Title:  Process and materials for the rapid detection of streptococcus pneumoniae employing purified antigen-specific antibodies

United States Patent:  6,824,997

Issued:  November 30, 2004

Inventors:  Moore; Norman James (North Berwick, ME); Fent; Mary Kathleen (Cumberland Center, ME); Koulchin; Vladimir Andrei (Portland, ME); Molokova; Elena Valentin (Portland, ME)

Assignee:  BINAX, Inc. (Portland, ME)

Appl. No.:  397110

Filed:  September 16, 1999

Abstract

A process is disclosed for obtaining a C-polysaccharide cell wall antigen containing not more than about 10% protein from Streptococcus pneumoniae bacteria. The antigen thus obtained is conjugated to a spacer molecule, and the free end of the latter is then conjugated to a chromatographic affinity column. The column is then utilized to purify raw antibodies to S. pneumonia bacteria, thereby producing antigen-specific antibodies. A portion of such antibodies is conjugated to a labeling agent which displays a visible color change upon reaction of the antibodies with their antigenic binding partner and embedded in a first zone of an immunochromatographic assay device. Another portion of such antibodies is bound to the reaction zone of the device which has a view window. When a liquid sample, such as patient urine, cerebrospinal fluid or blood is applied to the first zone, the conjugate of antibodies and labeling agent and the sample move along a flow strip of bibulous material to the reaction zone wherein, if the sample contains S. pneumoniae or its cell wall antigen, a sandwich is formed among the labeled conjugate, the antigen and the bound antibodies and a color change is observed. The immunochromatographic assay thus performed is completed within about 15 minutes. This assay affords a basis for rapid and reliable diagnosis of various pathogenic states caused by S. pneumoniae including pneumonia, bronchitis, otitis media, sinusitis, meningitis, and secondary disease states that commonly occur when primary pneumonic infection caused by this bacterium persists undiminished over a time period of 3-5 days.

Description of the Invention

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to a specific and sensitive immunochromatographic ("ICT") assay, performable within about 15 minutes, for the detection of Streptococcus pneumoniae in a bodily fluid, such as urine or cerebrospinal fluid, of a patient showing clinical signs of an infection caused by S. pneumoniae.

BACKGROUND OF THE INVENTION

Streptococcus pneumoniae ("S. pneumoniae") is a leading causative organism of pneumonia-type illnesses and other lower respiratory tract infections such as bronchitis, as well as of upper respiratory tract infections, including infectious otitis media and sinusitis and of disseminated invasive infections, including bacteremia and meningitis. When not properly diagnosed and treated, S. pneumoniae pneumonic infection may lead to any of pericarditis, empyema, purpura fulmiran, endocarditis or at least one type of arthritis, where S. pneumoniae is the causative organism in each instance. Such pneumonic infection is also often a precursor of bacteremia or meningitis. To now, it nevertheless is common for pneumonia arising from S. pneumoniae to be diagnosed and treated somewhat empirically.

To a significant extent, this is because the tests presently available for the detection of S. pneumoniae are either (1) time consuming, labor intensive and in need of instrumental assistance for reading results, or (2) lacking in sensitivity and/or specificity. Because of problems associated with lack of sensitivity and/or specificity, e.g., physicians tend toward conservatively prescribing expensive, broad spectrum antibiotics for patients with pneumonia-type respiratory infections in lieu of prescribing a less expensive antibiotic specific to S. pneumoniae where it would adequately cure the infection. This and other liberal prescribing of broad spectrum antibiotics is, of course, a major cause of today's well-publicized medical crisis consequent from the increasing resistance of many types of infectious bacteria to previously highly efficacious antibiotics. This crisis and the potential untoward consequences for at least some patients of empirical diagnosis and treatment are among many reasons why a reliable and rapid assay for detecting S. pneumoniae in human body fluids is needed.

Pneumonia caused by S. pneumoniae is a serious disease, estimated to occur at the rate of one to five cases per 1,000 persons per year in the United States alone. Depending upon the age and state of health (based on unrelated factors) of patients infected with S. pneumoniae-caused pneumonia, the disease has a mortality rate of between 4 percent and 30 percent of infected patients.

