Antibody

Abstract

The invention relates to antibodies to Aspergillus species and to methods of producing those antibodies. The invention also relates to the use of such antibodies in identifying the presence of the Aspergillus species and to methods of treating an infection with the Aspergillus species.

Claims

1. A hybridoma deposited under accession number ECACC 08120202.

2. An antibody capable of specifically binding to Aspergillus, which may be obtained by culture of the hybridoma of claim 1.

3. A method of assaying for the presence of an Aspergillus species in a sample, comprising: a) contacting the sample with labeled antibodies according to claim 2; and b) observing the sample for binding of the antibodies to epitopes in the sample; wherein binding of the antibodies is indicative of the presence of an Aspergillus species.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1 to 25 show amino acid sequences and nucleotide sequences of antibodies and fragments thereof according to the invention. FIGS. 1 to 25 disclose SEQ ID NOS 1-25, respectively, in order of appearance.

(2) FIG. 26. Analysis of affinity purified antigen by PAGE and Western blotting. M, represents the molecular weight marker.

(3) A. Western immunoblot with monoclonal antibody (mAb) JF5 after separation of purified antigen by SDS-PAGE under reducing conditions. Well was loaded with 0.2 μg of protein.

(4) B. Western immunoblot with mAb JF5 after treatment of purified antigen with peptide-N-glycosidase and separation by SDS-PAGE under denaturing conditions. Well was loaded with 0.2 μg of protein.

(5) FIG. 27. Photomicrographs of A. fumigatus AF293 cells immuno-stained with JF5 and anti-mouse polyvalent immunoglobulin FITC.

(6) A. Germlings examined under bright-field microscopy

(7) B. Same slide as in (A), but examined under epifluorescence. Note intense staining of the cell walls of germ tubes, but lack of staining in ungerminated conidia (arrowed)

(8) C. Hypha examined under bright-field microscopy

(9) D. Same slide as in (C), but examined under epifluorescence. Note intense staining of cell wall and secretion of the antigen at the growing tip (arrowed). Bar, 6 μm.

(10) FIG. 28. Immunogold localization of JF5 antigen in cells of A. fumigatus AF293. Longitudinal section of germling grown in human serum, showing localization of antigen in the cell walls of the germ tube (GT) and swollen conidium, in the septum (S), and in a surrounding capsular-like layer (C). Bar, 0.5 μm.

(11) FIG. 29. Serum LFD tests.

(12) A. LFD tests with normal human serum following inoculation with fungi and incubation for 48 h at 37° C. Negative reactions (single control line only) exhibited by Candida albicans (1), Pseudallescheria boydii (2), Rhizopus oryzae (3), Fusarium solani (4) and positive reaction (two lines) with Aspergillus fumigatus (5) are shown.

(13) B. LFD tests of normal human serum (1), and serum spiked with affinity purified antigen (2) at a concentration of 2.5 μg protein/m).

(14) C. Examples of results from LFD tests of serum samples from healthy individuals or patients confirmed with IA. Negative, weak and strong reactions exhibited with specimen numbers 9OHD (I), 1657 (2), and 1131 (3) are shown. Specimen numbers relate to those shown in Table 4.

(15) FIG. 30. Analysis of flow-through and eluate fractions from lectin spin columns by using denaturing SDS-PAGE and Western blotting. Lanes M, molecular mass markers; lane PA, untreated purified antigen; lanes LCH, ConA and GNA, flow-through or eluate fractions from lentil lectin (Lens culinaris hemagglutinin), Concanavalin A lectin or snowdrop lectin (Galanathus nivalis agglutinin) spin columns respectively. All wells were loaded with 0.5 μg of protein. Note strong binding of MAb JF5 to eluate fraction from GNA spin column showing that the JF5 glycoprotein antigen(s) comprises terminal non-reducing mannose residues linked α1-3 and α1-6.

(16) FIG. 31. Examples of results from (A) negative, (B) weakly positive, (C) moderately positive, and (D) strongly positive lateral-flow device assays. In the absence of the Aspergillus antigen, no complex was formed in the zone containing solid-phase JF5 antibody, and a single internal control line was observed (A).

(17) FIG. 32. Results from serial serum samples collected over time from the same guinea pigs with invasive pulmonary aspergillosis as measured by lateral-flow technology (A), the galactomannan assay (B), and the (1.fwdarw.3)-β-D-glucan assay (C). Each line represents the biomarker results from one animal at multiple time points. Serial samples were available for measurement of each biomarker at the multiple time points in 6 guinea pigs. For the y-axis of the lateral-flow device graph (A), + represents weakly positive results, ++ moderately positive results, and +++ strongly positive results.

DETAILED DISCLOSURE

Example 1

(18) Fungal Culture.

(19) All fungi were cultured on Sabouraud agar (SA) under a 16 h fluorescent light regime.

(20) Development of mAb, Preparation of Immunogen, and Immunization Regime.

