MXPA01002822A - Process and materials for the rapid detection of streptococcus pneumoniae - Google Patents

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. pneumoniae 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 commonlyoccur when primary pneumonic infection caused by this bacterium persists undiminished over a time period of 3-5 days.

Description

PROCESS AND MATERIALS FOR THE RAPID DETECTION OF STREPTOCOCCUS PNEUMONIAE USING ANTIBODIES SPECIFIC PURIFIES OF ANTIGEN The present invention relates to a sensitive immunochromatographic test ("PIC"), which is performed in a period of approximately 15 minutes, for the detection of Streptococcus pneumoniae in a body fluid, such as urine or cerebrospinal fluid, of a patient who shows clinical signs of an infection caused by S. pneumoniae. BACKGROUND OF THE INVENTION Streptococcus pneumoniae ("S. pneumoniae") is a causative organism of diseases of the pneumonia and other lower respiratory tract infections, such as bronchitis, as well as of the upper respiratory tract, including infectious otitis media and sinusitis. Invasive disseminated infections, including bacteremia and meningitis. When not diagnosed or treated appropriately, a pneumonic infection caused by S. pneumoniae can cause pericarditis, empyema, fulminating purpura, endocarditis or at least some type of arthritis, where S. pneumoniae is the causative organism in each Ref: 127867 - - case. Such pneumonic infections are often also precursors of bacteremia or meningitis. To date, although S. pneumoniae is a common cause of pneumonia, it has to be diagnosed and treated in some way empirically. To a significant degree, this is because the tests currently available for the detection of S. pneumoniae: (1) are time-consuming, laborious and require auxiliary instruments to read the results, or (2) lack sensitivity and / or specificity. Due to problems associated with lack of sensitivity and / or specificity, e.g. Doctors tend to prescribe conservatively expensive broad-spectrum antibiotics for patients suffering from pneumonia-type respiratory infections, instead of prescribing a less expensive antibiotic specifically against S. pneumoniae, when it would seem the proper cure for that infection. This and other liberal ways of prescribing broad-spectrum antibiotics, of course, are one of the main causes of the current well-publicized medical crisis that is a consequence of the increased resistance of many types of infectious bacteria to previously highly effective antibiotics. This crisis and the potential of its consequences for - at least some patients diagnosed and treated empirically, are among the many reasons why a reliable and rapid assay for the detection of S. pneumoniae in human body fluids is needed. Pneumonia caused by S. pneumoniae is a serious disease, which is estimated to occur at a rate of 1 to 5 cases per 1000 people per year in the United States alone. Depending on the age and health status (based on unrelated factors) of patients infected with pneumonia caused by S. pneumoniae, the disease has a mortality rate of between 4 and 30% of infected patients. The most traditional methods to try to diagnose diseases caused by S. pneumoniae and especially pneumonia, include Gram stain and expectorated sputum culture of patients who suspect that they are carriers of the disease, followed by biochemical methods of identification . This procedure requires 1 to 4 days to be done from start to finish. It has been found to be an unsatisfactory diagnostic tool because (1) other bacteria present in the patient's saliva often exceed growth in the sputum culture and (2) S. pneumoniae is often present in - - the human upper respiratory tract even when there are no signs of disease attributable to this bacterium in the individual. For example, it is estimated that approximately 30% of children in the United States are regular carriers of S. pneumoniae. Adults are also colonized by S. pneumoniae without entering a state of disease. The index of carriers of the organism in both children and adults increases when there are overcrowded conditions and in the winter months. Coagglutination methods have been developed, agglutination of latex particles and counter-immunoelectrophoresis to detect the capsular polysaccharide antigens of S. pneumoniae in sputum samples, which are rapid, but have not been shown to exhibit a reliable sensitivity or specificity, probably because there are approximately 83 serotypes of S. pneumoniae, each of which may vary in immunogenicity and in other aspects. The commercial polyvalent antiserum developed and used for these tests contains antibodies against the antigen of serotype of 5. pneumoniae, but nevertheless, it can fail to detect serotypes of less immunogenic antigens. This polyvalent antiserum has also shown reactivity - - crossed with other streptococci and with some other infectious bacteria, e.g. Haemophilus influenzae. Thus, false-negative reactions and false-positive reactions may occur randomly when these tests are used on sputum samples. Several enzyme immunoassays ("EIAs") have been developed, which are based on the detection of pneumococcal polysaccharide C antigen, which has been found to be present in the pneumococcal cell wall of all serotypes of S. pneumoniae. See e.g. Parkinson, A.J. Rabiego, M.E., Sepulveda, C, Davidson, M. and Johnson, C, 30 J. Clin. Microbiol. 318-322 (1992). This polysaccharide antigen C is a polysaccharide containing phosphocholine derived from teichoic acid. These EIA assays have acceptable specificity and sensitivity even though they are often performed on sputum samples. However, such tests require two to three hours for their completion after the sample has been collected, as well as the use of instruments that are normally available mainly in clinical laboratories. In addition, these tests must be performed or supervised by trained personnel. Relying on sputum samples for the diagnosis of S. pneumoniae infections with frequency is unsatisfactory to achieve a diagnosis of pneumonia caused by S. pneumoniae and not only due to the potential contamination of the sample by other bacteria in the mouth, but also by the presence in the upper respiratory tract of S. pneumoniae as normal flora. Often sputum is difficult to collect; In addition, once the patient's medication is started, the number of viable S. pneumoniae bacteria in the sputum drops rapidly. In particular, the presence of polysaccharide C antigen in sputum could quickly be difficult to detect if you are using an antibiotic therapy that attacks the cell wall of S. pneumoniae. When S. pneumoniae causes infectious otitis media, meningitis and several other previously mentioned infections, sputum samples are not helpful for diagnosis. The collection of blood cultures from patients suspected of having S. pneumoniae infection eliminates the contamination problems suffered by sputum samples. When it is found that samples of blood serum contain S. pneumoniae, the diagnosis of several diseases caused by it can be made. The drawback in this case is that only about 20% of patients with pneumonia - - infected by S. pneumoniae, present bacteremia; therefore, relying solely on blood samples to diagnose pneumonia caused by S. pneumoniae could produce false negative results. It has been found that urine samples are more reliable and convenient to use for the detection of pneumonia caused by S. pneumoniae, because they can be obtained non-invasively; will not be contaminated with oral microflora; and the presence of the bacteria persists in the urine, although at a constantly decreasing concentration level, even after the patient's therapy has been initiated, so that daily monitoring of the patient's urine samples could provide useful information for evaluate the effectiveness of a prescribed therapy. It should be noted that human carriers of S. pneumoniae showing no signs of disease often do not have a sufficient amount of the pathogen 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 antibody immunoglobulin A against phosphorylcholine was used to detect the polysaccharide antigen - - C. See Suertz, K. Merx, I, Eiffert, H., Schmutzhard, E., Mader, M. and Ñau, R., 36 J. Clin. Microbiol. 2346-2348. The results obtained compare 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 with an EIA in which two S. pneumoniae antibodies were used in cerebrospinal fluid - a horse antibody against the pneumococcal polysaccharide C antigen, bound to microplates, and a mixed rabbit antiserum against the capsular antigen polysaccharide in the liquid phase. BRIEF DESCRIPTION OF THE INVENTION In accordance with the present invention, antibodies against the polysaccharide C antigen of S. pneumoniae generated in rabbits, are purified by affinity with purified and isolated polysaccharide C antigen having less than about 10% protein content. These affinity purified antibodies are conjugated with an agent that produces a colorful reaction by forming a sandwich with the polysaccharide C antigen of S. pneumoniae from a test sample and the polysaccharide C antibody purified by additional affinity immobilized in a nitrocellulose matrix. The test is carried out in a disposable immunochromatographic test device and does not require instruments to interpret the result. This test can be done easily and successfully by people who do not have any training in laboratory techniques. The preferred test sample for the diagnosis of pneumonia caused by S. pneumoniae is the patient's urine, but also the test works with other body fluids samples containing S. pneumoniae, including serum and sputum. The diagnosis of meningitis caused by S. pneumoniae can be easily performed using the patient's cerebrospinal fluid as the test sample. The present invention, for the first time, offers the benefit of a test for S. pneumoniae that is carried out in a period of 15 minutes and that has a specificity and sensitivity at least equal to the EIA tests that require 8 to 12 times more Working time to get the result. The test is easy to perform, does not require special training, equipment or instruments and makes possible a rapid diagnosis of pneumonia caused by S. - - pneumoniae. It can be easily performed in a doctor's office, thus allowing the patient to be immediately placed on a specific therapeutic regimen against S. pneumoniae. Of course, the test can be performed by a clinical laboratory, but it can also be performed easily in a geriatric center, in the patient's home, or in any environment where pneumonia caused by S. pneumoniae or other pathogenic disorders is suspected to be epidemic . The test according to the present invention is important when diseases such as otitis media, bronchitis or sinusitis appear to have arisen and it can be established that any of these is due to S. pneumoniae instead of other infectious agents, and then it can be initiated quickly a therapy. Young children are especially prone to otitis media, due to the shorter length and smaller diameters of their eustachian tubes, so that early detection of S. pneumoniae, if present, can prevent the emergence of a state of more serious or even life-threatening illness. The 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 anemia with highly susceptible S. pneumoniae infection, where sepsis by S. pneumoniae is the most common invasive infection in this population and those who are infected in this way suffer from a high risk of recurrence and subsequent death. Clearly, the use of the PIC (immunochromatographic test) test of the present invention to test the urine of these patients on a regular basis may be of help in decreasing the need for prophylaxis with penicillin, which is recommended in the second of these documents. The ease of execution of the test and its ability to detect the polysaccharide C antigen of S. pneumoniae in the urine, suggests that this test should be performed prudently in patients without overt clinical signs of related infections, who report that they do not feel substantially well . Any such patient in whom it is established that S. pneumoniae is present in amounts significant enough to give a positive urine ICP test is a predictable candidate for developing a more serious infection - and the ability to anticipate the development of the disease before it becomes Severe administration of appropriate therapy is presented by this invention. DESCRIPTION OF THE DRAWINGS Figure 1 and related Figures IA, IB and 1C of the present show the structure of a typical PIC device that has been adapted to perform the S. pneumoniae assay, as will be described in more detail. DETAILED DESCRIPTION OF THE INVENTION Speaking in broad terms, the PIC assay for S. pneumoniae, as described herein, can be designed and configured to be performed on any disposable PIC device described in the art. Preferably, it is designed so that it can be made using a PIC device of the type described in co-pending US Patent No. 07/706, 639 by Howard Chandler or one of its continuation applications in part, which are assigned to Smith-Kline Diagnostics, Inc., but who have an exclusive license with Binax, Inc. (which has the right to assign this application), in a wide area of fields of use, which includes the diagnosis of diseases of the respiratory system human .

