glutardialdehyde, etc.
(b) Measurement_in the heteroqeneous phase Peptides of formula I/ e.g. of the R-D-Ala-D-Ala sequence, are bound via their N-terminal amino group to a solid 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.
(b.l) Quantitative determination of antibodies - against the peptide sub-unit pentapeptide of peptido-2~ glycans in a solid phase "sandwich" radioimmuno-assay (b.2) Quantitative determination of antibodies against the peptide sub-unit pentapeptide of peptido-glycans in a solid phase enzyme-immunoassay:
The test procedure of (b.l) above is modified by effecting the labelling of the anti-antibodies by means of an enzyme, such as those mentioned in (a.2) above, rather than by labelling with a radioactive isotope.
:~L218~9 (b.3) Quantitative determination of antibodies against the peptide sub-unit pentapeptide of peptido-glycans in a solid phase fluorescence immunoassay:
The test procedure oE (b.l) is modified by labelling the antibody by means of a fluorescent dye (see also the FI~X - StiQ system).
(b.4) Quantitative determination of antibodies against the peptide sub-unit pentapeptide of peptido-glycans by means of a latex agglutination test:
Peptides of formula I, e.g. of the sequence R-D-Ala-D-Ala, are coupled to latex particles either directly or via "spacers" such as proteins.
In the methods of quantitative determination discussed in (a) and (b) above the quantification is eEfected by means of a calibration curve which can be plotted with the aid of the standard solutions of antibodies described above. A suitable calibration curve is shot~n in the accompanying drawing~Fig. 1).
The drawing shows a calibration curve for quantitatively determining specific immunoglobulin G (IgG) against a pentapeptide of formula I, e.g.
R-D-Ala-D-Ala, using the peroxidase system as an indicator. In the figure, the extinction at 492 nm is plotted against the concentration of purified, specific IgG.
The following Examples are provided to illustrate the present invention without serving to restrict the scope of protection sought therefor:
Example l The determination of antibodies of class IqG against penta~ ptide sub-unit pentapeptide ~3 A. Preparation of the conjugate E'quimolar ~uantities oE peptides with the C-terminal amino acid D-Ala of the sequence R-D-Ala-D-Ala, preferably D-Ala-D-Ala-D-~la, and iodoace'tyl succinimide 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 in vac'uo and the reaction product formed is precipitated from the aq~ueous 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 dialysis liquid being changed six times) the peptide coupled to the albumin is lyophilised. With the process described, on average 10 mol of peptide per mole of earrier protein are covalently bound.
B. Adsorption of the conjugate on plastics surfaces In this e~ample, polystyrene tubes were used having a capacity of barely 2 ml, a height of 4 cm and a diameter of 1 cm. Microtitre plates, balls, dishes, ete. of similar plasties material are also suitable for binding.
lOO/ul of conjugate solution (corresponding to 50 ng of conjugate in 0.~ molar bicarbonate buffer, pH 9.6) are pipetted into the tubes and dried a-t 37C for 24 hours. The dry tubes are eaeh washed once with 200 mierolitres of 10 mmol/l phosphate buffer (pH 7.2) in isotonic common ;alt solution eontaining 0.1~ PBS (phosphate buffered saline)/Tween 80*and aft`er the wash solution has been removed by suction filtering the tubes are again rinsed with 200 microlitres of distilled water and then suetion filtered again. The tubes Trademark ~8;~
coated with the conjugate are stored in a drying cupboard at 37C.
C. Test procedure 100 microlitres of specific standard solution containing 0.1 ~g to lO~ug of IgG (see the preparation of the standard reEerred to herein) in ~PBS/Tween*
80 buffer are pipetted into the tubes coated as described above and incubated at 20C for 1 hour.
In 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 1:2000, depending on the titre, with PBS/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/Tween 80 buffer and then suction filtered again. lOOjul ~antihuman IgG) - immunoglobulin (e.g. from goats) labelled with peroxidase or alkaline phosphatase are pipetted into the tubes and incubated for 1 hour at a constant temperature of either 20C, 25C or 37C. Then the tubes are washed twice more, each time with 20 microlitres of PBS/Tween buffer. If conjugated peroxidase is used as the indicator system, o-phenylenediamine and H202 are added as substrate in a citrate buffer (0.1 mol/l, pH 5.0) and the tubes are incubated for 15 minutes at constant temperature.