(21) Mice were immunized with lyophilized mycelium (LM) of A. fumigatus AF293. Minimal medium (19 mM (NH.sub.4).sub.2PO.sub.4, 0.5% (wt/vol) yeast extract, 7 mM sodium citrate, 2 mM MgSO.sub.4.7H.sub.2O, 0.5 mM CaCl.sub.2H.sub.2O and 50 mM glucose, adjusted to pH 5.5 with 1 N HCl) was sterilized by autoclaving at 121° C. for 15 min. Three-wk-old SA Petri dish cultures of the fungus were flooded with 20 ml dH.sub.2O and the conidia suspended by gentle agitation using an inoculation loop. Spore suspensions were filtered through Miracloth to remove mycelium and the filtrate containing conidia transferred to 1.5 ml micro-centrifuge tubes. The conidia were washed three times with dH.sub.2O by repeated vortexing and centrifugation at 12 000 g for 5 min and finally suspended in dH.sub.2O to give a concentration of 10′ conidia/ml solution. Flasks containing 150 ml of media were inoculated with 200 μl of conidial suspension and incubated with shaking (150 rpm) for 24 h at 37° C. Mycelium was collected by filtering the contents of each flask through Miracloth, snap frozen in liquid N.sub.2, and lyophilized.

(22) One mg of LM was suspended in 1 ml of phosphate buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 8 mM Na.sub.2HPO.sub.4, and 1.5 mM KH.sub.2PO.sub.4 [pH7.2]). Six-week-old BALB/c female white mice were given four intraperitoneal injections (300-μl per injection) of immunogen at 2 wk intervals and a single booster injection five days before fusion.

(23) Production and Screening of Hybridomas and Determination of Antibody Specificity.

(24) Hybridoma cell were produced by the method described elsewhere (Thornton, C. R. 2001. Immunological methods for fungi, p. 227-257. In N.J. Talbot (ed.), Molecular and Cellular Biology of Filamentous Fungi, A Practical Approach. University Press, Oxford) and the supernatants were screened by enzyme-linked immunosorbent assay (ELISA) against soluble antigens extracted from LM in PBS and immobilized to the wells of MAXISORP™ ( a modified, highly charged polystyrene surface with high affinity to molecules with polar or hydrophilic groups) microtiter plates (50 μl per well). For antibody specificity tests, fungi were grown on SA and surface washings prepared in PBS as described in Thornton (Thornton, C. R. 2001. Immunological methods for fungi, p. 227-257. In N.J. Talbot (ed.), Molecular and Cellular Biology of Filamentous Fungi, A Practical Approach. University Press, Oxford). Protein concentrations, determined spectrophotometrically at 280 nm (NANODROP®, Agilent Technologies Limited, Berkshire, UK), were adjusted to 64 μg/ml buffer, and 50 μl volumes used to coat the wells of microtiter plates. After coating overnight at 4° C., wells were washed four times with PBST (PBS containing 0.05% [vol/vol] TWEEN® 20 ( a nonionic detergent)) and once each with PBS and dH.sub.2O and air-dried at 23° C. in a laminar flow hood. The plates were stored in sealed plastic bags at 4° C. in preparation for screening of hybridoma supernatants by ELISA as described below.

(25) ELISA.

(26) Wells containing immobilized antigens were incubated successively with hybridoma supernatant for 1 h, followed with goat anti-mouse polyvalent (immunoglobulin classes IgG, IgA, and IgM) peroxidase conjugate (Sigma Chemical Company, Poole, United Kingdom) diluted 1 in 1000 in PBST for a further hour. Bound antibody was visualised by incubating wells with tetramethyl benzidine substrate solution for 30 min and reactions were stopped by the addition of 3 M H.sub.2SO.sub.4. Absorbance values were determined at 450 nm with an MRX automated microplate reader (Dynex Technologies, Billingshurst, UK). Wells were given four 5-min rinses with PBST between incubations. Working volumes were 50 μl per well, and control wells were incubated with tissue culture medium (TCM) containing 10% (vol/vol) fetal calf serum. All incubation steps were performed at 23° C. in sealed plastic bags. The threshold for detection of antigen in ELISA, was determined from control means (2×TCM absorbance values) (Sutula, C. L., J. M. Gillett, S. M. Morrisey, and D. C. Ramsdell. 1986. Interpreting ELISA data and establishing the positive-negative threshold. Plant Dis. 70:722-726). These values were consistently in the range 0.050-0.100. Consequently absorbance values >0.100 were considered as positive for the detection of antigen.

(27) Determination of Ig Subclass and Cloning Procedure.

(28) The Ig class of mAbs was determined with a commercial mouse mAb isotyping kit (ISO-1) according to the manufacturers instructions (Sigma). Hybridoma cells lines were cloned by limiting dilution, and cell lines were grown in bulk in a non-selective medium, preserved by slowly freezing in fetal bovine serum/dimethyl sulfoxide (92:8 [vol/vol]), and stored in liquid nitrogen.