- The preferred device is suitably impregnated in a region with antigen-specific polyvalent antibodies against the polysaccharide C antigen of S. pneumoniae. Antigen-specific antibodies labeled in another area of the device are applied. The test sample suspected of containing S. pneumoniae is first contacted with the labeled antigen-specific antibodies, which then flow with the sample to the area of the device containing the unlabeled antigen-specific antibodies, where, if S. pneumoniae is present in the sample, the conjugate of labeled antibody: polysaccharide C antigen already formed comes into contact and binds with the unlabeled affinity-purified antibodies which are immobilized, thereby producing a visible colorful reaction. The label can be any substance known in the art that produces a visible color by reacting a labeled antibody: antigen complex with unlabeled antibodies bound. Such labels include various finely powdered metal compounds, various organic molecules and various molecular combinations such as enzymatic combinations with other color-producing molecules. In the present invention, the Colloidal gold particles constitute the preferred brand. It is of greater importance in the design of the test device, that the concentration of the antibody present in each of the two sites of the test device where the reaction occurs, is sufficient to ensure that the antigen present in the test sample will be captured. by the labeled antibodies when the test sample comes into contact with them and that the labeled antibody: antigen conjugate will be easily captured and maintained by the antibodies bound in the capture line of the sample. Experimental work carried out with reference to the present invention, has shown that the active antibody against the polysaccharide C antigen of S. pneumoniae must be present at each site of a test device where the antigen: antibody reaction will occur, in a concentration between 7.7 nanograms / mm2 of surface area and 385 nanograms / mm2 of surface area. If antibody concentrations lower than 77 ng / mm2 are present at the site where the reaction is intended to occur, false negative results are likely to be obtained.

