WO2020070461A1

WO2020070461A1 - Immunoassay for detection of streptococcus pneumoniae serotypes

Info

Publication number: WO2020070461A1
Application number: PCT/GB2018/052834
Authority: WO
Inventors: Carmen SHEPPARD; Seyi ELETU
Original assignee: Secretary of State for Health and Social Care
Priority date: 2018-10-04
Filing date: 2018-10-04
Publication date: 2020-04-09

Abstract

The present invention is directed to a method for detecting the presence or absence of a Streptococcus pneumoniae serotype-specific capsular polysaccharide in a sample; and a kit for use in such methods.

Description

IMMUNOASSAY FOR DETECTION OF STREPTOCOCCUS PNEUMONIAE SEROTYPES

The present invention relates to methods for detecting specific Streptococcus pneumoniae serotypes.

Streptococcus pneumoniae (pneumococcus) is a major cause of morbidity and mortality worldwide. Pneumococcal disease (PD) includes both invasive and non-invasive disease. The former includes bacteremia and meningitis; the latter, non-bacteremic pneumonia, sinusitis, and acute otitis media. To date, 97 “serotypes” of pneumococcus have been reported. These serotypes differ in virulence, prevalence, and antibiotic resistance and thus there is a need to detect and monitor the different serotypes.

To date, the most common serotypes that are vaccinated against are serotypes 1 , 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F, due to existing vaccines such as the 13-valent protein conjugate vaccine, Prevnar 13 (PCV13; Pfizer), which is used to vaccinate against each of these serotypes. As a result, surveillance studies on different continents have identified a decrease in the prevalence of these conjugate vaccine serotypes.

However, subjects vaccinated with PCV13 are still susceptible to infection with non-PCV13 serotypes and a concomitant increase in the prevalence of such serotypes has developed. It is therefore of significant importance, from a public health point of view, that assays and methods be developed for monitoring the distribution of a broad spectrum of pneumococcal serotypes, for example in order to determine the effectiveness of available vaccines for the prevention of pneumococcal disease, for detecting non-vaccine serotypes whose prevalence is increasing and for elucidation of the appropriate treatment to be administered. Conventional laboratory identification of specific S. pneumoniae serotypes (e.g. non-PCV13 serotypes) is laborious, slow and expensive, and the present invention is a dramatic improvement in this regard.

For the first time, the present inventors have developed an extended-specificity multiplex immunoassay, which has utility in detecting (greater than) 24 distinct serotypes/serogroups, including the PCV13 serotypes as well as more than 11 further non-PCV13 vaccine serotypes (e.g. serotypes becoming more prevalent concomitantly with the vaccination of vaccine serotypes). More particularly, the present inventors have determined that a number of monoclonal antibodies which target an S. pneumoniae capsular polysaccharide exhibit cross reactivity with other S. pneumoniae capsular polysaccharides, and have found that antibodies having cross reactivity may be combined to confirm the presence or absence of a single, particular capsular polysaccharides.

The method of the present invention is suitably performed in vitro on a sample (e.g. urine) taken from a subject, and may be performed directly on said sample (i.e. culturing of the bacterium is not necessary). The method relies on detection of serotype-specific capsular polysaccharides using monoclonal antibodies (e.g. human mAb). This assay provides excellent inter-serotype discrimination, for example allowing detection of one serotype in a sample separately comprising another serotype.

Monoclonal antibodies regularly have some inherent binding affinity for antigens other than their target antigen (e.g. cross reactivity). The multiplex approach of the present invention has the further advantage of allowing the detection of specific serotypes (or serogroups) based on analysis of the cross reactive properties of two or more mAbs (see e.g. Example 3 and Figure 1).

Furthermore, although antibody cross reactivity is generally considered undesirable in the art, the present inventors have advantageously and surprisingly adapted such cross reactivity for detecting the presence or absence of a number of further S. pneumoniae serotype-specific capsular polysaccharides. For example, in one embodiment wherein a 9N polysaccharide cross-reacts with the mAb targeting serotype 14, a sample producing a positive result with both a serotype 9N and a serotype 14 mAb could be interpreted as actually being positive for the serotype 9N polysaccharide antigen - this combination could also be positive for the 9L antigen, but in this case, the 9L antigen also cross-reacts with the mAb targeting 9V and 19A, and therefore, for a sample containing the 9L antigen, a positive result for the 9V and 19A assays as well as for the serotype 9N and 14 assays would be observed (thus, a 9L serotype would be detected) - see e.g. Figure 1. This is highly surprising, as none of these antibodies have 9L as a direct target.

In one aspect, there is provided a method for detecting the presence or absence of a Streptococcus pneumoniae 9L capsular polysaccharide in a sample, said method comprising:

a) contacting a sample with:

I. a first monoclonal antibody (mAb) that binds a S. pneumoniae capsular polysaccharide (CP) to form a first mAb-antigen complex, wherein said first mAb comprises the following six CDRs: i. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 1 , or a functional variant thereof;

ii. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 2, or a functional variant thereof;

iii. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 3, or a functional variant thereof;

iv. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 4, or a functional variant thereof;

v. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 5, or a functional variant thereof; and

vi. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 6, or a functional variant thereof;

wherein said first mAb binds a 9N CP; and

II. a second mAb that binds a S. pneumoniae capsular polysaccharide (CP) to form a second mAb-antigen complex, wherein said second mAb comprises the following six CDRs:

i. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 7, or a functional variant thereof;

ii. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 8, or a functional variant thereof;

iii. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 9, or a functional variant thereof;

iv. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 10, or a functional variant thereof;

v. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 11 , or a functional variant thereof; and

vi. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 12, or a functional variant thereof;

wherein said second mAb binds a 9V CP; and

b) detecting the presence or absence of said first mAb-antigen complex and detecting the presence or absence of said second mAb-antigen complex;

c) wherein the presence of the first mAb-antigen complex, and the presence of the second mAb-antigen complex confirms the presence of a 9L CP;

d) wherein the absence of the first mAb-antigen complex, and/or the absence of the second mAb-antigen complex is indicative of the absence of a 9L CP. In another aspect, there is provided a method for detecting the presence or absence of a Streptococcus pneumoniae 11 D capsular polysaccharide in a sample, said method comprising:

a) contacting a sample with:

I. a third monoclonal antibody (mAb) that binds a S. pneumoniae capsular polysaccharide (CP) to form a third mAb-antigen complex, wherein said third mAb comprises the following six CDRs:

i. a light chain CDR13 comprising an amino acid sequence of SEQ ID NO: 13, or a functional variant thereof;

ii. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 14, or a functional variant thereof;

iii. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 15, or a functional variant thereof;

iv. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 16, or a functional variant thereof;

v. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 17, or a functional variant thereof; and

vi. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 18, or a functional variant thereof;

wherein said third mAb binds an 11A CP; and

II. a fourth mAb that binds a S. pneumoniae CP to form a fourth mAb-antigen complex, wherein said fourth mAb comprises the following six CDRs: i. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 19, or a functional variant thereof;

ii. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 20, or a functional variant thereof;

iii. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 21 , or a functional variant thereof;

iv. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 22, or a functional variant thereof;

v. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 23, or a functional variant thereof; and

vi. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 24, or a functional variant thereof;

wherein said fourth mAb binds a 15B CP; and

b) detecting the presence or absence of said third mAb-antigen complex and detecting the presence or absence of said fourth mAb-antigen complex; c) wherein the presence of the third mAb-antigen complex, and the presence of the fourth mAb-antigen complex confirms the presence of an 11 D CP;

d) wherein the absence of the third mAb-antigen complex, and/or the absence of the fourth mAb-antigen complex is indicative of the absence of an 11 D CP.

In a further aspect, there is provided a method for detecting the presence or absence of a Streptococcus pneumoniae 18A capsular polysaccharide in a sample, said method comprising:

a) contacting a sample with:

I. a first monoclonal antibody (mAb) that binds a S. pneumoniae capsular polysaccharide (CP) to form a first mAb-antigen complex, wherein said first mAb comprises the following six CDRs:

i. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 1 , or a functional variant thereof;

v. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 5, or a functional variant thereof; and vi. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 6, or a functional variant thereof;

wherein said first mAb binds a 9N CP;

II. a third monoclonal antibody (mAb) that binds a S. pneumoniae capsular polysaccharide (CP) to form a third mAb-antigen complex, wherein said third mAb comprises the following six CDRs:

iv. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 16, or a functional variant thereof; V. a heavy chain CDR2 comprising an amino acid sequence of SEQ

ID NO: 17, or a functional variant thereof; and

wherein said third mAb binds an 11A CP; and

III. a fourth mAb that binds a S. pneumoniae CP to form a fourth mAb-antigen complex, wherein said fourth mAb comprises the following six CDRs: i. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 19, or a functional variant thereof;

wherein said fourth mAb binds a 15B CP; and

b) detecting the presence of or absence of said first mAb-antigen complex, detecting the presence of or absence of said third mAb-antigen complex and detecting the presence of or absence of said fourth mAb-antigen complex;

c) wherein the presence of two or more selected from the first mAb-antigen complex, the third mAb-antigen complex and the fourth mAb-antigen complex confirms the presence of 18A CP;

d) wherein the absence of two or more selected from the first mAb-antigen complex, the third mAb-antigen complex and the fourth mAb-antigen complex confirms the absence of 18A CP.

In one aspect, there is provided a method for detecting the presence or absence of a Streptococcus pneumoniae 6C capsular polysaccharide in a sample, said method comprising:

a) contacting a sample with:

I. a tenth monoclonal antibody (mAb) that binds a S. pneumoniae capsular polysaccharide (CP) to form a tenth mAb-antigen complex;

wherein tenth first mAb binds a 6A CP; II. a ninteenth mAb that binds a S. pneumoniae capsular polysaccharide (CP) to form a second mAb-antigen complex;

wherein said nineteenth mAb binds a 22F CP; and

III. an eleventh mAb that binds a S. pneumoniae capsular polysaccharide (CP) to form an eleventh mAb-antigen complex;

wherein said eleventh mAb binds a 6B CP; and

b) detecting the presence or absence of said tenth mAb-antigen complex, detecting the presence or absence of said nineteenth mAb-antigen complex, and detecting the presence of absence of said eleventh mAb-antigen complex;

c) wherein the presence of the tenth mAb-antigen complex, the presence of the nineteenth mAb-antigen complex, and the absence of the eleventh mAb-antigen complex confirms the presence of a 6C CP;

d) wherein the absence of the tenth mAb-antigen complex, the absence of the nineteenth mAb-antigen complex and the presence of the eleventh mAb-antigen complex is indicative of the absence of a 6C CP.

In one aspect, there is provided a method for detecting the presence or absence of a Streptococcus pneumoniae 6D capsular polysaccharide in a sample, said method comprising:

a) contacting a sample with:

wherein said tenth mAb binds a 6A CP; and

II. an eleventh mAb that binds a S. pneumoniae capsular polysaccharide (CP) to form an eleventh mAb-antigen complex;

wherein said eleventh mAb binds a 6B CP; and

b) detecting the presence or absence of said tenth mAb-antigen complex and detecting the presence or absence of said eleventh mAb-antigen complex;

c) wherein the presence of the tenth mAb-antigen complex, and the presence of the eleventh mAb-antigen complex confirms the presence of a 6D CP;

d) wherein the absence of the tenth mAb-antigen complex, and/or the absence of the eleventh mAb-antigen complex is indicative of the absence of a 6D CP.

In one aspect, there is provided a method for detecting the presence or absence of a Streptococcus pneumoniae 18F capsular polysaccharide in a sample, said method comprising:

a) contacting a sample with: I. a fifteenth monoclonal antibody (mAb) that binds a S. pneumoniae capsular polysaccharide (CP) to form a fifteenth mAb-antigen complex; wherein fifteenth mAb binds a 18C CP;

II. a third mAb that binds a S. pneumoniae capsular polysaccharide (CP) to form a third mAb-antigen complex;

wherein said third mAb binds a 11A CP; and

b) detecting the presence or absence of said fifteenth mAb-antigen complex and detecting the presence or absence of said third mAb-antigen complex; c) wherein the presence of the fifteenth mAb-antigen complex, and the absence of the third mAb-antigen complex confirms the presence of a 18F CP;

d) wherein the absence of the fifteenth mAb-antigen complex, and/or the presence of the third mAb-antigen complex is indicative of the absence of a 18F CP.

The present invention encompasses the antibodies defined herein having the recited CDR sequences or variable heavy and variable light chain sequences (reference antibodies), as well as functional variants thereof. A functional variant binds to the same target antigen as the reference antibody, and preferably exhibits the same antigen cross-reactivity as the reference antibody. The functional variants may have a different affinity for the target antigen when compared to the reference antibody, but substantially the same affinity is preferred.

In one embodiment functional variants of a reference antibody show sequence variation at one or more CDRs when compared to corresponding reference CDR sequences. Thus, a functional antibody variant may comprise a functional variant of a CDR. Where the term “functional variant” is used in the context of a CDR sequence, this means that the CDR has at most 2, preferably at most 1 amino acid differences when compared to a corresponding reference CDR sequence, and when combined with the remaining 5 CDRs (or variants thereof) enables the variant antibody to bind to the same target antigen as the reference antibody, and preferably to exhibit the same antigen cross-reactivity as the reference antibody.

In one embodiment a variant antibody comprises:

a light chain CDR1 having at most 2 amino acid difference when compared to a corresponding reference CDR sequence;

a light chain CDR2 having at most 2 amino acid difference when compared to a corresponding reference CDR sequence;

a light chain CDR3 having at most 2 amino acid difference when compared to a corresponding reference CDR sequence; a heavy chain CDR1 having at most 2 amino acid difference when compared to a corresponding reference CDR sequence;

a heavy chain CDR2 having at most 2 amino acid difference when compared to a corresponding reference CDR sequence; and

a heavy chain CDR3 having at most 2 amino acid difference when compared to a corresponding reference CDR sequence;

wherein the variant antibody binds to the same target antigen as the reference antibody, and preferably exhibits the same antigen cross-reactivity as the reference antibody.