(29) Antigen Purification, Polyacrylamide Gel Electrophoresis and Western Blotting.

(30) Antigen was purified from PBS extracts of LM by affinity chromatography using a Protein A IgG Plus Orientation Kit (Pierce Biotechnology, Rockford, Ill., USA) containing immobilized JF5 mAb. Ascites fluid was prepared from JF5 hybridoma cells in female BALB/c mice (Eurogentec s.a., Belgium). Mice were injected with 10.sup.6 hybridoma cells washed in PBS and, after 3 wk, approximately 5 ml of ascites fluid was recovered from each mouse and was stored at −20° C. prior to use. For preparation of the affinity column, 15 μl of ascites fluid was mixed with 2 ml of binding buffer and the solution applied to the Protein A-agarose matrix. Crude PBS antigen extract was then incubated with the immobilized antibody and bound antigen was eluted with 0.1 M glycine-HCl (pH2.8) buffer. Polyacrylamide gel electrophoresis (PAGE) was carried out using the system of Laemmli (Laemmli, U. K. 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680-685), with 4-20% (wt/vol) gradient polyacrylamide gels (Bio-Rad Laboratories Limited, Hemel Hempstead, UK), under denaturing conditions. Purified antigen was mixed with Laemmli buffer and denatured by heating at 95° C. for 10 min in the presence of β-mercaptoethanol prior to gel loading. Proteins were separated for 1.5 h at 23° C. (165V). Pre-stained, broad range, markers (Bio-Rad) were used for molecular mass determinations. For westerns, separated proteins were transferred electrophoretically to a PVDF membrane (Bio-Rad). Membranes were washed three times with PBS and then blocked for 16 h at 4° C. with PBS containing 1% (wt/vol) bovine serum albumin (BSA). Blocked membranes were incubated with JF5 mAb supernatant diluted 1 in 2 with PBS containing 0.5% (wt/vol) BSA (PBSA) for 2 h at 23° C. After washing three times with PBS, membranes were incubated for 1 h with goat anti-mouse IgG (whole molecule) alkaline phosphatase conjugate (Sigma) diluted 1 in 15,000 in PBSA. Membranes were washed twice with PBS, once with PBST and bound antibody visualized by incubation in substrate solution. Reactions were stopped by immersion in dH.sub.2O and air-dried between sheets of WHATMAN® filter paper (a cotton liner treated to achieve a minimum alpha cellulose content of 98%). (Modification of the JF5 antigen using peptide-N-glycosidase (PNGase) was carried out prior to electrophoresis and western blotting according to procedures described elsewhere (Bleddyn Hughes, H., R. Carzaniga, S. L. Rawlings, J. R. Green, and R. J. O'Connell. 1999. Spore surface glycoproteins of Colletotrichum lindemuthianum are recognized by a monoclonal antibody which inhibits binding to polystyrene. Microbiol. SGM. 145:1927-1936).

(31) Immunofluorescence and Immunogold Electron Microscopy of A. fumigatus Conidia and Germlings.

(32) Immunogold labelling was performed with germlings of A. fumigatus AF293. Germlings were prepared by incubating washed conidia in normal human serum (BIOSERA®, Ringmer, UK) or in sterile filtered (0.2 μM) 1% (wt/vol) glucose solution for 16 h at 37° C. with gentle mixing. Germlings were pelleted by centrifugation and low temperature embedding of material was carried out as described elsewhere (Thornton, C. R., and N. J. Talbot. 2006. Immunofluorescence microscopy and immunogold EM for investigating fungal infections of plants. Nat. Prot. 5:2506-2511). Immunolabeling was carried with mAb JF5 and goat anti-mouse 20-nm gold conjugate (British Biocell International, Cardiff, Wales) as the secondary reporter molecule. Control grids were incubated with TCM instead of mAb supernatant, but were otherwise treated the same. For IF studies, washed conidia were suspended in glucose solution and transferred to the wells of multiwell slides. After incubation at 37° C. for 16 h, slides were air-dried and fixed as described in Thornton (Thornton, C. R. 2001. Immunological methods for fungi, p. 227-257. In N.J. Talbot (ed.), Molecular and Cellular Biology of Filamentous Fungi, A Practical Approach. University Press, Oxford). Wells were incubated for 1 h with 50 μl of mAb JF5 supernatant or TCM only. Slides were washed three times with PBS with gentle agitation and incubated for a further 30 min with goat anti-mouse polyvalent FITC conjugate (Sigma) diluted 1 in 40 in PBS. Slides were given three 5-min rinses with PBS and the wells overlaid with coverslips mounted in PBS-glycerol mounting medium (Sigma). Slides were examined with a Zeiss Axiophot microscope fitted with epifluorescence, using a UV excitation filter of 365 nm and an absorption filter of 420 nm. All incubation steps were performed at 23° C. in a moist environment and slides were stored at 4° C. in the dark in Petri dishes containing moistened WHATMAN® filter paper no. 1.