- - Several methods of affinity purification of antibodies against the polysaccharide c antigen of S. pneumoniae are known. The one described below is preferred in the present invention, but may be substituted by others. However, it is noted that the affinity purified antibodies of the present invention are distinguished from the "affinity purified antibody preparation" which is described in Sjogren and Hol e 102, J. Immunol. Methods 93-100 (1987). These authors describe the obtaining of a polysaccharide C antigen of S. pneumoniae purified with hot phenol, containing 17% protein and absorbing it in a DEAE-Sepharose CL6B ion exchange gel. After 48 hours of incubation, this preparation was packed in columns at approximately neutral pH of 7.2. The binding efficiency of the antigen to the gel was approximately 60%. The antibodies found in these columns were incubated for 30 minutes, followed by elution of the columns with 0.5M NaCl in phosphate buffer (SRF). It is known that there is an escape of antigen from the ion exchange columns and it occurs frequently. In this system, it is reasonable to hypothesize that the product eluted from the gel was an immune complex formed in its antibody and antigen, rather than a Preparation of the purified antigen of the present invention. It should be particularly noted that, in the present invention, the purified antigen contains less than 10% of proteins and is covalently coupled with a spacer molecule, such as bovine serum albumin (BSA), conjugated to hydrazine and that the labeled antigen: conjugated ligand conjugate is covalently coupled to a chromatographic gel -eg the formyl spherilose of Example 4, which is then applied to a column. The antibodies are added and eluted with a strongly acidic regulatory solution of the antigen immobilized on the column. The preferred antibody herein is obtained by conventional injection of a rabbit with the strain of S. pneumoniae R6, which is an unencapsulated strain of S. pneumoniae available from the North American Collection of Type Culture (American Type Culture Collection) with the ATCC access number no. 39938, subjecting these bacteria to heat inactivation before injecting them into the animal. After an appropriate period of time, the animal is bled to obtain serum containing the desired antibodies, followed by purification thereof. Other antibodies against the polysaccharide C antigen of S. pneumoniae can be substituted by those specifically described - in the present, without departing from the scope of the present invention. The antibodies must initially be tested for cross-reactivity with other common infectious bacteria. The preferred antibody of the present was tested using the ELISA method for cross-reactivity with each of the following microorganisms: Citrobacter freundii, Staphylococcus aureus, Enterobacter cloacae, Enterobacter faecalis, Streptococcus, group B, Type III, E. coli, Neisseria meningitidis , Salmonella cubana, Salmonella paratyphi A, Klebsiella 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, Acínetobacter baumannii, Klebsiella oxytoca, Acinetobacter lwoffii, Pseudomonas aeruginosa, Staphylococcus saphrophyticus, Enterococcus durans, Corynebacterium bovis, Proteus mirabilis, Pseudomonas stutzeri, Pseudomonas cepacia, Salmonella typhi, Streptococcus, Group F, Streptococcus, Group B, type la, Candida stellatoides, Streptococcus parasanguis, Streptococcus, Group G , Streptococcus, Group C, Streptococcus mutans, Morganella morganii, Staphylococcus haemolyticus, Haemophilus infl uenzae Type B, Stenotropho onas maltophilia, Haemophilus influenzae type D, Gardnerella vaginalis, Streptococcus mytis, Haemophilus parainf luenzae, Streptococcus sanguis and nontypable H. influenzae. The only significant cross-reactivity that was found was with Streptococcus mytis and with Staphylococcus aureus. The first, S. mitis, is a causative agent of endocarditis, whose symptoms in a patient doctors can easily distinguish from those caused by a lung infection with S. pneumoniae. S. mytis contains the same polysaccharide C antigen as S. pneumoniae and the two share the ability to cause endocarditis, although S. pneumoniae usually does so in patients whose primary pneumonia was not treated properly and who can then develop bacteremia and / or endocarditis or some other secondary pathogenic infection. S. my tis, in contrast, is not a causative agent of pneumonia; Endocarditis attributable to S. mitis usually develops independently of any other infection. Consistent with this, it is considered that suspected cases of primary endocarditis caused by S. mitis can be confirmed, when necessary, using the assay of the present invention. However, it should be noted that S. mytis is less likely to be present in the urine than S. pneumoniae andtherefore, a serum assay would be more likely to produce confirmatory information in this case. Some strains of S. aureus are known to secrete protein A, which is a non-specific protein that binds indiscriminately to IgG and, therefore, to all antibodies. The suspicious presence of these S. aureus entities can be easily confirmed or can be excluded by performing other simple tests known in the art (as shown in Example 9, strains of S. aureus in which the protein is not present A, do not show any cross reaction with the antibodies of the present invention). A less cross-reaction was observed with Haemophilus influenzae, but it is not considered to be significant enough to cause a problem in the detection of S. pneumoniae in urine samples. The following examples illustrate the preferred mode of affinity purification of the antibody, including preliminary separation and purification of the antigen used to effect purification. of the antibody, thereby obtaining an antigen-specific polyvalent antibody preparation. EXAMPLE 1 - Bacterial Growth Conditions The S. pneumoniae R6 strain (ATCC No. 39938) was grown in S. pneumoniae broth supplemented with 20 mM HEPES buffer. The broth had the following composition per liter: Casein pancreatic digest 17.0 g Glucose 10.0 g NaCl 5.0 g Digested with soy papain 3.0 g Yeast extract 3.0 g K2HP04 2.5 g HEPES 20 M This broth had an initial p H of 7.2 ± 0.2 at 26 ° C. It was sterilized by autoclaving for 15 minutes at 15 psi (1.05 Kg / cm2) and 121 ° C and allowed to cool. Frozen aliquots of the strain of S. pneumoniae R6 (ATCC No. 39938) were inoculated on agar plates with 5% sheep blood and allowed to grow. The growth of the plates was harvested in similar aliquots of the seed broth and this seed broth was inoculated in three flasks, each containing 1700 L of S. pneumoniae broth supplemented with the composition shown above and allowed to grow at 37 ° C in an atmosphere with 5% C02, with agitation, but without aeration. When the pH of the broth fell below 5.5 (its late log phase), the flasks were removed from the incubator, the cells were inactivated with 0.1 percent sodium azide and the pH was adjusted to a value above 7.0 for prevent autolysis. The flasks were stored at 4 ° C overnight. The next day, the suspension of each flask was centrifuged at 8,000 r.p.m. for 60 minutes. The pellets were then combined and centrifuged again at 13,000 r.p.m. for 30 minutes. The wet weight of the pellet was recorded and stored at -20 ° C. EXAMPLE 2 - Isolation of Polysaccharide C Antigen from S. pneumoniae Containing Less than 10% Protein Cells were grown and treated and stored according to Example 1, thawed at room temperature and suspended in regulatory saline. of phosphates ("SRF") with a pH of 7.2, with 0.2 percent sodium azide in a ratio of 1.2 mL of buffer solution per 1 gram of wet cells and left at room temperature for 2 days. Then 11 mL of NaOH 0. IN per gram of wet cells was added to the suspension of S. pneumoniae (in phosphate buffer saline), obtaining a pH of 12.34 (measured with pH meter) and incubated for 45 minutes at approximately 30 ° C. The pH of the suspension was adjusted to 2.75 (measured with pH meter) with 2N HCl, followed by centrifugation at 3,500 r.p.m. for 25 minutes. The supernatant was separated and its pH adjusted to 7.0-7.1 with IN NaOH. This essentially neutralized supernatant was dialyzed at 4 ° C against water for 2 days in a dialysis tube (obtained in Spectra / Por) having a molecular weight cutoff of 12,000 to 14,000. The dialyzed supernatant was concentrated 25 to 40 times in a vacuum rotary evaporator. Proteinase K (from Boehringer Mannheim) was added in an amount of 0.20 mg per gram of wet cells and the mixture was allowed to stand at 37 ° C for three and a half to 4 hours and then at room temperature overnight and the next day . After digestion with proteinase K, the resulting supernatant was dialysed at 4 ° C against water in the dialysis tube of Spectra / Por having a molecular weight cutoff of 12,000 to 14,000. The dialyzed supernatant was divided into 12 aliquots, each of which was placed in a 30 mL glass tube and mixed with an equal volume of 90% phenol. The - - tubes were closed and incubated for 23 minutes at 68-72 ° C in a water bath, where the water level was slightly above the level of the liquid in the tubes. The suspension of each tube was occasionally stirred with a glass Pasteur pipette to make the suspension more homogeneous with the naked eye. After this incubation, the suspension was allowed to stand at room temperature for 30 minutes and then centrifuged at 5,000 r.p.m. for 40 minutes, at a temperature of 15 ° C. The upper aqueous phase of each tube was carefully removed with a glass syringe; its volume was measured carefully, in each individual tube, and replaced by an equal volume of fresh water. The incubation steps of the suspension at 68-72 ° C followed by a centrifugation at 5,000 r.p.m. for 40 min at 15 ° C, it was carried out again and then repeated once more. The lower phenolic phase of each tube was carefully removed with a glass syringe, leaving the intermediate (mixed with phenol-water) and the upper (water) phase in the tubes. Meanwhile, a flask containing cold ethanol with a volume ratio of approximately 10: 1 in relation to the combined extracted phenolic phase of the tubes, was placed in an ice bath. To this flask the phenolic phase was added slowly, by dripping, with intense agitation. After the whole phenolic phase was added, stirring continued for 10 to 15 minutes, after which the mixture was placed in a refrigerator at 4 ° C and left overnight for the polysaccharide C antigen pellet to form. The next day, the mixture was subjected to a centrifugation at 12,000 rpm for 20 minutes at 4 ° C. The resulting pellet of polysaccharide C antigen was suspended in approximately 0.4 mL per gram of wet cells of water and dialyzed against distilled water at 4 ° C overnight, using the Spectra / Por tube with a molecular weight cutoff of 12,000 to 14,000 previously referred. The resulting aqueous solution of the polysaccharide C antigen was lyophilized and weighed. The concentration of proteins was evaluated by the Lowry method; its composition was verified by SDS-PAGE (12% gel) with a Western blot assay and the activity of the polysaccharide antigen was verified by ELISA. This operation was repeated a number of times. It was found that the overall yield of polysaccharide C antigen of S. pneumoniae was about 1.2 to 1.4% per gram of wet cells of strain S. - pneumoniae R6, while its protein content was between approximately 5 and approximately 8%. It should be noted that, in general, polysaccharide C antigen preparations with a protein content exceeding 10%, are less likely to perform satisfactorily in the present invention, compared to preparations having a lower protein content. to 10%. EXAMPLE 3 - Preparation of ASB Conjugate of the Antigen For the coupling of the polysaccharide C antigen of purified S. pneumoniae R6 to a chromatographic column, to allow affinity purification of anti-S rabbit antibodies. pneumoniae R6, a conjugate with ASB-hydrazine was selected. Other materials that are known to have similar functions can be selected and conjugated to carry out this coupling function. The ASB-hydrazine conjugate was prepared in the following manner: Hydrazine dihydrochloride obtained at Aldrich Chemical Co. was dissolved in water to produce a 0.5M solution. The pH was adjusted to 5.2 with dehydrated NaOH and bovine serum albumin ("ASB") was added from Sigma Chemical Co. to produce a concentration - - final ASB of 25 mg per mL of solution. After complete dissolution of the ASB, N- (dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride (from Fluka Chemical Co.) was added in an amount sufficient to produce a final concentration of 2.5 mg per L of solution. This reaction mixture was incubated at room temperature, with continuous stirring, overnight. The next day, it was intensively dialyzed against distilled water at 4 ° C. The concentration of the conjugate was measured (as ASB concentration) at 280 nm in a Beckman DU 640 spectrophotometer. To couple this conjugate with the polysaccharide C antigen of S. pneumoniae R6, the procedure was as follows: The dehydrated preparation of the antigen was dissolved in an amount of 1.1 mg per mL of distilled water. Using a dilute HCl solution, the pH of the solution was adjusted to 5.0-6.0. The conjugate ASB: hydrazine in aqueous solution, at a concentration of 23 mg per L, was treated with dilute HCl to bring its pH to a value between 4.0 and 5.0 and this solution was slowly added to the antigen solution, in a ratio of volume of about 1: 6.65 (about 3: 1 by weight). After 3 minutes of stirring, N- hydrochloride was added - - (dimethylaminopropyl) -N-ethylcarbodiimide (from Fluka Chemical Co.), in an amount of 100 to 200 μL of distilled water, to the reaction mixture in α-ethylcarbodiimide hydrochloride, at a weight ratio with the polysaccharide C antigen of about 1 to 1.92. After stirring for 2 hours at room temperature, the pH of the resulting mixture was adjusted to approximately 9.0 with diluted a? OH. Incubation was continued at room temperature for 1 hour and then at 4 ° C overnight. EXAMPLE 4 - Preparation of the Affinity Column and Purification of the Antibody To the solution of the ligand of Example 3, dilute HCl was added to bring its pH to 7.0. Isil spherylose from Isco, Inc. was selected as the matrix for the immunoadsorbent gel. The polysaccharide C antigen ligand was coupled to this matrix using the known procedures, e.g., as described in spherilose applications, ISCO Applications Bulletin 78, on p. 28-35. Other known matrices and coupling methods can be used. The gel was packed in a column and washed alternately with distilled water and with a glycine solution 0.2M-HC1 of pH 2.5, a buffer solution of - - phosphate of triple strength pH 7.2 and DBS of regular strength pH 7.2, using 5 to 10 volumes per volume of gel, in the case of each solution. The resulting activated column was used for affinity purification of the antibodies, thereby producing antigen-specific antibodies, in the following manner: Rabbit antiserum against S. pneumoniae R6 (ATCC No. 39938) directed against killed whole cells by heat, it was mixed with dehydrated NaCl to a final concentration of 0.5M. This mixture was centrifuged at 8,500 r.p.m. for 20 minutes and the supernatant was filtered through cotton. The filtrate was applied in the affinity column. The unbound components were washed from the column with triple-strength phosphate buffer salt solution pH 7.2 and regular strength phosphate regulating saline pH 7.2. The antibodies were eluted from the column with glycine buffer 0.2M-HC1, pH 2.5. The eluent was monitored at 280 n in the Beckman spectrophotometer and the fractions containing antibodies were mixed in a flask, which was placed in an ice bath. The mixed fractions were neutralized with 0.5M NaH2P04 in aqueous solution, pH 9.0.

