FI102217B - Method for the detection of Pseudomonas aeruginosa serotypes in a sample by in vitro monoclonal antibodies, the monoclonal antibodies used in the method and the test kit used in the method - Google Patents

Description

1 102217

Method for the detection of Pseudomonas aeruginosa serotypes in a sample by in vitro monoclonal antibodies, the monoclonal antibodies used in the method and the test kit useful in the method 5

Divided separated from application 865 027.

The invention relates to an immunological method useful in the diagnosis of bacterial infections, in particular in the diagnosis of Pseudomonas aeruginosa serotypes. More specifically, the invention relates to a method for detecting Pseudomonas aeruginosa serotypes in a sample in vitro, comprising using human monoclonal antibodies or binding fragments thereof that specifically react with at least two Pseudomonas aeruginosa IATS serotypes, and detecting the complex formed. The invention also relates to human monoclonal antibodies for use in this method and to a test kit that can be used in this method.

20 The disease caused by Gram-negative bacteria and its most serious complications, such as bacteremia and endotoxemia, cause significant morbidity and mortality in human patients. This applies in particular to the Gram-negative organism, Pseudomonas aeruginosa. ' 25 which has been increasingly associated with bacterial infections, particularly nosocomial infections, over the past fifty years.

The literature presents a number of serotyping programs that could be used to investigate the presence of anti-Pseudomonasvas 30 agents in cell supernatants.

: The programs are mainly based on thermostable somatic major antigens of this organism (see Zierdt, C.

H. in Glucose Nonfermenting Gram-Negative Bacteria in Clinical Microbiology, ed. Gilardi, G. L., CRC Press 35 1978, pp. 213-238). The increase in serotyping guidelines 2 102217 has made it quite difficult to compare serological tests for P. aeruginosa, and thus the choice of a particular system for testing supernatants appears to be somewhat random. Se-5 convenience between typing systems has recently been reduced by the International Antigenic Typing Scheme (IATS) proposed by the Subcommittee on Pseudominadaceae of the International Committee on Systematic Bacteriology as a framework for more detailed serological testing of P. aeruginosa. This 10 system, which contains 17 different serotypes designated IATS type 1, IATS type 2, etc., covers all thermostable somatic major antigens identified in previous systems. See Liu, P. V., Int. J. Syst. Bacteriol. 33 (1983) 256-264, which is incorporated herein by reference.

When developing monoclonal antibodies that cross-react with P. aeruginosa strains, it is also advantageous to include a typing program based on P. aeruginosa protective antigens. Such a program is designed to develop a vaccine for clinical use and is described in detail in Fisher, M. V. et al., J. Bacteriol. 93 (1969) 835-836.

This system, commonly referred to as the Fisher typing system, classifies the majority of known P. 25 aeruginosa strains into seven types, denoted Fisher immunotype 1, Fisher immunotype 2, etc. The correlation between the IATS and Fisher typing systems has been elucidated in the literature. (see Liu, PV et al., supra), and is shown in Table I. As shown in Table I, there are no corresponding Fisher immunotypes for certain IATS serotypes, although each Fisher immunotype corresponds to a particular IATS serotype. In the case of the IATS and Fisher typing systems, antigenic determinants important for both serotyping systems are believed to be located on P. aeruginosa surface 3 -102217 ta-LPS molecules [Liu, P.V. et al., Supra; Hanessien; F et al., Nature 229 (1979) 209-210].

Table I

5 Gating and correlation of IATS and Fisher typing programs for Pseudomonas aeruginosa IATS Fisher 1 4 2 3 10 3 4 5 7 6 1 7 15 8 6 • · 9 10 5 11 2 12 20 13 14 15 16 17

In 1975, Kohler and Milstein published their evolutionary finding that certain mouse cell lines could be fused to mouse spleen cells to produce hybridomas, each of which secretes an antibody specific for one agent, i.e., monoclonal antibodies [Kohler, G., and Milstein]. , C., Nature 256 (1975) 495-5497]. With the invention of this technique, it became possible in some cases to produce large amounts of fully specific murine antibodies to one or more determinants on antigens.

Using the hybridoma technique, Sadoff et al. have reported 10 produced a murine IgM monoclonal antibody against the O-chain determinant of LPS molecules of a particular serotype of P. aeruginosa (Abstracts of the 1982 Interscience Conference on Antimicrobial Agents and Chemotherapy, No. 253). They further report that this mouse antibody protected mice from a lethal attack by P. aeruginosa of the same serotype as that against which the antibodies were directed (i.e., a homologous serotype). A few subsequent articles detail 20 murine and human monoclonal anti-Ps with different specificities. development of aeruginosa LPS antibodies; for example, Sawada, S. et al., J. Inf. Dis. 150 (1984) 570-576; Sadoff, J. et al., Antibiot. Chemother. 36 (1985) 134-146; Hancock, R. et al., Infect. Immun. 37 (1982) 166-171; Siadak, A. W. 25, and Lonstrom, M. E. in Human Hybridomas and Monoclonal Antibodies, eds. Engleman, EG et al., Plenum Publishing Corp. 185, (pp. 167-185; and Sawada, S. et al., J. Inf. Dis. 152 (1985) 965-970. Serotype-specific human monoclonal anti-P. The production and immunotherapeutic use of aeruginosa LPS antibodies is described in open U.S. Patent Applications 734,642 and 828,005, which are incorporated herein by reference.

Although in some situations, such as when an infection can be traced to one particular serotype, there may be certain advantages in using monoclonal antibodies specific for one P. aeruginosa s 102217 serotype, they would not be advantageous in many other situations.

Thus, there is a significant need to provide 5 human monoclonal anti-β. aeruginosa antibodies with the ability to recognize multiple P. aeruginosa serotypes.

The present invention relates to a method for detecting Pseudomonas aeruginosa serotypes in a sample in vitro, the method being characterized by contacting the sample with optionally labeled human monoclonal antibodies that specifically react less than all but at least two Pseudomonas aeruginosa IATS serotypes. 15 binding fragments, and the complex formed is detected, whereby monoclonal antibodies can be prepared by cell line ATCC No. CRL 8941, 9171 or 9258.