(33) Configuration of the LFD. The LFD consisted of G&L Diecut 1734 backing card, WHATMAN® 17chr and 1281 top and sample pads respectively, and WHATMAN® IMMUNOPORE 5 μM nitrocellulose membrane. Monoclonal antibody JF5 was conjugated to 40 nm gold particles, applied to the release pad at 100 units of conjugate/cm, and dried for 16 h at 37° C. The test line antibody consisted of JF5 mAb at 0.5 mg protein/ml of PBS containing 1% (wt/vol) BSA while a commercial rabbit anti-mouse Ig acted as the control line.

(34) Sensitivity and specificity of the LFD. Affinity purified antigen (protein concentrations determined as described) was diluted into normal human serum or PBS and 100 μl samples applied to the LFD. Unspiked serum and PBS acted as the negative controls. Results were recorded after 15 min as positive for the presence of Aspergillus antigen (two lines) or negative (single control line only). Specificity of the LFD was determined by growing fungi in normal human serum. Replicate 1 ml serum samples contained in 1.5-ml eppendorf tubes, were inoculated with 10.sup.4 washed conidia from filamentous fungi (Aspergillus flavus, A. fumigatus, A. niger, A. terreus, Fusarium solani, Pseudallescheria boydii and Rhizopus oryzae), or an equivalent number of washed yeast cells (Candida albicans and Cryptococcus neoformans). Tubes were incubated at 37° C. with shaking (100 rpm) for 48 h and fungal propagules precipitated by centrifugation. One hundred-μl samples of neat, cell-free, supernatants were applied to LFD devices and results recorded as described. Growth of filamentous fungi and the yeast Candida albicans was determined by visual appraisal of hyphal development or by increases in turbidity of serum samples (C. neoformans). Unspiked serum incubated under the same conditions acted as the negative control.

(35) Further tests of LFD specificity were conducted using serum containing the β-lactam antibiotics penicillin-G (Melford Laboratories Limited, Ipswich, UK), amoxicillin (Fluka) and piperacillin (Sigma), the β-lactamase inhibitor tazobactam (Sigma), the cancer prodrug cyclophosphamide (Sigma), and lipoteichoic acids from the bacteria Enterococcus faecalis and Staphylococcus aureus (both from Sigma). Following reconstitution, 100 μl volumes of solutions containing 5 mg of solid/ml serum (lipoteichoic acids) or 50 mg solid/ml serum (antibiotics, tazobactam and cyclophosphamide) were applied to LFD devices and results recorded as described. Unspiked serum acted as the negative control, while serum samples containing purified antigen and test chemicals acted as positive controls. Three replicates were performed for each test.

(36) LFD detection of antigen in IA sera. The ability of the LFD to detect circulating antigen in humans with IA was tested with sera collected from known or suspected IA patients and from healthy controls. The samples were kindly provided during a blind assessment of assay sensitivity and specificity conducted in collaboration with Dr Elizabeth Johnson (Bristol Health Protection Agency). The samples had previously been tested using the PLATELIA™ GM (galactomannan) EIA (enzyme immunoassay) and a pan-fungal β-glucan test (FUNGITELL®). One hundred-μl samples of undiluted scrum or serum diluted 1 in 10 in normal human serum were applied to LFD devices and the results recorded as described. Three replicates were performed for each sample.

(37) Production of Hybridoma Cell Lines and Isotyping of mAbs.

(38) A single fusion was performed. Cell lines were selected for further study based on the strength of mAb reaction in ELISA. The cell line JF5 was selected and was sub-cloned three times. The monoclonal antibody from the sub-cloned cell line IFS belonged to the immunoglobulin class G3 (IgG3).

(39) Monoclonal Antibody Specificity Tests.

(40) Monoclonal antibody JF5 was tested for specificity against a wide range of related and unrelated fungi (Table 1). It reacted with antigens from Aspergillus species and related fungi from the teleomorphic genera Emericella, Eurotium and Neosartorya. It cross-reacted with antigens from certain Penicillium species, but not with Penicillum species in the subgenus Biverticillium or teleomorphic Talaromyces species whose Penicillium anamorphs belong to this subgenus. It cross-reacted weakly with antigens from the closely related fungus Paecilomyces variotti, but did not react with antigens from a wide range of unrelated fungi including the well-documented invasive pathogens Candida albicans, Cryptococcus neoformans, and the emerging pathogens Fusarium solani, Pseudallescheria boydii and Rhizopus oryzae (Groll, A. H, and T. J. Walsh. 2001. Uncommon opportunistic fungi: new nosocomial threats. Clin. Microbiol. Infect. 7:8-24, Ribes, J. E., C. L. Vanover-Sams, DJ. Baker. 2000. Zygomycetes in human disease. Clin. Microbiol. Rev. 13:236-301, Walsh, T. J., and A. H. Groll. 1999. Emerging fungal pathogens: evolving challenges to immunocompromised patients for the twenty-first century. Transpl. Infect. Dis. 1: 247-261, Walsh, T. J., A. Groll, and J. Hiemenz. 2004. Infections due to emerging and uncommon medically important fungal pathogens. Clin. Microbiol. Infect. 10: 48-66).

