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

Abstract

Disclosed is a cell wall C-polysaccharide antigen of Streptococcus pneumoniae which contains not more than 10% by weight of protein, and preferably less which has been purified with 0.1N NaOH prior to deproteinizing. Also disclosed are polyvalent antibodies raised against Streptococcus pneumoniae which have been affinity purified by passing them over a chromatographic affinity matrix to which is coupled the purified and at least partially deproteinized antigen to render them antigen-specific.

Description

PARENT APPLICATION[0001]This application is a division of U.S. application Ser. No. 09 / 397,110 filed Sep. 16, 1999, which is in turn a continuation in part of U.S. application Ser. No. 09 / 156,486 filed Sep. 18, 1998 in the names of the same inventors.BRIEF DESCRIPTION OF THE INVENTION[0002]The present invention relates to a purified carbohydrate antigen of S. pneumoniae and its use in affinity purifying antibodies raised in an animal against S. pneumoniae bacteria or against the carbohydrate antigen. The affinity purified antibodies are especially useful in 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[0003]Streptococcus pneumoniae (“S. pneumoniae”) is a leading causative organism of pneumonia-type illnesses and other lower re...

AI Technical Summary

Benefits of technology

[0016]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. pneumoniae-caused meningitis may be readily made using patient cerebrospinal fluid as the test sample.

[0017]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.

[0018]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.

[0019]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.

Problems solved by technology

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.

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.

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. pneumoniae, 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.

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.

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< /

i>. Sputum is often difficult to collect; more-over, once medication of the patient is commenced, the number of viable S. pneumoniae in sputum rapidly decre

ases. 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 microor

ganism. When S. pneumoniae causes infectious otitis media, meningitis and various other aforementioned infectious disease states, sputum samples are of no aid in d

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.

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.

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