The invention also relates to human monoclonal antibodies and a test kit for use in detecting Pseudomonas aeruginosa serotypes as claimed.

The present invention provides novel cells capable of producing human monoclonal antibodies, and compositions comprising such antibodies, which have the ability to selectively recognize multiple, and in some cases all, strains of P. aeruginosa, wherein the individual antibodies typically recognize multiple IATS serotypes of P. aeruginosa and none or one or more Fisher immunotypes. The cells in question have identifiable chromosomes in which their or the precursor cell's cell line DNA is arranged to encode an antibody with a binding site for certain P. aeruginosa serotypes on a common epitope. These human monoclonal antibodies can be used in many ways for diagnosis.

The cells used in this invention are typically transformed human lymphocytes that produce mono-5 clonal human antibodies against achievable LPS molecules of P. aeruginosa. By "achievable" is meant that the LPS molecules are physically available in the environment of use to directly interact with the monoclonal antibodies.

The production of monoclonal antibodies can be accomplished by stabilizing the expression of nucleic acid sequences encoding antibodies specific for an epitope on multiple serotypes of P. aeruginosa on LPS molecules. Monoclonal antibodies are typically produced by cell-directed transformation with Epstein-Barr virus (EBV) of B lymphocyte cells obtained from human donors who are or have been exposed to one or more appropriate P. aeruginosa serotypes. The antibody-secreting cell lines thus produced are characterized as continuously growing lymphoblastoid cells with a diploid karyotype that are Epstein-Barr nuclear antigen positive and that secrete a monoclonal antibody that is IgG, IgM, IgA, or IdD isotype, including various subtypes such as IgG1, IgG2, IgG3 and IgG4. The cell-directed transformation process itself is described in detail in U.S. Patent 4,464,465, which is incorporated herein by reference. Monoclonal antibodies can be used whole or in fragments, such as Fv, Fab and F (ag ') 2 fragments, but usually whole.

Alternatively, antibody-producing cell lines could be produced by cell fusion between appropriately drug-labeled human myeloma, mouse myeloma, human-mouse heteromyeloma, or human lymphoblasting dis cells and human B lymphocytes to yield human hybrid cell lines.

Lymphoblastoid or hybrid cell lines can be cloned and probed by conventional methods with antibodies capable of binding to epitopes of various P. aeruginosa serotypes observed in cell supernatants. One aspect of the invention is human monoclonal antibodies capable of specifically binding to lipopolysaccharide determinants present in several but not all IATS serotypes of P. aeruginosa. For example, determinants may be present in two or three IATS serotypes, and not in any one or at least two Fisher immunotypes.

The monoclonal antibodies of this invention can be used in a variety of ways in vitro. By way of example, monoclonal antibodies may be used for typing, isolation of certain P. aeruginosa strains, selective removal of P. aeruginosa cells in a heterogeneous cell mixture, or the like.

When monoclonal antibodies are used for diagnostic purposes, they may be either labeled or unlabeled. Diagnostic assays typically detect a complex formed by the binding of a monoclonal: * 25 antibody to LPS of P. aeruginosa. Unlabeled antibodies can be used for agglutination studies. In addition, unlabeled antibodies can be used in conjunction with other labeled antibodies (other antibodies) that react with the monoclonal antibody, such as in combination with immunoglobulin-specific antibodies. Alternatively, monoclonal antibodies can be labeled directly. A wide variety of markers can be used, such as radionuclides, fluorescent agents, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (especially hapten), etc. Many types of immunoassays are available. 827, 3,850,752, 3,901,654, 3,935,074, 5,998,533, 3,996,345, 4,034,074 and 4,098,876, all of which are incorporated herein by reference.

The monoclonal antibodies of this invention are commonly used in enzyme immunoassays in which the antibodies in question, or other antibodies from another species, are conjugated to the enzyme. When a sample containing P. aeruginosa of a particular serotype, such as human blood or a lysate thereof, is combined with the antibodies in question, binding occurs between the antibodies and the molecules containing the desired epitope. Such cells can then be separated from unbound reagents and another (enzyme-labeled) antibody added. The presence of an antibody-enzyme conjugate specifically bound to the cells is then determined. Other conventional methods well known to those skilled in the art may also be used.

The test kits of the invention are suitable for use in conjunction with the antibodies of the invention to detect the presence of P. aeruginosa infection or P. aeruginosa antigen. Thus, the monoclonal antibody composition of this invention may be supplied, usually lyophilized, either alone or in combination with other antibodies specific for other gram-negative bacteria. Kits include antibodies, which may be attached to the label or unlabeled, as well as buffers such as: Tris, phosphate, carbonate, etc. buffers, stabilizers, biocides, inert proteins, e.g., bovine serum albumin, and the like. the agents are present in less than about 5% by weight of the amount of active antibody, and generally have a total amount of at least about 9 102217 0.001% by weight of the antibody. It is often desirable to include an inert excipient in the composition to dilute the active ingredients, and the excipient may be present in an amount of about 1-99% by weight of the total composition. When a second antibody capable of binding to a monoclonal antibody is used, it is usually in a separate container. The second antibody is typically attached to a label and is formulated in a manner similar to the antibody formulations described above.

The monoclonal antibodies of this invention can be lyophilized for storage and mixed back into a suitable carrier prior to use. This method has been shown to be effective with conventional immunoglobulins, and freeze-drying and working solution mixing methods known in the art can be used. Those skilled in the art will recognize that lyophilization and reconstitution into a re-use solution may result in varying degrees of loss of antibody activity (e.g., with conventional immunoglobulins, IgM antibodies tend to suffer greater activity losses than IgG antibodies) and that application rates may need to adjust this loss to compensate.

Example I

Example I illustrates methods for producing human monoclonal antibodies (IgM isotype) reactive with IATS serotypes: 2.5 and 16 and Fisher immunotypes 3 and 7.

The source of human B cells was a peripheral blood sample obtained from a patient with cystic fibrosis known to have a chronic P. aeruginosa infection. Mononuclear cells were separated from blood by standard Ficoll-Paque centrifugation methods [Boyum, A; Scand. J. Clin. Lab. Invest. 21, Suppl. 97 (1968) 77-98] and washed twice in calcium / magnesium-free phosphate-buffered saline (PBS).