(41) Characterisation of the Antigen.

(42) Polyacrylamide gel electrophoresis and Western blotting. The affinity purified antigen eluted from the column as a single peak containing 0.340 mg protein/ml of buffer. The diffuse binding pattern in Western blotting studies (FIG. 26A) showed that antigen bound by JF5 is glycosylated and is a pattern consistent with binding of mAbs to extracellular glycoproteins in A. fumigatus (Stynen, D., J. Sarfati, A. Goris, M.-E. Prevost, M. Lesourd, H. Kamphuis, V. Darras, and J.-P. Latgé. 1992. Rat monoclonal antibodies against Aspergillus galactomannan. Inf. Immun. 60:2237-2245). De-glycosylation of the antigen with the enzyme PNGase showed that the protein moiety of the glycoprotein bound by mAb JF5 has an approximate molecular weight of 40 kDa and has an N-glycosylated component (FIG. 26B).

(43) Immunofluorescence and immunogold electron microscopy of conidia and germlings. Immunofluorescence studies showed that the antigen was absent from the surface of ungerminated spores, but was present on the hyphal surface of germlings and was secreted from the hyphal tip (FIG. 27). Immunogold electron microscopy showed that the antigen was present in the hyphal cell wall and in septa and in a capsule-like layer surrounding cells (FIG. 28).

(44) Sensitivity and specificity of the LFD. There was strong detection of the affinity purified antigen in LFD tests (FIG. 29) with an assay sensitivity of 37 ng protein per ml of serum. In PBS only, the sensitivity of the assay was 1.25 ng protein per ml. After 48 h growth of fungi in human serum, there was strong detection of the antigen in serum spiked with 104 conidia of A. fumigatus AF293 (FIG. 29) and with other Aspergillus species (results not shown). No antigen was detected in serum inoculated with the other fungi tested (FIG. 29), despite prolific growth. No false-positive reactions were exhibited with the S-lactam antibiotics tested or with tazobactam, cyclophosphamide, and bacterial lipoteichoic acids. The chemicals did not inhibit detection of purified antigen (results not shown).

(45) Detection of antigen in IA sera. The JF5 antigen was detected in sera from patients with known or probable IA infection (Table 2). No false negatives were found with sera from healthy individuals. LFD test results were similar to those for GM detection using the PLATELIA™ EIA. However, three of the samples (1655, 1665 and 1667) from patients diagnosed with IA on the basis of clinical symptoms gave positive reactions with the LFD but were negative in the GM test. One of these samples (1655) and two others (samples 1537 and 1538) gave negative LFD reactions when used undiluted but gave positive reactions when diluted ten-fold in normal serum. This was likely due to a high-dose hook effect where high serum antigen concentrations impaired antigen-antibody binding. Results for all other samples were the same when used neat or diluted. Examples of negative and positive reactions with sera are shown in FIG. 29.