- - The concentration of the antibodies was evaluated by the absorbance value at 280 nm in the spectrophotometer. The antibody solution was dialyzed again against SRF pH 7.2 and concentrated on a PM-10 filter obtained in 7Amicon, until an antibody concentration of 0.8-1.5 mg / mL was reached. It was found that 18 to 20 mg of affinity purified antibodies were recovered for each 25 mL of rabbit antiserum against S. pneumoniae R6. These antibodies, affinity purified, were used in a PIC test specific for polysaccharide C antigen of S. pneumoniae, as described in the following example. EXAMPLE 5 - PIC Device and Its Preparation A. Preparation of the Test Device: A test device comprising a cardboard card comprising a hinge card housing equipped with a window to allow viewing of the test results was prepared. and the results of the control, as shown in Figure 1. The device has a well-shaped plastic recess in which a pad is placed to receive the wet pad with the sample on the right side (marked as 1 in he - - drawing) . Then a label shown on the Figure IA on the entire right side of the device.

The label has two perforations - a lower one (marked as B in Figure IA) in which the saturated pad is inserted, and an upper one (marked as B in Figure IA) to which the pad will be pushed after inserting it in bore B. The position of the label with its perforations A and B and the well with the pad cooperate to hold the pad in an appropriate position during the test and promote the expulsion of the pad of the liquid sucked. A preassembled test strip (marked C in Figure 1) described below is inserted into a recess (marked 2 in Figure 1) and held in place with an adhesive applied to the bottom of the recess. A label shown in Figure B is placed up on the left side. It is equipped with a single perforation (marked D in Figure IB), which houses with perforation A on the right side when the device is closed to perform the test. The assembled device is stored in a sealed bag with desiccant until it is used. Before sealing the bag and storing the product, a - - lightly adhesive tape on the outer edge of the right half of the device. B. Construction and Preparation of the Test Strip Figure lc shows the construction of the preassembled strip. It is comprised of a conjugate pad of sorbent material with a conjugate of gold particles and is impregnated with the antigen-specific rabbit antibodies against the polysaccharide C antigen of S. pneumoniae. An Ahlstrom 1281 pad (not shown) that serves as a bridge, connects to the conjugate pad with a nitrocellulose pad on which a capture line has been established for the sample that reacts with the conjugate, soaking a strip of the antigen-specific rabbit antibodies against the polysaccharide C antigen of S. pneumoniae, prepared in the manner described above. The nitrocellulose pad also has a downward control line, which is established by placing a strip on the pad with goat serum against rabbit immunoglobulin (IgG). After the nitrocellulose pad, the strip ends with an absorbent pad that serves as a receptacle for the liquid.

- - All these pads have an adhesive strip on the back and are placed on a device. The conjugate pad is usually made of non-spun polyester or an extruded cellulose acetate material. To prepare this pad for use in the assay, gold particles of 50 nm in diameter are conjugated with the rabbit antibodies against the polysaccharide C of S. pneumoniae prepared in the manner described above. The conjugation is carried out using a known method, such as that described by DeMay in Polak, J.M. and Van Norden, S. (Eds)., Immunochemistry: Modern Methods and Applicati on, (Wright, Bristol, England, 1986). , The conjugate particles of gold are mixed with a drying agent consisting of 5mM sodium tetraborate pH 8.0 containing 1.0% ASB, 0.1% Triton X-100, 2.0% Tween 20, 6.0% sucrose and sodium azide. 0.02% The pad heats up enough to remove all the liquid present and is stored in a low humidity environment until the reactive strip is assembled. These pads and their treatment are specially selected in such a way that the pads keep the conjugate dry and is released only when the latter is wetted with the sample.

- - The nitrocellulose pad is first treated by placing a strip of rabbit antibodies against the S. pneumoniae polysaccharide C purified by affinity in a first position, using a vehicle solution of phosphate buffering saline. These antibodies act as the capture line. In a second portion of the pad below the first in the assembled test device, the control line is established by placing a strip of goat serum against rabbit IgG in the same vehicle solution, on the surface of the pad. Then the nitrocellulose pad is subjected to a drying at 18-25 ° C to promote permanent absorption of the protein. The absorbent pad used is a commercial pad of cellulose material sold under the name Ahlstrom 939. This pad does not require any special treatment. C. Package Preparation To be sold commercially, the test device containing the finished test strip is assembled. In practice, a number of devices are packed with a proportional number of isotopes of fibrous Dacron and a bottle of "reagent A" equipped with a dropper to supply reagent A.

- - "Reagent A" is a solution of 2.0% Tween 20, 0.05% sodium azide and 0.5% sodium dodecyl sulfate, in a buffer solution of 0.05M sodium citrate-sodium phosphate, pH 6.5. Also included are the positive control and the negative control in each package. EXAMPLE 6 - Realization of the PIC Test In practice, the isopode provided in each device is immersed in the sample liquid, completely introducing the head of the isope. The isopo serves to act as a filter of undissolved solids, semisolids and colloids present in biological liquid samples, such as urine, blood, lymphatic fluid, etc., and also in samples of environmental liquids, where this iso is subject to copending Patent Application Serial No. 09 / 044,677 by Norman Moore and Vincent Sy, filed on March 19, 1998, which is assigned to Binax, Inc. The isopen is inserted into the perforation of the bottom of the device (perforation B of Figure IA) and pressed gently so that the tip of the isopo is visible in the upper part of the perforation (perforation A of Figure IA). The reagent bottle A is kept vertical over perforation B and 3 drops of reagent A are added slowly. Then the line of adhesive - - detaches from the right edge of the device and the device is closed and sealed, thus pressing the isope in the well against the gold conjugate pad. After 15 minutes, you can read the result in the device window. A negative sample -i.e., one that does not contain identifiable S. pneumoniae C polysaccharide antigen-will exhibit only the control line in the upper half of the window. A positive sample containing the white antigen will show two lines, the lower one being the one corresponding to the patient (or sample); even a very weak line in the sample indicates the presence of the white antigen in the sample. If no line appears in the window after 15 minutes, or if only one line appears in the sample, in the lower part of the window, the test is invalid and must be repeated.

Using the procedure described above, the devices prepared in the manner described in Example 5 were tested using the immunochromatographic test (PIC) described in 146 patient urine samples obtained from the US Center for Disease Control. As the diagnoses of the patients regarding the presence of S. pneumoniae infection were based on a variety of different indications, including - - blood culture, Gram stain, sputum culture, PCR with autolysin and PCR with pneumolysin, but no tests had been done in urine, each of these samples was tested in our laboratory with the PIC as described here and also by an ELISA technique; looking for the presence of polysaccharide C antigen of S. pneumoniae. The personnel who performed the PIC and ELISA tests were not informed of the classifications of the Center for Disease Control of the urine samples that had been collected from patients with positive or negative diagnosis of S. pneumoniae infection. It was found that the results of PIC and ELISA, at least were comparable in terms of sensitivity and specificity in 134 cases. However, it is observed that in some cases neither the ELISA nor the PIC test were completely correlated with the diagnosis of the patient provided by the Center for Disease Control. It is thought that there are some recognized shortcomings in the technique on the evaluation of cultures as a basis for the diagnosis of S. pneumoniae infections and the fact that there was no information regarding the therapy administered to the patients, or the time in which they were administered. which therapy had begun in relation to time in the - - that the urine sample was taken, are factors that exclude any significant comparison of all the results. The substantial comparability of the results of PIC and ELISA in 134 cases confirms the considerable advantages of the 15 minute PIC test of the present invention, which offers a rapid diagnosis of infections caused by S. pneumoniae and consequently the possibility of instituting a more effective therapy for the patient. EXAMPLE 7 - Clinical Studies Using the devices prepared in the manner described in Example 5 and the PIC procedure described in Example 6, first paragraph, clinical studies were conducted at three sites, using a bank of characterized specimens. These included 273 urine samples collected from inpatients and outpatients. Among the 273 patients, 35 had positive blood culture results and 238 had negative blood culture results (it should be noted that culture methods often vary substantially from one place to another). Urine samples from patients with positive blood culture were presumed to have been collected within 24 hours after collection of the sample of blood and the initial administration of antibiotics. Of the 238 urine samples from patients with a negative blood culture, 28 were collected from patients with bacteremia, 4 from patients with empyema, 53 from patients with pneumonia and 153 from patients with urinary tract infections. In addition, 100 urine samples that were collected from individuals without known infection, were analyzed with the test of the present invention, involving the devices prepared in the manner described in Example 5 and the PIC procedure described in Example 6. The samples of blood of these individuals gave negative results in the culture tests. Of the 35 urine samples from patients with blood culture positive for S. pneumoniae, 30 gave positive results in the test of the present invention and 5 gave negative results. Of the 338 urine samples from patients where all of them gave negative blood culture and 100 of which were presumably negative, 21 were positive in the PIC test of the present invention and 317 were negative. The sensitivity of the PIC test was calculated as 86%, the specificity as 94% and the accuracy as 93%.