Preferably a variant antibody comprises:

a light chain CDR1 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence;

a light chain CDR2 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence;

a light chain CDR3 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence;

a heavy chain CDR1 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence;

a heavy chain CDR2 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence; and

a heavy chain CDR3 having at most 1 amino acid difference when compared to a corresponding reference CDR sequence;

For example, a variant of the first antibody may comprise:

a light chain CDR1 having at most 2 amino acid difference when compared to SEQ ID

NO: 1 ;

a light chain CDR2 having at most 2 amino acid difference when compared to SEQ ID

NO: 2;

a light chain CDR3 having at most 2 amino acid difference when compared to SEQ ID

NO: 3;

a heavy chain CDR1 having at most 2 amino acid difference when compared to SEQ ID NO: 4;

a heavy chain CDR2 having at most 2 amino acid difference when compared to SEQ ID NO: 5; and a heavy chain CDR3 having at most 2 amino acid difference when compared to SEQ ID NO: 6;

wherein the variant antibody binds to a 9N CP, and preferably exhibits the same antigen cross-reactivity as the first antibody.

For example, a variant of the first antibody may (preferably) comprise:

a light chain CDR1 having at most 1 amino acid difference when compared to SEQ ID

NO: 1 ;

a light chain CDR2 having at most 1 amino acid difference when compared to SEQ ID

NO: 2;

a light chain CDR3 having at most 1 amino acid difference when compared to SEQ ID

NO: 3;

a heavy chain CDR1 having at most 1 amino acid difference when compared to SEQ ID NO: 4;

a heavy chain CDR2 having at most 1 amino acid difference when compared to SEQ ID NO: 5; and

a heavy chain CDR3 having at most 1 amino acid difference when compared to SEQ ID NO: 6;

The foregoing can be applied analogously to variants of the other antibodies described herein (e.g. the second, third, etc.), wherein the amino acid differences are defined relative to the CDR sequences thereof, and wherein the variant antibody binds to the same target antigen as said antibodies, and preferably exhibits the same antigen cross-reactivity.

In one embodiment a variant antibody may have at most 5, 4 or 3 amino acid differences total in the CDRs thereof when compared to a corresponding reference antibody, with the proviso that there is at most 2 (preferably at most 1) amino acid differences per CDR. Preferably a variant antibody has at most 2 (more preferably at most 1) amino acid differences total in the CDRs thereof when compared to a corresponding reference antibody, with the proviso that there is at most 2 amino acid differences per CDR. More preferably a variant antibody has at most 2 (more preferably at most 1) amino acid differences total in the CDRs thereof when compared to a corresponding reference antibody, with the proviso that there is at most 1 amino acid difference per CDR. The amino acid difference may be an amino acid substitution, insertion or deletion. In one embodiment the amino acid difference is a conservative amino acid substitution as described herein.

In one embodiment a variant antibody has the same framework sequences as the exemplary antibodies described herein. In another embodiment the variant antibody may comprise a framework region having at most 2, preferably at most 1 amino acid difference (when compared to a corresponding reference framework sequence). Thus, each framework region may have at most 2, preferably at most 1 amino acid difference (when compared to a corresponding reference framework sequence).

In one embodiment a variant antibody may have at most 5, 4 or 3 amino acid differences total in the framework regions thereof when compared to a corresponding reference antibody, with the proviso that there is at most 2 (preferably at most 1) amino acid differences per framework region. Preferably a variant antibody has at most 2 (more preferably at most 1) amino acid differences total in the framework regions thereof when compared to a corresponding reference antibody, with the proviso that there is at most 2 amino acid differences per framework region. More preferably a variant antibody has at most 2 (more preferably at most 1) amino acid differences total in the framework regions thereof when compared to a corresponding reference antibody, with the proviso that there is at most 1 amino acid difference per framework region.

Thus, a variant antibody may comprise a variable heavy chain and a variable light chain as described herein, wherein:

the heavy chain has at most 14 amino acid differences (at most 2 amino acid differences in each CDR and at most 2 amino acid differences in each framework region) when compared to a heavy chain sequence herein; and

the light chain has at most 14 amino acid differences (at most 2 amino acid differences in each CDR and at most 2 amino acid differences in each framework region) when compared to a light chain sequence herein;

Said variant heavy or light chains may be referred to as“functional equivalents” of the reference heavy or light chains. In one embodiment a variant antibody may comprise a variable heavy chain and a variable light chain as described herein, wherein:

the heavy chain has at most 7 amino acid differences (at most 1 amino acid difference in each CDR and at most 1 amino acid difference in each framework region) when compared to a heavy chain sequence herein; and

the light chain has at most 7 amino acid differences (at most 1 amino acid difference in each CDR and at most 1 amino acid difference in each framework region) when compared to a light chain sequence herein;

Suitably, each of said mAb may be contacted with the sample is a discrete compartment.

The capsular polysaccharides detectable by a method of the present invention are serotype- specific. Thus, for example, where a serotype 9L capsular polysaccharide is detected in a sample, this indicates the presence of the S. pneumoniae serotype ‘9L’. The capsular polysaccharide may be independent of a bacterium (e.g. no longer integral to the bacterial membrane), yet may provide an indication of the presence or absence of said bacterium within a sample (e.g. presence or absence of an infection with said bacterium). This is highly advantageous, as direct detection of the bacterium (e.g. intact bacterium) is not required, and a free capsular polysaccharide may be used as a proxy/indicator of the presence of the bacterium.

The present invention allows for the detection of specific serotypes in the sample, or serotyping of a S. pneumoniae (e.g. isolated S. pneumoniae). Advantageously, this allows for diagnosis of a subject with an infection of specific serotype of S. pneumoniae , and thus administration of a therapy suitable for treating said serotype. The method of the invention may be used for diagnosing infection of a subject with a S. pneumoniae (e.g. a specific serotype of S. pneumoniae).

One key prior art problem that has been addressed by the present invention is the provision of a robust set of assays (e.g. comprising mAbs) that are mutually compatible (i.e. mAbs retain accurate binding specificity) within a single set of assay conditions. A particular advantage associated with methods of the invention is speed. By way of example, a method of the invention is typically performed in (less than) one day, preferably within 6 hours or 4 hours. Thus, a method of the present invention enables rapid determination of the S. pneumoniae serotype present in a sample. Similarly, the invention provides a rapid method for the confirmation that all of said serotypes are absent from the sample by way of a multiplex method. A multiplex method means that a plurality of assays are performed, preferably under the same assay conditions and/or substantially at the same time. Alternatively, the assays may be performed at separate times.

Furthermore, the methods do not require culturing of bacteria isolated from a subject, and can be performed on samples (e.g. crude samples) directly isolated from a subject. The existing‘gold standard’ assay for serotyping a broad spectrum of serotypes is the‘Quellung reaction’. While this method is capable of identifying numerous pneumococcal serotypes, it requires the use of many specific pneumococcal antisera (e.g. polyclonal antibodies) and is costly and laborious. A significant drawback of this method of typing is that it requires the recovery of a viable pneumococcal culture and thus precludes any case where an isolate is not obtainable - for example, when antimicrobial treatment has been administered prior to specimen collection, or in cases of non-invasive disease. The BinaxNOW pneumococcal test (Alere) can detect pneumococcal cell wall C polysaccharide (CWP) in samples (via a CWP polyclonal antibody). However, this test is not capable of reporting any serotype-specific information. Molecular techniques (e.g. PCR) for serotyping suffer from requiring the conditions for each assay to be individually optimised, and from requiring a multitude of complex component parts.

A wide spectrum of S. pneumoniae serotype-specific capsular polysaccharides can be detected by methods of the present invention due to the provision of an array of monoclonal antibodies which bind said serotype-specific capsular polysaccharides. The inventors have demonstrated that the presence or absence of more than 24 S. pneumoniae serotype- specific capsular polysaccharides can be detected by methods of the present invention.

In one embodiment, a method of the invention may further comprise administering to said subject a therapy suitable for treating an infection with a S. pneumoniae serotype comprising said capsular polysaccharide.

Suitable treatments include one or more an antibiotic selected from penicillin, cefotaxime, erythromycin and co-trimoxazole.

In one embodiment, a method of the invention further comprises contacting the sample with one or more of a fifth to thirteenth mAb, to form a fifth to thirteenth mAb-antigen complex wherein the fifth to thirteenth mAb binds (at least) CP 1 , 2, 3, 4, 5, 6A, 6B, 8, or 14, respectively. In such embodiments, the presence of mAb-antigen complex is indicative of the presence of said CP, and the absence of mAb-antigen complex is indicative of the absence of said CP.

In one embodiment, a method of the invention further comprises contacting the sample with one or more selected from a fourteenth to twenty-first mAb, to form a fourteenth to twenty- first mAb-antigen complex, wherein the fourteenth to twenty-first mAb binds CP 17F, 18C, 19A, 19F, 20, 22F, 23F, or 33F, respectively. In such embodiments, the presence of mAb- antigen complex is indicative of the presence of said CP, and the absence of mAb-antigen complex is indicative of the absence of said CP.

In one embodiment, a method of the invention further comprises contacting the sample with one or more of a twenty-second to twenty-fourth mAb, to form a twenty-second to twenty- fourth mAb-antigen complex, wherein the twenty-second to twenty-fourth mAb binds CP 7F, 10A, or 12F, respectively. In such embodiments, the presence of mAb-antigen complex is indicative of the presence of said CP, and the absence of mAb-antigen complex is indicative of the absence of said CP.

In one embodiment, a method of the invention comprises:

a) contacting a sample with:

I. a first mAb that binds a S. pneumoniae capsular polysaccharide (CP) to form a first mAb-antigen complex;

wherein said tenth mAb binds a 9N CP; and

II. a second mAb that binds a S. pneumoniae capsular polysaccharide (CP) to form an second mAb-antigen complex;

wherein said second mAb binds a 6B CP; and

b) detecting the presence or absence of said first mAb-antigen complex and detecting the presence or absence of said second mAb-antigen complex; c) wherein the presence of the first mAb-antigen complex, and the absence of the second mAb-antigen complex confirms the presence of a 9N CP;

d) wherein the absence of the first mAb-antigen complex, and/or the presence of the second mAb-antigen complex is indicative of the absence of a 9N CP.

In one embodiment, a method of the invention comprises:

a) contacting a sample with: I. a thirteenth mAb that binds a S. pneumoniae capsular polysaccharide (CP) to form a thirteenth mAb-antigen complex;

wherein said thirteenth mAb binds a 14 CP; and

II. a first mAb that binds a S. pneumoniae capsular polysaccharide (CP) to form a first mAb-antigen complex;

wherein said first mAb binds a 9N CP; and

b) detecting the presence or absence of said thirteenth mAb-antigen complex and detecting the presence or absence of said first mAb-antigen complex; c) wherein the presence of the thirteenth mAb-antigen complex, and the absence of the first mAb-antigen complex confirms the presence of a 14 CP;

d) wherein the absence of the thirteenth mAb-antigen complex, and/or the presence of the first mAb-antigen complex is indicative of the absence of a 14 CP.

In one embodiment, a method of the invention comprises:

a) contacting a sample with:

I. a seventeenth mAb that binds a S. pneumoniae capsular polysaccharide (CP) to form a seventeenth mAb-antigen complex;

wherein said seventeenth mAb binds a 19F CP; and

II. a sixteenth mAb that binds a S. pneumoniae capsular polysaccharide (CP) to form a sixteenth mAb-antigen complex;

wherein said sixteenth mAb binds a 19A CP; and

b) detecting the presence or absence of said seventeenth mAb-antigen complex and detecting the presence or absence of said sixteenth mAb-antigen complex;

c) wherein the presence of the seventeenth mAb-antigen complex, and the absence of the sixteenth mAb-antigen complex confirms the presence of a 19F CP;

d) wherein the absence of the seventeenth mAb-antigen complex, and/or the presence of the sixteenth mAb-antigen complex is indicative of the absence of a 19F CP.

In one embodiment, a method of the invention comprises:

a) contacting a sample with:

I. a nineteenth mAb that binds a S. pneumoniae capsular polysaccharide (CP) to form a nineteenth mAb-antigen complex;

wherein said nineteenth mAb binds a 22F CP; and

II. a tenth mAb that binds a S. pneumoniae capsular polysaccharide (CP) to form a tenth mAb-antigen complex;

wherein said tenth mAb binds a 6A CP; and III. an eighteenth mAb that binds a S. pneumoniae capsular polysaccharide (CP) to form an eighteenth mAb-antigen complex;

wherein said eighteenth mAb binds a 20 CP; and

b) detecting the presence or absence of said nineteenth mAb-antigen complex, detecting the presence or absence of said tenth mAb-antigen complex, and detecting the presence or absence of said eighteenth mAb-antigen complex; c) wherein the presence of the nineteenth mAb-antigen complex, the absence of the tenth mAb-antigen complex, and the absence of the eighteenth mAb-antigen complex confirms the presence of a 22F CP;

d) wherein the absence of the nineteenth mAb-antigen complex, the presence of the tenth mAb-antigen complex, and the presence of the eighteenth mAb-antigen complex is indicative of the absence of a 22F CP.

It is often sufficient to detect specific S. pneumoniae serogroups, as serotypes within the same serogroup often comprise similar levels of virulence, prevalence, and antibiotic resistance.

All aspects, embodiments and definitions relating to detecting‘serotypes’ are also applicable to analogous aspects/embodiments, wherein the “serotype” is simply replaced by “serogroup”.

Thus, in one embodiment of methods of the invention the presence of capsular polysaccharide-mAb complex is indicative of the presence of a serogroup to which said capsular polysaccharide belongs, and the absence of capsular polysaccharide-mAb complex is indicative of the absence of a serogroup to which said capsular polysaccharide belongs.

In one embodiment, the presence of the first mAb-antigen complex (e.g. bound capsular polysaccharide-9N mAb complex), and the absence of one or more selected from the thirteenth mAb-antigen complex (bound capsular polysaccharide-14 mAb complex) and the absence of fourth mAb-antigen complex (e.g. bound capsular polysaccharide-15B mAb complex) is indicative of the presence of 47A and/or 43 capsular polysaccharide.