10 102217

Unicellular cells were purified from T cells using a modified E-rosette procedure. Briefly, cells were first suspended at a concentration of 1 x 10 7 cells / ml in PBS containing 20% fetal bovine serum (FCS) at 4 ° C. 1 ml of this suspension was then placed in a 17 x 100 mm round-bottomed polystyrene tube to which 1 x 10 9 2-aminoisothiouronium bromide (EET)-treated sheep erythrocytes were added as a 10% (v / v) solution in Iscove 10 modified form. In Dulbecco's medium (Iscoven's medium) [Madsen, M. and Johnson, HE, J. Immun. Methods 27 (1979) 61-74]. The suspension was stirred very gently for 5-10 min at 4 ° C, and the E-rosette cells were then removed by centrifugation for 8 min at Ficoll-Paque at 15,500 x g at 4 ° C. E-rosette-negative single peripheral blood cells (E'PBMC) that formed a zone at the interface were collected, washed once with Iscoven's medium, and resuspended in the same medium containing 15% v / v FCS, L-glutamine 20 (2 mmol / l). , penicillin (100 IU / ml), streptomycin (100 μg / ml), hypoxanthine (1 x 1C4 mol / l), aminopterin (4 x 10'7 mol / l) and thymidine (1.6 x 10'5) mol / 1). This medium is hereinafter referred to as HAT medium.

Cell-directed transformation of E'PBMC was performed in culture; 25 together with the transforming cell line. The transforming cell line was an Epstein-Barr nuclear anti-antigen-positive (EBNA-positive) human lymphoblast cell line derived from ethyl methanesulfonate (EMS) mutagenesis of the GM 1500 lymphoblastoid cell line followed by 30 selection of 6-thioguanine (30 μg / ml). ) ', in which case the cells became hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient and thus sensitive to HAT. This cell line has been designated the 1A2 cell line and deposited in the American Type Culture 35 Collection (ATCC) on March 29, 1983 under ATCC accession number u 102217 CRL 8119. Logarithmically growing IA2 cells were suspended in HAT medium and then combined with E "PBMCs relative to 8 1A2 cells / 1 E ~ PBMC cell.

The cell mixture was inoculated into eight round-bottom 96-5-well microtiter plates (Costar 3799) at a concentration of 72,000 cells / well at a volume of 200 μΐ / well and incubated at 37 ° C in a humidified atmosphere containing 5% CO 2. Cultures were supplemented on days 5 and 8 after inoculation with a cor-10 dowel, half of the supernatant with fresh HAT medium. Cells were monitored every other day with an inverted microscope for signs of cell proliferation. Twelve days after inoculation, it was found that 100% of the wells contained proliferating cells and 15 that most of the wells had cells at a sufficiently dense removal and examination of the supernatants for anti-P. aeruginosa antibody.

Supernatants were tested for anti-P. the presence of aeruginosa antibodies using an ELISA method. The antigen plates were flat-bottomed 96-well microtiter plates (Immulon II, Dynatech) containing various live bacteria adsorbed to the bottom of the well. To absorb the bacteria into the plastic, 50 μΐ / well of poly-L-lysine (PLL) (1 μg / ml in PBS, pH 7.2) was incubated for 30 min at room temperature. The adsorbed PLL was then removed, the plates were washed once with PBS, 50 μl of washed bacterial suspension (00 = 0.2) in PBS was added to each well, and the plates were incubated for 1 hour at 37 ° C. Unadsorbed bacteria were removed by washing the plates three times with physiological saline containing Tween (0.9% NaCl, 0.05% v / v Tween-20). The various antigen plates used in the study were: (1) a mixture of P. aeruginosa Fisher immunotypes 1, 2, and 4 (ATCC Nos. 27312, 27313, and 27315); (2) a mixture of 35 P. aeruginosa Fisher immunotypes 3, 5, 6, and 7 12 102217 (ATCC Nos. 27314, 27316, 27317, and 27318, respectively); and (3) a microtiter plate without bacteria.

The ELISA was started by first protecting the plates with protection buffer [200 μΐ / well, PBS, pH 7.2, containing 5% 5 (w / v) skim milk powder, 0.01 ti] auxiliary% Antifoam A (Sigma) and 0.01% (w / v) thimerosal], treatment time 60 min at room temperature. After protection, the plates were washed three times with physiological saline containing Tween. All wells 10 were then charged with 50 μΐ PBS, pH 7.2 containing 0.1% Tween-20 and 0.2% (w / v) bovine serum albumin (BSA). Supernatants from the wells of the culture plates were replicated inoculated into the corresponding wells of the antigen plates (50 μΐ / well), and the plates were incubated for 30 min at 15 room temperature. The supernatants were then removed, the wells were washed three times with Tween-containing saline, and 50 μ ja of appropriately diluted horseradish peroxidase (HRP) -contained goat anti-human (IgG + IgN) antibody (American Qualex International # A1114 # #) was added to the wells. A1124). In this example, the final dilution ratio of HRP (goat anti-IgG) and HRP (goat anti-IgM) was 1: 5,000 and 1: 3,000 respectively in PBS, pH 7.2, containing 0.05% Tween -20 and 0.1% BSA. After incubation for 30 min at room temperature, excess goat antibodies attached to the enzyme were removed and the wells were washed three times with physiological saline containing Tween. 100 μ 100 of substrate (0.8 mg / ml o-phenylenediamine dihydrochloride in 100 mM citrate buffer, pH 0.5 + 0.03% H 2 O 2 in deionized water) was then added to each well, mixed in equal volumes just prior to transfer to the plate. ). After incubation in the dark for 30 min, 50 μΐ 3 N H 2 SO 4 was added to all wells to stop the reactions. Culture supernatants containing antibodies reactive with 35 plates of antigen, 13 × 02217, were detected by color development in the corresponding wells, and the intensity of the reaction was quantified by measuring the absorbance at 490 nm on a Bio-Tek EL-310 micro-ELISA meter.