To stop the reaction, 50jul of 5N H2SO4 are added The extinction at 492 nm is proportional to the quantity of immunoglobulin used (see the accompanying drawing).
In human sera, concentrations of from 1 ~g per ml up to the mg range are found.
The coefficient of variation for determining the specific IgG's is in the central area of the calibration curve at 6.3~.
* Trademark - l3 -Example 2 Obtaining a_specific human immunoglobulin-G standard A. Coupling the albumin-D-Ala-3-conjugate to _epharose*4 b Synthesis is carried out as described for the test. 10 ml of sepharose 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 is kept constant at 11.0 by the addition of 2 mol/1 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 of 0.1 mol/l of sodium hydrogen carbonate solution. The sepharose is stirred into 7.5 ml of 0.1 mol/l oE sodium hydrogen carbonate containing 50 mg of albumin-D-Ala-3 conjug~ate.
Coupling is effected overnight at 4C with gentle stirring. After the reaction time, the substance is suction filtered again and the cou~led 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/l of PBS, pH 7.2. 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 enriched immuno-globulin fraction is added. The charged column is washed with PBS buffer (without Tween) until the extinction at 2~0 nm in the eluate falls to below 0.03 (consumption of about twice as much buffer as serum) * Trademark ~2~829~
Elution is carried out with acetic acid (0.1 to l mol/l). The antibody is eluted with the first fractions of the acetic acid, the fractions containing antibodies are combined and neutralised. The combined eluates are dialysed with a sufficient quantity of PBS, then concentrated and subsequently chromato-graphed on Sephadex G 200.*
C. Characterisation of the antibody fraction The purified immunoglobulin was eluted as a symmetrical peak, measured at 280 nm, ~rom a calibrated Sephadex G 200 column. The molecular weight of this fraction was about 150,000. The molecular weight and homogeneity were further checked by analytical ultracentrifugation, measured with the UV optical equipment at 280 nm. The protein sedimented as a uniform band. Under standard conditions, an S20W of 7.8 was obtained. In cellulose acetal electro~horesis, a uniform band was obtained in the region of the gammaglobulins. In immunoelectro-phoresis, a precipitation line in the immunoglobulin--G range was found when a polyvalent anti-serum was used.
* Trademark
With regard to the hapten binding test itself, there is the objection that it is impossible to differ-entiate between the classes of immunoglobulin in the form in which the test is carried out.
Moreover, the hapten binding test in the form described can easily be disrupted by sùbstances occurring in the test material, e.g. any free antigen present or any antibiotics of similar structure.
This high degree of unreliability is a result of the fact that substances of this kind can very easily influence the state of equilibrium of the soluble antibody/hapten complexes.
However, the main objection to the use of the hapten binding test as a routine method of detecting antibodies against peptidoglycans is the fact that 22Na is used as an internal standard, in addition to 125I for labelling the antigen.
The use of a second isotope (22Na, a hard ~-emitter) used with some considerable radioactivity may be justified in exceptional cases for scientific investi~-- gation, but such a method cannot be recommended for routine tests.
According to one aspect of the present invention there are thus provided antibodies against the peptide sub-unit pentapeptide L-Ala-D-Glu-(L-Lys-D-Ala-D-Ala) of peptidoglycans, said antibodies being from the class of immunoglobulins G. The antibodies of the invention are preferably isolated from other immunoglobulins and/or are in sterile composition and are suitably those characterised a) by the ability to bind to pentapeptides of formula I
-3L2~
R - A - D - Ala (I) (wherein R represents an amino acid component with l, 2 or 3, preferably 3 amino acid residues selected from Gly, L-Ala, D-Glu and L-Lys, and Al represents one of the amino acid residues D-Ala, D-Ser, D-Val and D-Leu); and/or b) by the ability to bind to bacterial cell walls or cell wall material which contain pentapeptides of formula I; and~or c) by the ability to bind to Gram-`positive bacteria which contain pentapeptides of formula I.
According to a further aspect of t~he invention there is provided a method of extracting antibodies according to the invention from human plasma which method comprises: (a) coupling a pentapeptide of formula I to a solid phase, e.g. using coupling techniques known to the art; (b) contacti~g said solid phase with immunoglobulin G-containing human plasma, preferably an immunoglobulin fraction from human plasma which is enriched in immunoglobulin G by a precipitation reaction, whereby said antibodies selectively bind to said pentapeptide coupled to said soiid phase; and (c) subsequently eluting said antibodies selectively from said solid phase.
