AU721873B2 - Process for binding monosaccharides, oligosaccharides or polysaccharides to a solid phase - Google Patents

Description

WO 96/32644 PCT/EP96/00706 Process for binding monosaccharides, oligosaccharides or polysaccharides to a solid phase The present invention relates to a process for binding monosaccharides, oligosaccharides or polysaccharides to a solid phase which is carrying secondary amino groups, to solid phases which are prepared by this process, and to test methods for detecting substances which bind to monosaccharides, oligosaccharides or polysaccharides.

Owing to their severity and frequency, Hemophilus influenzae (Hi) infections occupy a special position in the study of pediatric infections. Serologically, Hemophilus influenzae is subdivided into two strains, i.e. capsulated and non-capsulated strains. As commensals, the non-capsulated strains colonize the mucous membranes of the upper airways and are pathogenic, in particular in association with previously damaged epithelium. Accordingly, they are frequently detected as causative agents of sinusitis, otitis and chronic bronchitis. It is predominantly children who are between the 4th month of life and the 5th year of life who contract infections with capsulated strains.

The capsule is composed of polyribosylribitol phosphate (PRP) and is crucial for virulence since only capsulated strains are able to start an infection hematogenically.

In more than 95% of cases, this infection is caused by the b serotype (Hib). The other serotypes, a to f, are only responsible for an infection in very rare cases.

Before Hib vaccination was introduced in Germany in 1990, the incidence of severe Hib infections was 33 for every 100,000 children under 5 years of age. This corresponds roughly to the frequency in other European countries. In about 2/3 of cases, infection with Hib causes meningitis or epiglottitis. The more unusual manifestations of Hib infections include bacteremia, septic arthritis, phlegmon, osteomyelitis, pericarditis and pneumonia.

WO 96/32644 2 PCT/EP96/00706 However, the frequency of Hib being the causative agent of primary pneumonia is not clear. If the results from lung biopsies are taken as a basis, Hib can also be demonstrated to be the causative agent in 10% of pneumonia cases.

Hib meningitis is by far and away the most dangerous.

While the majority of cases recover completely without sequelae, about 20% of children only survive the disease with severe consequential neurological damage, such as deafness, cerebral fit disorders and mental disability.

According to data from the literature, the mortality in Hib meningitis is between 1.6 and 7%.

In animal experiments, antibodies against the capsule.PRP possess a protective function, either by passive or active immunization, and produce a complement-dependent lysis of thebacterium. PRP antibody levels of greater than 0.15 Ag/ml are regarded as protecting against invasive Hemophilus infections. The connection between low PRP antibody levels and the probability of contracting a Hib infection is also apparent from the natural course taken by antibody levels: Owing to the maternal antibodies which have been loaned to them, newborn babies possess relatively high antibody titers. These antibodies fall continuously until the 6th month of life and then remain low until the 18th month of life. The antibodies then increase once again in babies who have passed the 18th month of life, which is the time at which ontogenesis of the immune system has come to an end and natural exposure induces an immune response. For this reason, a Hib infection is relatively rare in children younger than 3 months and older than 5 years.

However, there are considerable ethnic differences in the age-dependent frequency of Hib infection. For example, Hib meningitis occurs significantly later in German children as compared with Eskimo children.

WO 96/32644 3 PCT/EP96/00706 Investigations of Hemophilus-specific antibody levels following natural infection demonstrated that there are large age-dependent differences in the development of specific immunity. The results obtained by several investigators highlight the fact that the overwhelming majority of patients who were younger than 18 months did not develop Hemophilus-specific antibody concentrations greater than 0.15 ;g/ml. The development of immunity proceeded in a much more favorable manner in patients older than 18 months. These results indicate clearly to the pediatrician that patients younger than 18 months constitute an at-risk group following Hib infection and that active immunization with subsequent serological monitoring should be performed.

Because the activity of the pure PRP vaccines was unsatisfactory, new conjugate vaccines have been developed in recent years which are based on carrier/hapten complexes and which are also active in babies. The principle of these new conjugate vaccines is that of coupling the weak immunogen PRP to a powerfully antigenic protein. This results in a switch from a T cell-independent antigen to a T cell-dependent antigen.

This leads to a change in the immune reaction: The antibody titers are markedly higher A second vaccination results in a booster reaction The IgM antibody response switches to the IgG antibody response.

Several new conjugate vaccines have been developed: PRP-CRM (CRM and CRM 197 modified diphtheria toxin), PRP-D (D diphtheria toxoid), PRP-OMP (OMP Neisseria meningitidis protein complex) and PRP-T (T tetanus toxoid).