(46) TABLE-US-00001 TABLE I Details of organisms and results of ELISA specificity tests Absorbance Organism Isolate no. Source.sup.a (450 nm).sup.b Absidia corymbifera 101040 CBS 0.027 A. glauca 1 CRT 0.032 A. spinosa 3 CRT 0.000 Acremonium atrogriseum 306.85 CBS 0.083 A. blochii 424.93 CBS 0.006 Alternaria alternata 42 CRT 0.000 Apophysomyces elegans 658.93 CBS 0.007 Subgenus Aspergillus Section Aspergillus Eurotium amstelodami 34 CRT 0.866 Section Restricti Aspergillus restrictus 116.50 CBS 0.938 Subgenus Fumigati Section Fumigati Aspergillus fumigatus 181 CRT 1.020 AFC CRT 0.935 AF293 SK 1.213 Neosartorya fischeri var. fischeri 681.77 CBS 1.105 Section Cervini A. cervinus 537.65 CBS 0.667 Subgenus Ornati Section Ornati A. ornatus 184 CRT 1.381 (Hemicarpenteles ornatus) Subgenus Clavati Section Clavati A. clavatus 514.65 CBS 1.307 Subgenus Nidulantes Section Nidulantes A. nidulans 542.83 CBS 1.133 (Emericella nidulans var. nidulans) A4 FGSC 1.237 A26 FGSC 1.075 Emericella quadrilineata 591.65 CBS 1.045 Section Versicolores Aspergillus versicolor 599.65 CBS 1.120 Section Usti A. ustus 209.92 CBS 0.510 Section Terrei A. terreus var. terreus 601.65 CBS 1.186 Section Flavipedes A. niveus (Fennelia nivea) 261.73 CBS 1.085 Subgenus Circumdati Section Wentii A. wentii 229.67 CBS 0.000 Section Flavi A. flavus 91856iii IMI 1.053 A. oryzae 29 CRT 0.963 Section Nigri A. niger 102.40 CBS 1.433 121.49 CBS 1.155 522.85 CBS 1.057 553.65 CBS 1.066 Section Circumdati A. ochraceous 625.78 CBS 1.249 Section Candidi A. candidus 266.81 CBS 0.541 Aureobasidium pullulans 657.76 CBS 0.015 Botrytis cinerea R2 CRT 0.077 Candida albicans SC5314 SB 0.000 C. dubliniensis 8500 CBS 0.015 C. glabrata 4692 CBS 0.000 Chaetomium globosum 147.51 CBS 0.013 Cladosporium herbarum 159.59 CBS 0.067 Cryptococcus neoformans 5728 CBS 0.010 C. neoformans 7779 CBS 0.009 Cunninghamella bertholletiae 182.84 CBS 0.012 Exophiala dermatitidis 153.94 CBS 0.024 Fusarium oxysporum f. sp. melonis 422.90 CBS 0.000 F. oxysporum f. sp. pisi 260.50 CBS 0.005 F. solani 224.34 CBS 0.034 F. solani 80 CRT 0.056 F. solani var. petrophilum 102256 CBS 0.006 F. verticillioides 539.79 CBS 0.000 Geotrichum capitatum 327.86 CBS 0.014 Mucor fragilis 4 CRT 0.033 M. hiemalis var. silvaticus 50 CRT 0.002 Paecilomyces variotii 339.51 CBS 0.163 P. variotii 17.1 CRT 0.143 Penicillium brevicompactum 210.28 CBS 0.571 P. cinnabarinum 39 CRT 0.885 P. chrysogenum 105 CRT 1.248 P. citrinum 139.45 CBS 0.556 P. cyclopium 123.14 CBS 0.630 P. dierckxii 250.66 CBS 0.629 P. expansum 106 CRT 1.141 P. jensenii 43 CRT 1.115 P. islandicum 338.48 CBS 0.004 P. marneffei 101038 CBS 0.093 669.95 CBS 0.057 P. melinii 218.30 CBS 0.486 P. pupurogenum 364.48 CBS 0.006 P. roqueforti 221.30 CBS 0.347 P. simplicissimum 220.30 CBS 0.500 P. spinulosum 108 CRT 1.290 P. variabile 385.48 CBS 0.037 Phialophora verrucosa 225.97 CBS 0.021 Pseudallescheria boydii 835.96 CBS 0.004 Rhizomucor miehei 360.92 CBS 0.005 Rhizopus microsporus var. 102277 CBS 0.020 rhizopodiformis R. oryzae 146.90 CBS 0.016 R. oryzae 395.54 CBS 0.010 R. sexualis var. sexualis 209090 IMI 0.000 R. stolonifer G1 CRT 0.000 Saksenaea vasiformis 133.90 CBS 0.030 Scedosporium prolificans 742.96 CBS 0.010 S. prolificans 100391 CBS 0.025 Stachybotrys chartarum 485.48 CBS 0.017 Talaromyces flavus 437.62 CBS 0.051 T. stipitatus 266.91 CBS 0.046 Trichoderma longibrachiatum 446.95 CBS 0.000 T. pseudokoningii 500.94 CBS 0.000 Verticillium coccosporum GD2/B8 CRT 0.000 Wallemia sebi 196.56 CBS 0.043 .sup.aCBS = Centraalbureau voor Schimmelcultures, PO Box 85167, 3508 AD Utrecht, The Netherlands; FGSC = Fungal Genetics Stock Centre, University of Missouri, 5007 Rockhill Road, Kansas City, MO 64110, USA; CRT = C. R. Thornton; IMI = International Mycological Institute, Egham, England; SB = S. Bates, School of Biosciences, University of Exeter; SV = S. Krappman, Institute of Microbiology and Genetics, Department of Molecular Microbiology and Genetics, Georg-August-University, Gottingen, Germany. .sup.bEach value represents the mean of replicated values. Threshold absorbance value for detection of antigen: ≥0.100.