In one embodiment, the absence of the first mAb-antigen complex (e.g. bound capsular polysaccharide-9N mAb complex) confirms the absence of 47A and/or 43 capsular polysaccharide. In one embodiment the presence of the tenth mAb-antigen complex (e.g. bound capsular polysaccharide-6A mAb complex), and the absence of the nineteenth mAb-antigen complex (e.g. bound capsular polysaccharide-22F mAb) is indicative of (e.g. confirms) the presence of a 6C and/or 6B capsular polysaccharide (e.g. a serogroup 6 capsular polysaccharide) in the sample.

In one embodiment, the presence of the twenty-second mAb-antigen complex (e.g. bound capsular polysaccharide-7F mAb complex) is indicative of (e.g. confirms) the presence of a 7 A and/or 7F capsular polysaccharide (e.g. a serogroup 7 capsular polysaccharide).

In one embodiment, the presence of the second mAb-antigen complex (e.g. bound capsular polysaccharide-9V mAb complex) and the absence of the first mAb-antigen complex (e.g. bound capsular polysaccharide-9N mAb complex) is indicative of (e.g. confirms) the presence of a 9A and/or 9V capsular polysaccharide (e.g. a serogroup 9 serotype).

In one embodiment, the presence of the twenthy-third mAb-antigen complex (e.g. bound capsular polysaccharide-10A mAb) complex is indicative of (e.g. confirms) the presence of a 10A, 10B, 10B, 10C, 33C and/or 39 capsular polysaccharide.

In one embodiment, the presence of the third mAb-antigen complex (e.g. bound capsular polysaccharide-11A mAb complex), the absence of the fourth mAb-antigen complex (e.g. bound capsular polysaccharide-15B mAb complex), and the absence of the fifteenth mAb- antigen complex (e.g. bound capsular polysaccharide-18C mAb complex) is indicative of (e.g. confirms) the presence of a 11A, 11C, 11 E, 16A, and/or 16F capsular polysaccharide.

In one embodiment, the presence of the twenty-fourth mAb-antigen complex (e.g. bound capsular polysaccharide-12F mAb complex), the absence of the fourth mAb-antigen complex (e.g. bound capsular polysaccharide-15B mAb complex), and the absence of the ninteenth mAb-antigen complex (e.g. bound capsular polysaccharide-22F mAb complex) is indicative of (e.g. confirms) the presence of a 12F and/or 44 capsular polysaccharide.

In one embodiment, the presence of the fourth mAb-antigen complex (e.g. bound capsular polysaccharide-15B mAb complex), the absence of the first mAb-antigen complex (e.g. bound capsular polysaccharide-9N mAb complex), the absence of the third mAb-antigen complex (e.g. bound capsular polysaccharide-11 A mAb complex) and the absence of the twenty-fourth mAb-antigen complex (e.g. bound capsular polysaccharide-12F mAb complex) is indicative of (e.g. confirms) the presence of 15A, 15B, 15C, and/or 15F capsular polysaccharide (e.g. a serogroup 15 capsular polysaccharide).

In one embodiment, the presence of the fifteenth mAb-antigen complex (e.g. bound capsular polysaccharide-18C mAb complex) is indicative of (e.g. confirms) the presence of 18B, 18C, and/or 18F capsular polysaccharide (e.g. a serogroup 18 capsular polysaccharide).

In one embodiment, the presence of the sixteenth mAb-antigen complex (e.g. bound capsular polysaccharide-19A mAb complex) is indicative of (e.g. confirms) the presence of 19A and/or 19F capsular polysaccharide (e.g. a serogroup 19 capsular polysaccharide).

In one embodiment, the presence of the seventeenth mAb-antigen complex (e.g. bound capsular polysaccharide-19F mAb complex) is indicative of (e.g. confirms) the presence of 19A, 19B, 19C and/or 19F capsular polysaccharide (e.g. a serogroup 19 capsular polysaccharide).

In one embodiment, the presence of the twentieth mAb-antigen complex (e.g. bound capsular polysaccharide-23F mAb complex) is indicative of (e.g. confirms) the presence of 23F, 32A and/or 32F capsular polysaccharide.

In one embodiment, the presence of the twenty-first mAb-antigen complex (e.g. bound capsular polysaccharide-33F mAb complex) is indicative of (e.g. confirms) the presence of 33A, 33B, 33D and/or 33F capsular polysaccharide (e.g. a serogroup 33 serotype).

In one embodiment, said fifth mAb (which binds CP 1 ) comprises the following six CDRs: i. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 175, or a functional variant thereof;

ii. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 176, or a functional variant thereof;

iii. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 33, or a functional variant thereof;

iv. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 34, or a functional variant thereof;

v. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 35, or a functional variant thereof; and

vi. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 36, or a functional variant thereof. In one embodiment, said sixth mAb (which binds CP 2) comprises the following six CDRs: i. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 37, or a functional variant thereof;

ii. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 38, or a functional variant thereof;

iii. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 39, or a functional variant thereof;

iv. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 40, or a functional variant thereof;

v. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 41 , or a functional variant thereof; and

vi. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 42, or a functional variant thereof.

In one embodiment, said seventh mAb (which binds CP 3) comprises the following six CDRs: i. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 43, or a functional variant thereof;

ii. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 44, or a functional variant thereof;

iii. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 45, or a functional variant thereof;

iv. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 46, or a functional variant thereof;

v. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 47, or a functional variant thereof; and

vi. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 48, or a functional variant thereof.

In one embodiment, said eight mAb (which binds CP 4) comprises the following six CDRs: i. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 49, or a functional variant thereof;

ii. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 50, or a functional variant thereof;

iii. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 51 , or a functional variant thereof; iv. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 52, or a functional variant thereof;

v. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 53, or a functional variant thereof; and

vi. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 54, or a functional variant thereof.

In one embodiment, said ninth mAb (which binds CP 5) comprises the following six CDRs: i. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 55, or a functional variant thereof;

ii. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 56, or a functional variant thereof;

iii. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 57, or a functional variant thereof;

iv. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 58, or a functional variant thereof;

v. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 59, or a functional variant thereof; and

vi. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 60, or a functional variant thereof.

In one embodiment, said tenth mAb (which binds CP 6A) comprises the following six CDRs: i. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 61 , or a functional variant thereof;

ii. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 62, or a functional variant thereof;

iii. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 63, or a functional variant thereof;

iv. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 64, or a functional variant thereof;

v. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 65, or a functional variant thereof; and

vi. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 66, or a functional variant thereof.

In one embodiment, said eleventh mAb (which binds CP 6B) comprises the following six

CDRs: i. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 67, or a functional variant thereof;

ii. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 68, or a functional variant thereof;

iii. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 69, or a functional variant thereof;

iv. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 70, or a functional variant thereof;

v. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 71 , or a functional variant thereof; and

vi. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 72, or a functional variant thereof.

In one embodiment, said twelfth mAb (which binds CP 8) comprises the following six CDRs: i. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 73, or a functional variant thereof;

ii. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 74, or a functional variant thereof;

iii. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 75, or a functional variant thereof;

iv. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 76, or a functional variant thereof;

v. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 77, or a functional variant thereof; and

vi. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 78, or a functional variant thereof.

In one embodiment, said thirteenth mAb (which binds CP 14) comprises the following six

CDRs:

i. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 79, or a functional variant thereof;

ii. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 80, or a functional variant thereof;

iii. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 81 , or a functional variant thereof;

iv. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 82, or a functional variant thereof; V. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 83, or a functional variant thereof; and

vi. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 84, or a functional variant thereof.

In one embodiment, said fourteenth mAb (which binds CP 17F) comprises the following six CDRs:

i. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 85, or a functional variant thereof;

ii. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 86, or a functional variant thereof;

iii. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 87, or a functional variant thereof;

iv. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 88, or a functional variant thereof;

v. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 89, or a functional variant thereof; and

vi. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 90, or a functional variant thereof.

In one embodiment, said fifteenth mAb (which binds CP 18C) comprises the following six CDRs:

i. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 91 , or a functional variant thereof;

ii. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 92, or a functional variant thereof;

iii. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 93, or a functional variant thereof;

iv. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 94, or a functional variant thereof;

v. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 95, or a functional variant thereof; and

vi. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 96, or a functional variant thereof.

In one embodiment, said sixteenth mAb (which binds CP 19A) comprises the following six CDRs: i. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 97, or a functional variant thereof;

ii. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 98, or a functional variant thereof;

iii. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 99, or a functional variant thereof;

iv. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 100, or a functional variant thereof;

v. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 101 , or a functional variant thereof; and

vi. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 102, or a functional variant thereof.

In one embodiment, said seventeenth mAb (which binds CP 19F) comprises the following six CDRs:

i. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 103, or a functional variant thereof;

ii. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 104, or a functional variant thereof;

iii. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 105, or a functional variant thereof;

iv. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 106, or a functional variant thereof;

v. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 107, or a functional variant thereof; and

vi. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 108, or a functional variant thereof.

In one embodiment, said eighteenth mAb (which binds CP 20) comprises the following six CDRs:

i. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 109, or a functional variant thereof;

ii. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 110, or a functional variant thereof;

iii. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 111 , or a functional variant thereof; iv. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 112, or a functional variant thereof;

v. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 113, or a functional variant thereof; and

vi. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 114, or a functional variant thereof.

In one embodiment, said nineteenth mAb (which binds CP 22F) comprises the following six CDRs:

i. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 115, or a functional variant thereof;

ii. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 116, or a functional variant thereof;

iii. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 117, or a functional variant thereof;

iv. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 118, or a functional variant thereof;

v. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 119, or a functional variant thereof; and

vi. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 120, or a functional variant thereof.

In one embodiment, said twentieth mAb (which binds CP 23F) comprises the following six CDRs:

i. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 121 , or a functional variant thereof;

ii. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 122, or a functional variant thereof;

iii. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 123, or a functional variant thereof;

iv. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 124, or a functional variant thereof;

v. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 125, or a functional variant thereof; and

vi. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 126, or a functional variant thereof. In one embodiment, said twenty-first mAb (which binds CP 33F) comprises the following six CDRs:

i. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 127, or a functional variant thereof;

ii. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 128, or a functional variant thereof;

iii. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 129, or a functional variant thereof;

iv. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 130, or a functional variant thereof;

v. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 131 , or a functional variant thereof; and

vi. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 132, or a functional variant thereof.

Detection of a serotype specific capsular polysaccharide can be used to indicate the presence of the corresponding bacterium serotype in the sample, or in the subject from which the sample is isolated. For example, a free capsular polysaccharide (released from a bacterium) may be detected in the sample, indicating the presence of bacterium serotype in the subject.

In one embodiment, the method may be used to diagnose a subject with an infection with a S. pneumoniae serotype, wherein the presence of mAb-antigen complex is indicative of the presence of an infection with a serotype comprising said capsular polysaccharide, and wherein the absence mAb-antigen complex is indicative of the absence of an infection with a serotype comprising said capsular polysaccharide.

The term “diagnosis” as used herein encompasses identification, confirmation and/or characterisation of S. pneumoniae serotype infection. Methods of diagnosis according to the invention are useful to confirm the existence of an infection. Methods of diagnosis are also useful in methods for assessment of clinical screening, prognosis, choice of therapy, evaluation of therapeutic benefit, i.e. for drug screening and drug development. Efficient diagnosis allows rapid identification of the most appropriate treatment (thus lessening unnecessary exposure to harmful drug side effects), and reducing relapse rates.

In another aspect, there is provided a method for determining prognosis of an infection with a S. pneumoniae serotype in a subject, comprising detecting the presence or absence of a serotype-specific capsular polysaccharide through a method of the invention. In such aspects, the presence of mAb-antigen complex is indicative of (e.g. correlates with) a poor prognosis for an infection with a S. pneumoniae serotype comprising said capsular polysaccharide, and the absence of mAb-antigen complex is indicative of (e.g. correlates with) a good prognosis for an infection with a S. pneumoniae serotype comprising said capsular polysaccharide.

Methods of the invention may comprise further assays permitting confirmation of pneumococcal capsular polysaccharide detection. In one embodiment, a method of the invention further comprises contacting the sample with a twenty-fifth mAb which binds Streptococcus pneumoniae CWP. Suitably, said contacting provides a twenty-fifth mAb- antigen complex. The CWP (cell wall polysaccharide) antigen is common to all S. pneumoniae serotypes, and is absent from non-pneumococcal bacteria.

Thus, in one embodiment the presence of said twenty-fifth mAb-antigen complex is indicative of the presence of a S. pneumoniae bacterium in the sample, and the absence of said twenty-fifth mAb-antigen complex is indicative of the absence of a S. pneumoniae bacterium in the sample.

In one embodiment, said twenty-fifth mAb (which binds CWP) comprises the following six CDRs:

i. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 169, or a functional variant thereof;

ii. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 170, or a functional variant thereof;

iii. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 171 , or a functional variant thereof;

iv. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 172, or a functional variant thereof;

v. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 173, or a functional variant thereof; and

vi. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 174, or a functional variant thereof.

In one embodiment, said first mAb comprises:

a) a light chain variable region comprising an amino acid sequence of SEQ ID: NO 25, or functional equivalent thereof; and b) a heavy chain variable region comprising an amino acid sequence of SEQ ID: NO

26, or functional equivalent thereof.

In one embodiment, said second mAb comprises:

a) a light chain variable region comprising an amino acid sequence of SEQ ID: NO

27, or functional equivalent thereof; and

b) a heavy chain variable region comprising an amino acid sequence of SEQ ID: NO

28, or functional equivalent thereof.

In one embodiment, said third mAb comprises:

29, or functional equivalent thereof; and

30, or functional equivalent thereof.

In one embodiment, said fourth mAb comprises:

31 , or functional equivalent thereof; and

32, or functional equivalent thereof.

In one embodiment, said fifth mAb comprises:

133, or functional equivalent thereof; and

134, or functional equivalent thereof.

In one embodiment, said sixth mAb comprises:

135, or functional equivalent thereof; and

136, or functional equivalent thereof.

In one embodiment, said seventh mAb comprises:

137, or functional equivalent thereof; and b) a heavy chain variable region comprising an amino acid sequence of SEQ ID: NO

138, or functional equivalent thereof.

In one embodiment, said eight mAb comprises:

139, or functional equivalent thereof; and

140, or functional equivalent thereof.

In one embodiment, said ninth mAb comprises:

141 , or functional equivalent thereof; and

142, or functional equivalent thereof.