- - It should be noted that of the patients whose urine test was positive for S. pneumoniae by PIC and whose blood cultures gave negative results for S. pneumoniae, it was established by other tests that those with urinary tract infections 5 presented infections by E. coli, 2 Enterobacter cloacae infections, 3 infections by Lactobacillus, 1 infection by Providencia stuartii, 1 infection by Staphylococcus aureus, 1 infection by Streptococcus (no A, no B) and 1 infection by Streptococcus (no D). Two of those who had pneumonia were also infected with Mycobacterium tuberculosis and one was infected with Mycobacterium um kansasii. A patient who had bacteremia was also infected by Proteus mirabili s. Four patients without known infection showed positive results in the urine sample by means of the PIC test of the present invention. EXAMPLE 8 - Clinical Studies Tests were performed in 7 hospitals, 6 in the United States and 1 in Spain, to evaluate 215 urine samples from inpatients and outpatients with at least one symptom of lower respiratory tract infection and symptoms of sepsis or who were otherwise suspected to be carriers of urine. - - pneumococcal pneumonia. In these tests, the device prepared according to Example 5 was used in the procedure of Example 6 and the results were compared with the blood culture results performed on blood samples from the patients. No effort was made to ensure the uniformity of farming methods among participating institutions. The blood cultures gave 31 positive results for S. pneumoniae and 184 negative results. Of the 31 patients whose blood culture was positive, the PIC test of the present invention performed on urine samples showed 28 positive and 3 negative results. The 184 patients whose blood culture results were negative, 45 showed positive results in urine samples by the PIC test of the present invention, while 139 urine samples from these patients gave a negative result. The sensitivity in this study of the test of the present invention was calculated in 90%, the specificity in 76% and the accuracy in 78%. The results obtained with the PIC tests of the present invention in Examples 7 and 8, should be considered in light of the well-known problems associated with culture tests and the possibility - known that approximately 80% of patients infected with pneumococcal pneumonia will not produce blood samples containing S. pneumoniae. It is thought that further experiments with the assay of the present invention will convincingly demonstrate that its specificity, sensitivity and accuracy are underestimated in Examples 7 and 8 due to the use of blood culture tests for comparison purposes. EXAMPLE 9 - Additional Tests of Cross Reactions By using the device prepared according to Example 5 and the method according to Example 6, approximately 144 microorganisms were tested at concentrations of 10 ° to 109 CFU / mL. Each of the tested organisms was grown on an appropriate agar and incubated at 37 ° C overnight with 5% C02, after which the plates were checked for purity and well isolated colonies were selected from each one of the microorganisms tested. Of the 144 organisms, only one -S. my tis, A.T.C.C. # 49456, gave a positive result and therefore presented cross-reactivity. This was to be expected, as noted above, because S. mytis is known to contain the wall antigen.

- Cellular polysaccharide C which is the one detected according to the design of the test of the present invention.

Negative results were obtained in the assay of the present invention with each of the following organisms: Acinetobacter ani tratus (A.T.C.C. # 49139), Acinetobacter baumanii (A.T.C.C. # 1906-T), Acinetobacter calcoaceticus (A.T.C.C. # 49466), Acinetobacter haemolyticus (A.T.C.C. # 19002), Adenoviruses 2 and 3 (pure culture mixture obtained at the Center for Disease Control), Alcali faecalis (ATCC # 6633), Bordetella pertussis (ATCC # 3467), Branhamella catarrhalis (ATCC # 25238-T), Blastomyces dermati tidis (pure culture obtained at the Center for Disease Control, unknown strain number), Candida albicans (ATCC # 's el0231, page 22 orig 14053 and 60193, each tested separately), Candida stellatoides (ATCC # 11006 ), C trobacter freundii (ATCC # 375GT), Coccidioides immi tis (pure culture obtained at the Center for Disease Control, number of unknown strain), Corynebacterium kutscheri (ATCC # 15677-T), Corynebacterium ma truchotii (ATCC # 14266-T), Corynebacterium pseudodipheri ticum (A.T.C.C. # 10700-T), Enterobacter cloacae (A.T.C.C. # ^ s 13047-T, 23355, 35030 and 49141, each tested separately), - - Enterococcus avi um (ATCC # 49462), Enterobacter durans (ATCC # 49135), Enterococcus faecali s (ATCC # ^ s 19433-T, 29212, 49477, 49478, 49149 and 51299, each tested separately), Escherichia coli (ATCC # Ns 23513, 8739, 23514, 25922, 35218, 1173GT, 35421 and 15669 and an unnumbered sample, each tested separately), Escherichia hermannii (ATCC # "s 33650-T and 4648GT, each tested separately), Flavobacterium indologenes (ATCC # 49471), Flavobacterium um meningosepticum (ATCC # 49470), Gardnerella vaginalis (ATCC # 14018-T), Haemophilus infl uenzae, a (ATCC # 9006), Haemophilus infl uenzae, b (ATCC # s 9795 and 33533, each tested separately), Haemophilus infl uenza, c (ATCC # 9007), Haemophilus infl uenzae, d (ATCC # 9008), Haemophilus infl uenzae, e (ATCC # 8142), Haemophilus infl uenzae , f (ATCC # 9833), Haemophilus infl uenzae, NT (ATCC # * s 49144, 49247 and 49766, each tested separately), Haemophilus parainf l uenzae (ATCC # 3339Z-T, obtained in the form of a pure culture at the Center for Disease Control), Histoplasma capsulatum ( two pure cultures separately from the Center for Disease Control, unknown strains, each tested separately), Klebsiella oxytoca (ATCC # s 43086 and 49131, each tested by - separated), Klebsiella pneumoniae (A.T.C.C. # ~ S 13882, 13883-T and 49472, each tested separately), Lactobacillus acidophilus (A.T.C.C. # 4356), Lactobacillus casei (ATCC # 393), Lactobacillus gasseri (ATCC # 33323), Lactobacillus jensenii (ATCC # 25258), Legi onella pneumophila (ATCC # 33152), Listeria monocytogenes (ATCC # 7644), Micrococcus l uteus (ATCC #ss 9341 and 49732, each tested separately), Moraxella osloensis (ATCC # 15276), Morganella morganii (ATCC # 25830-T), Mycoplasma geni talium (ATCC # 33530, obtained in the form of a pure culture at the Center for the Disease Control), Mycoplasma hominis (ATCC # 27545, obtained in the form of a pure culture at the Center for Disease Control), Mycoplasma pneumoniae (FH type 2, obtained as a pure culture at the Center for Disease Control ), Neisseria cinerea (A.T.C.C. # 14685), Neisseria gonorrheae (A.T.C.C. #% s 8660, 19424-T and 27631, each tested separately), Neisseria lactamica (A.T.C.C. # 23970-T), Neisseria meningi tidis (A.T.C.C. # 13077-T), Neisseria subflava (A.T.C.C. # 49275), Nocardia farcinia (obtained in the form of a pure culture at the Center for the Control of Diseases), Paracoccidioides brasiliensis (number of unknown strain, obtained in the form of a pure culture in - - the Center for Disease Control), Parainf l uenzae Type 1 (strain C 39, obtained in the form of a pure culture at the Center for Disease Control), Parainf l uenzae Type 2 (strain HA 47885, obtained in the Center for Disease Control in the form of pure culture), Proteus mirabili s (ATCC # "s7002 and 12453, each tested separately), Proteus vulgaris (ATCC # sl3315-T and 49132, each tested separately), Providencia stuartii (ATCC # 49809), Pseudomonas aeruginosa (ATCC # * s 15442 and 27853, each tested separately ), Pseudomonas cepacia (ATCC # 25416-T), Pseudomonas pickettii (ATCC # 49129), Pseudomonas putida (ATCC # 49128), Pseudomonas putrefaciens (ATCC # 49138), Pseudomonas stutzeri (ATCC # 17588-T), Syncytial Virus Respira torio , mixed (sample as a mixture of strain A2 and strain ATCC # 18573, each one obtained at the Center for Disease Control in the form of pure culture), Rhinovirus (ATCC # "s 088 and 077, each obtained in the form of a pure culture at the Center for Disease Control, and each tested separately), Cuban Salmonella (ATCC # 12007), Salmonella enteri tidis (ATCC # 13076-T), Salmonella paratyphi A (ATCC # 9150) , Salmonella typhi (ATCC # 6539), Serra tia marcescens (ATCC - - # 13880-T), Sphingobacterium mul tivorum (ATCC # 35656), Staphylococcus aureus (ATCC # * s 12598, 6538P, 25923, 29213, 43300 and 49476, each tested separately), Staphylococcus epidermidis (ATCC # s 12228, 14990-T, 49134 and 49461, each tested separately), Staphyl ococcus haemolyticus (ATCC # 29970-T), Staphyl ococcus saprophyticus (ATCC # ^ s 15305-T and 49907, each tested separately), Staphylococcus xylosis ( ATCC # 49148), Stenotrophomonas mal tophilia (ATCC # 13637-T), Streptococcus anginosus (A.T.C.C. # 9895), Streptococcus bovis (A.T.C.C. # 49133), Streptococcus, Group A (ATCC # ^ s 1357 and 19615, each tested separately), Streptococcus, Group B (ATCC # "s 13813-T, 12386, 12400, 12401, 27591, 12973, 12403 and 31475, each tested separately), Streptococcus, Group C (ATCC # 12388), Streptococcus, Group F (ATCC # 12392), Streptococcus, Group G (ATCC # 12394), Streptococcus mutans (strain Shockman), Streptococcus parasanguis (ATCC # 15909), Streptococcus sanguis (A.T.C.C. # 10556-T), Tri chomonas vaginalis (A.T.C.C. # ~ s 085 and 520, each obtained in the form of a pure culture at the Center for Disease Control and tested separately).

