WO1996032644A1

WO1996032644A1 - Process for binding mono, oligo or polysaccharides to a solid phase

Info

Publication number: WO1996032644A1
Application number: PCT/EP1996/000706
Authority: WO
Inventors: Michael Bröker; Stefan Zielen
Original assignee: Behring Diagnostics Gmbh
Priority date: 1995-04-10
Filing date: 1996-02-21
Publication date: 1996-10-17
Also published as: JPH11503525A; AU4878796A; EP0821792A1; DE19512707A1; CA2217713A1; AU721873B2

Abstract

The present invention relates to a process for binding mono, oligo or polysaccharides to a solid phase bearing secondary amino groups, solid phases which can be produced by this process and test methods for detecting substances binding to mono, oligo or polysaccharides.

Description

Process for binding mono-, oligo- or polysaccharides to a solid phase

The present invention relates to a method for binding mono-, oligo- or polysaccharides to a solid phase bearing secondary amino groups, solid phases which can be prepared by this method and test methods for the detection of substances which bind to mono-, oligo- or polysaccharides.

Haemophilus influenzae (Hi) infections occupy a special position within pediatric infectious diseases because of their severity and frequency. Serologically Haemophilus influenzae is divided into two strains, encapsulated and non-encapsulated. The unencapsulated strains colonize the mucous membranes of the upper respiratory tract as commensals and are particularly pathogenic if the epithelium is damaged. Accordingly, they are often found to cause sinusitis, otitis and chronic bronchitis. Infections with encapsulated strains mainly affect children between the 4th month and the 5th year of life.

The capsule is made of polyribosyl ribitol phosphate (PRP) and is crucial for virulence, since only encapsulated strains can cause a hematogenous infection. This is caused by serotype b (Hib) in more than 95% of cases. The other serotypes a to f are only responsible for infection in very rare cases.

Before the introduction of Hib vaccination in Germany in 1990, the incidence of severe Hib infections was 33 to 100,000 children under the age of 5. This speaks roughly the frequency in other European countries. In about 2/3 of the cases, Hib infection causes meningitis or epiglottitis. The rarer manifestations of Hib infections include bacteremia, septic arthritis, phlegmon, osteomyelitis, pericarditis and pneumonia. However, the frequency of Hib as the causative agent of primary pneumonia is unclear. Based on the results of pulmonary puncture, Hib is also detectable as a pathogen in pneumonia in 10% of cases.

By far the most threatening is Hib meningitis. The majority of the cases heal without consequences, but about 20% of the children survive the disease only with severe neurological damage, such as deafness, cerebral seizures and intellectual disability. The mortality rate of Hib meningitis is between 1, 6 and 7% according to the literature.

Antibodies against PRP of the capsule have a protective function in experimental animal experiments either through passive or active immunization and produce complement-dependent lysis of the bacterium. PRP antibody levels above 0.15 μg / ml are considered protective against invasive hemophilus infections. The relationship between low PRP antibody levels and the likelihood of developing a Hib infection is also evident from the natural course of the antibody:

Newborns have relatively high antibody titers due to maternal loan antibodies. These decrease continuously until the 6th month of life and remain low until the 18th month. In children beyond the age of 18, the antibodies then rise again, a point in time at which the ontogeny of the immune system is complete and natural exposure induces an immune response. Therefore, Hib infection is rather rare in children under 3 months and beyond 5 years. However, there are significant ethnic differences in the age-related frequency of Hib infection. Compared to Eskimo children, Hib meningitis, for example, occurs much later in German children.

The investigation of the haemophilus-specific antibody levels after natural infection showed that there are large age-dependent differences in the development of a specific immunity. The results of several examiners show indicated that the vast majority of patients under the age of 18 months did not develop haemophilus-specific antibodies above 0.15 μg / ml. Immunity development was significantly more favorable in patients over 18 months. For the pediatrician, this clearly shows that patients under 18 months after Hib infection represent a risk collective and that an active immunization with subsequent serological control should be carried out.

Due to the insufficient effectiveness of the pure PRP vaccines, new conjugate vaccines based on carrier-hapten complexes have been developed in recent years, which are also effective in young children. The principle of these new conjugate vaccines is the coupling of the weak immunogen PRP to a strong antigenic protein. This results in a "switch" from a T cell-independent to a T cell-dependent antigen. This leads to a changed immune response:

- »The antibody titers are significantly higher

- A "booster reaction" occurs with a second vaccination.

- »There is a change from the IgM to the IgG antibody response.

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

4 Hib conjugate vaccines are currently approved in Germany. There are differences in immunogenicity between the individual Hib conjugate vaccines. Thus, the PRP-OMP vaccine achieves by far the highest antibody levels after a single vaccination. On the other hand, regardless of the basic immunization, the highest booster response occurs when a new vaccination with the PRP-D vaccine has been carried out. The protective titer after immunization is considered at> 1 μg / ml anti PRP in contrast to the> 0.15 μg / ml after a natural infection by Hib.