According to a yet further aspect of the invention there is provided a process for the quanti-tative determination of immunoglubulin G antibodiesagainst the peptide subunit pentapeptide L-Ala-D-Glu-(L-Lys-D-Ala-D-Ala) of peptidoglycans in a biological test material, in particular immunoglobulin containing human plasma, which process comprises contacting said test material with an antigen of formula I which is optionally labelled or substrate b~und. The process oE the invention, which is suitably perormed in vitro, is conveniently performed .
12~8~9 according to immunochemical techniques known to the art, such as the double antibody radioimmunoassay, the double antibody enzyme-immunoassay, the nephelo-metric method, the solid-phase sandwich radioimmuno-assay, the solid-phase enzyme immunoassay, the solid-phase fluorescence immunoassay and the latex agglutination test.
According to a still further aspect of the invention there is provided the use of antibodies of the invention, or of antibodies extracted according to the method of the invention, as standards for or as positive controls in the quantitative determination process of the invention.
Antibodies with a specificity against the peptide sub-unit pentapeptide ~-Ala~D-Glu(L-Lys-D-Ala-D-Ala), i.e. antibodies according to the invention, may be obtained by afEinity chromatography (with matrix-bonded peptides of formula I, e.g.
of the sequence R-D-Ala-D-Ala) from patients' sera in which such antibodies have been detected. 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 solid matrix may be effected by specific desorption of the bound immunoglobulins with peptides of formula I e.g.
of the sequence R-D-Ala-D-Ala. Desorption may also be effected by methods which have been published in principle, such as the use of low pH values or high salt concentrations. After dialysis with buffer solutions, the content of specific antibodies against the peptide sub-unit pentapeptide of peptido-glycans is quantified by means of the immunoglobulin content of the purified fraction.
- Quantitative determination of specific antibodies of the various immunoglobulin classes against the peptide sub-unit pentapeptide of peptidoglycans -~L8;~9 can be effected by two fundamentally different methods.
a) Quantitatlve determination in a soluble system (measurement in a homogeneous phase), bj Quantitative determination by measurement in a heterogeneous system (measurement in a heterogeneous phase; solid-phase immunoassay).
Examples of these two methods of quantitative determination are discussed below a) easurement in the homoqeneous'phase (a.l) quantitative determination of antibodies against the peptide sub-unit pentapeptide of peptidoglycans in a double antibody radioimmunoassay:
Peptides of formula I, e.g. of the R-D~Ala-D-Ala sequence, are radioactively labelled and , the test mixture is prepared according to the following scheme:
*Pept. + Ab ~' *Pept.-Ab twherein *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 3Q precipitation of the immunoglobulins using a second anti-antibody according to the following scheme:
*Pept.-Ab ~ Anti-~b ---~ *Pept.-Ab m~(Anti-Ab)m-complexes (where Anti-Ab represents the anti-antibody) Radioactive labelling of the antigen may be efEected, for example:
iz~
i) by coupling tyrosine to the N-terminal end of the peptide and subsequently iodinating with 125I
by conventional methods ii) by iodinating with (N-succinimidyl-3-(4-hydroxy 5-( I)iodophenyl)propionate iii~ by tritiation of the antigen with N-succinimidyl (2,3-3H)propionate.
By suitable selection o the specificity of the second antibody it is possible to determine separate different classes o immunoglobulin.
It is advisable to purify the anti-antibodi-es used by affinity chromatography on matrix-bonded peptides of formula I, e.g. of the sequence R-D-Ala-D-Ala, in order to remove any antibodies against peptidoglycans which they may contain.
(a.2) Quantitative determination of antibodies against the peptide sub-unit pentapeptide in a double antibody enzyme-immunoassay:
The test procedure of (a.lj above is modified by effecting the labelling of the anti-antibodies by means of an enzyme such as alkaline phosphatase, peroxidase, glucose-oxidase-peroxidase, glucose-6-phosphate-dehydrogenase, malate-dehydrogenase, etc., rather than labelling with a radioactive isotope.