Currently, 4 Hib conjugate vaccines are authorized in Germany. There are differences with regard to 3 immunogenicity between the individual Hib conjugate WO 96/32644 4 PCT/EP96/00706 vaccines. Thus, after a single inoculation, the PRP-OMP vaccine achieves by far and away the highest antibody level. On the other hand, the highest booster response is obtained, irrespective of the basal immunization, when repeat vaccination is carried out with the PRP-D vaccine.

The protective titer following immunization is regarded as being a 1 gg of anti-PRP/ml, in contrast to the a 0.15 gg/ml following a natural infection with Hib.

Within the context of developing combination vaccines with the aim of reducing the frequency of immunizations, care must be taken to ensure that the individual proportions of the combination vaccine do not exert any negative interference. For example, when combining the Hib vaccine with the existing combination DPT in order to form the tetravalent vaccine DPTHib, the seroconversion titer against the individual vaccine antigens must be of the same order of size as that achieved with a chronologically staggered vaccination with a DPT vaccine on the one hand and a Hib vaccine on the other.

Hitherto, only a radioantigen-binding test (RIA) has been regarded as being meaningful, and accepted by the relevant committees and licensing bodies, for assessing the immunogenicity of a Hib vaccine and measuring the anti-Hib antibodies in the serum of patients.

RIA suffers from the disadvantage that it is not able to distinguish between the different isotypes and subclasses of the antibodies which bind to the PRP and are able to precipitate it. Furthermore, work with radionuclides is cost-intensive, problematic from the point of view of medical safety at work, and environmentally damaging.

As an alternative to RIA, various groups have attempted to construct test systems in an ELISA format for determining the antibody titers. A problem in this respect is that the Hib-specific polysaccharide, i.e. the PRP, does not bind adequately to conventional microtitration plates WO 96/32644 5 PCT/EP96/00706 made of polystyrene). For this reason an attempt was made, for example, to increase the binding rate of the PRP by conjugating it with human serum albumin (Phipps et al., J. Immunol. Methods 135, 1I1-128 [1990]).

Another attempt consisted of modifying PRP by tyraminizing it and binding this chemically modified polysaccharide to customary microtitration plates (Kristensen and Bentzon, Acta Pathologica et Microbiologica Scandinavica 100, 142-146 [1992]).

However, the chemical modification results in the formation of neoantigens, which can lead both to falsely positive and falsely negative results. Another method consists in preincubating the microtitration plates with poly-L-lysine or protamine, for example, and binding the PRP, which reacts chemically as an acid, to the microtitration plates indirectly by way of these basic components.

However, ELISAs which are constructed in accordance with the abovementioned principles are very trouble-prone, are of low sensitivity and frequently also lead to nonspecific binding of the sample antibodies or the conjugate.

The present invention was therefore based on the object of making available improved test systems for detecting monosaccharides, oligosaccharides or polysaccharides, which test systems no longer exhibit the abovementioned disadvantages.

This object was achieved by an advantageous process for more efficiently binding monosaccharides, oligosaccharides or polysaccharides to a solid phase and by the provision of an advantageous solid phase, for example in the form of a microtitration plate, which can be obtained by means of this process.

It has been found, surprisingly, that, in contrast to known solid phases which are used in customary micro- 23. MAY. 2000 12:19 WATERMARK 613 98196010 NO. 7789 P. titration plates. a solid p~mase, such as a polystyrene surface, to whichb secondary amine groups have been bounmd by way of spacers (secondary ami~o group-carrying solid phase i-SASP) have a high binding capacity for different sacebaride struictural coponents such as PRP. The preparation of SASPs is descrIbed, for example, in European Patent Applic~ation 0 319 957.

For example, while PRP can ca= bind directly to the SASP, it cannotE bind directly to customary solid phases, such as conventional microtitration plates made of polystyren1e, which do not carry any secondary amino groups on the surface, specifically nor when more than 6 jig of PRP per well are used for coating a microtitration plate: The osco=ercially available microtitration plate CovaLix~c NE" 15 (registered trade mark of !(uac A/S, P.O. Box 280, Kamstrup, DXi-4000 Roskilde, Denmark) is an example of a very suitable SASF. The standard commercial Nunc A/S U8 microtitration plate is an example of a conventional 5005 solid phase. The SASPs are therefore very well suited for producing a microtitration plate which is appropriate for determining PRP-specific anti~bodies and which can also be stored without loss of acti-vity. A PRP ELISA which has been prepared in this way is vrery suitable f or the precise determination of antibodies against PRP, and the values which are obtained correlate with the values which .5 are obtained in an RIA such that the ELISA can be used as *an alternative to the RIA.