The most time-honored methods of attempting to diagnose S. pneumoniae-caused diseases, and especially pneumonia, involve the Gram stain and culture of expectorated sputum of patients suspected of harboring the disease, followed by biochemical identification methods. This procedure requires in the order of one to four days from start to finish. It has proved to be an unsatisfactory diagnostic tool because (1) other bacteria present in the patient's saliva often overgrow the sputum culture, and (2) S. pneumoniae frequently is present in the human upper respiratory tract even when no sign of disease attributable to this bacterium is present in the individual. For example, it is estimated that some 30 percent of U.S. children are habitual carriers of S. pneumoniae. Adults, too may become colonized by S. pneumoniae without themselves entering a disease state. The carriage rates of the organism by both children and adults increase with crowding conditions and during winter months.

Co-agglutination, latex particle agglutination and counter-immunoelectrophoresis methods for detecting the polysaccharide capsular antigens of S. pneumoniae in sputum specimens have been developed and are rapid, but they have not been shown to exhibit reliable sensitivity or specificity, probably because there are some 83 serotypes of S. pneunoniae, each of which may vary in immunogenicity and in other respects. The commercial polyvalent anti-serum developed and used for these tests contains antibodies to all 83 of the S. pneumoniae serotype antigens, but it nevertheless may fail to detect the less immunogenic antigen serotypes. This polyvalent antiserum also has shown cross-reactivity with other streptococci and some other infectious bacteria, e.g., Haemophilus influenzae. Hence both false-negative and false-positive reactions may occur randomly when these tests are used on sputum samples.

Several enzyme-inmmunoassays ("EIA") have been developed which are based on detection of the pneumococcal C-polysaccharide antigen that has been found to be present in the pneumococcal cell wall of all of the S. pneumoniae serotypes. See, e.g., Parkinson, A. J., Rabiego, M. E., Sepulveda, C., Davidson, M. and Johnson, C., 30 J. Clin. Microbiol. 318-322 (1992). This C-polysaccharide antigen is a phosphocholine-containing polysaccharide derived from teichoic acid. These EIA assays are of acceptable specificity and sensitivity even though most often performed on sputum samples. Each such assay, however, requires two to three hours performance time after sample collection as well as the use of instrumentation normally available primarily in clinical laboratories. In addition, these assays need to be run by, or under close supervision of, trained personnel.

Reliance upon sputum samples to diagnose S. pneumoniae infections is frequently less than satisfactory in achieving a diagnosis of S. pneumoniae-caused pneumonia, and not just because of the potential for contamination of the sample by other bacteria in the mouth and/or by indigenous upper respiratory tract S. pneumoniae. Sputum is often difficult to collect; moreover, once medication of the patient is commenced, the number of viable S. pneumoniae in sputum rapidly decreases. In particular, the presence of the C-polysaccharide antigen in sputum may rapidly become difficult to detect if an antibiotic therapy is used that attacks the cell wall of the S. pneumoniae microorganism. When S. pneumoniae causes infectious otitis media, meningitis and various other aforementioned infectious disease states, sputum samples are of no aid in diagnosis.

Collection of blood cultures from patients suspected of S. pneumoniae infection eliminates the contamination problems that attend sputum samples. Where blood serum samples are found to contain S. pneumoniae, diagnosis of various diseases of which it is causative may readily be made. The drawback here is that only about 20 percent of all pneumonia patients infected by S. pneumoniae become bacteremic; therefore, relying solely on blood samples to diagnose S. pneumoniae-caused pneumonia may yield false-negative results.

Urine samples have been found to be the most reliable and convenient ones to use in detecting S. pneumoniae-caused pneumonia because they can be non-invasively obtained; they will not be contaminated with oral microflora; and the presence of the bacterium in urine persists, albeit at a constantly decreasing level of concentration, even after patient therapy has been initiated, so that daily monitoring of patient urine samples to assess the efficacy of a prescribed therapy may yield useful information. It should be noted that human carriers of S. pneumoniae who show no disease symptoms often do not have sufficient pathogen present to have S. pneumoniae antigens present in their urine.

A very recent article describes the successful diagnosis of meningitis caused by S. pneumoniae using an EIA method to test samples of cerebrospinal fluid. In the EIA, a monoclonal immunoglobulin A antiphosphoryl-choline antibody was employed to detect the C-polysaccharide antigen. See Stuertz, K., Merx, I., Eiffert, H., Schmutzhard, E., Mader, M. and Nau, R., 36 J. Clin. Microbiol. 2346-2348. The results obtained compared favorably with those reported by Yolken, R. H., Davis, D., Winkelstein, J., Russell, H. and Sippel, J. E., 20 J. Clin. Microbiol. 802-805 (1984) obtained in an EIA in which two antibodies for S. pneumoniae in cerebrospinal fluid were used--a horse antibody to the pneumococcal C-polysaccharide antigen, bound to microtiter plates, and a pooled rabbit antiserum to the polysaccharide capsular antigen in the liquid phase.