~a.3) Quantitative determination of antibodies against the peptide sub-unit pentapeptide of peptido-glycans by nephelometric methods:
Peptides of formula I, e.g. of the R-D-Ala-D-Ala sequence, are bound, via their N-terminal amino group, to a soluble matrix, e.g. a protein, polyvinyl pyrrolidone, etc., and the test is carried out according to the following scheme:
~2~8~
g Matrix-(Pept.)n + Ab ---~DMatrix-(Pept.)n-Ab-complexes turbidity The coupling of the peptide!s to the carrier material may be effected, for example:
l. by binding iodoacetylated peptides-to the matrix 2. by the carbodiimide method 3. by means of bifunctional reagents, e.g. diiso-cyanates, glutardialdehyde, etc.
(b) Measurement_in the heteroqeneous phase Peptides of formula I/ e.g. of the R-D-Ala-D-Ala sequence, are bound via their N-terminal amino group to a solid 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.
(b.l) Quantitative determination of antibodies - against the peptide sub-unit pentapeptide of peptido-2~ glycans in a solid phase "sandwich" radioimmuno-assay (b.2) Quantitative determination of antibodies against the peptide sub-unit pentapeptide of peptido-glycans in a solid phase enzyme-immunoassay:
The test procedure of (b.l) above is modified by effecting the labelling of the anti-antibodies by means of an enzyme, such as those mentioned in (a.2) above, rather than by labelling with a radioactive isotope.
:~L218~9 (b.3) Quantitative determination of antibodies against the peptide sub-unit pentapeptide of peptido-glycans in a solid phase fluorescence immunoassay:
The test procedure oE (b.l) is modified by labelling the antibody by means of a fluorescent dye (see also the FI~X - StiQ system).
(b.4) Quantitative determination of antibodies against the peptide sub-unit pentapeptide of peptido-glycans by means of a latex agglutination test:
Peptides of formula I, e.g. of the sequence R-D-Ala-D-Ala, are coupled to latex particles either directly or via "spacers" such as proteins.
In the methods of quantitative determination discussed in (a) and (b) above the quantification is eEfected by means of a calibration curve which can be plotted with the aid of the standard solutions of antibodies described above. A suitable calibration curve is shot~n in the accompanying drawing~Fig. 1).
The drawing shows a calibration curve for quantitatively determining specific immunoglobulin G (IgG) against a pentapeptide of formula I, e.g.
R-D-Ala-D-Ala, using the peroxidase system as an indicator. In the figure, the extinction at 492 nm is plotted against the concentration of purified, specific IgG.
The following Examples are provided to illustrate the present invention without serving to restrict the scope of protection sought therefor:
Example l The determination of antibodies of class IqG against penta~ ptide sub-unit pentapeptide ~3 A. Preparation of the conjugate E'quimolar ~uantities oE peptides with the C-terminal amino acid D-Ala of the sequence R-D-Ala-D-Ala, preferably D-Ala-D-Ala-D-~la, and iodoace'tyl succinimide 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 in vac'uo and the reaction product formed is precipitated from the aq~ueous 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 dialysis liquid being changed six times) the peptide coupled to the albumin is lyophilised. With the process described, on average 10 mol of peptide per mole of earrier protein are covalently bound.
B. Adsorption of the conjugate on plastics surfaces In this e~ample, polystyrene tubes were used having a capacity of barely 2 ml, a height of 4 cm and a diameter of 1 cm. Microtitre plates, balls, dishes, ete. of similar plasties material are also suitable for binding.
lOO/ul of conjugate solution (corresponding to 50 ng of conjugate in 0.~ molar bicarbonate buffer, pH 9.6) are pipetted into the tubes and dried a-t 37C for 24 hours. The dry tubes are eaeh washed once with 200 mierolitres of 10 mmol/l phosphate buffer (pH 7.2) in isotonic common ;alt solution eontaining 0.1~ PBS (phosphate buffered saline)/Tween 80*and aft`er the wash solution has been removed by suction filtering the tubes are again rinsed with 200 microlitres of distilled water and then suetion filtered again. The tubes Trademark ~8;~
coated with the conjugate are stored in a drying cupboard at 37C.
C. Test procedure 100 microlitres of specific standard solution containing 0.1 ~g to lO~ug of IgG (see the preparation of the standard reEerred to herein) in ~PBS/Tween*
80 buffer are pipetted into the tubes coated as described above and incubated at 20C for 1 hour.
In 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 1:2000, depending on the titre, with PBS/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/Tween 80 buffer and then suction filtered again. lOOjul ~antihuman IgG) - immunoglobulin (e.g. from goats) labelled with peroxidase or alkaline phosphatase are pipetted into the tubes and incubated for 1 hour at a constant temperature of either 20C, 25C or 37C. Then the tubes are washed twice more, each time with 20 microlitres of PBS/Tween buffer. If conjugated peroxidase is used as the indicator system, o-phenylenediamine and H202 are added as substrate in a citrate buffer (0.1 mol/l, pH 5.0) and the tubes are incubated for 15 minutes at constant temperature.