As part of the development of combination vaccines with the aim of reducing the frequency of immunizations, it must be ensured that the individual rates of the combination vaccine do not cause any negative interference. For example, with a combination of the Hib vaccine with the existing combination DPT to the tetravalent vaccine DPTHib, the seroconversion titer against the individual vaccine antigens must be in the same order of magnitude as with a delayed vaccination with a DPT vaccine on the one hand and a Hib vaccine on the other.

To assess the value of the immunogenicity of the Hib vaccine and the anti-Hib antibody in the serum of patients, only a radioantigen binding test (RIA) has so far been considered meaningful and has been accepted by the relevant committees and approval bodies.

The disadvantage of the RIA is that it cannot distinguish between the different isotypes and subclasses of the antibodies that bind to the PRP and are able to precipitate it. In addition, working with radionuclides is cost-intensive, problematic for occupational health and safety and polluting the environment.

To determine the antibody titers as an alternative to the RIA, various groups have attempted to set up test systems in ELISA format. A problem here is that the Hib-specific polysaccharide, the PRP, binds insufficiently to conventional microtitration plates (e.g. made of polystyrene). For this reason, attempts have been made, for example, to increase the binding rate by conjugating the PRP with human serum albumin (Phipps et al., J. Immunol. Methods 135, 111-128 [1990]). Another attempt was to modify PRP by tyraminization and to bind this chemically modified polysaccharide to conventional microtitration plates (Kristensen and Bentzon, Acta Pathologica et Microbiologica Scandinavica 100, 142-146 [1992]). However, chemical modification leads to the formation of neoantigens, which can lead to both false positive and false negative results. Another method is to use the microtitration plates e.g. pre-incubate with poly-L-lysine or protamine and indirectly bind the chemically acidic PRP to the microtitration plates via these basic components.

However, ELISAs based on the abovementioned construction principles are very susceptible to interference, have a low sensitivity and often also lead to unspecific binding of the sample antibody or the conjugate. The object of the present invention was therefore to provide improved test systems for the detection of mono-, oligo- or polysaccharides which no longer have the disadvantages mentioned above.

This object was achieved by an advantageous method for the more efficient binding of mono-, oligo- or polysaccharides to a solid phase and the provision of an advantageous solid phase obtainable by this method, for example in the form of a microtitration plate.

Surprisingly, it has been found that a solid phase (solid phase), for example a polystyrene surface, to which secondary amino groups have been bound via spacers (secondary amino group-bearing solid phase = SAFP), in contrast to known solid phases, is used in conventional microtitration plates has a high binding capacity to different saccharide components, such as PRP. The production of SAFPs is described, for example, in European patent application 0319957.

For example, PRP can bind directly to the SAFP, but not to a conventional solid phase, such as conventional polystyrene microtitration plates, which do not have secondary amino groups on the surface, even if more than 6 μg PRP for coating one well Microtitration plate can be used. An example of a very suitable SAFP is the commercially available microtitration plate CovaLink NH® (registered trademark of Nunc A / S, PO Box 280, Kamstrup, DK-4000 Roskilde, Denmark). An example of a conventional solid phase is the commercially available U8 microtitration plate from Nunc A / S. The SAFP are therefore very suitable, for example, for producing a microtitration plate suitable for PRP-specific antibody determination, which can also be stored without loss of activity. A PRP-ELISA produced in this way is well suited for the precise determination of antibodies against PRP and the values determined correlate with the values determined in the RIA such that it can be used as an alternative to the RIA. The present invention accordingly relates to a method for binding a polysaccharide to a solid phase carrying secondary amino groups (SAFP), characterized in that the polysaccharide is reacted with the secondary amino groups of SAFP in the presence of a bifunctional reagent.

Preferred embodiments are those which relate to the binding of polysaccharides from pneumococci, for example types 3, 6, 8, 14, 19 or 23, or Haemophilus influenzae (Hi), particularly preferably Hib, to a SAFP. A particularly preferred embodiment of the above-mentioned method according to the invention relates to the diagnostically important polysaccharide polyribosyl ribitol phosphate (PRP) from the capsule of Hib.

Preferred methods according to the invention are furthermore those which use either a carbodiimide or N-hydroxysuccinimide or both substances as the bifunctional reagent. Suitable methods for this are described for example in Rasmussen, S.E., Ann. Biol. Clin. (1990), 48, 647-650.

The present invention further relates to solid phases (SAFP), preferably in the form of microtitration plates, which can be produced by one of the methods according to the invention.

Using the solid phases according to the invention, test methods for the detection of substances, preferably antibodies, which bind to one of the abovementioned mono-, oligo- or polysaccharides can be established. According to the invention, for example, an ELISA for detecting antibodies against PRP from Hib is very particularly preferred.

In the following, the invention on which the present patent application is based is also illustrated with the aid of examples which describe the structure of an anti-PRP test in ELISA format. Examples are given for the determination of anti-PRP antibodies in human sera, but also the determination of antibodies from other human lesions, such as cerebrospinal fluid or umbilical cord blood, and also the determination of antibodies from organs of non-human origin, such as mice, Rabbit sheep and especially all (other) common experimental animals explained. The ELISA for the detection of PRP described in the present patent application can also be used to measure the value of monoclonal antibodies against PRP. An ELISA with an analog structure can be used to determine antibodies against other mono-, oligo- or polysaccharides if at least one corresponding sugar antigen, such as a polysaccharide from pneumococci, is bound to CovaLink NH® microtitration plates, for example, analogously to PRP.