Analysis of the culture supernatants by the method described above resulted in the identification of two wells (1C1 and 2H12) containing anti-P. aeruginosa antibodies that react with a plate containing Fisher immunotypes 3, 5, 6, and 7, but not with a plate containing Fisher immuno-10 types 1, 2, and 4, nor with a bacterial-free plate. In order to identify one or more specific Fisher immunotypes identified, antigen plates containing only PLL-attached bacteria of one Fisher immunotype were prepared from each immunotype. Performing an ELISA assay on culture supernatants obtained from wells 1C1 and 2H12 in plates containing individual immunotypes as described above showed that these two wells contained an antibody specific for both Fisher immunotype 20 3 and 7. A similar ELISA was performed in series P.

aeruginosa IATS-type strains (obtained from the ATCC and contained Nos. 33348-33364) showed that the antibody in wells 1C1 and 2H12 reacted specifically with IATS strains 2, 5, and 16.

I! The isotype of one or more reactive antibodies in wells 1C1 and 2H12 was otherwise determined by an ELISA similar to that in the specificity assays, but HRP (goat anti-human IgG) and HRP (goat anti-human IgM) were used separately in the second step. reagent-30 wall and not combined. A positive reaction of one or more antibodies of wells 1C1 and 2H12 with Fisher immunotypes 3 and 7 was observed only with anti-IgM reagents, indicating that one or more relevant antibodies of each well are of the IgM isotype. .

14 102217

To determine whether one or more antibodies from wells 1C1 and 2H12 caused the anti-Fisher immunotype 3 and 7 reaction model <i.e. one antibody reacting with both Fisher immunotype 3 and 7, or two antibodies, one reacting with Fisher immunotype 3 and the other with Fisher immunotype 7), cells of each well were cultured at low density, and proliferating cells were examined. antibody activity on separate Fisher immunotype 3 and Fisher-im-10 egg type 7 bacterial antigen plates. Subculture was performed in 96-well round-bottom plates at a density of 5 cells / well in a total of 100 μΐ of HAT medium lacking the aminopterin component (HT medium). Non-transformed HAT-sensitive lymphoblastoid cells were included in all wells at 500 cells / well as feeder cells. Four days after inoculation, 100 μΐ HAT medium was added to all wells to selectively kill mast cells. On day 9 after inoculation, supplemental food was added to the wells by replacing 20 half of the supernatant with HAT medium. Thereafter, HT medium was added to the wells in the same manner every 4-5 days until the density of lymphoblastoid cells in the wells was sufficient to analyze the supernatant by ELISA. When examined on single Fisher immunotype 3 25 and Fisher immunotype 7 antigen-containing plates, all supernatants that reacted with Fisher immunotype 3 also reacted with Fisher immunotype 7, indicating that one antibody caused activity on both Fisher immunotypes. When randomly selected supernatants with IATS strains 2, 5, and 16 were examined, they were found to react with all three strains instead of individual strains, further supporting the conclusion that one antibody cross-reacted with Fisher immunotypes 3 and 7 and IATS strains. 2, 5 and 16 with 35.

1β 102217

Cloning of cells producing specific antibody in wells 1C1 and 2H12 was performed by performing several limiting dilution clonings on cells in each well until all 5 clone supernatants analyzed by the above ELISA program gave a positive reaction with Fisher immunotypes 3 and 7 and IATS strains 2, 5 and 2. Feed cells were used for cloning as described above for subculture. In this way, two cloned transformed human cell-10 lines (1C1 and 2H12) were obtained, which are continuous (immortal) and both secrete a human monoclonal antibody reactive with Fisher immunotypes 3 and 7 and IATS strains 2, 5 and 16. In this example, the same label is used for the cell line and the antibody it produces.

Example II

Example II illustrates methods for producing a human monoclonal antibody (IgG isotype) 20 that reacts with IATS serotypes 2, 5, and 16 and Fisher immunotypes 3 and 7. The production programs are approximately the same as in Example I. Briefly, the source of human B cells was a peripheral blood sample obtained from a patient with cystic fibrosis who had a chronic P. aeruginosa infection. Unicellular cells differential; 25 was otherwise used as described in Example I, but 1.6 ml of PBS suspension with 1.6 x 10 9 AET-treated sheep erythrocytes was used. Cell-directed transformation was also performed in the same manner with the following exceptions: the ratio of 1A2 to E "PBMCs was 7.5; 15 plates with a cell density of 17,000 / well were used; cultures were supplemented on days 6 and 10 after inoculation. and 16 days after inoculation, almost all wells contained proliferating cells.

Examination of specific antibodies from culture supernatants was performed as described and resulted in the discovery of a single well (9D1) containing an antibody that reacted with a plate containing Fisher immunotypes 3, 5, 6, and 7, but not With a plate containing Fisher immunotypes 1, 2 and 4 and not a bacterial-free plate. Performing the described 5 ELISA assay on plates containing a single immunotype or serotype bacterial antigen using 9D1 culture supernatant showed an antibody specific for both Fisher immunotypes 3 and 7 as well as IATS strains 2, 5 and 16. According to Example I-10. further experiments performed showed that the antibody specificity observed in well 9D1 was associated with one clone secreting an IgG isotype antibody.

Example III

Example III demonstrates the antigen specificity of some of the antibodies of this invention.

To determine whether antibodies 1C1, 2H12, and 9D1 reacted with the same antigenic target and were thus identical, additional experiments were performed. Antibodies were first compared in an ELISA using a series of reference chickens and clinically isolated P. aeruginosa samples.

The ELISA program was as described above with the following modifications: 1) Instead of bacteria adsorbed on the surfaces of PLL-coated plates, the wells of the plate contained various whole bacteria fixed with ethanol at the bottom of the well. Plates were prepared by adding a 50 μΐ suspension of washed bacteria (00 ^ = 0.2) in PBS to the wells, centrifuging the plates for 20 min at 500 x g, aspirating off PBS, adding 75 μΐ ethanol for 10 min, removing ethanol and then air drying. The antigen plates contained IATS strains 2, 5, 11, and 16 (ATCC Nos. 33349, 33352, 33358, and 33363, respectively) and 16 clinically isolated samples pre-typed and agglutinated with Bacto-Pseudomonas aeruginosa from Difco (Detroit, Michigan). with the antiserum according to the manufacturer's instructions that the ELISA (as described in it 102217) is serotypes 2, 5, 16 or a combination of these serotypes; 2) rabbit typing antisera were used diluted 1: 500 in PBS with the exception of anti-IATS 16 diluted 1: 250.