In one embodiment, said tenth mAb comprises:

143, or functional equivalent thereof; and

144, or functional equivalent thereof.

In one embodiment, said eleventh mAb comprises:

145, or functional equivalent thereof; and

146, or functional equivalent thereof.

In one embodiment, said twelfth mAb comprises:

147, or functional equivalent thereof; and

148, or functional equivalent thereof.

In one embodiment, said thirteenth mAb comprises:

149, or functional equivalent thereof; and b) a heavy chain variable region comprising an amino acid sequence of SEQ ID: NO

150, or functional equivalent thereof.

In one embodiment, said fourtheenth mAb comprises:

151 , or functional equivalent thereof; and

152, or functional equivalent thereof.

In one embodiment, said fifteenth mAb comprises:

153, or functional equivalent thereof; and

154, or functional equivalent thereof.

In one embodiment, said sixteenth mAb comprises:

55, or functional equivalent thereof; and

56, or functional equivalent thereof.

In one embodiment, said seventeenth mAb comprises:

157, or functional equivalent thereof; and

158, or functional equivalent thereof.

In one embodiment, said eighteenth mAb comprises:

159, or functional equivalent thereof; and

160, or functional equivalent thereof.

In one embodiment, said nineteenth mAb comprises:

161 , or functional equivalent thereof; and b) a heavy chain variable region comprising an amino acid sequence of SEQ ID: NO

162, or functional equivalent thereof.

In one embodiment, said twentieth mAb comprises:

163, or functional equivalent thereof; and

164, or functional equivalent thereof.

In one embodiment, said twenty-first mAb comprises:

165, or functional equivalent thereof; and

166, or functional equivalent thereof.

In one embodiment, said twenty fifth mAb comprises:

167, or functional equivalent thereof; and

168, or functional equivalent thereof.

In one embodiment, a sample may be one or more selected from saliva, blood (e.g. whole blood, blood serum or blood plasma), mucous, sputum, cerebrospinal fluid, synovial fluid, a lesion, bodily fluid isolated from a lesion, eye fluid, lymphatic fluid, seminal fluid, cerebrospinal fluid, sebaceous secretions, and/or sputum.

In one embodiment, the sample is obtained from surgical or other medical equipment. In one embodiment, the sample is an environmental sample (e.g. water, soil and/or sediment).

In a preferable embodiment, the sample is urine. Suitably, said urine sample may be isolated from a subject suspected of having an infection with a S. pneumoniae serotype. In some embodiments, the sample is isolated from a subject diagnosed as having an S. pneumoniae infection.

A key advantage to using a urine sample in methods of the present invention is that this sample is readily obtainable from a subject having or suspected of having an infection with a S. pneumoniae serotype and is obtained without the need for invasive techniques. In one embodiment, a sample may be processed to isolate a S. pneumoniae serotype from a sample prior to detecting the presence or absence of S. pneumoniae serotype capsular polysaccharide. In one embodiment, the S. pneumoniae is cultured from a bacterium isolated from a subject and the resulting culture is applied to an assay in methods of the invention.

The terms“subject”,“individual” and“patient” are used interchangeably herein to refer to a mammalian subject. In one embodiment the“subject” is a human, a companion animal (e.g. a pet such as dogs, cats, and rabbits), livestock (e.g. pigs, sheep, cattle, and goats), and horses. In a preferable embodiment, the subject is a human. In methods of the invention, the subject may not have been previously diagnosed as having an S. pneumoniae infection. Alternatively, the subject may have been previously diagnosed as having an S. pneumoniae infection. The subject may also be one who exhibits disease risk factors, or one who is asymptomatic for an S. pneumoniae infection. The subject may also be one who is suffering from or is at risk of developing an S. pneumoniae infection. Thus, in one embodiment, a method of the invention may be used to confirm the presence of an S. pneumoniae (serotype) infection in a subject. For example, the subject may previously have been diagnosed with S. pneumoniae (serotype) infection by alternative means. In one embodiment, the subject has been previously administered an S. pneumoniae (serotype) therapy.

A“mAb-antigen complex” means a complex (e.g. macromolecular complex) comprising a capsular polysaccharide antigen which has become bound to a mAb (e.g. a mAb with binding affinity for said capsular polysaccharide antigen). The term “mAb-antigen complex” is synonymous with the terms“bound capsular polysaccharide-mAb complex” and“mAb bound to a capsular polysaccharide”.

A mAb-antigen complex may be detected by any means known to the skilled person. In one embodiment, a mAb-antigen complex is detected by means of a secondary (e.g. detection) antibody which binds the capsular polysaccharide and/or capsular polysaccharide-mAb complex.

Suitably, said secondary antibody comprises a detection means, such as a tag/label to aid detection. Said detection means is preferably conjugated to the secondary antibody. Examples of suitable labels include detectable labels such as radiolabels or fluorescent or coloured molecules, enzymatic markers or chromogenic markers - e.g. dyes that provide a visible colour change upon binding of the detection antibody to an antigen. By way of example, the label may be fluorescein-isothiocyanate (FITC), R-phycoerythrin, Alexa 532, CY3 or digoxigenin. The label may be a reporter molecule, which is detected directly, such as by detecting its fluorescent signal, or by exposure of the label to photographic or X-ray film. Alternatively, the label is not directly detectable, but may be detected, for example, in a two-phase system. An example of indirect label detection is binding of an antibody to the label.

In a preferable embodiment, said secondary antibody comprises a fluorescent tag, and a mAb-antigen complex is detected by the florescence emitted from a mAb-antigen-secondary antibody complex. A “mAb-antigen-secondary antibody complex” means a complex comprising a capsular polysaccharide antigen which has become bound to a mAb (e.g. a mAb with binding affinity for said capsular polysaccharide antigen), wherein said complex has further become bound by a secondary antibody which binds said capsular polysaccharide and/or capsular polysaccharide-mAb complex.

Suitably, a mAb-antigen complex is detected when the signal (preferably fluorescence) emitted from the detection label is greater than the signal detected in a control comprising no mAb (e.g. no mAb which binds a capsular polysaccharide). Said control may alternatively comprise a mAb, but the sample is not applied to said control.

Suitably, a mAb-antigen complex is detected when the ratio of signal (preferably fluorescence) detected compared to the signal detected in the control is at least about 1.5, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8 or 3, preferably at least about 2.

In a preferable embodiment, a mAb of the present invention is a human mAb, humanised mAb, chimeric mAb and/or resurfaced mAb. Preferably, the mAb is a human (e.g. fully human) mAbs.

In one embodiment, the mAb is immobilised on a surface. Preferably, the mAb is immobilised on (e.g. absorbed to) the surface of a bead. Suitably, said bead may be fluorescent (e.g. to allow for its detection). In a preferable embodiment, said bead is constructed with/from a carboxylated polystyrene material. Preferably said bead is a carboxylated polystyrene microsphere. In one embodiment, the mAb is immobilised on the surface of a discrete compartment. Said discrete compartment may be a well of a plate (e.g. a multiwell plate). Said discrete compartment may be a test tube (e.g. a glass test tube) or an‘eppendorf tube.

In one embodiment, a method of the invention is a multiplex method wherein said contacting step with each mAb is performed simultaneously and preferably under the same conditions.

Preferably, each mAb is present within a discrete compartment, and the urine sample is contacted with the mAb within said discrete compartment. Thus, each mAb may be contacted with the urine to provide a plurality of discrete assays.

Conditions (e.g. assay conditions) during the method are preferably kept consistent, preferably without the need for optimisation of conditions for individual assays. For example, the volume of sample applied to each assay is preferably the same, as are the time (e.g. incubation) and temperature conditions, etc.

In a preferable embodiment, a detection step is preceded by an incubation step, wherein the sample is incubated with the mAb to allow capsular polysaccharide in the sample to contact with the mAb within the assay. Said incubation may be for any time between 1 hour and 48 hours (e.g. 1 hour, 3 hours, 6 hours, 12 hours, 24 hours or 48 hours). Suitably, said incubation is for about 12 hours.

Suitably, contacting, incubating and/or detecting steps of methods of the invention are performed as an immunoassay. In one embodiment, an immunoassay comprises (i) contacting the sample with a mAb which binds a S. pneumoniae capsular polysaccharide described herein, (ii) allowing capsular polysaccharide present in the sample to bind to a mAb which binds said capsular polysaccharide, (iii) removing (e.g. washing off) non-bound material (e.g. non mAb-antigen complex), (iii) contacting the assay with a secondary detection antibody (preferably conjugated to a detection means such as a fluorescent dye) which binds the S. pneumoniae capsular polysaccharide and/or a mAb-antigen complex, (iv) allowing said secondary antibody to bind to capsular polysaccharide and/or a mAb-antigen complex, (v) removing (e.g. washing off) any non-bound material (e.g. non mAb-antigen- secondary antibody complex). Preferably, the mAb which binds a S. pneumoniae capsular polysaccharide is immobilised on a surface within the assay (e.g. on the surface of a bead).

Suitably, eight or more of said assays may be conducted. In a preferable embodiment, a method of the invention comprises performing each assay in a discrete compartment (e.g. of an immunoassay apparatus), preferably wherein said sample is applied to each of said wells.

Any means (e.g. apparatus) in which an assay may be performed may constitute a ‘compartment’ (e.g. of an immunoassay apparatus) as described herein. In one embodiment, a‘compartment’ is a discrete well (e.g. of a multi-well plate). In another embodiment, a ‘compartment’ is a tube (e.g. a test tube or Eppendorf tube).

In a preferable embodiment, each compartment comprises only one mAb capable of binding a S. pneumoniae serotype capsular polysaccharide.

Methods of the invention have high levels of sensitivity and specificity.

In one embodiment, a capsular polysaccharide is detected at a concentration of greater than or equal to (>) about 0.001 ng/ml. In one embodiment, a capsular polysaccharide is detected at a concentration of > about 0.003 ng/ml, 0.01 ng/ml, 0.1 ng/ml, 0.3 ng/ml, or 1 ng/ml.

Preferably, a 1 , 3, 8, 9V, 14, and/or 19A capsular polysaccharide is detected at a concentration of (e.g. a concentration as low as) > about 0.001 ng/ml. Preferably, a 4, 5, 6A, 10A, 11A, 15B, 18C and/or 20 capsular polysaccharide is detected at a concentration of (e.g. a concentration as low as) > about 0.003 ng/ml. Preferably, a 2, 12F, 19F and/or 22F capsular polysaccharide is detected at a concentration of (e.g. a concentration as low as) > about 0.01 ng/ml. Preferably, a 6B, 7F, 23F, or 33F capsular polysaccharide is detected at a concentration of (e.g. a concentration as low as) > about 0.03 ng/ml. Preferably, a 9N or 17F capsular polysaccharide is detected at a concentration of (e.g. a concentration as low as) > about 0.1 ng/ml.

In one embodiment, a capsular polysaccharide is detected with a specificity of at least 85% (e.g. at least 90%, 95% or 100%). In one embodiment, a capsular polysaccharide is detected with a specificity of about 98% (e.g. 98.4%, 95% confidence interval, 91.5%, 99.7%). In one embodiment, a capsular polysaccharide is detected with a specificity of about 95% (e.g. 95.2%, 95% confidence interval, 86.7%, 99%). Specificity is typically determined by detecting the rate of mAb binding to non-target antigens (e.g. antigens other than the capsular polysaccharide comprising their target epitope). Said non-target antigens are suitably non- pneumococcal antigens. Specificity may be determined as set out in Example 2 below. Preferably, a monoclonal antibody of the present invention has an affinity (Kd) of at least about 1X10 ⁸ M, 1X10 ⁹ M, 1X1 O ¹⁰ M, or 1x10 ¹¹ M. Suitably, said affinity is measured by way of ELISA.

In one embodiment, a method of the invention is performed with Luminex x-MAP technology.

In another embodiment, a method of the invention comprises applying the urine sample to a control assay comprising no mAb. Alternatively, said control assay may comprise a mAb, but no urine sample is applied to it.

In another aspect, there is provided a kit for detecting the presence or absence of a Streptococcus pneumoniae 9L capsular polysaccharide in a sample, wherein said kit comprises:

a) a first monoclonal antibody (mAb) that binds a S. pneumoniae capsular polysaccharide (CP) to form a first mAb-antigen complex, wherein said first mAb comprises the following six CDRs:

IV. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO:

4, or a functional variant thereof;

V. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO:

5, or a functional variant thereof; and

VI. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO:

6, or a functional variant thereof;

wherein said first mAb binds a 9N CP; and

b) a second mAb that binds a S. pneumoniae capsular polysaccharide (CP) to form a second mAb-antigen complex, wherein said second mAb comprises the following six CDRs:

II. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 8, or a functional variant thereof; III. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 9, or a functional variant thereof;

IV. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO:

10, or a functional variant thereof;

V. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO:

11 , or a functional variant thereof; and

VI. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO:

12, or a functional variant thereof;

wherein said second mAb binds a 9V CP; and

c) instructions to use said kit to detect the presence or absence of said first mAb- antigen complex and detect the presence or absence of said second mAb-antigen complex;

i. wherein said instructions indicate that the presence of the first mAb- antigen complex, and the presence of the second mAb-antigen complex confirms the presence of a 9L CP; and

ii. wherein said instructions indicate that the absence of the first mAb-antigen complex, and/or the absence of the second mAb-antigen complex is indicative of the absence of a 9L CP.

In a further aspect, there is provided a kit for detecting the presence or absence of a Streptococcus pneumoniae an 11 D capsular polysaccharide in a sample, wherein said kit comprises:

a) a third monoclonal antibody (mAb) that binds a S. pneumoniae capsular polysaccharide (CP) to form a third mAb-antigen complex, wherein said third mAb comprises the following six CDRs:

I. a light chain CDR13 comprising an amino acid sequence of SEQ ID NO:

13, or a functional variant thereof;

II. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO:

14, or a functional variant thereof;

III. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO:

15, or a functional variant thereof;

IV. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO:

16, or a functional variant thereof;

V. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO:

17, or a functional variant thereof; and

VI. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO:

18, or a functional variant thereof; wherein said third mAb binds an 11A CP; and

b) a fourth mAb that binds a S. pneumoniae CP to form a fourth mAb-antigen complex, wherein said fourth mAb comprises the following six CDRs:

I. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO:

19, or a functional variant thereof;

II. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO:

20, or a functional variant thereof;

III. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO:

21 , or a functional variant thereof;

IV. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO:

22, or a functional variant thereof;

V. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO:

23, or a functional variant thereof; and

VI. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO:

24, or a functional variant thereof;

wherein said fourth mAb binds a 15B CP; and

c) instructions to use said kit to detect the presence or absence of said third mAb- antigen complex and detect the presence or absence of said fourth mAb-antigen complex;

i. wherein said instructions indicate that the presence of the third mAb- antigen complex, and the presence of the fourth mAb-antigen complex confirms the presence of an 11 D CP;

ii. wherein said instructions indicate that the absence of the third mAb- antigen complex, and/or the absence of the fourth mAb-antigen complex is indicative of the absence of an 11 D CP.