The present invention consequently relates to a process for binding a polysaccharide to a solid phase which is carrying secondary ainino groups (SASP), characterized in that the polysaccharide is reacted with the secondary amino groups of the SASP in the presence of carbodjimide and/or Nhydroxysuccinimide.

Those embodiments are preferred which relate to the bintding of polysaccharides from Pneiuiococci, for example types 3, 6, 8, 14, 19 or 23, or Hemophilus iif luenzae 23/05 '00 TUE 13:08 [TX/RX NO 8956] 23. MAY, 2060 12:19 WATERMARK 613 98196010 NO, 7789 P. 6 particularly preferably Rib, to an SAsP. An embodiment of the abovementioned process which is very particularly preferred according to the invention relates to the diagnostically importan~t polysaccharide polyribouylribitol phosphate (PRP) from the Hi~b capsule.

Suitable methods for this Purpose are described, for example, in Rasmussen, Ann. Biol. Clin. (1990), 48, 647-650.

Th rsn neto ute*mr eae osldpae (SS) prfrbyi hSomof=coirto lts whc a0epoucdb n fteprcse codn toteivnin see The prsedpassacodntt h invention futanmr rebee tosl u sed t :stali: testP) mefbl nthod for~ ofetc ati plbsances, rfeal 0ibdis which bian topoucdb one of thess aovdiprfre codlgto the in-wention Intht whiidhse fordngs, the invention eryng ehue t0 prstatlshet ppctiod s fordeetingll depbctadcesh othe reablyo eamtipes, which rben toe controf iothe ae giovsen a~es n LS for determting at-R antibodies ua ea suc a as ceibrpia, fluio emile ord batlarlyI arefso c acoding to the determinton o nioisfo a nd tha phichifollor, tle ithetontuer yinusthar **IS 2sdscie0i h present patent application, i!:diinalnepcedt alhe aieo elise forc esrie the concetrctions of 23/05 '00 TUlE 13:08 ITX/RX NO 89561 WO 96/32644 8 PCT/EP96/00706 monoclonal antibodies against PRP. An ELISA which is constructed in an analogous manner can be used for determining antibodies against other monosaccharides, oligosaccharides or polysaccharides when at least one appropriate sugar antigen, such as a pneumococcal polysaccharide, is bound to CovaLink NH® microtitration plates, for example, in analogy with PRP.

The invention underlying the present patent application is also described in the patent claims.

23. MAY. 2000 12:19 WATERMARK 613 98196010 NO. 7789 P. 7 9- Coating microtitratimi Plates PRP antigen is dissolved in 1% N- (3 -dime thylaminopropyl) N-ethylcarbodiimide hydrochloride solution at a concentration of 1 g.g/ml. 1"Zcrotitration plates (to which secondary amino groups have been bound by way-of spacers) are coated with 100 RI of the antigen solution per well and, after having been sealed off, are incubated at 37*C for 18 hours. The suicrotitration plates are then washed twice with POD washing solution (200 1/we11) and dried over silica gel for two 6edays. The microtitration plates which have been prepared in 0.0.this way are welded into flat aluminum bags, together with two drying capsules in each case, and stored at 4'C until subsequent use.

Ezaay1e 2 Test implementation 100 Al of PBS, pH 7.2, containing 20% sheep serum are pipetted into each well, in order to block non-specific binding of antibodies to the microtitration plates, after which the plates axe sealed off, incubated at 370C for 1 hour and 'then washed three times in PBS, 7.2, *0.05% Tweezio- The antibody samples are preferably prediluted from 1:10 to 1±100 iii PBS, pH 7-2 2% sheep ser-um and loaded onto the umicrotitration platen in doubling dilution steps, as a duplicate determination, up to the eighth row. The plates are sealed off and incubated at 37*C for 60 5 minutes. They are then washed -three times with PBS, pH 7.2 0.05% Tweeno After that, 100 Al of anti-human IgG-POD conjugate (POD peroxcidase)+ (diluted 1:10000 in PBS, pH 7.2 2% sheep seru=) are pipetted into each well, and the plates are sealed off and incubated at 370C for 60 5 minutes- The smicrotitration plates are washed three times with PBS, pH 7.2 +i 0. 05% Tween ,and 100 yl of chromogen solution 23/05 '00 TUE 13:08 [TX/RX NO 8956] WO 96/32644 10 PCT/EP96/00706 are then added. After incubating at room temperature for minutes in the dark, the reaction is terminated by adding 100 ip of stop solution per well. The plates are evaluated photometrically at 450 nm.