Culture supernatants containing 1Cl, 2H12 and 9D1 antibodies were used as such; 3) biotinylated protein A (B-2001, Vector Laboratories, Inc., Burlingame, CA) diluted 1: 500 and biotinylated goat anti-10 human Ig (4703, Tago, Inc., Surlingame, CA) diluted 1: 500 for the detection of rabbit and human antibodies, respectively. 50 μΐ of reagent was added to the appropriate wells, unbound reagent was removed after incubation for 30 min at room temperature, and 15 wells were washed three times. 50 μ 50 of a preformed complex of avidin and biotinylated horseradish peroxidase (Vectastain ABC Kit, Vector Laboratories, Inc., Burlingame, CA) was then added to each well. After incubation for 30 min at room temperature, the excess 20 Vectastain ABC reagent was removed and the wells were washed again three times before the substrate was added.

As shown in Table II, the results of the experiment showed that although antibodies 1C1 and 9D1 reacted with each clinically isolated sample that was; 25 typed as IATS serotypes 2, 5, or 6, antibody 2H12 did not react with three such samples. This suggests that antibody 2H12 recognized a different epitope than 1C1 and 9D1, and that the two epitopes appear to co-exist in most but not all of the 30 clinically isolated samples corresponding to IATS, serotypes 2, 5, or 16.

1β 102217

Table II

Dlfcon Bacto-P. Reaction of aeruginosa antisera and xnonoclonal human antibodies 2H12, 1C1 and 9D1 with type strains and clinically secreted P. aeruginosa 5 samples

Type strain or Rabbit____Human _ clinically isolated-——.

tv sample IATS IATS IATS IATS MAb MAb MAb __a2 a5 ai 6 all 2H12 1C1 9D1 10 IATS 2 __ + __-__-__-__ + __ + __ + _ IATS 5 __ (+) * + __-__-__ + __ + __ + _ IATS 16 __ (+) a (a) * + __-__ + __ + ___ + _ IATS 11 __: __-__-__ + __: __- _ A5 23 + + - + + + 15 B406 + + ( +) * - _ + _ + + C27 __ + __ + __-__-__ + __ + __ + D26 __ + __ + __-__-__ + __ + __ + F155 __ + __ + __ + _____ + __ + __ + F225__ + __ (+) a + __-__ + __ + __ + _ 20 F250 + + -_ ~ _ + + + F253 __: __ + __: __-__ + __ + __ + _ F255 __ + __ + __: __: __-__ + ____ + _ F256 __ + __-__: __-__-__ + __ + _ F396 __ + __ + __ + __-__ + __ + __ + 25 H217 __ + __ + __ + __-__ + __ + __ + _ H218 __ + __ + __-__-__ + __ + + _ H219 __-__ + __-__: __ + __ + + _ H220 + + - + + + H221 + + --- + + 30, a (+ ) = ELISA reaction very weakly positive.

The results further suggested that the molecular target identified by antibodies 1C1 and 9D1 was likely present in all clinically isolated P. aeruginosa samples that could be typed into 19 102217 IATS serotypes 2, 5, or 16, whereas antibody 2H12 the identified target appeared in a subset of such isolated samples.

To determine whether 1C1, 2H12, and 59D1 antibodies react with different antigens or alternatively

different epitopes of the same antigen, in which case these epitopes would be present in most but not all IATS.

For serotypes 2, 5, and 16, immunoblot analysis was performed.

Because the common antigenicity of IATS strains 2, 5, and 16 is caused by 10 apparently thermostable antigens (Liu, PV et al., Supra) and because thermal stability is an inherent property of lipopolysaccharide molecules mentioned earlier, LPS preparations from IATS strains 2, 5, and 16 were selected. antigen preparations for analysis. Crude LPS was prepared from type strains by extraction with physiological saline at 60 ° C [Orskov, F. et al., Acta. Path. Microbiol. Scand. Section B 79 (1971) 142 - 152]. LPS for each serotype (10 μg, determined from 2-keto-3-deoxyoctonate (KDO) content) 20 [Karkhanis, Y. D. et al., Anal. Biochem. 85 (1978) 595-601] was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) [Hancock, R. E. W. and Carey, A. M., J. Bacteriol. 149 (1977) 901-910] on a gel with a concentration gradient of 10- * 20%. The separated Mole-25 native specimens were transferred from the gel to a nitrocellulose membrane (NCM) as described in the literature [Towbin, H. et al., Proc. Natl. Acad. Sci. USA 76 (1979) 4350-4354], and the NCM spot was protected by holding for 1 hour in PBS containing Tween [Batteiger, B.et et al., J. Immunol. Meth. 55 30 (1982) 297-307]. The spots were then incubated for 1 hour at room temperature in 20 ml of spent culture supernatant obtained from cell line 1C1, 2H12 or 9D1. Each NCM spot was rinsed for 5 min with PBS containing Tween and then incubated for 1 hour at 25 ° C with goat anti-human (IgG + IgA + IgM) 20 ° 102217 (Zymed) attached to alkaline phosphate-35, which was diluted 1: 1,000 or 1: 1,500 with PBS-Tween. The spots were then washed for 5 min in PBS-Tween, after which the antigen-antibody interactions were visualized by incubating the spots for 15-20 min at 25 ° C in 30 ml of nitrosininetetrazolium / 5-bromo-4-chloro-3-indolyl phosphate (NBT- BCIP) substrate according to Learyn et al. (Proc. Natl. Acad. Sci. USA 80 (1983) 4045-4049]. Color development was stopped by rinsing the spot several times with deionized water. The spot patterns obtained with these three antibodies were clearly different. Antibody 2H12 recognized mainly a short a series of regularly spaced (i.e., ladder-shaped) small molecule molecules in antigen preparations prepared from serotypes 2, 5, and 16. This antibody also weakly recognized some of the larger spaced molecules that occur at regular intervals. using LPS preparations pretreated with proteinase 20 K at 60 ° C to destroy protein antigens did not alter the patterns.The small molecule bands corresponded exactly to the smaller forms of LPS molecules visualized by similarly performed SDS-PAG By E-gel electrophoresis in which antigens were not transferred to 25 NCM but stained specifically to detect the presence of LPS [Tsai, C.M. and Farsch, C.E., Anal. Biochem. 119 (1982) 115-119)], except that the lowest zone of the silver-stained gel (corresponding to the core region + lipid A of LPS) was not identified. Antibody 1C1 identified the same series of regularly spaced small molecules from antigen preparations made from serotypes 2, 5, and 16, but not 11, although the reaction intensity was not as high as that of 2H12. However, this antibody also strongly recognized a series of regularly spaced 35 larger molecules of serotypes 21 102217 2, 5, and 16, which, together with the identified smaller molecular bands, resulted in distinct ladder-like patterns. Pretreatment of antiserum preparations with proteinase K did not alter these patterns. Figures vas-5 again aligned the ladder-like zone patterns observed with LPS-specifically stained gels (i.e., they appeared to correspond to zone by zone), except that the zone corresponding to core + lipid A was not identified. Antibody 9D1 had a similar reaction pattern to antibody 1C1 for IATS strains 2, 5, and 16, but not 11. Again, the lowest zone of the silver-stained gel ladder pattern of the various LPS preparations was not identified, and pretreatment of the LPS preparations with proteinase K did not alter the overall pattern. Together, these findings indicated that LPS of serotypes 2, 5, and 16 was a molecular target recognized by antibodies 2H12, 1C1, and 9D1.