In another aspect, there is provided a kit for detecting the presence or absence of a Streptococcus pneumoniae serotype-specific capsular polysaccharide in a sample, wherein said apparatus comprises:

II. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 2, or a functional variant thereof; III. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 3, or a functional variant thereof;

wherein said first mAb binds a 9N CP;

b) a third monoclonal antibody (mAb) that binds a S. pneumoniae capsular polysaccharide (CP) to form a third mAb-antigen complex, wherein said third mAb comprises the following six CDRs:

IV. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO:

16, or a functional variant thereof;

V. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO:

17, or a functional variant thereof; and

VI. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO:

18, or a functional variant thereof;

wherein said third mAb binds an 11A CP; and

c) a fourth mAb that binds a S. pneumoniae CP to form a fourth mAb-antigen complex, wherein said fourth mAb comprises the following six CDRs:

I. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 19, or a functional variant thereof;

IV. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO:

22, or a functional variant thereof;

V. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO:

23, or a functional variant thereof; and VI. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO:

24, or a functional variant thereof;

wherein said fourth mAb binds a 15B CP; and

d) instructions to use said kit to detect the presence or absence of said first mAb- antigen complex, detect the presence or absence of said third mAb-antigen and detect the presence or absence of said fourth mAb-antigen complex;

i. wherein said instructions indicate that the presence of two or more selected from the first mAb-antigen complex, the third mAb-antigen complex and the fourth mAb-antigen complex confirms the presence of 18A CP; and

ii. wherein said instructions indicate that the absence of two or more selected from the first mAb-antigen complex, the third mAb-antigen complex and the fourth mAb-antigen complex confirms the absence of 18A CP.

Said kit may further comprise a fourth to twenty-fifth mAb as described herein.

In one embodiment, said kit comprises a twenty-fifth mAb which binds Streptococcus pneumoniae CWP.

In one embodiment, said mAbs are provided within a discrete compartment.

Preferably, a kit of the present invention is for use in a method of the present invention.

In another embodiment, the immunoassay apparatus further comprises a secondary antibody. Said secondary antibody is suitably a detection antibody capable of binding to a capsular polysaccharide and/or capsular polysaccharide-mAb complex described herein. In a preferable embodiment, said secondary antibody is conjugated to a detection means, preferably a fluorescent dye. Such secondary antibody is preferably used for detecting for the presence or absence of capsular polysaccharide and/or capsular polysaccharide-mAb complex within one or more assays in methods of the present invention.

An immunoassay apparatus may suitably be constructed with Luminex x-MAP technology. Luminex x-MAP technology involves the use of magnetic, spectrally distinct carboxylated polystyrene microspheres, or“beads,” that can be coated with a broad range of molecules, including nucleic acids or proteins, and used in various assay formats, such as PCR-based assays and immunoassays. Coating such beads with antibodies specific to pneumococcal serotypes allows for the simultaneous detection of different pneumococcal serotype antigens in a single sample, thus reducing the amount of sample required. To date, Luminex microsphere technologies have been used to aid in the detection of pneumococcal serotypes using antibody detection of polysaccharide, competitive inhibition assays, PCR-based multiplex assays, and serological assays to detect antibody responses to pneumococcal serotypes.

In embodiment, the monoclonal antibody is immobilised (e.g. adsorbed) on the surface of a bead such as a carboxylated polystyrene microsphere. Preferably said bead is fluorescent.

The term "antibody" covers monoclonal antibodies and fragments thereof (e.g. exhibiting the desired biological activity). In a preferable embodiment, an antibody of the present invention is a monoclonal antibody. In a more preferable embodiment, the antibody is a fully human monoclonal antibody. In one embodiment, methods of the invention may employ polyclonal antibodies.

In particular, an antibody is a protein including at least one or two, heavy (H) chain variable regions (abbreviated herein as VHC), and at least one or two light (L) chain variable regions (abbreviated herein as VLC). The VHC and VLC regions can be further subdivided into regions of hypervariability, termed "complementarity determining regions" ("CDR"), interspersed with regions that are more conserved, termed "framework regions" (FR). The extent of the framework region and CDRs has been precisely defined (see, Kabat, E.A., et al. Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991 , and Chothia, C. et al, J. Mol. Biol. 196:901-917, 1987). Preferably, each VHC and VLC is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRI, CDRI, FR2, DR2, FR3, CDR3, FR4. The VHC or VLC chain of the antibody can further include all or part of a heavy or light chain constant region. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are interconnected by, e.g., disulfide bonds. The heavy chain constant region includes three domains, CH1 , CH2 and CH3. The light chain constant region is comprised of one domain, CL. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The term "antibody" includes intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof), wherein the light chains of the immunoglobulin may be of types kappa or lambda. The term antibody, as used herein, also refers to a portion of an antibody that binds to one of the above-mentioned markers, e.g., a molecule in which one or more immunoglobulin chains is not full length, but which binds to a marker. Examples of binding portions encompassed within the term antibody include (i) a Fab fragment, a monovalent fragment consisting of the VLC, VHC, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fc fragment consisting of the VHC and CH1 domains; (iv) a Fv fragment consisting of the VLC and VHC domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, Nature 341 :544-546, 1989), which consists of a VHC domain; and (vi) an isolated complementarity determining region (CDR) having sufficient framework to bind, e.g. an antigen binding portion of a variable region. An antigen binding portion of a light chain variable region and an antigen binding portion of a heavy chain variable region, e.g., the two domains of the Fv fragment, VLC and VHC, can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VLC and VHC regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science IAI-ATί-AIb; and Huston et al. (1988) Proc. Natl. Acad. ScL USA 85:5879-5883). Such single chain antibodies are also encompassed within the term antibody. These may be obtained using conventional techniques known to those skilled in the art, and the portions are screened for utility in the same manner as are intact antibodies.

In one embodiment, a mAb of the invention is a scFV.

In a preferable embodiment, a mAb of the invention is a fully human mAb. Suitably, said fully human mAb is an IgG.

Antibody preparation

The antibodies of the present invention can be obtained using conventional techniques known to persons skilled in the art and their utility confirmed by conventional binding studies. By way of example, a simple binding assay is to incubate the cell expressing an antigen with the antibody. If the antibody is tagged with a fluorophore, the binding of the antibody to the antigen can be detected by FACS analysis.

Antibodies of the present invention can be raised in various animals including mice, rats, rabbits, goats, sheep, monkeys or horses. Antibodies may be raised following immunisation with individual capsular polysaccharides, or with a plurality of capsular polysaccharides. Blood isolated from these animals contains polyclonal antibodies - multiple antibodies that bind to the same antigen. Antigens may also be injected into chickens for generation of polyclonal antibodies in egg yolk. To obtain a monoclonal antibody that is specific for a single epitope of an antigen, antibody-secreting lymphocytes are isolated from an animal and immortalized by fusing them with a cancer cell line. The fused cells are called hybridomas, and will continually grow and secrete antibody in culture. Single hybridoma cells are isolated by dilution cloning to generate cell clones that all produce the same antibody; these antibodies are called monoclonal antibodies. Methods for producing monoclonal antibodies are conventional techniques known to those skilled in the art (see e.g. Making and Using Antibodies: A Practical Handbook. GC Howard. CRC Books. 2006. ISBN 0849335280). Polyclonal and monoclonal antibodies are often purified using Protein A/G or antigen-affinity chromatography.

Human mAb preparation

Human mAbs may be prepared following the method of Smith et. al. “Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen” (Nat Protoc. 2009; 4(3): 372-384., which is incorporated herein by reference.

Briefly, a human subject is immunised with a capsular polysaccharide. Antibody variable heavy chain and variable light chain sequences of are amplified from cells (typically peripheral blood mononuclear cell / ASC cells) of said subject following immunisations. Said heavy and light chain sequences are cloned into suitable expression vectors for expression of fully human IgG antibodies - the sequences of suitable expression vectors are available on GenBank (accessions numbers: FJ475055, FJ475056, FJ517647). Said vectors are available through AddGene. The expression vectors (comprising the variable heavy chain and variable light chain sequences) are transfected into a suitable cell for expression (e.g. 293 A cells).

SEQUENCE HOMOLOGY

Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et al. , CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position- Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. Mol. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences. Non-limiting methods include, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501 -509 (1992); Gibbs sampling, see, e.g., C. E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131 ) Science 208-214 (1993); Align- M, see, e.g., Ivo Van Walle et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics: 1428-1435 (2004).

Thus, percent sequence identity is determined by conventional methods. See, for example, Altschul et al. , Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1 , and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown below (amino acids are indicated by the standard one-letter codes). The "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, % identity may be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids, multiplied by 100. Calculations of % sequence identity may also take into account the number of gaps, and the length of each gap that needs to be introduced to optimize alignment of two or more sequences. Sequence comparisons and the determination of percent identity between two or more sequences can be carried out using specific mathematical algorithms, such as BLAST, which will be familiar to a skilled person.

ALIGNMENT SCORES FOR DETERMINING SEQUENCE IDENTITY

A R N D C Q E G H I L K M F P S T W Y V

A 4

R-1 5

N -2 06

D-2-2 1 6

C 0-3 -3 -3 9

Q-1 1 0 0-3 5

E -1 0 02-42 5

G 0-2 0-1-3 -2 -2 6

H -2 0 1 -1 -3 0 0 -2 8

I -1 -3 -3 -3 -1 -3 -3 -4 -34

L -1 -2 -3 -4 -1 -2 -3 -4-32 4

K-1 20-1-3 1 1-2-1 -3-2 5

M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2-1 5

F -2 -3 -3 -3 -2 -3 -3-3-1 0 0-3 06

P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7

S 1 -1 1 0-1 0 00-1 -2-2 0-1 -2-1 4

T 0 -1 0-1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2-1 1 5

W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4-3-211

Y -2 -2 -2 -3 -2 -1 -2 -32 -1 -1 -2 -1 3 -3 -2 -2 2 7

V 0-3-3 -3 -1 -2 -2 -3-3 3 1 -2 1 -1 -2 -2 0-3-1 4

The percent identity is then calculated as:

Total number of identical matches

x 100

[length of the longer sequence plus the

number of gaps introduced into the longer

sequence in order to align the two sequences]

Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino- terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag. CONSERVATIVE AMINO ACID SUBSTITUTIONS

Basic: arginine

lysine

histidine

Acidic: glutamic acid

aspartic acid

Polar: glutamine

asparagine

Hydrophobic: leucine

isoleucine

valine

Aromatic: phenylalanine

tryptophan

tyrosine

Small: glycine

alanine

serine

threonine

methionine

In addition to the 20 standard amino acids, non-standard amino acids (such as 4- hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline and a -methyl serine) may be substituted for amino acid residues of the polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for polypeptide amino acid residues. The polypeptides of the present invention can also comprise non-naturally occurring amino acid residues.

Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4- methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo- threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro- glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3- azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991 ; Ellman et al., Methods Enzymol. 202:301 , 1991 ; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271 :19991-8, 1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the polypeptide in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues of polypeptides of the present invention.

Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related components (e.g. the translocation or protease components) of the polypeptides of the present invention.

Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241 :53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al. , Biochem. 30:10832-7, 1991 ; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241 :53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991 ; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide the skilled person with a general dictionary of many of the terms used in this disclosure.

This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspects or embodiments of this disclosure.

Amino acids are referred to herein using the name of the amino acid, the three letter abbreviation or the single letter abbreviation. The term“protein", as used herein, includes proteins, polypeptides, and peptides. As used herein, the term“amino acid sequence” is synonymous with the term“polypeptide” and/or the term“protein”. In some instances, the term“amino acid sequence” is synonymous with the term“peptide”. In some instances, the term“amino acid sequence” is synonymous with the term“enzyme”. The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, the conventional one-letter and three-letter codes for amino acid residues may be used. The 3- letter code for amino acids as defined in conformity with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

Other definitions of terms may appear throughout the specification. Before the exemplary embodiments are described in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be defined only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a monoclonal antibody" includes a plurality of such monoclonal antibodies and reference to "the capsular polysaccharide" includes reference to one or more capsular polysaccharides and equivalents thereof known to those skilled in the art, and so forth. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto. BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples.

Figure 1 shows results of assays comprising each of the mAbs described herein, outlined in a‘checkerboard’ format. The sign‘+’ indicates that the mAb displays binding for the capsular polysaccharide indicated on the left hand column. The sign (+/-) indicates that such binding may not always be detected (e.g. absent optimisation of conditions to allow for binding and/or detection thereof).

EXAMPLES

MATERIALS & METHODS

Bacterial strains

Bacterial isolates and control strains, including 13 non-pneumococcal streptococcal species (n, 42 strains) and a selection of various bacteria that are associated with respiratory infections or that may be found in the urogenital tract (n, 111 strains) were obtained from laboratories in the Bacteriology Reference Department, Public Health England (PHE) National Infection Service, Colindale, United Kingdom. A panel of 91 pneumococcal serotype reference strains obtained from SSI Diagnostica AG, Hillerod, Denmark, and the reference strain for serotype 6D, kindly provided by The National Institute of Health and Welfare, Helsinki, Finland, were also included in order to assess the serotype specificity of the assay.