The invention on which the present patent application is based is also described in the patent claims.

example 1

Coating of microtitration plates

PRP antigen is dissolved in a concentration of 1 μg / ml in 1% N- (3-dimethylamino-propyl) -N-ethylcarbodiimide hydrochloride solution. Microtitration plates are coated with 100 μl of the antigen solution per well and, after the plates have been taped off, incubated at 37 ° C. for 18 hours. The microtitration plates are then washed twice with POD washing solution (200 μl / well) and dried over silica gel for two days. The microtitration plates prepared in this way are sealed with two dry capsules in flat aluminum bags and stored at 4 ^° C. until further use.

Example 2

Test implementation

For blocking non-specific binding of antibodies to the microtiter plates are pipetted into each well 100 ul of PBS pH 7.2 with 20% serum Schaf¬, taped the plates incubated for 1 hour at 37 C and ^β anschlie¬ ßend three times in PBS 7.2 0 , 05% Tween® washed. The antibody samples are preferably prediluted 1:10 to 1: 100 in PBS, pH 7.2 + 2% sheep serum and applied in two dilution stages as a double determination to the eighth row of the microtitration plates. The plates are taped and incubated at 37 ° C for 60 ± 5 minutes. It is then washed three times with PBS pH 7.2 + 0.05% Tween ® 20.

Depending on the well, 100 μl anti-human-IgG-POD conjugate (POD = peroxidase) (diluted 1: 10,000 in PBS pH 7.2 + 2% sheep serum) is pipetted, the plates are taped and 60 ± 5 minutes at 37 ° C incubated. The microtitration plates are washed three times with PBS pH 7.2 + 0.05% Tween® and 100 μl chromogen solution are added. After 30 minutes of incubation at room temperature in the dark, the reaction is stopped by adding 100 μl stop solution per well. The photometric evaluation takes place at 450 nm. Example 3

Comparison between RIA and ELISA values

The antibody titers of human sera are listed by way of example in the following table, which were determined on the one hand by means of a radioimmunoassay (RIA) or with the ELISA described here.

The ELISA presented here does not measure false positive values. This means that human sera with an anti PRP titer of <0.1 μg / ml according to the RIA, but a high antibody content against other bacterial polysaccharides, such as pneumococci, do not result in any cut-off values in the ELISA either lying titer.

Example 4

The determination of the PRP-specific antibodies is not only limited to human test material; Mouse anti-PRP antibodies can also be determined in this test system, for example. It is advantageous to use a different incubation buffer for the determination of the mouse anti-PRP antibodies in this ELISA than for the determination of human PRP antibodies, namely PBS, pH 7.2 with 2% bovine serum albumin (Boviserin®) or PBS, pH 7.2 with 2% Haemaccel®.

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

Example 5

If, in analogy to Examples 1 and 2, the polysaccharide antigens, which are specific for type 6, type 8, type 19 or type 23 pneumococci, are bound to the CovaLink NH® microtitration plate, surprisingly, here too, similar to the binding from PRP, a much better binding of the pneumococcal polysaccharides is observed than if one binds them to conventional microtitration plates, for example made of polystyrene. The binding of polysaccharides of type 3 and type 14 pneumococci, on the other hand, is also sufficient on conventional ELISA plates, but the binding on CovaLink NH® plates is improved by a further 10 to 30%.

The determination of antibodies against type-specific pneumococcal polysaccharide antigens is not adversely affected by the addition of other type-specific polysaccharides.

Claims

claims

1. A method for binding a mono-, oligo- or polysaccharide to a secondary amino-bearing solid phase (SAFP), characterized in that the mono-, oligo- or polysaccharide is reacted with the secondary amino groups of the SAFP in the presence of a bifunctional reagent.

2. The method according to claim 1, characterized in that the mono-, oligo- or polysaccharide carries at least one phosphate group.

3. The method according to claim 1, characterized in that the mono-, oligo- or polysaccharide comes from Haemophilus influenzae.

4. The method according to claim 3, characterized in that the mono-, oligo- or polysaccharide comes from Haemophilus influenzae b.

5. The method according to claim 4, characterized in that the polysaccharide is polyribosyl ribitol phosphate (PRP).

6. The method according to claim 1, characterized in that the mono-, oligo- or polysaccharide comes from pneumococci.

7. The method according to claim 6, characterized in that the pneumococci are assigned to one or more of types 3, 6, 8, 14, 19 or 23.

8. The method according to claim 1, characterized in that the bifunctional reagent is a carbodiimide and / or N-hydroxysuccinimide.

9. The method according to claim 8, characterized in that N- (3-dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride is used.

10. Solid phase coated with a mono-, oligo- or polysaccharide, producible by the process according to one or more of claims 1 to 9.

11. Use of the solid phase according to claim 10 in a test method for the determination of antibodies against mono-, oligo- or polysaccharides.