Example IV

Example IV describes a method for preparing a human monoclonal antibody reactive with P. aeruginosa IATS-20 serotypes 4 and 11 and Fisher immunotype 2. The procedure otherwise described in Examples I to III was repeated, but it was necessary to make certain modifications to isolate, sharpen, and examine the antibody described in this example. The following are changes in the procedures made and the results obtained with the monoclonal antibody described herein.

The source of human B cells was a person previously immunized with a high molecular weight polysaccharide prep-30 parate isolated from Fisher immunotype 3; [Pier, g. B., et al., Infect. Immun. 45 (1984) 309-313, which is incorporated herein by reference].

Cell-directed transformation of E-PBMC was performed by co-culturing these cells with the transforming cell line 1A2 35. Logarithmic growth phase IA2 cells 102217 22 were suspended in HAT medium and then combined with E-PBMCs at a ratio of 15 IA2 cells / l E ”PBMCs. The cell mixture was seeded in 14 microtiter plates at a concentration of 62,000 cells / well at a volume of 200 μΐ / well-5 nys. Cultures were supplemented with food on days 7 and 11 after inoculation, and it was found that by day 15, all wells contained proliferating cells.

Anti-P. aeruginosa antibodies, two antigen plates were used: 10 (1) a plate containing a mixture of P. aeruginosa Fisher immunotypes 1-7 (ATCC Nos. 27312, 27313, 27314, 27315, 27317 and 27318); and (2) a PLL-treated, bacteria-free microtiter plate.

By analyzing the culture supernatants according to the method of the above examples, about 100 wells containing anti-P were identified. aeruginosa antibodies that react with a plate containing Fisher immunotypes 1-7 but not with a PLL-treated, bacteria-free plate. In order to identify the identified one or more Fisher immunotypes, antigen plates were constructed as described above, in which each row contained only PL Fisher-attached bacteria of one Fisher immunotype. An ELISA assay was performed as described above for each anti-P. the culture supernatant from the aeruginosa-positive well was plated in rows on new antigen plates, identifying a number of wells containing antibodies specific for Fisher immunotype 2. To further analyze the serological specificity of anti-Fisher immunotype 2 antibodies, supernatants were examined by a similar ELISA assay performed on antigen plates containing each of 17 aerobinosa IATS serotypes in 35 separate wells. The results of this experiment showed that the vast majority of supernatants were specific for IATS serotype 11, although one supernatant, in well 6D6, was cross-reactive specific for IATS serotypes 4, 11, 13, and 14.

5 To determine whether the reaction pattern against IATS serotypes 4, 11, 13, and 14 was elicited by one or more antibodies in well 6D6, additional samples of the supernatant from well 6D6 were separately adsorbed against IATS serotypes 4, 7 (negative control). 11, 13 and 14, and the absorbed supernatants were then tested on PLL-attached bacteria of each of the five IATS serotypes by the ELI-SA method described above. Absorbations were performed by resuspending the concentrated bacterial cell pellet in an equal volume of supernatant for one hour in an ice bath, after which the supernatant was separated from the bacteria by centrifugation. The results of this experiment showed that IATS serotypes 4 and 11 adsorbed anti-mutant antibody activity but not activity against IATS-se-20 rototypes 7, 13, and 14, respectively. This indicated that at least two different anti-Ps were present in well 6D6. aeruginosa antibody, at least one of which cross-reacts with IATS serotypes 4 and 11.

Isolation and cloning of cells of well 25-producing wells producing the appropriate antibody were performed in three steps. In the first step, cells were subcultured at a low density (20 cells / well), and a second low-density subculture (5 cells / well) was performed on cells obtained from anti-IATS serotypes from a 4 + 11-positive well formed in the first min. -continuous subculture (20 cells / well). Each sub-culture was performed in 96-well round-bottom plates at a stated density for a total of 100 μΐ of HAT medium lacking the aminopterin component (HT intermediate 35). Untransformed HAT-sensitive lymphoblastoid cells were added to all wells as mast cells at a density of 500 cells / well. Four days after the last inoculation, 100 μΐ HAT medium was added to all wells to selectively kill mast cells. Cells were repopulated 9 days after inoculation by replacing half of the supernatant with HAT medium. The cells were then similarly fed at 4-5 day intervals with the addition of HAT medium until a sufficient lymphoblastoid cell density was present in the wells to analyze the supernatant by ELISA as described above.