To stop the reaction, 50jul of 5N H2SO4 are added The extinction at 492 nm is proportional to the quantity of immunoglobulin used (see the accompanying drawing).
In human sera, concentrations of from 1 ~g per ml up to the mg range are found.
The coefficient of variation for determining the specific IgG's is in the central area of the calibration curve at 6.3~.
* Trademark - l3 -Example 2 Obtaining a_specific human immunoglobulin-G standard A. Coupling the albumin-D-Ala-3-conjugate to _epharose*4 b Synthesis is carried out as described for the test. 10 ml of sepharose 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 is kept constant at 11.0 by the addition of 2 mol/1 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 of 0.1 mol/l of sodium hydrogen carbonate solution. The sepharose is stirred into 7.5 ml of 0.1 mol/l oE sodium hydrogen carbonate containing 50 mg of albumin-D-Ala-3 conjug~ate.
Coupling is effected overnight at 4C with gentle stirring. After the reaction time, the substance is suction filtered again and the cou~led 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/l of PBS, pH 7.2. 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 enriched immuno-globulin fraction is added. The charged column is washed with PBS buffer (without Tween) until the extinction at 2~0 nm in the eluate falls to below 0.03 (consumption of about twice as much buffer as serum) * Trademark ~2~829~
Elution is carried out with acetic acid (0.1 to l mol/l). The antibody is eluted with the first fractions of the acetic acid, the fractions containing antibodies are combined and neutralised. The combined eluates are dialysed with a sufficient quantity of PBS, then concentrated and subsequently chromato-graphed on Sephadex G 200.*
C. Characterisation of the antibody fraction The purified immunoglobulin was eluted as a symmetrical peak, measured at 280 nm, ~rom a calibrated Sephadex G 200 column. The molecular weight of this fraction was about 150,000. The molecular weight and homogeneity were further checked by analytical ultracentrifugation, measured with the UV optical equipment at 280 nm. The protein sedimented as a uniform band. Under standard conditions, an S20W of 7.8 was obtained. In cellulose acetal electro~horesis, a uniform band was obtained in the region of the gammaglobulins. In immunoelectro-phoresis, a precipitation line in the immunoglobulin--G range was found when a polyvalent anti-serum was used.
* Trademark
Claims (9)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. Antibodies against the peptide sub-unit pentapeptide L-Ala-D-Glu(L-Lys-D-Ala-D-Ala) of peptidoglycans, said antibodies being from the class of immunoglobulins G.
2. Antibodies as claimed in claim 1 possessing at least one of the following abilities:
a) the ability to bind to pentapeptides 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);
b) the ability to bind bacterial cell walls or cell wall material which contain pentapeptides of formula I; and c) the ability to bind to Gram-positive bacteria which contain pentapeptides of formula I.
a) the ability to bind to pentapeptides 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);
b) the ability to bind bacterial cell walls or cell wall material which contain pentapeptides of formula I; and c) the ability to bind to Gram-positive bacteria which contain pentapeptides of formula I.
3. Antibodies as claimed in claim 2 posessing the ability to bind to pentapeptides of formula I (as defined in claim 2) wherein R represents an amino acid component with 3 amino acid residues selected from Gly, L-Ala, D-Glu and L-Lys.
4. Antibodies as claimed in claims 1, 2 or 3 isolated from other immunoglobulins and/or in sterile compositions.
5. Antibodies as claimed in claims 1, 2 or 3 which are isolated from other immunoglobulins by affinity chromatography.
6. A method of extracting antibodies against the peptide sub-unit pentapeptide L-Ala-D-Glu(L-Lys-D-Ala-D-Ala) of peptido-glycans from human plasma, which method comprises: (a) coupling a pentapeptide of formula I (as defined in claim 2) to a solid phase; (b) contacting said solid phase with immunoglobulin G-containing human plasma whereby said antibodies selectively bind to said pentapeptide coupled to said solid phase; and (c) sub-sequently selectively eluting said antibodies from said solid phase.
7. A method as claimed in claim 6 wherein said plasma com-prises an immunoglobulin fraction from human plasma which has been enriched by a precipitation reaction.