BRIEF DESCRIPTION OF THE INVENTION

According to the present invention, antibodies to the C-polysaccharide antigen of S. pneumoniae raised in rabbits are affinity purified with isolated and purified C-polysaccharide antigen having less than about 10% protein content.

These affinity purified antibodies are conjugated to an agent which produces a color reaction upon the formation of a sandwich with S. pneumoniae C-polysaccharide antigen from a test sample and additional affinity purified C-polysaccharide antibody immobilized upon a nitrocellulose matrix.

The test is conducted in a disposable immnunochromatographic test device and requires no instrumentation to interpret the result. It can easily and successfully be performed by persons who have no training in laboratory techniques.

The preferred test sample for diagnosis of S. pneumoniae-caused pneumonia is patient urine, but the test also works with other bodily fluid samples that contain S. pneumoniae, including serum and sputum. Diagnosis of S. pneunoniae-caused meningitis may be readily made using patient cerebrospinal fluid as the test sample.

This invention for the first time offers the benefit of a test for S. pneumoniae that is performable within a 15-minute time span and is of at least equal specificity and sensitivity to EIA tests requiring eight to twelve times as long and much more work, to obtain a result. The test is easy to perform, requires no special training, equipment, or instrumentation and it enables a rapid diagnosis of pneumonia caused by S. pneumoniae. It can be readily performed in a doctor's office, thus permitting the patient to be immediately placed on a S. pneumoniae-specific therapeutic regimen. It can, of course, be performed in a clinical laboratory, but it can also easily be performed in a geriatric center, in a patient's home or in any environment where S. pneumoniae-caused pneumonia or other pathogenic condition is suspected to be epidemic.

The test of this invention is important to administer when disease states such as otitis media, bronchitis or sinusitis appear because once it can be established that any of these is due to S. pneumoniae rather than another infectious agent, appropriate therapy can promptly be initiated. Small children are especially prone to otitis media because of the shorter length and smaller diameter of their Eustachian tubes, so that early detection of S. pneumoniae if present may well forestall the onset of a more serious, or even life-threatening, disease state. Papers by Norris et al, J. Pediatrics, 821-827 (1966) and Hongeng et al, 130 J. Pediatrics, No. 5 (May 1997) indicate that children with sickle cell disease are highly susceptible to S. pneumoniae infection, with S. pneumoniae sepsis being the most common invasive infection among this populace and those once so infected having a much heightened risk of recurrence and subsequent death. Clearly, employing the ICT test of this invention to test the urine of these patients on a regular basis may be helpful in diminishing the need for the unremitting penicillin prophylaxis that the second of these papers recommends.

The ease of performance of the test and its ability to detect the C-polysaccharide antigen of S. pneumoniae in urine suggests that this test should prudently be performed on patients without overt clinical signs of related infection who report feeling substantially under par. Any such patient in whom it is established that S. pneumoniae is present in significant enough quantities to give a positive-urine ICT test is a predictable candidate for developing a more severe infection--and the ability to forestall the disease development before it becomes severe by administering appropriate therapy is newly presented by this invention.

DETAILED DESCRIPTION OF THE INVENTION

Broadly speaking, the ICT assay for S. pneumoniae as herein described may be designed and configured to be run on any known disposable ICT device disclosed in the art. Preferably it is designed to be conducted, and is conducted, using an ICT device of the type disclosed in copending U.S. patent application Ser. No. 07/706,639 of Howard Chandler, or one of its continuation-in-part applications, all of which are assigned to Smith-Kline Diagnostics, Inc. but are exclusively licensed to Binax, Inc. (which is entitled to assignment of this application), in a wide area of use fields that includes diagnoses of human respiratory system diseases.