- - EXAMPLE 10 - Clinical Study with Healthy and Sick Children A clinical study, still not completed, with healthy and sick children as participants is in progress. Preliminary results show that, by using the devices of the present invention prepared in the manner described in Example 6 and following the procedure described in Example 7, S. pneumoniae was detected in urine of 2 of 3 children diagnosed with sinusitis . It is thought that the case of sinusitis in which the child's urine was negative may involve a different causative agent.

In the same studies, S. pneumoniae was detected with the device and the method of the present invention in the cerebrospinal fluid of only children who exhibited open signs of meningitis, making possible a fast and effective therapeutic treatment in this individual. EXAMPLE 11 - Detection of S. pneumoniae Antigen in Urine of Patients with Meningitis Two patients who exhibited general clinical symptoms of meningitis were hospitalized. One had received antimicrobial treatment before admission; the other, no. Cerebrospinal fluid was obtained from each one and was subjected to a culture test.

- The results of the test were negative and also the results of the blood culture. As a last resort of the diagnosis, the devices prepared according to Example 5 were used in the procedure described in Example 6 in the urine samples obtained from each patient. In each case the urine samples gave a positive test for the antigen of the cell wall polysaccharide C of S. pneumoniae. These preliminary results strongly suggest that urine samples can be routinely used instead of cerebrospinal fluid to test for meningitis caused by S. pneumoniae. The ability to replace urine with cerebrospinal fluid as a test medium, if confirmed by additional clinical trials, will be of great benefit to patients as well as to medical personnel. The spinal puncture, by which the cerebrospinal fluid must be obtained, is painful for the patients and also somewhat dangerous. For the medical staff, the spinal punctures are delayed and require concentration and attention to detail. The technicians in the field of immunochemistry and especially those experts in immunoassays, - - recognize that other materials and ingredients and times, as well as other procedural steps, may be easily substituted, other than those specifically recommended herein. A wide range of literature, both patent and non-patent, discusses the design and use of disposable immunoassay testing devices, reliable and single-use, which could be replaced by the preferred PIC device as described and recommended herein. The present invention is not intended to be limited with respect to substitutable test devices, materials, ingredients or process steps, except as limited by the following claims. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (30)