(47) TABLE-US-00002 TABLE 2 Results of LFD tests of serum samples from healthy individuals or from patients with known or suspected invasive aspergillosis Specimen Invasive Platelia GM Platelia Fungitell β-glucan Fungitell LF number Aspergillosis.sup.a index value GM result concentration (pg/mL) result res 6OHD No — — 45.90 Negative − 7OHD No — — 42.40 Negative − 8OHD No — — 44.30 Negative − 9OHD No — — 44.09 Negative − 813 Yes 0.12 Negative 128.35 Positive − 815 Yes 0.36 Negative 360.49 Positive − 1263 Yes 0.16 Negative 111.72 Positive − 1652 Yes 0.32 Negative 111.94 Positive − 1655 Yes 0.35 Negative 104.13 Positive +.sup.c 1657 Yes 0.71 Positive 122.23 Positive +/− 1665 Yes 0.16 Negative 108.28 Positive +/− 1667 Yes 0.30 Negative 142.19 Positive +/− 1130 Probable 2.04 Positive 85.51 Equivocal + 1131 Probable 1.52 Positive 219.61 Positive + 1537 Probable 4.64 Positive 782.95 Positive +.sup.b 1538 Probable 4.64 Positive >500 Positive +.sup.c .sup.aProven or probable cases of disease formally classified according to EORTC criteria .sup.bReactions in LFD tests: − (no antigen detected), +/− (weak reaction), + (strong reaction). Results from specimens 9OHD, 1657 and 1131 are shown in FIG. 29. .sup.cSamples with a strong reaction at a 1 in 10 dilution in normal serum, but negative undiluted

Example 2

(48) Summary

(49) Lectin binding studies show that the antigen(s) bound by MAb JF5 is/are immunogenic N-linked mannoprotein(s) comprising terminal non-reducing mannose residues linked α1-3 and α1-6. Insensitivity of the antigen(s) in ELISA to mild alkaline hydrolysis (β-elimination) shows that the MAb does not bind to glycan structures O-linked through serine and threonine.

(50) Methodology

(51) Lectin binding studies. Antigen(s) were purified from Aspergillus fumigatus using the method described. Purified antigen solution was subjected to glycoprotein fractionation using a QPROTEOME™ Mannose Glycoprotein Kit (Catalog no. 37551; Qiagen Ltd., Crawley, UK) according to the manufacturer's instructions. The ConA, GNA, and LCH lectin spin columns in the kit allow specific enrichment of glycoproteins with mannose-rich glycan moieties. The three lectins each bind different subclasses of these moieties. ConA binds biantennary and triantennary complex type N-glycans; LCH binds biantennary and triantennary complex type N-glycans with core fucose. GNA binds α1-3 and α1-6 linked high mannose structures.

(52) Flow-through and eluted fractions from the lectin spin columns were assayed by Western blotting. Polyacrylamide gel electrophoresis (PAGE) was carried under denaturing conditions, with 4-20% (wt/vol) gradient polyacrylamide gels (Bio-Rad Laboratories Limited, Hemel Hempstead, UK). Fractions were mixed with Laemmli buffer and denatured by heating at 95° C. for 10 min in the presence of 3-mercaptoethanol prior to gel loading. Each well was loaded with 0.5 mg of protein. Glycoproteins were separated for 1.5 h at 23° C. (165V) and pre-stained, broad range, markers (Bio-Rad) were used for molecular mass determinations. For Westerns, separated proteins were transferred electrophoretically to a PVDF membrane (Bio-Rad). The membranes were blocked for 16 h at 4° C. with PBS containing 1% (wt/vol) bovine serum albumin (BSA) and incubated with JF5 MAb supernatant diluted 1 in 2 with PBS containing 0.5% (wt/vol) BSA (PBSA) for 2 h at 23° C. After washing three times with PBS, the membrane was incubated for 1 h with goat anti-mouse IgG (whole molecule) alkaline phosphatase conjugate (Sigma) diluted 1 in 15,000 in PBSA. The membrane was washed twice with PBS, once with PBST and bound antibody visualized by incubation in substrate solution. Reactions were stopped by immersion in dH.sub.2O and air-dried between sheets of WHATMAN® filter paper.

(53) Mild alkaline hydrolysis (β-elimination). Mild alkaline hydrolysis results in cleavage of glycans O-linked through the β-hydroxy amino acids serine and threonine. It does not cleave glycans N-linked through asparagine. Chemical modification of the purified antigen was carried out according the procedure described in Thornton (Thornton, C. R. 2001. Immunological methods for fungi, p. 227-257. In NJ. Talbot (ed.), Molecular and Cellular Biology of Filamentous Fungi, A Practical Approach. University Press, Oxford). Briefly, purified antigen was immobilized to the wells of MAXISORP™ microtitre plates. The wells were incubated with 50 μl of a 50 mM solution of NaOH or were incubated with 50 μl of dH.sub.2O only (control). After incubation for 24 h at 23° C., the wells were washed three times (3 min each time) with PBS and assayed by Enzyme-Linked Immunosorbent Assay (ELISA) with MAb JF5 as described.