Preparation of crude bacterial antigen for specificity testing

Pure bacterial cultures were suspended in phosphate-buffered saline (PBS), and colony counts were performed on a selection of the bacterial suspensions covering a range from the least to the most optically dense. All samples counted were estimated to have >1.2 x 10⁹ CFU/ml. The bacteria were typically heat killed in a heat block (Grant Instruments) at 100°C. An additional preparation of killed Legionella pneumophila was obtained by overnight treatment of a suspended L. pneumophila culture in 2% formalin, followed by centrifugation at 13,000 x g for 10 min before resuspension of the bacterial pellet in PBS. All of the killed bacterial antigen preparations were stored at 20°C. Prior to testing, the antigen preparations were thawed and were spun in a centrifuge at 13,000 x g for 3 min, and an aliquot of the supernatant was diluted 1 :100, 1 :2,000, and/or 1 :1 ,000 in 25 mM HEPES (Sigma)-buffered polysaccharide antigen-negative urine prior to testing by the UAD assay. Aliquots of the pneumococcal antigen preparations were diluted 1 :2,500 in 25 mM HEPES-buffered antigen negative urine.

Panel of purified polysaccharides

Purified capsular polysaccharides from 25 different pneumococcal serotypes (serotypes 1 , 2, 3, 4, 5, 6B, 6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F) and the pneumococcal cell wall polysaccharide (CWP) were obtained from the ATCC, Manassas, VA, USA. The purified capsular polysaccharide for serotype 6A was acquired from SSI Diagnostica. For specificity testing, each polysaccharide was typically diluted in 25 mM HEPES-buffered antigen negative urine to a final concentration of 10 ng/ml. Preparation of controls and standards

A pneumococcal polysaccharide antigen-negative urine sample was kindly supplied by a healthy donor for use as a negative control. The same negative-control urine sample was also used to dilute a standard titration consisting of a mixture of purified capsular polysaccharides for each of the 25 antibody targets. The standards were titrated at concentrations of 10, 3, 1 , 0.3, 0.1 , 0.03, and 0.01 ng/ml for all assay runs except for the repeatability experiments, where further dilutions of 0.003, 0.001 , and 0.0003 ng/ml were also included.

Panel of urine specimens

Urine samples were obtained from individuals presenting to secondary care centers in the United Kingdom, either as part of a prospective cohort study of patients with community- acquired pneumonia (CAP) or for confirmation testing following a positive BinaxNOW result. All urine samples were anonymized for use in specificity testing.

A panel of 588 urine samples were obtained from patients diagnosed with CAP between 2006 and 2015 from two UK regional NHS hospital trusts. Patients treated by the admitting team with the presence of new or progressive infiltrates on a chest radiograph and at least one symptom of acute lower respiratory tract infection, such as cough, fever, dyspnea, sputum, or pleuritic chest pain, were diagnosed with CAP. These urine samples were frozen prior to transportation to PHE-Colindale for testing by the UAD assay.

A further 28 urine samples from patients with suspected PD based on clinical symptoms, contact links with outbreak cases, and/or a positive BinaxNOW test were obtained from the Respiratory and Vaccine Preventable Bacteria Reference Unit (RVPBRU) at PHE-Colindale. Three of these urine samples were from a family cluster with severe pneumonia, and seven were from a suspected outbreak of pneumococcal serotype 6C and influenza on a hospital ward. In addition to the negative-control urine samples, a further two urine samples were obtained from healthy donors who had tested negative for pneumococcal urinary antigen by the BinaxNOW test. The RVPBRU urine samples were stored at 2 to 8°C. All other urine samples were frozen at < 70°C for long-term storage and were then refrigerated at 2 to 8°C prior to testing.

Antibodies used for bead coupling

Purified human monoclonal antibodies (h-MAbs) to the target serotypes and CWP were supplied by Pamlico Biopharma/Oklahoma Medical Research Foundation. These antibodies were fully human, full-length monoclonal antibodies produced from single antibody-secreting cells sorted 7 days after vaccination with either Pneumovax 23 (Merck & Co., Inc.) or Prevnar 13 (Wyeth Pharmaceuticals, Inc.).

Bead-coupling method

Spectrally distinct Luminex carboxylated magnetic beads (Luminex) are mapped to unique bead regions. Each h-MAb preparation was coupled to a distinct bead region by use of the method described in the manufacturers instructions, with the following adaptations: the post activation reagents were typically kept on ice to maintain a cold temperature; a coupling protein concentration of 3.2 pg of antibody per million beads was typically used; and the beads were blocked overnight in PBS-1 % bovine serum albumin (BSA) (Sigma) with mixing by rotation at room temperature. The bead regions used and the antibodies coupled to them are shown in Table 1. The coupled beads were stored in StabilGuard (SurModics) at 2 to 8°C in the dark and were stable for 1 year. Prior to use, the beads were counted using a Bio-Rad TC10 automated cell counter (Bio-Rad), and aliquots of bead mixtures containing approximately 15.25 x 10⁴ beads of each bead type/mI were prepared. Bead mixtures were stored in light-protective amber microcentrifuge tubes (Alpha Laboratories) at 2 to 8°C until required. One batch of each coupled bead preparation was used for testing the 588 CAP urine samples, but different batches were used for sensitivity, specificity, and reproducibility experiments. Each batch of beads was subject to a quality control check before use to ensure equivalence of results.

Multiplex serotype-specific antigen detection assay

Each bead mixture was diluted in PBS prior to use to produce final concentrations of 100 beads of each bead type/mI. Twenty-five microliters of the bead mixture was then added to each test well of a Greiner black 96-well assay plate (Greiner Bio-One), resulting in approximately 2,500 beads of each bead type per well. At least one antigen-negative control urine well and a standard titration were included on each test plate; for sensitivity, specificity; and reproducibility experiments, the samples, controls, and standards were added in the replicates stated in each section. For routine sample testing, samples were added in duplicate, and controls and standards were added in single wells. HEPES buffer (Sigma) was added to all clinical, negative-control, and standard urine samples at a final concentration of 25 mM, and urine samples were spun in a centrifuge at 16,000 x g for 2 min to remove any debris before use. All samples, controls, standards, and reagents were added at 100 mI per well to the appropriate wells of the assay plate, and for all incubations, the plate was sealed with a foil-backed plate lid (Corning) and was placed on a plate shaker (VWR International) set to shake at 500 rpm at room temperature. The samples, controls, and standards, along with the h-MAb-coupled beads, were typically allowed to incubate on a plate shaker overnight (although much shorter incubation periods are possible). After incubation, the assay plate was washed with PBS-0.05% Tween 20 (PHE-Colindale Media Services) three times using a Bioplex Pro 2 magnetic plate washer (Bio-Rad). This washing method was used for all subsequent wash steps. Omni antiserum (SSI Diagnostica), previously purified for IgG using the Melon Gel IgG Spin purification kit (Thermo Fisher Scientific), was diluted 1 :1 ,000 in the assay diluent (PBS-2% BSA and a negative human reference serum diluted to produce a total IgG concentration of 10 pg/ml) and was added to the assay plate, incubated on the plate shaker for 1 h, and then washed. After washing, a polyclonal goat anti-rabbit-R-phycoerythrin (RPE) conjugate solution (Thermo Fisher Scientific) diluted to 1 :400 in the assay diluent was added to the assay plate. The plate was covered and was incubated for 30 min. The plate was then washed and the beads resuspended in 150 pi PBS-0.05% Tween 20. The assay plate was then loaded onto the Bioplex 200 suspension array system (Bio-Rad) and the resulting fluorescence intensities read with the system set to read > 100 beads per region with a low photomultiplier tube (PMT) setting.

Interpretation of results

Results were interpreted by observing the signal-to-noise ratio of the median FI, referred to as the t/n (test sample-to-negative control) ratio. In order to enable results to be comparable between test samples, these ratios were typically normalized using the method described in Sheppard et al. Journal of Medical Microbiology (2011), 60, 49-55. This method assumes that there are no more than 3 positive results for each sample. The normalized data were then normalized further by calculating the median ratio result for each assay (e.g. in cases where the majority of samples were negative, and therefore, the median would be representative of a negative result). Each of the individual ratios for that assay were then divided by the median ratio for the same assay. Any results above the normalized t/n ratio cutoff of 2.5 were considered positive. To establish the cutoff threshold, negative-control ratios (negative/negative - expected to be t/n = 1) were normalized.

Repeatability

To test the repeatability of the assay, three standard curves, consisting of a mixture of purified capsular polysaccharides for each of the 25 antibody targets at individual concentrations of 10, 3, 1 , 0.3, 0.1 , 0.03, 0.01 , 0.003, 0.001 , and 0.0003 ng/ml, were tested over 6 days. Twenty-one urine samples were included in the repeatability runs. Fourteen of these were urine samples obtained between 2008 and 2013 from patients with culture- confirmed or suspected PD based on clinical symptoms, contact links, and/or BinaxNOW results (Table 4), two were donated urine samples from healthy donors who had previously tested negative for pneumococcal cell wall C polysaccharide by the BinaxNOW test, and five were spiked samples in which antigen-negative urine was spiked with purified polysaccharide antigen to produce a final concentration just above the estimated sensitivity cutoff. The spiked urine samples consisted of polysaccharides of serotype 19F (at 1 ng/ml), 18C (at 0.4 ng/ml), 6B (at 0.1 ng/ml), 33F (at 0.04 ng/ml), or 7F (at 0.4 ng/ml). Each urine sample was tested in triplicate over 5 days and was tested once on day 6.

EXAMPLE 1

Determination of positivity cutoff

Analysis of the fluorescence intensity (FI) data produced from the repeatability runs in the novel 25-plex S. pneumoniae polysaccharide assay revealed that a normalized signal-to- noise ratio (test sample/negative control [t/n] ratio) of 2 is >3 standard deviations above the mean FI of the negative controls in all 25 assays and, therefore, based on these data, is >99% accurate for calling positivity compared to the negative-control urine standard.

Furthermore, a normalized ratio of 2.5 is >3 standard deviations above the mean of the normalized ratios for the negative-control samples in all 25 assays (Table 1). Therefore, a t/n ratio of 2.5 was used as the general positivity cutoff for the analysis of the urine results. Particularly, Table 1 presents calculated cutoff values for each serotype, based on results from negative control urine in repeatability assays³. Calculated as 3 standard deviations greater than the mean of the normalized negative-control ratios; ^bMean t/n ratio, from six repeatability runs, of negative urine mFI/negative urine mFI (initially 1.0) following the plate normalization procedure; ^cCWP, cell wall polysaccharide antigen target.

Table 1

EXAMPLE 2

Analytical sensitivity

The limit of detection for the purified capsular polysaccharides for each assay was estimated as 3 standard deviations above the mean FI of the negative controls in the repeatability runs. Based on these values, it was estimated that serotypes 1 , 8, 14, 19A, and 22F can be detected at or below a concentration of 0.0003 ng purified polysaccharide/ml, serotypes 2, 3, 4, 5, 6A, 9V, 10A, 1 1A, 12F, 15B, 20, and 33F at concentrations as low as 0.001 ng/ml, serotypes 6B, 9N, 17F, and 23F at concentrations as low as 0.01 ng/ml, serotype 7F at 0.03 ng/ml, and serotypes 18C and 19F at 0.1 ng/ml. Table 2 lists the minimum concentration for reporting the positive detection of each of the purified polysaccharides in the standard curves used in the repeatability runs when a normalized t/n ratio cutoff of 2.5 is applied; the mode value is shown to avoid artificially high or low mean values caused by outlier results.

Particularly, Table 2 presents the modal minimum concentration for detection of each of the purified capsular polysaccharides used in the standard curves in the repeatability runs when a normalized t/n ratio of 2.5 is used as the positivity cutoff.

Table 2

EXAMPLE 3

Analytical specificity

The pneumococcal-serotype specificity of the assay was tested using a panel of purified polysaccharides and a panel of 92 crude pneumococcal serotype reference strain antigens. Pneumococcal specificity tests indicate that 16 of 25 human MAbs (h-MAbs) exhibit cross reactivity for other non-targeted pneumococcal serotypes, many of which are in the same serogroup (Table 3). Taking the cross reactions into account, assay results positive for serotype 6A, 6B, 7F, 9V, 10A, 11 A, 12F, 15B, 18C, 23F, or 33F are reported as serotype 6 A/C, 6B/D, 7A/F, 9A/V, group 10/33C/39, 11A/C/E/45/group 16, 12F/44, group 15, group 18/35C/42, 23F/group 32, or 33A/B/D/F, respectively. However, when an h-MAb cross-reacts with a polysaccharide also targeted by another h-MAb in our UAD assay, this can be taken into consideration in interpreting the results. For example, since the 9N polysaccharide cross-reacts with the h-Mab targeting serotype 14, a sample producing a positive result with both the serotype 9N and serotype 14 MAbs could be interpreted as actually being positive for the serotype 9N polysaccharide antigen. This combination could also be positive for the 9L antigen, but in this case, the 9L antigen also cross-reacts with the h-MAbs targeting 9V and 19A, and therefore, for a sample containing the 9L antigen, a positive result for the 9V and 19A assays as well as for the serotype 9N and 14 assays would be observed. In this manner, results can be interpreted using a checkerboard system similar to that used for the serotyping of isolates by the Quellung reaction and by slide agglutination with the SSI antisera.

Particularly, Table 3 shows h-MAbs that cross-react with non-targeted pneumococcal serotypes. ^aSerotypes in boldface indicate a cross-reaction with a different serogroup. ^ftCross-reaction with a polysaccharide antigen detected in the 25-plex assay.

Table 3

Analytical specificity with regard to non-pneumococcal antigens was tested using antigens prepared from 42 non-pneumococcal streptococci, comprising 13 different species, and 111 pure bacterial cultures associated with respiratory infections or the urogenital tract, comprising 21 genera and 59 different species (see Table 4). When tested at >1.2 x 10⁶ CFU/ml, eight of the nine Lancefield group B streptococcus (GBS) serotype II isolates gave t/n results of >2.0 for the pneumococcal serotype 14 assay, and six of these had t/n ratios of >2.5. Advantageously, the CWP assay was negative for all eight of these serotype 14- positive GBS serotype II samples such that the CWP assay may be used to confirm the presence or the absence of said serotype. Among the other streptococci, the Streptococcus pseudopneumoniae, Streptococcus mitis, and Streptococcus peroris (a member of the S. mitis group) isolates tested positive for the pneumococcal CWP. None of the other bacterial isolates in the panel generated a positive result in this UAD assay, leading to analytical specificities of 98.4% (95% confidence interval [Cl], 91.5%, 99.7%) and 95.2% (95% Cl, 86.7%, 99.0%) for the serotype and CWP assays, respectively, against non-pneumococcal bacteria. Advantageously, bacteria testing positive for both the serotype and CWP assays were not detected.