1. - - CLAIMS Having described the invention as an antecedent, the content of the following claims is claimed as property: 1. A method for obtaining a cell wall polysaccharide C antigen that contains no more than about 10% protein of the bacterium Streptococcus pneumoniae, characterized in that it comprises the steps of: (a) culturing the bacteria for a time necessary to obtain a sample of the desired size and harvesting the bacterial cells in the form of a moist cell pellet; (b) suspend the wet cell pellet in an alkaline solution and mix; (c) adjusting the pH to an acidic pH with a strong acid and centrifuging; (d) separating the supernatant from the step of part () and adjusting its pH to approximately neutrality; (e) digest this product with a broad-spectrum protease enzyme preparation, to destroy residual proteins; (f) adjusting the pH to the alkaline side with a weak alkaline aqueous solution; - - (g) separating the carbohydrate esentially free of protein or polysaccharide antigen, in a size exclusion column equilibrated with a weakly alkaline solution; and (h) mixing the eluted material in the first peak and adjusting its pH to approximately neutrality.
2. 2. A cell wall polysaccharide C antigen, characterized in that it contains no more than about 10% protein, obtained by the method according to claim 1.
3. 3. A method according to claim 1, characterized in that the alkaline solution of the stage of part (b) comprises approximately 20 mL per gram of wet cell pellet of 0.1M aqueous sodium hydroxide.
4. 4. A method according to claim 1, characterized in that in the step of item (c) the pH is adjusted to approximately 3.0
5. 5. A method according to claim 1, characterized in that in the step of subsection (f) the pH is adjusted to a pH between about 10 and about 11.
6. 6. A method according to claim 1, characterized in that, after the - step of section (h), a lyophilization step is carried out.
7. 7. A method of purification of crude antibodies against S. pneumoniae, characterized in that it comprises the steps of: (a) separating from the S. pneumoniae bacterium, in accordance with the method of claim 1, a cell wall polysaccharide C antigen that it does not contain more than about 10% protein; (b) conjugating the antigen to one end of a double-ended spacer molecule, to form a conjugate of the antigen with the spacer molecule; (c) coupling the conjugate to an activated chromatographic column; (d) subjecting the crude antibodies to affinity chromatography on the column of the step of part (c), to obtain purified antigen-specific antibodies; (e) eluting purified antigen-specific antibodies from the column.
8. 8. Antigen-specific antibodies purified against the polysaccharide C of the cell wall of S. pneumoniae, characterized in that they are obtained by the method of claim 7. - -
9. 9. A chromatographic column for the affinity purification of crude antibodies against S. pneumoniae that have covalently coupled thereto, by means of a separating molecule, a cell wall antigen polysaccharide C of S. pneumoniae according to claim 2.
10. 10. A method for testing the presence of S. pneumoniae or its cell wall polysaccharide C antigen in a fluid, characterized in that it comprises the steps of: (a) extracting the S bacteria. pneumoniae the polysaccharide C antigen of the cell wall according to claim 2, (b) coupling the antigen to a spacer molecule to form a conjugate, (c) coupling the conjugate from the step of part (b) to an affinity chromatography column , (d) purifying the crude antibodies against S. pneumoni bacterium ae in the affinity chromatography column of step (c), to produce purified antigen-specific antibodies; and (e) using the antibodies purified from the step of part (d) to detect the presence or absence of S. pneumoniae or its cell wall antigen polysaccharide C, in a fluid .. - -
11. 11. The method according to claim 10, characterized in that the separating molecule of the step of part (b) is a protein molecule.
12. 12. The method according to claim 10, characterized in that the separating molecule of the step of part (b) is a conjugate of hydrazine and bovine serum albumin.
13. The method according to claim 10, characterized in that the fluid from the passage of part (e) is a natural fluid of mammalian origin.
14. 14. The method according to claim 13, characterized in that the fluid is human urine.
15. 15. The method according to claim 13, characterized in that the fluid is obtained from a patient exhibiting clinical signs of a pneumonia-type disease.
16. 16. The method according to claim 15, characterized in that the step of subsection (e) is an immunoassay process.
17. 17. The method according to claim 15, characterized in that the step of - - subsection (e) is an immunochromatographic test process ("PIC").
18. 18. The method according to claim 17, characterized in that a portion of the antigen-specific antibodies purified from the step of part (d), are conjugated with a labeling agent that is known to show a visible color when the antibodies react with its corresponding antigen.
19. 19. The process according to claim 18, characterized in that the marking agent is finely powdered metallic gold.
20. 20. A PIC assay for the detection of the S bacterium. pneumoniae or the polysaccharide C cell wall antigen thereof, characterized in that it comprises: (a) contacting a sample of a liquid suspected of containing the bacterium or its antigen with a PIC device comprising a strip of a absorbent material, wherein the strip has (i) a first zone in which a conjugate of (1) a labeling agent that shows a visible color change after the reaction, is fixed. - the antibodies with their corresponding antigenic binding partner and (2) antigen-specific antibodies purified against the polysaccharide C cell wall antigen of S. pneumoniae, wherein these antibodies have been purified by passing them through an affinity chromatography column, such as described in claim 9, (ii) a second zone having attached thereto the same antigen-specific antibodies purified in unconjugated form, wherein this zone is equipped with a window to observe the color changes, (b) allow that the sample flows laterally along the test strip towards the first zone, (c) allowing the sample, together with the conjugate of antigen-specific antibodies and tag, to flow laterally along the test strip into the second zone, and (d) within a period of approximately 15 minutes from the start from step (a), observe through the window if a colored line appears in the second zone, which indicates the presence of S. pneumoniae or its cell wall polysaccharide C antigen in the sample.
21. 21. The method according to claim 20, characterized in that the fluid suspected of containing S. pneumoniae bacteria or its polysaccharide C cell wall antigen is a natural fluid of mammalian origin.
22. 22. The method according to claim 21, characterized in that the natural fluid of mammalian origin is human urine.
23. 23. The method according to claim 21, characterized in that the natural fluid of mammalian origin is blood or serum.
24. 24. The method according to claim 21, characterized in that the natural fluid of mammalian origin is cerebrospinal fluid.
25. 25. The method according to claim 22, characterized in that the marking agent that shows a visible color change after the reaction of the antibodies with their corresponding antigen pairs, is a finely powdered metal.
26. 26. The method according to claim 25, characterized in that the marking agent is finely powdered gold.
27. 27. The method according to claim 20, characterized in that the agent of - - Marking is gold finally pulverized and the fluid is blood or human serum.
28. 28. The method according to claim 20, characterized in that the marking agent is finely powdered gold and the fluid is human cerebrospinal fluid.
29. 29. The method according to claim 20, characterized in that the marking agent is finely powdered gold and the fluid is human urine.
30. 30. A PIC device for the detection of S. pneumoniae or its cell wall antigen polysaccharide C, characterized in that it comprises a strip of an absorbent material having (i) a first zone having fixed therein a conjugate of (1) a labeling agent showing a visible color change after the reaction of the antibodies with their corresponding antigenic pair and (2) Antigen-specific antibodies purified against S-cell polysaccharide C-cell antigen. pneumoniae, wherein the antibodies were purified by passing them through an affinity chromatography column according to claim 9; Y . - - (ii) a second zone that has the same anti-antigen-specific antibodies purified in unconjugated form, where this zone is equipped with a window to observe the color changes. SUMMARY OF THE INVENTION The present invention relates to a process for obtaining a polysaccharide C cell wall antigen that contains no more than about 10% protein of the bacterium Streptococcus pneumoniae. The antigen obtained in this way is conjugated with a separating molecule and the free end of the latter is subsequently conjugated with an affinity chromatography column. The column is then used to purify crude antibodies against S. pneumoniae bacteria, thereby producing antigen-specific antibodies. A portion of these antibodies is conjugated with a labeling agent that shows a visible color change after the reaction of the antibodies with their respective antigen and is included in a first zone of an immunochromatographic assay device. Another portion of such antibodies binds to the reaction zone of the device, which has a window. When a liquid sample, such as urine, cerebrospinal fluid or blood of a patient is applied to the first zone, the conjugated antibodies and the labeling agent and the sample are moved along a strip of an absorbent material towards the area of reaction, in which, if the sample contains S. pneumoniae or some antigen from its cell wall, a sandwich is formed between the labeled conjugate, the antigen and the bound antibodies, and a color change is observed. The inocromatographic assay carried out in this way concludes a period of approximately 15 minutes. This assay provides 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 a primary pneumonic infection caused by this bacterium persists without yielding for a period of 3 to 5 days.