Supernatants that reacted with IATS serotype 4 also reacted with IATS serotype 11 and Fisher immunotype 2 in each experiment. In addition, antibody activity against IATS serotype-15 small 13 and 14 had been lost, confirming previous specificity definitions.

Cells producing a specific antibody were cloned by first inoculating the cells to a very low density (calculated at 1 cell / well) in 72-well Terasaki plates (Nunc # 1-36538) at a volume of 10 μΐ / well. The plates were placed in an incubator for 3 hours to allow the cells to settle to the bottom of the plate, and then examined by two different individuals to identify wells containing a single cell. Each of these cells ’25 was then individually placed in wells of a 96-well round bottom plate along with the feeder cells, and cultured as described above for low density subculture. Supernatants from all resulting clones were positive against IATS serotypes 4 and 30 11 when tested by the ELISA method described above.

Thus, a cloned transformed human cell was obtained that grows continuously (is immortal) and secretes a human monoclonal antibody that reacts with Fisher-im-35 egg type 2 and is cross-reactive with IATS serotypes 10 and 11. In this example, the same label is used for the cell line and the antibody it produces (i.e., 6D6).

The isotype 5 of the antibody in well 6D6 was determined by an ELISA method otherwise similar to the specificity assays described above, but using HRP goat anti-human IgG and HRP goat anti-human IgM separately as the second step reagent, and combined. A positive reaction of the 6D6 antibody with Fisher immunotype 2 and IATS serotypes 4 and 11 was observed only with the anti-IgM reagent, indicating that this antibody is of the IgM isotype.

Biochemical characterization of the molecular specimen recognized by the 6D6 antibody was performed by immunoblotting as otherwise described in Example 3 above, but LPS preparations from Fisher immunotype 2 and IATS serotypes 4 and 11 were selected as antigen preparations for analysis. An LPS preparation of Fisher immunotype 1 was included as a negative control.

Each impure LPS preparation was diluted 1: 1 with digestion buffer [0.125 mol / l Tris, 4% (w / v) sodium dodecyl sulfate (SDS), 20% v / v glycerol, 10% v / v β-mercap-25 ethanol, 0 , 4% (w / v) bromophenol blue, PH 6.87], and sonicated the bath for 5 min. To degrade the proteins in the samples, a solution of proteinase K (1 mg / ml in water) at an enzyme content of 40% by weight of LPS was added to each sample, and incubated at 60 ° C for 2 hours with sonication of the bath for 5 minutes for 1 hour. after incubation. The samples were then kept at 100 ° C for 5 min and centrifuged in a microcentrifuge for 2 min.

Clarified samples corresponding to 10 mg of 35 LPS from each bacterial strain were subjected to SDS-polyacrylic-102217 26 amide gel electrophoresis (SDS-PAGE) as described above. After transferring the separated molecular specimen to the NCM, the NMCs were incubated for 2 hours at room temperature together with the culture supernatant 5 of the 6D6 cell line. The procedure was otherwise as described above.

Positive results were observed only in those bands containing Fisher immunotype 2, IATS serotype 4, or IATS serotype 11 LPS. In the bands corresponding to Fisher immunotype 2 and IATS serotype 11, antibody 6D6 identified a short series of small molecules at regular intervals (ladder) that correspond exactly to the smaller forms of LPS molecules that were seen on similarly performed SDS-PAGE, in which antigens were not transferred to the NCM, but were stained specifically to detect the presence of LPS; the only difference was that the lowest zone present on the silver-stained gel (corresponding to the core region + lipid A of LPS.) was not identified. In contrast, in the band containing IPS serotype 4 LPS, the antibody identified a full set of 20 regularly spaced bands covering nearly the entire length of the gel; the most intense reactions occurred in the zones corresponding to higher molecular weights. Again, the pattern corresponded to the ladder-like zone pattern observed with the LPS-specifically stained gel (i.e., the patterns corresponded zone by zone); the only exception was that no zone representing the core + lipid A was observed. These results clearly demonstrate that the LPS O-side chain of the molecular-kyylikohde which the antibody 6D6 identified Fisher immuno-30 type 2 and IATS serotypes 4 and 11.

Example V

Example V illustrates a method for preparing a human monoclonal antibody that reacts with P. aeruginosa IATS serotypes 6 and 13 and Fisher immunotype 1. The procedure otherwise described in Examples I to IV was repeated, but it was necessary to make certain changes to isolate, characterize, and examine the antibody described in this example. The following are changes in the procedures and results obtained with the mono-5 clonal antibody described herein.

The source of human B cells was a person previously immunized with a preparation containing high molecular weight polysaccharides isolated from Fisher immunotype 2 [Pier et al., Infect. Immun. 34 (1981) 461]. After collecting E'PBMC as described above, the cells were frozen in FCS containing 10% v / v DMSO in a liquid nitrogen tank. These cells were subsequently rapidly thawed at 37 ° C, washed once with Iscoven's medium, and suspended in HAT medium. Cell-directed transformation was performed at a ratio of 15 IAl cells / 1 E ~ PBMC cell. The cell mixture was seeded in 20 microtiter plates at a concentration of 78,500 cells / well. Cultures were supplemented every 3-5 days, and after 11 days, all wells were found to contain proliferating cells.

20 Anti-P. an antigen plate containing a mixture of P. aeruginosa Fisher immunotypes 1-7 (clinically isolated sample PSA 1277 (Genetic Systems Corporation Organism Bank, "GSCOB") was used to detect the presence of aeruginosa antibodies in the supernatants by ELISA, ATCC 27313, PSA G98 (GSCOB), ATCC 27315, PSA F 625 (GSCOB), ATCC 27317 and ATCC 27318, respectively). A PLL-treated microtiter plate that did not contain bacteria was also used in the experiment.