Example 3 Comparison between RIA values and ELISA values By way of example, the antibody titers of human sera which were determined by means of a radioimmunoassay (RIA), on the one hand, or using the ELISA which is described here, on the other, are listed in the following table.

Serum No. Antibody titer (gg/ml) RIA

ELISA

3 1.1 3 4 24.6 7.2 6 1.2 0.65 7 6.6 6.6 8 1.1 0.8 9 3.0 3.2 2.8 11 0.24 13 3.3 1.7 10 7.6 16 9.8 6.7 1.68 1.1 21 1.65 1.3 22 0.54 0.35 23 8.62 15.9 24 5.7 6.1 26 1.9 2.3 27 97.2 S28 1.93 4.1 WO 96/32644 11 PCT/EP96/00706 29 21.8 29.1 0.33 0.56 The ELISA which is presented here does not measure any falsely positive values. This means that human sera which have an anti-PRP titer of 0.1 Ag/ml by RIA but have a high content of antibodies against other bacterial polysaccharides, such as pneumococcal polysaccharides, do not give titers which are above the cut-off value in the ELISA either.

Example 4 The determination of the PRP-specific antibodies is not restricted simply to human investigative material; mouse anti-PRP antibodies, for example, can also be determined in this test system. An incubation buffer other than that used for determining human PRP antibodies, i.e. PBS, pH 7.2 containing 2% bovine serum albumin (Boviserin or PBS, pH 7.2 containing 2% Haemaccel is more advantageously used for determining mouse anti-PRP antibodies in this ELISA.

(Boviserin® and Haemaccel® are registered trade marks of Behringwerke AG, D-35001 Marburg, Germany).

Example If the polysaccharide antigens which are specific for the Pneumococci types 6, 8, 19 or 23 are bound to the CovaLink NH® microtitration plate in analogy with Examples 1 and 2, the binding of the pneumococcal polysaccharides is also, surprisingly, observed, in this case too, to be substantially improved, as was the case with the binding of PRP, as compared to the binding achieved when these polysaccharides are bound to conventional microtitration plates made, for example, of polystyrene.

By contrast, polysaccharides of the Pneumococci types 3 and 14 bind satisfactorily to conventional ELISA plates S as well; nevertheless, the binding of these poly- WO 96/32644 12 PCT/EP96/00706 saccharides to CovaLink NHO plates is improved by a further 10 to The determination of antibodies against type-specific pneumococcal polysaccharide antigens is not negatively affected by adding other type-specific polysaccharides.

Claims (7)

1. Process for binding a monosaccharide; oligosaccharide or polysaccharide to a solid phase which is carrying secondary amino groups (SASP), characterized in that the monosaccharide, oligosaccharide or polysaccharide is reacted with the secondary amino groups of the SASP in the presence of carbodiimide and/or N- hydroxysuccinimide. 0*0

2. Process according to claim 1, characterized in that the monosaccharide, oligosaccharide or polysaccharide carries at least one phosphate group. S: 3. Process according to claim 1, characterized in that the monosaccharide, oligosaccharide or polysaccharide is derived from Hemophilus influenzae.

4. Process according to claim 3, characterized in that the monosaccharide, oligosaccharide or polysaccharide is derived from Hemophilus influenzae b. *000 00*0

5. Process according to claim 4, characterized in that the polysaccharide is polyribosylribitol phosphate (PRP).

6. Process according to claim 1, characterized in that the monosaccharide, oligosaccharide or polysaccharide is derived from Pneumococci. 00 7, Process according to claim 6, characterized in that the Pneumococci are to be assigned to one or more of the types 3, 6, 8, 14, 19 or 23.

8. Process according to claim 1, characterized in that N- (3- dimethylaminopropyl) N-ethylcarbodiimide hydrochloride is employed. 23/05 '00 TUE 13:08 [TX/RX NO 8956]

23. MAY. 2000 12:20 WATERMARK 613 98198010 NO. 7789 P. 9 14 91 Solid phase which is coated with a monosaccharide, an oligosaccharide or a polysaccharide and which has been produced by the process acco rding to one or more of claims 1 to s. Use of the solid phase according to claim 9 in a test method for determining antibodies against monosaccha rides, oligosaccharides or polysaccharides. S.d DATED this 22n day of May 2000 e~g. *DADE BEHRING MARBURG GMBH WATERMARK PATENT TRADEMARK ATTORNEYS 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 :0:..:AUSTRALIA KJSIMH/MEH P8515AUooflOC 23/05 '00 TUE 13:08 ITX/RX NO 8956]