Table 4 - List some of the non-pneumococcal organisms used in specificity tests

EXAMPLE 4

Repeatability

All the results obtained for the clinical urine test samples and those spiked with purified polysaccharide antigen were repeatable over the 6 days, supporting the qualitative repeatability of the assay. Table 5 lists the UAD assay results for the urine samples used in the repeatability runs. The interassay coefficient of variation (CV) for each of the negative controls was below 20%.

Particularly, Table 5 presents Bioplex assay results for urine samples tested in the repeatability experiments. ^aSerotype result for pneumococcal culture of blood or a bronchoalveolar lavage specimen from the same patient as the tested urine sample. ^b For urine samples spiked with antigen, the concentration (in nanograms per milliliter) at which the serotype was detected is given in parentheses. ^cTaking into account any potential cross reactions as listed in Table 3. ^dSample with a positive CWP assay result but negative for all other assay serotypes.

Table 5

EXAMPLE 5

Clinical testing

The assay was used to test 616 urine samples. Of these, 520 were also tested for S. pneumoniae urinary antigen by the BinaxNOW test. All 95 (100%) of the BinaxNOW-positive urine samples tested positive for a pneumococcal serotype and/or the cell wall polysaccharide antigen by our assay. Seventy-four of the urine samples were from patients who had pneumococci isolated from their blood cultures or bronchoalveolar lavage (BAL) specimens, and the UAD assay was able to detect pneumococcal antigen in 69 (93.2%) of these 74 urine samples. Serotype information was available for 65 of these pneumococcal cultures. Six of the culture-identified serotypes are not currently typeable with our multiplex immunoassay; however, the CWP was detected in all six of these matched nontypeable urine samples.

In total, of the 616 urine specimens tested, 132 (21.4%) were obtained from patients with pneumococcal disease as defined by a positive culture isolate from blood or BAL fluid and/or a positive BinaxNOW test result. Our assay identified 127 of the 132 (96.2%) urine samples as positive for pneumococcal antigen, and serotype information was available for 110 of these. Of the 484 urine samples that were negative for pneumococci by the BinaxNOW test and had no associated pneumococcal blood or BAL culture, the present method was able to identify a further 144 as positive for pneumococcal antigen, and 115 of these were serotyped. Multiple (more than one) serotypes were detected in 27 (4.4%) of the 616 urine samples.

A further method was used to investigate specificity, which calculated the specificity of a pneumococcal-serotype-specific urine antigen test (UAT) using urine samples from community-acquired pneumonia (CAP) patients by comparing the number of negative UAT samples with the number of“true negatives,” defined as CAP cases with bacteremia caused by another pathogen or CAP cases with only a positive Legionella urinary antigen test. Using this method, 69 of the urine samples tested by our UAD assay could be described as true negatives, and of these, 62 samples produced a negative result when tested in our assay, resulting in a clinical specificity of 89.9%. Thirty-six of the urine samples tested were from CAP patients defined as“true negative” due to bacteremia caused by a pathogen other than S. pneumoniae ; of these, 29 were negative when tested by our UAD assay, with a resulting specificity of 80.6%.

EXAMPLE 5

Detecting 9L capsular polysaccharide

As shown here (together with Figure 1), the first mAb which binds 9N also cross reacts with 9L, 18A, 43 and 47A. The second mAb which binds 9V also cross reacts with 9A and 9L. By subtractive analysis, it is possible to determine the presence of a 9L capsular polysaccharide.

A sample comprising a 9L capsular polysaccharide was added to a first assay with the first mAb, and a second assay with the second mAb. Both assays showed a positive result (for capsular polysaccharide binding). Thus, it was possible to detect the presence of a 9L capsular polysaccharide in the sample.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.

Sequences

SEQ ID NO: 25: EIVLTQSPGTLSLSPGERATLSCRASQSISPH LA

WYQQKPGQSPRLLIYDASNRATGI PARFSGSESGTDFTLSISSL EPEDFAVYYCQQSGDWPLTFGGGTKVEIK

SEQ ID NO: 26: EVQLLESGGALVQPGGSLRLSCAASGFTFTSFA

MGWVRQAPGKGLEWVSAVTGSGYYKNYADSVKGRFTISRDNS DNTLYLQM NSLRGDDTALYYCAKAHRGDWNNFFDYWGQGTLV TVSS SEQ ID NO: 27: D I VM TQ S P DS LA VS LG E R AT I SC KSSQS L LYSSS

N KNYLAWFQQKPGQAPKLL I Y WA ST R ESGVPDRFSGSGSGTDF TLTISSLQTEDVAVYYCLQYRSAPFTFGGGTKVEI K

SEQ ID NO: 28: EVQLVESGGG LM KPGGSLR LSCAVSG FTFTNA

WLSWVRQPPG KGLEWVGRAYSSSGGWTM DYSSPVRG RFTITR DDSKNTLYLQM N N LKTEDTAVYYCTTDIG KG WYT H YPDLWGQG T LVTVSS

SEQ ID NO: 29: D I QLTQSPSF LSASVG DRVTITCRASQG I DTR LI

WYQQKPG EAPKLLIYEASTLQSGAPSRFSGSG FGTEFTLTI SSL QPEDFATYYCQQFKGYPLTFGGGTKVEI K SEQ ID NO: 30: EVQLVESGGG LVQPGGSLRLSCAASG FTFSDYR

M DWVRQAPG RG LEWIA RI R H R DAGYSTEYAASVRG RFTVSR D DSQSTLYLQM NSLKADDTAVYI CLKDSSQYSFDAWGQGTMVTV SS SEQ ID NO: 31: DIVMTQSPDSLAVSLGE RATI N CKSSQSVLYSS

N N KNYLAWYQQKPGQPPKM LIYWASTRESGVPD RFSGSGSGT DFTLTI SSLQAEDVAVYYCQQYYTTPPITFGQGTRLEI K

SEQ ID NO: 32: EVQLVESGGG LVQPGGS LR LSCAASG FS F DTS WMTWVRQAPG KG LEWVATI NQGGSD KYYVDSVKG R FTI SR DN AKN SLYLQM N S LRAE DTAVYYCARAGGCSSTRC HTTPG F DYW GQGA LVTVSS

SEQ ID NO: 133: EIVMTQS PAS LSVSPG ETATLSC RASQSVGSTL AWYQQKPGQAPRLLI YNVFTRAAGVPARFSGSGSRTEFTLTISS LQSEDFAVYYCQQYSTWLWTFGQGTKVEI K

SEQ ID NO: 134: EVQ LVESGGG LVQPGGSLR LSCVASG FTFSTY

WM HWVRQPPG KG LVWVSR I N PDGSSTNYADSVNG RFTI SR DN AKNTLYLEM NSLRVEDTALYYCARSPGGYFDYWGHSTLVTVSS

SEQ ID NO: 135: DIQMTQSPSSLSASVGDRVTITCQASQDI RKLLN

WYQQ R PG KAPN LLIYDASN LETGVPSRFSGSGSGTH FSFTITSL QPEDIATYYCQQFESFPRTFG PGTKVDI K

SEQ ID NO: 136: EVQLVESGGG LVQI GGSLRLSCAASGFTFSTYW MSWVRQAPG KGLECVASI KEDGSERYYVDSVKG R FTI SR DNAK NSLH LQM DSLRAADTAVYFCARGRN N FRHWGQGTLVTVSS SEQ ID NO: 137: EIVMTQSPATLSVSPG ERATLSC RASQSVSG D L VWYQQKPGQAPRLLIYGATTRASGVPARFSGSGSGTEFTLTISS LQSEDFAIYYCQQYN NWPRTFGQGTKVEI K

SEQ ID NO: 138: EVQ LVESGGG LVQ PGGS LR LSCAASG FTFSSY WMSWVRQAPG KG L E WV G KI KEDGSEKYYVDSVKG RFAI SR DN AKNSLSLQM NSLRAEDTAVYYCARGQSYPGIWGQGTMVTVSS

SEQ ID NO: 139: EIVLTQSPATLSLSPG ERATLSCRASQTVSRYLA

WYQQKPGQAPRLLIYAASN RATG I PTRFSGSGSGTDFTLTI SSL EPEDFAFYYCQQRSNWPATFGGGTKVEI K SEQ ID NO: 140: EVQLLESGG DLVQPGGSLRLSCAASG F DFSIYG

M NWVRQA PG KG LEWVSVI SG DGTI IYYADSVKG RFTI SR D N SK NTLFLQVNSVRAEDTAVYYCAKGGYYESGTM RAFDIWGQGTMV TVSS

SEQ ID NO: 141: D I Q LTQSPSF LSASVG D RVTI TC RASQ DMTH S L

A WYQ Q KP G KAPN LLIYNAYTLQSGVPSRFSGSGSGTEFTLTI SS LQPEDFATYYCQQI NSYPRTFGQGTKVEI K SEQ ID NO: 142: QVQ LQESG PG LVKPSETLSLTCTVSGGSI SSHY WSWI RQPPAKG LEWI GYIYHSGMTNYN PSLKSRVTI SI DTSKNQ FSLKLSSVTAADTAVYYCARGDGYN FFWGQGTLVTVSS

SEQ ID NO: 143: EIVLTQSPGTLSLSPG ERATLSCRASQSVSSTYL NWYQQKPGQAPRLLIYGASTRATGI PDRFSGSGSGADFTLTI SR LEPEDFAVYYCQQYDDSRWTFGQGTKVEI K

SEQ ID NO: 144: EVQLVESGGG LVKPG ESLRLSCATSGVN F N IAW

MTWVRQAPG KGLEWVG RI KSKI GGGTTDYAAPVKG RFTM SI D D SKNTLYLQM NSLKTEDTAVYYCTTVRN MADLSLN HWGQGTLVT VSS

SEQ ID NO: 145: D I Q M TQ S PSS LSAS VG D S VT I TC R AS Q S I S S Y L N

WYHQKPG KAPKLLI YGASTLQSGVPSRFGGSGSGTDFTLTI SSL QPDDFATYYCQQSHSSPLTFGGGTKVEI K

SEQ ID NO: 146: EVQ LVESGGG LVQ PG GS LR LSCAASG FT FSTY

WM HWVRQTP EKG LVWVS RI H PDGS NTAYADSVKG R FTI SR DN AKNTLYLQM N SLRVEDTAFYYCTRGGSGATI NYWGQG I LVTVS S

SEQ ID NO: 147: D I VM TQ S P DS LA VS LG E R AT I N C KSS LS VLSSS N

N EN YLAWYQQ KPGQP PKLLI YWASTRGSGVPG R FSGSGSGTD FTLTI SSLQAEDVAVYYCHQYYTTPFAFGPGTKVDI K

SEQ ID NO: 148: EVQLVESGGGLVKPG ESLRLSCATSGLTFSNVW

MSWVRQAPG KG LEWVG R LKN KPDGGTTDYAAPVKG RFTI SRD DSKTTLYLEM N SLKVEDTAVYYCTTD NGVKAFD IWGQGTMVTV SS

SEQ ID NO: 149: EIVLTQSPGTLSLSPG ERATLSCRASQN I GTALA WYQ Q KPGQAPRLI IYETSN RATDVPARFSGSGSGTDFTLTI SSL EREDFALYYCQQRADWPLTFGGGTKVEI K SEQ ID NO: 150: QVQLVESGGGVVQPGGSLR LSCAASG LTFS N Q D F H WV R Q A P G KG L E WVA F I RYD GG F KN YADS VKG R FT I S R D N S QKM LYLQM DSLRVEDTAVYYCAKCGAEDSTTVWLNWFDPWGQ GTLVTVSS

SEQ ID NO: 151: EIVMTQSPATLSVSPG ERATLSCRASQSVG N N L AWFQQKPGQAPRLLIYGASTRATG I PARFSGSGSGTEFTLTI SS LQSEDFAVYHCQHYH NWPPTFGQGTKVEI K

SEQ ID NO: 152: QVQLQ ESG PG LVKPSETLS LTCTVSGGS ITNYY WGWI RQ PPG EG LEWI GYIYYSGSTNYN PSLKSRVTI SVDTS KN QFSLKLTSVTAADTAVYYCAG RAYSSGYYYLI DYWGQGTLVTV SS SEQ ID NO: 153: E I VLTQS P G I LS LS PG E R AT LS C R ASQ S VSS RS L SWYQQR PG LAPR LLIYAASSRAAVTPDR FTASGSGTDFTLTI SS LEPEDFAVYYCQHYGTSPPRYTFGQGTKVEI K

SEQ ID NO: 154: EVQLVESGGALVQPGGSLRLSCAASG FI FSN SW MGWFRQAPGKRPEFVAN I KPDGSEKFHVDSVKG RFTI SRDNAE NSLYLLM NSLRAEDTAVYYCARDSTSPARFGYWGQGTLVTVSS SEQ ID NO: 155: E I V LTQS PGT LS LS PG E R AT LSC R ASQS VYS I Y F

AWYQQKPGQAP RPLIYGVSN RATGI PDRFSGSGSGTDFTLTI SR LEPEDFAVYYCQQYGSLPRTFGQGTKVEI K

SEQ ID NO: 156: QVQLQESG PG LVKPSETLSLTCSVSADSFSPYK WSWI RQPPG KG LEWIGYIYSSG NTNYN PPLKSRVTI SLDTSKNQ VSLRLSSVAAADTAMYYCAREWSG FDFWGQGTMVTVSS

SEQ ID NO: 157: EIVLTQSPGTLSFSPG DRATLSC RASQSVSSSA

LAWYQQKPGQAPSLLIYGASSRATG I PD RFSGSGSGTDFTLTI S RLEPEDFAVYYCQQYGNSPRTFGQGTKVEI K

SEQ ID NO: 158: EVQLVESGGG LVQPGGS LR LSCAASG FTFS NY WM RWVRQSPG KG LVWVSH I N P DGSFTNYADSVKG R FTI SRD N TKNTLYLQM NSLRAEDTAVYYCVN FQLGWGQGTLVTVSS