By analyzing the culture supernatants by the method described above, about 200 wells containing anti-β were identified. aeruginosa antibodies that react with a plate containing Fisher immunotypes 1-7. To identify wells containing antibody reactive with two or more IATS serotypes, antigen plates were generated as described above, in which the rows of plates contained only one PLL-attached bacterium of the IATS serotype. In each anti-P. The supernatant of the added culture in an aeruginosa-positive well was subjected to an ELISA-5 assay as described above. Supernatants were plated in rows on new antigen plates to identify a number of wells containing antibody that reacted with several IATS serotypes. One well, designated 8H7, contained antibodies specific for IATS sero-10 types 6 and 13. When the supernatant obtained from this well was examined by ELISA 7

Regarding the Fisher immunotype as described in Example V, it was found that the antibodies in well 8H7 were specific for Fisher immunotype 1.

To determine whether the reaction pattern against IATS serotypes 6 and 13 was elicited by several antibodies in well 8H7, additional samples of the supernatant were adsorbed separately with IATS serotypes 6, 13, and 17 (negative control), and the adsorbed supernatants each of the three IATS serotypes was then examined with PLL-attached bacteria by the ELISA method described above. The results showed that IATS serotypes 6 and 13 absorbed antibody activity against each other. IATS serotype 17 did not absorb 25 activity against IATS 6 or IATS 13. These results indicate that the reaction pattern against IATS serotypes 6 and 13 was induced by a single antibody contained in well 8H7.

Isolation and cloning of antibody-producing cells contained in well 8H7 was performed in three steps approximately as described in Example IV above. Thus, a cloned transformed human cell line was obtained that grows continuously (i.e., is immortal) and secretes a human monoclonal antibody that reacts with 35 Fisher immunotype 1 and cross-reacts with IATS sero 29 102217 types 6 and 13. In this example, the same designation 8H7 is used for the cell line and the antibody it produces. By the procedure described in Example IV, the antibody isotype of well 8H7 was determined to be IgM.

Biochemical characterization of the molecular specimen recognized by antibody 8H7 was performed by immunoblotting as otherwise described in Examples III and IV, but LPS preparations from Fisher immunotype 1 and IATS serotypes 6 10 and 13 were selected as antigen preparations for analysis. The LPS preparation from IATS serotype 10 was used as a negative control.

Analysis of the obtained immunoblots showed positive results only in those NCM bands containing Fisher immunotype 1, IATS serotype 6, or IATS serotype 13 LPS. In the bands corresponding to Fisher immunoassay type 1 and IATS serotype 6, antibody 8H7 identified a series of regularly spaced (i.e., ladder-like) bands covering nearly the entire length of the gel. These bands corresponded exactly to the many different molecular weight forms of the LPS molecules seen in the similarly performed SDS-PAGE, in which the antigens were not transferred to the NCM but were specifically stained to detect the presence of LPS. In lane 25 containing IPS serotype 13 LPS, antibody 8H7 identified a shorter series of regularly spaced bands that were medium to large molecular weight in the corresponding gel region. The identified bands again corresponded in position to the bands detected on the LPS-specific 30 stained gel; however, the smallest and large molecular forms of LPS seen on the stained gel did not become well identified in Western blot analysis. The results clearly show that the molecular target identified by antibody 8H7 with Fisher immunotype 1 and IATS-35 serotypes 6 and 13 was LPS.

3 “102217

Based on the above, it is concluded that the prepared cell lines produce human monoclonal antibodies and fragments thereof that are cross-reactive against various P. aeruginosa IATS serotypes. Cell-5 lines produce antibodies that find use in immunoassays and other known procedures.

Although the present invention has been described in some detail in an illustrative and exemplary manner to facilitate its understanding, it will be apparent that certain changes and modifications may be made therein without departing from the scope of the appended claims.

Claims (8)

3i '102217 A method for detecting Pseudomonas aeruginosa serotypes in a sample in vitro, comprising contacting the sample with potentially labeled human monoclonal antibodies that specifically react less than all but at least two Pseudomonas aeruginosa IATS serotypes, or binding fragments thereof, and the complex formed is detected, wherein the monoclonal antibodies can be prepared by cell line ATCC No. CRL 8941, 9171 or 9258. 2. Human monoclonal antibodies or binding fragments thereof, characterized in that they are capable of reacting specifically with accessible lipopolysaccharide determinants of Pseudomonas aeruginosa IATS serotypes 2 and 5 and Fisher immunotypes 3 and 7 and are produced by a continuously cultured cell line, said monoclonal antibody, such as the cell line ATCC No. CRL 8941, wherein the cell line is obtained by transforming human B lymphocytes. Human monoclonal antibodies or binding fragments thereof according to claim 2,. 25 characterized in that they are produced by the cell line ATCC No. CRL 8941. 4. Human monoclonal antibodies or binding fragments thereof, characterized in that they are capable of reacting specifically with Pseudomonas aeruginosa 30 with available lipopolysaccharide determinants of IATS serotypes 6 and 13 and Fisher immunotype 1 and that they are produced by a continuously cultured cell line secreting said monoclonal antibody, such as the cell line ATCC No. CRL 9258, from which a cell line 35 was created. obtained by transformation of human B-lymphocytes. 102217 Human monoclonal antibodies or binding fragments thereof according to claim 4, characterized in that they are produced by the cell line ATCC No. CRL 9258. 6. Human monoclonal antibodies or binding fragments thereof, characterized in that they are capable of reacting specifically with accessible lipopolysaccharide determinants 10 of Pseudomonas aeruginosa IATS serotypes 4 and 11 and Fisher immunotype 2 and are produced by a continuously cultured cell line, such as for example, ATCC No. CRL 9171, which secretes said monoclonal antibody, wherein the cell line is obtained by transforming human B lymphocytes. Human monoclonal antibodies or binding fragments thereof according to claim 6, characterized in that they are produced by the cell line ATCC No. CRL 9171. 8. A test kit for use in detecting Pseudomonas aeruginosa serotypes, characterized in that it comprises a composition comprising at least one human monoclonal antibody that specifically reacts less than all but at least two Pseudomonas aeruginosa IATS serotypes, or a binding agent thereof. fragment, and detectable signaling markers covalently bound to said antibodies or to other antibodies that react with all of said monoclonal antibodies, wherein the monoclonal antibodies can be prepared from cell line ATCC No. CRL 8941, 9171 or 30 9258. 33 102217