The preferred device is suitably impregnated in one region thereof with antigen-specific polyvalent antibodies to the C-polysaccharide antigen of S. pneumoniae. Labeled antigen-specific antibodies are applied to another area of the device. The test sample suspected of containing S. pneumoniae is contacted first with the labeled antigen-specific antibodies, which then flow with the sample to the device area containing unlabeled bound antigen-specific antibodies, whereupon if S. pneumoniae is present in the sample, the labeled antibody: C-polysaccharide antigen conjugate already formed by contact binds to the immobilized unlabeled affinity purified antibodies, whereupon a visible color reaction is produced. The label may be any substance known in the art to produce visible color upon the reaction of a labeled antibody:antigen complex with bound unlabeled antibodies. Such labels include various finely divided metallics, various organic molecules, and various molecular combinations such as enzyme combinations with another color-producing molecule. In this invention, colloidal gold particles constitute the preferred label.

It is of major importance in designing the test device, that the concentration of antibody present at each of the two sites of the test device where reaction occurs be sufficient to insure that antigen present in the test sample will be captured by the labelled antibodies as the test sample contacts them and that labelled antibody: antigen conjugate will be readily captured and held by the bound antibodies at the sample capture line. Experimental work undertaken in connection with this invention has shown that active antibody to the C-polysaccharide antigen of S. pneumoniae must be present at each site of a test device at which antigen: antibody reaction is to occur in a concentration of between 7.7 nanograms/sq. mm. of surface area and 385 nanograms/sq. mm. of surface area. If antibody concentrations lower than 7.7 nanograms/sq. mm. are present at a site where reaction is intended to occur, false negative results are likely.

Various methods of affinity purification of antibodies to the C-polysaccharide antigen of S. pneumoniae, are known. The one hereinafter described is preferred in the present invention, but others may be substituted. It is noted, however, that the affinity-purified antibodies of this invention are to be sharply distinguished from the "affinity-purified antibody preparation" which is described by Sjogren and Holme, 102 J. Immunol. Methods 93-100 (1987). These authors describe obtaining a hot phenol-purified C-polysaccharide antigen of S. pneumoniae containing 17% protein and absorbing it on an ion exchange gel, DE AE--Sepharose CL6B. After 48 hours incubation this preparation was packed into columns at approximately neutral pH of 7.2. The binding efficiency of the antigen to the gel is said to be about 60%. Antibodies were passed over these columns and incubated for 30 minutes, followed by elution of the columns with 0.5 M Na Cl in PBS. It is known that leakage of antigen from ion-exchange columns is a frequent occurrence. In this system, it is reasonable to hypothesize that the product eluted from the gel was an in situ--formed immune complex of antibodies and antigen rather than a preparation of purified antigen to this invention. It should particularly be noted that, in this invention, the purified antigen containing less than 10% protein is covalently coupled to a spacer molecule such as BSA--hydragine conjugate, and the resulting labile antigen: conjugate ligand then covalently coupled to a chromatographic gel--e.g. the Formyl Sperilose of Example 4, which is then applied to a column. The antibodies are added and eluted with strongly acidic buffer from the immobilized antigen on the column.

The antibody herein preferred is raised by conventionally injecting a rabbit with S. pneumoniae strain R6, a non-encapsulated S. pneumoniae strain available from the American Type Culture Collection under ATCC No. 39938 which is subjected to heat-killing of the cells before injection into the animal. After an appropriate time period, the animal is bled to obtain serum containing the desired antibodies, followed by purification thereof. Other antibodies to the S. pneumoniae C-polysaccharide antigen may be substituted for those specifically described herein without departing from this invention.

The antibody should initially be tested for cross reactivity to other common infectious bacteria. The preferred antibody referred to herein was tested, using the ELISA method, for cross-reactivity with each of the following: Citrobacter freundii, Staphylococcus aureus, Enterobacter cloacae, Enterobacter faecalis, Streptococcus, group B, Type III, E. coli, Neisseria meningitidis, Salmonella cubana, Salmonella paratyphi A, Klebsielia pneumoniae, Streptococcus, Group B, type II, Staphylococcus epidermidis, Salmonella enteritidis, Streptococcus, Group A, Serratia marcescens, Candida albicans, Haemophilus influenzae, Moraxella catarrhalis, Corynebacterium kutscheri, Pseudomonas putida, Proteus vulgaris, Enterococcus avium, Acinetobacter baumannii, Klebsiella oxytoca, Acinetobacter lwoffli, Pseudomonas aeroginosa, Staphylococcus saphrophyticus, Enterococcus durans, Corynebacterium bovis, Proteus mirabilis, Pseudomonas stutzeri, Pseudomonas cepacia, Salmonella typhi, Streptococcus, Group F, Streptococcus, Group B, type 1a, Candida stellatoides, Streptococcus parasanguis, Streptococcus, Group G, Streptococcus, Group C, Streptococcus mutans, Morganella morganii, Staphylococcus haemolyticus, Haemophilus influenzae type B, Stenotrophomonas maltophilia, Haemophilus influenzae type D, Gardnerella vaginalis, Streptococcus mitis, Haemophilus parainfluenzae, Streptococcus sanguis, and H. influenzae nontypeable.