SEQ ID NO: 159: EIVLTQS PGTLSLS PG E RATLSC RASQSVTYLA WYQQ KPGQAPR LLFYGTSSRATG I P DR FSGSGSGTD FTLTI SR VEPEDFAVYYCQQFGSSPPDTFGGGTKVEI K SEQ ID NO: 160: EVQ LLESGGG LVQ PG GS LRVSCAASG FT FSN S

GMSWVRQAPG KG LEWVSG I GGGGGSAYYADSVKG RFTI SRDN SKNTLYLQM N N LRAEDTAVYYCAKGVTSFDYWGQGI LVTVSS

SEQ ID NO: 161: E IVMTQS PATLSVS PG ETATLSC RASQSVN S F L AWYQQKPGQAPRLLIYAASTRATGVPARFSGSGSGTEFTLTISS LQSEDFAVYYCHQYKNWPPMGTFG PGTKVDI K

SEQ ID NO: 162: EVQLVESGGG LVQPGGSLR LSCAASG FAI SGNY

MSWVRQAPG KG LEWVSLI YWTDDTVYADSVKG RFTISRDVSKN MVH LQMSSLRVEDTAVYYCARELGVFHSGG DQWLG PLDCWGQ GTLVTVSS

SEQ ID NO: 163: E IVLTQSPATLS LS PG ERATLSC RASQSVSSS L

A WYQ Q KPGQAPRLLIYDASKRATD I PARFSGSGSGTDFTLTI SS LEPEDFAVYYCQH RGEWPPGATFG PGTKVDI K

SEQ ID NO: 164: QVQ LVESGGGVVQ PG RS LR LSCAASG LTFS NY GM HWVRQAPG KG LEWVAVVSARGGTTYYADSVKG R FTI SR DN SKNTM SLQM N G LR PD DTAVYFCTKEGAP PG KYAFD IWGQGTM V T V S S

SEQ ID NO: 165: D I QMTQS PSTLSASVG DRVTITC RASQS I SGWL

AWYQQKAGKAPKLLI YKASSLESGVPSRFSGSGSGTEFTLTISS LQPDDFATYYCQQYYSWGTFGQGTKVEI K SEQ ID NO: 166: EVQLVESGGG LVQPGGSLRLSCAASG FTVSSI F

MSWVRQAPGQG LEWVSVI Y T D G K T Y Y A H S V E G R F T I S R D D S K N MVYLQLSSLRTEDTAVYYCARDI PTTFG IG EAFDIWGQGTMVTV SS

SEQ ID NO: 167: D I V M TQS P D S LA VS LG E R AT I N C KS S QS I LS R S N

N KNYLAWYQQKPGQPPKLLLYWASTR ESGVP DR FSVSGSGSD FTLTI SSLQAEDVAVYYCQQYYNAPLTFGGGTKVEI K SEQ ID NO: 168: QVQ LVESGGGVVQPG RS LR LSCAASG FTFSN H

G M HWLRQTPG KG LEWVAVI SYDGSTKYYA DSVKG RCTLSR DN SKETVFLQM NSLRPEDTAVYYCAKGCSNGG NCFLI DYWGPGTL VTVSS

Claims

1. A method for detecting the presence or absence of a Streptococcus pneumoniae 9L capsular polysaccharide in a sample, said method comprising:

a) contacting a sample with:

wherein said first mAb binds a 9N CP; and

vi. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 12, or a functional variant thereof; and

wherein said second mAb binds a 9V CP; and b) detecting the presence or absence of said first mAb-antigen complex and detecting the presence or absence of said second mAb-antigen complex; c) wherein the presence of the first mAb-antigen complex, and the presence of the second mAb-antigen complex confirms the presence of a 9L CP;

d) wherein the absence of the first mAb-antigen complex, and/or the absence of the second mAb-antigen complex is indicative of the absence of a 9L CP.

2. A method for detecting the presence or absence of a Streptococcus pneumoniae 11 D capsular polysaccharide in a sample, said method comprising:

a) contacting a sample with:

wherein said third mAb binds an 11A CP; and

iv. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 22, or a functional variant thereof; V. a heavy chain CDR2 comprising an amino acid sequence of SEQ

ID NO: 23, or a functional variant thereof; and

wherein said fourth mAb binds a 15B CP; and

3. A method for detecting the presence or absence of a Streptococcus pneumoniae 18A capsular polysaccharide in a sample, said method comprising:

a) contacting a sample with:

wherein said first mAb binds a 9N CP; and

i. a light chain CDR13 comprising an amino acid sequence of SEQ ID NO: 13, or a functional variant thereof; ii. a light chain CDR2 comprising an amino acid sequence of SEQ ID

NO: 14, or a functional variant thereof;

wherein said third mAb binds an 11A CP;

wherein said fourth mAb binds a 15B CP; and

b) detecting the presence of or absence of said first mAb-antigen complex, detecting the presence of or absence of said third mAb-antigen and detecting the presence of or absence of said fourth mAb-antigen complex;

4. The method according to claim 1 or claim 3, wherein said first mAb comprises: a) a light chain variable region comprising an amino acid sequence of SEQ ID: NO

25, or functional equivalent thereof; and

26, or functional equivalent thereof.

5. The method according to claim 1 , wherein said second mAb comprises:

27, or functional equivalent thereof; and

28, or functional equivalent thereof.

6. The method according to claim 2 or claim 3, wherein said third mAb comprises:

29, or functional equivalent thereof; and

30, or functional equivalent thereof.

7. The method according to claim 2 or claim 3, wherein said fourth mAb comprises: a) a light chain variable region comprising an amino acid sequence of SEQ ID: NO

31 , or functional equivalent thereof; and

32, or functional equivalent thereof.

8. The method according to claim 1 , further comprising contacting the sample with:

v. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO: 17, or a functional variant thereof; and vi. a heavy chain CDR3 comprising an amino acid sequence of SEQ

ID NO: 18, or a functional variant thereof;

wherein said third mAb binds an 11A CP; and/or

II. a fourth mAb that binds a S. pneumoniae CP to form a fourth mAb-antigen complex, wherein said fourth mAb comprises the following six CDRs: i. a light chain CDR1 comprising an amino acid sequence of SEC ID NO: 19, or a functional variant thereof;

ii. a light chain CDR2 comprising an amino acid sequence of SEC ID NO: 20, or a functional variant thereof;

wherein said fourth mAb binds a 15B CP.

9. The method according to claim 2, further comprising contacting the sample with:

wherein said first mAb binds a 9N CP; and/or II. a second mAb that binds a S. pneumoniae capsular polysaccharide (CP) to form a second mAb-antigen complex, wherein said second mAb comprises the following six CDRs:

vi. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO: 12, or a functional variant thereof.

wherein said second mAb binds a 9V CP.

10. The method according to claim 3, further comprising contacting the sample with:

wherein said first mAb binds a 9N CP.

11. The method according to any one of the preceding claims, further comprising contacting the sample with one or more selected from a fifth to thirteenth mAb, to form a fifth to thirteenth mAb-antigen complex, wherein the fifth to thirteenth mAb binds CP 1 , 2, 3, 4, 5, 6A, 6B, 8, or 14, respectively;

wherein the presence of mAb-antigen complex is indicative of the presence of said CP, and wherein the absence of mAb-antigen complex is indicative of the absence of said CP.

12. The method according to any one of the preceding claims, further comprising contacting the sample with one or more selected from a fourteenth to twenty-first mAb, to form a fourteenth to twenty-first mAb-antigen complex, wherein the fourteenth to twenty- first mAb binds CP 17F, 18C, 19A, 19F, 20, 22F, 23F, or 33F, respectively;

13. The method according to any one of the preceding claims, further comprising contacting the sample with one or more of a twenty-second to twenty-fourth mAb, to form a twenty-second to twenty-fourth mAb-antigen complex, wherein the twenty-second to twenty-fourth mAb binds CP 7F, 10A, or 12F, respectively;

14. The method according to any one of the preceding claims, further comprising contacting the sample with a twenty-fifth mAb which binds Streptococcus pneumoniae CWP.

15. The method according to any one of the preceding claims, wherein said contacting step comprises applying the sample to an immunoassay comprising said mAb.

16. The method of any one of the preceding claims, wherein said contacting step is performed in a discrete compartment comprised within an immunoassay apparatus.

17. The method of any one of the preceding claims, wherein said sample is an isolated sample obtained from a subject.

18. The method of any one of the preceding claims, wherein said capsular polysaccharide is detected at a concentration of greater than or equal to about 0.001 ng/ml, 0.003 ng/ml, 0.01 ng/ml, 0.1 ng/ml, 0.3 ng/ml, or 1 ng/ml.

19. The method of any one of the preceding claims, wherein said capsular polysaccharide and/or said CWP is an integral component of a membrane of a Streptococcus pneumoniae bacterium.

20. The method according to any one of the preceding claims, wherein said method is a multiplex method wherein said contacting step with each mAb is performed simultaneously and preferably under the same conditions.

21. The method according to any one of the preceding claims, wherein said sample is a urine sample.

22. A kit for detecting the presence or absence of a Streptococcus pneumoniae 9L capsular polysaccharide in a sample, wherein said kit comprises:

IV. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO:

4, or a functional variant thereof;

V. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO:

5, or a functional variant thereof; and

VI. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO:

6, or a functional variant thereof;

wherein said first mAb binds a 9N CP; and

b) a second mAb that binds a S. pneumoniae capsular polysaccharide (CP) to form a second mAb-antigen complex, wherein said second mAb comprises the following six CDRs: I. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 7, or a functional variant thereof;

IV. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO:

10, or a functional variant thereof;

V. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO:

11 , or a functional variant thereof; and

VI. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO:

12, or a functional variant thereof;

wherein said second mAb binds a 9V CP; and

iii. wherein said instructions indicate that the presence of the first mAb- antigen complex, and the presence of the second mAb-antigen complex confirms the presence of a 9L CP; and

iv. wherein said instructions indicate that the absence of the first mAb-antigen complex, and/or the absence of the second mAb-antigen complex is indicative of the absence of a 9L CP.

23. A kit for detecting the presence or absence of a Streptococcus pneumoniae an 11 D capsular polysaccharide in a sample, wherein said kit comprises:

I. a light chain CDR13 comprising an amino acid sequence of SEQ ID NO:

13, or a functional variant thereof;

II. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO:

14, or a functional variant thereof;

III. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO:

15, or a functional variant thereof;

IV. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO:

16, or a functional variant thereof; V. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO:

17, or a functional variant thereof; and

VI. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO:

18, or a functional variant thereof;

wherein said third mAb binds an 11A CP; and

I. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO:

19, or a functional variant thereof;

II. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO:

20, or a functional variant thereof;

III. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO:

21 , or a functional variant thereof;

IV. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO:

22, or a functional variant thereof;

V. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO:

23, or a functional variant thereof; and

VI. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO:

24, or a functional variant thereof;

wherein said fourth mAb binds a 15B CP; and

iii. wherein said instructions indicate that the presence of the third mAb- antigen complex, and the presence of the fourth mAb-antigen complex confirms the presence of an 11 D CP;

iv. wherein said instructions indicate that the absence of the third mAb- antigen complex, and/or the absence of the fourth mAb-antigen complex is indicative of the absence of an 11 D CP.

24. A kit for detecting the presence or absence of a Streptococcus pneumoniae serotype- specific capsular polysaccharide in a sample, wherein said apparatus comprises:

I. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 1 , or a functional variant thereof; II. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 2, or a functional variant thereof;

IV. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO:

4, or a functional variant thereof;

V. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO:

5, or a functional variant thereof; and

VI. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO:

6, or a functional variant thereof;

wherein said first mAb binds a 9N CP;

I. a light chain CDR13 comprising an amino acid sequence of SEQ ID NO:

13, or a functional variant thereof;

II. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO:

14, or a functional variant thereof;

III. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO:

15, or a functional variant thereof;

IV. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO:

16, or a functional variant thereof;

V. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO:

17, or a functional variant thereof; and

VI. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO:

18, or a functional variant thereof;

wherein said third mAb binds an 11A CP; and

I. a light chain CDR1 comprising an amino acid sequence of SEQ ID NO:

19, or a functional variant thereof;

II. a light chain CDR2 comprising an amino acid sequence of SEQ ID NO:

20, or a functional variant thereof;

III. a light chain CDR3 comprising an amino acid sequence of SEQ ID NO:

21 , or a functional variant thereof;

IV. a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO:

22, or a functional variant thereof; V. a heavy chain CDR2 comprising an amino acid sequence of SEQ ID NO:

23, or a functional variant thereof; and

VI. a heavy chain CDR3 comprising an amino acid sequence of SEQ ID NO:

24, or a functional variant thereof;

wherein said fourth mAb binds a 15B CP; and

iii. wherein said instructions indicate that the presence of two or more selected from the first mAb-antigen complex, the third mAb-antigen complex and the fourth mAb-antigen complex confirms the presence of 18A CP; and

iv. wherein said instructions indicate that the absence of two or more selected from the first mAb-antigen complex, the third mAb-antigen complex and the fourth mAb-antigen complex confirms the absence of 18A CP.

25. The kit according to any one of claims 22-24, further comprising a twenty-fifth mAb which binds a Streptococcus pneumoniae CWP.

26. The kit according to any one of claims 22-25, wherein said mAb is present within a discrete compartment of an immunoassay apparatus.

27. The kit according to any one of claims 22-26, wherein said kit is used in a method according to any one of claims 1-21.

28. The method or kit according to any one of the preceding claims, wherein said mAb is a human mAb.

29. The method or kit according to any one of the preceding claims, wherein said mAb is immobilised (e.g. adsorbed) on a surface.

30. The method or kit according to any one of the preceding claims, wherein said mAb is immobilised on a bead; preferably wherein said bead is a carboxylated polystyrene microsphere.

31. The method or kit according to any one of claims 1-30, wherein said mAb is immobilised on the surface of a discrete compartment.

32. The method or kit according to any one of the preceding claims, wherein the presence or absence of mAb-antigen complex is detected by means of a secondary antibody which binds the CP and/or mAb-antigen complex, preferably wherein said secondary antibody is conjugated to a detection means (e.g. a fluorescent dye).