8. A process for the quantitative determination of immuno-globulin G antibodies against the peptide sub-unit pentapeptide L-Ala-D-Glu(L-Lys-D-Ala-D-Ala) of peptidoglycans in a biological test material which process comprises contacting said test material with a pentapeptide of formula I (as defined in claim 2) which is optionally labelled and optionally substrate bound.
9. A process as claimed in claim 8 wherein in said penta-peptide of formula I R represents an amino acid component with 3 amino acid residues selected from Gly, L-Ala, D-Glu and L-Lys.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19823231204 DE3231204A1 (en) | 1982-08-21 | 1982-08-21 | ANTIBODIES AGAINST BACTERIAL PEPTIDOGLYCANES, METHODS FOR THEIR PRODUCTION AND METHODS FOR THEIR QUANTITATIVE DETERMINATION |
| DEP3231204.0 | 1982-08-21 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1218299A true CA1218299A (en) | 1987-02-24 |
Family
ID=6171441
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000434968A Expired CA1218299A (en) | 1982-08-21 | 1983-08-19 | Antibodies against bacterial peptidoglycans, processes for preparing them and methods of quantitively measuring them |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP0101886A3 (en) |
| JP (1) | JPS5990055A (en) |
| AU (1) | AU557044B2 (en) |
| CA (1) | CA1218299A (en) |
| DE (1) | DE3231204A1 (en) |
| IL (1) | IL69531A (en) |
| ZA (1) | ZA836125B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020118042A1 (en) * | 2018-12-05 | 2020-06-11 | Jacobs Wagner Christine | Borrelia burgdorferi peptidoglycan as a diagnostic and target for therapeutic intervention of lyme disease-related pathologies |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3328832A1 (en) * | 1983-08-10 | 1985-02-28 | Boehringer Ingelheim Diagnostika GmbH, 8046 Garching | REAGENT AND METHOD FOR THE QUANTITATIVE DETERMINATION OF ANTIBODY |
| DE3402739A1 (en) * | 1984-01-27 | 1985-08-01 | Boehringer Ingelheim Diagnostika GmbH, 8046 Garching | ANTIBODIES AGAINST THE GLYCAN STRAND OF PEPTIDOGLYCAN, METHODS AND REAGENTS FOR THEIR PRODUCTION AND METHODS FOR THEIR QUANTITATIVE DETERMINATION |
| DE3580882D1 (en) * | 1984-09-14 | 1991-01-24 | Asahi Medical Co | A PROTEIN SPECIFICALLY PURE FOR RHEUMATOID ARTHRITIS AND AN ANTIBODY AGAINST IT. |
| JPS6170463A (en) * | 1984-09-14 | 1986-04-11 | Medekusu:Kk | Refining method of protein specific to rheumatism |
| DE102010048382B4 (en) * | 2010-10-13 | 2012-07-12 | Vag-Armaturen Gmbh | Corrosion protected fitting |
Family Cites Families (1)
| 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 |
-
1982
- 1982-08-21 DE DE19823231204 patent/DE3231204A1/en not_active Withdrawn
-
1983
- 1983-07-20 EP EP83107087A patent/EP0101886A3/en not_active Withdrawn
- 1983-08-19 IL IL69531A patent/IL69531A/en unknown
- 1983-08-19 JP JP58151471A patent/JPS5990055A/en active Pending
- 1983-08-19 AU AU18136/83A patent/AU557044B2/en not_active Ceased
- 1983-08-19 CA CA000434968A patent/CA1218299A/en not_active Expired
- 1983-08-19 ZA ZA836125A patent/ZA836125B/en unknown
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020118042A1 (en) * | 2018-12-05 | 2020-06-11 | Jacobs Wagner Christine | Borrelia burgdorferi peptidoglycan as a diagnostic and target for therapeutic intervention of lyme disease-related pathologies |
Also Published As
| Publication number | Publication date |
|---|---|
| IL69531A0 (en) | 1983-11-30 |
| AU1813683A (en) | 1984-02-23 |
| JPS5990055A (en) | 1984-05-24 |
| AU557044B2 (en) | 1986-12-04 |
| IL69531A (en) | 1987-10-30 |
| DE3231204A1 (en) | 1984-03-01 |
| ZA836125B (en) | 1985-04-24 |
| EP0101886A2 (en) | 1984-03-07 |
| EP0101886A3 (en) | 1986-07-02 |
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