The only significant cross reactivity found was with Streptococcus mitis and Staphylococcus aureus. The first, S. mitis, is a causative agent for endocarditis, the overt patient symptoms of which physicians can readily distinguish clinically from those of an S. pneumoniae lung infection. S. mitis contains the same C-polysaccharide antigen as S. pneumoniae and the two share the ability to cause endocarditis, albeit S. pneumoniae normally does so in patients whose primary pneumonia has not been appropriately treated and who may then develop bacteremia and/or endocarditis or another pathogenic secondary infection. S. mitis, by contrast, is not a causative agent for pneumonia; endocarditis attributable to S. mitis normally develops independently of any other infection. It is accordingly believed that suspected cases of primary endocarditis caused by S. mitis can be confirmed, when needed, using the assay of this invention. It should be noted, however, that S. mitis is less likely to be present in urine than S. pneumoniae and hence, an assay of blood serum may be more likely to yield confirmatory information in that instance.

Some strains of S. aureus are known to secrete Protein A, a non-specific protein which indiscriminately binds IgG, and hence, all antibodies. The suspected presence of these S. aureus entities may be readily confirmed or ruled out by running other simple tests well known in the art. (As shown in the example of this invention , S. aureus strains in which protein A is not present show no cross reactivity to the antibody of this invention.) A minor cross-reaction with Haemophilus influenzae was observed, but is not believed to be significant enough to cause a problem in the detection of S. pneumoniae in urine samples.

Claim 1 of 18 Claims

What is claimed is:

1. A method of detecting the presence of the cell wall C-polysaccharide antigen of Streptococcus pneumoniae, in a liquid sample, which method comprises the following steps:

a) culturing Streptococcus pneunoniae bacteria, to obtain a desired size of culture and harvesting therefrom cells therof as a wet cell pellet;

b) separating from the wet cell pellet the cell wall C-polysaccharide antigen containing not more than 10% protein by performing a series of steps which comprises;

(i) suspending the wet cell pellet in an alkaline solution and nixing;

(ii) adjusting the pH to an acid pH with a strong acid;

(iii) separating the mixture from step (ii) into two layers;

(iv) removing the upper layer and adjusting its pH to approximate neutrality;

(v) adding to the product from step (iv) a broad spectrum protease enzyme and digesting to destroy residual proteins;

(vi) adjusting the pH of the product from step (v) to alkaline pH with a weakly alkaline aqueous solution: and (vii) separating out the cell wall C-polysaccharide antigen containing not more than 10% proteing;

c) coupling to a chromatographic column through a spacer molecule the cell wall C-polysaccharide antigen containing not more than 10% protein obtained in step (b);

d) passing polyvalent antibodies to Streptococcus pneumoniae over the chromatographic affinity column of step (c) to produce purified antigen-specific antibodies; and

e) conducting an immunoassay upon a liquid sample suspected of containing Streptococcus pneumoniae and/or its C-polysaccharide cell wall antigen which immunoassay comprises the steps of

(i) contacting the liquid sample with conjugates of purified antigen specific antibodies from step (d) hereof and a labelling agent capable of manifesting a color or a detectable signal upon completion of the immunoassay, whereupon C-polysaccharide cell wall antigen of Streptococcus pneumoniae in the sample, whether or not in free form, will react with said conjugates to form labelled antibody-antigen conjugates,

(ii) further contacting the liquid and all of the conjugates it contains with a solid surface upon which a mass of unlabeled antigen-specific antibodies from step (d) hereof have been immobilized, whereupon any labelled antibody-antigen conjugates present will react with the immobilized antibodies on the surface to form labelled antibody-antigen-immobilized antibody sandwiches, and

(iii) detecting any label thereby accumulated on the solid surface by a detection means appropriate to the nature of the label so as to confirm the presence of the Streptococcus pneumoniae C-polysaccharide cell wall antigen in the sample.

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