(54) Results and Discussion

(55) Lectin binding studies. Western blotting analysis of flow-through and eluate fractions from lectin spin columns show that the JF5 antigen(s) has/have a high affinity for the mannose-binding lectin (GNA) from Galanthus nivalis (snowdrop) (FIG. 30). No binding or very weak binding only was found with the other two mannose-binding lectins, ConA from Canavalia ensiformis (jack bean) or LCH from Lens culinaris (lentil), respectively. GNA lectin is unique in that it is specific for D-mannose groups only (unlike ConA that is a mannose/glucose-specific lectin), especially those possessing Man(α1-3)Man units. It displays selective reactivity with mannans or mannose-containing glycoproteins and has a strict requirement for non-reducing terminal mannose units (Shibuya, N., I. J. Goldstein, E. J. M. Van Damme, and W. J. Peumans, 1988. Binding Properties of a Mannose-specific Lectin from the Snowdrop (Galanthus nivalis) Bulb. J. Biol. Chem. 263:728-734). Poor binding of the JF5 antigen to LCH and ConA lectins shows that the antigen(s) is/are not hybrid type or bi- and tri-antennary complex type N-linked glycoproteins.

(56) Mild alkaline hydrolysis (0-elimination). ELISA studies using chemical modification of the purified antigen with mild alkali show that the MAb does not bind to glycan structures O-linked through serine and threonine. There was no significant difference (Student's t-test; t=0.113, not significant) between the absorbance values obtained with treated antigen(s) (1.378*0.009) compared to the control (1.376±0.013).

(57) N-terminal sequencing. The N-terminal sequence (ALFALAKXV) of the protein component of the purified antigen was shown to have significant homology to the protein Cwplp from the yeast Saccharomyces cerevisiae (GenBank accession number EEU05173.1). Cwplp is a cell wall mannoprotein, linked to a β-1,3- and β-1,6-glucan heteropolymer through a phosphodiester bond (Van Der Vaart, J. M., L. H. P. Caro, J. W. Chapman, F. M. Klis, and C. T. Verrips, 1995. Identification of three mannoproteins in the cell wall of Saccharomyces cerevisiae. J. Bacteriol. 177:3104-3110). Using protein subcellular localization prediction software, the Cwplp glycoprotein is predicted to have a signal peptide, to be secreted and to be extracellular. Despite homology of the protein component to yeast Cwplp, monoclonal antibody JF5 retains its specificity for Aspergillus species. It does not cross-react with S. cerevisiae.

Example 3

(58) Detection of Invasive Pulmonary Aspergillosis by Lateral Flow Technology Compared to Galactomannan and (1.fwdarw.3)-β-D-Glucan

(59) Early diagnosis of invasive aspergillosis is critical for the initiation of appropriate antifungal therapy and may improve outcomes in high-risk patients. The use of sensitive biomarkers, including the non-invasive assays for galactomannan and (1.fwdarw.3)-β-D-glucan, also reduces the use of unnecessary antifungal agents. Despite their advantages, the galactomannan and the (1.fwdarw.3)-β-D-glucan assays are confined to laboratories equipped for these tests or require samples be sent to reference laboratories. Lateral-flow technology incorporates immunochromatographic assays into simple devices for point-of-care diagnosis. When coupled to a monoclonal antibody specific to an extracellular glycoprotein of Aspergillus this technology is a sensitive and specific biomarker (Thornton, C. R. 2008. Development of an Immunochromatographic Lateral-Flow Device for Rapid Serodiagnosis of Invasive Aspergillosis. Clin. Vacc. Immunol. 15:1095-1105). Our objective was to evaluate the time to positivity and sensitivity of a lateral-flow device in an established guinea pig model of invasive pulmonary aspergillosis, and directly compare these results to those obtained using the galactomannan and (1.fwdarw.3)-β-D-glucan assays.

(60) Immunosuppressed male Hartley guinea pigs (Charles River Laboratories) were exposed to conidia for 1 hour in an aerosol chamber. Serum samples were collected on days 3, 5, and 7 post-inoculation. A previously described lateral-flow device was used for the serodiagnosis of invasive aspergillosis (Thornton, C. R. 2008. Development of an Immunochromatographic Lateral-Flow Device for Rapid Serodiagnosis of Invasive Aspergillosis. Clin. Vacc. Immunol. 15:1095-1105). Briefly, an IgG monoclonal antibody (JF5) to an epitope on an extracellular antigen secreted constitutively during active growth of Aspergillus was immobilized to a capture zone on a porous nitrocellulose membrane. JF5 IgG was also conjugated to colloidal gold particles to serve as the detection reagent. Serum was added to a release pad containing the antibody-gold conjugate, which bound the target antigen, then passed along the porous membrane and bound to JF5 IgG monoclonal antibody immobilized in the capture zone. Test results were available within 10-15 minutes after loading the sample. Bound antigen-antibody-gold complex were observed as a red line with an intensity proportional to the antigen concentration, and were classified as negative, weakly positive, moderately positive, or strongly positive (FIGS. 31A, B, C, and D). Anti-mouse immunoglobulin immobilized to the membrane in a separate zone served as an internal control.

(61) The (1.fwdarw.3)-β-D-glucan assay was performed using a commercially available kit (FUNGITELL®, Associates of Cape Cod). Serum was transferred in duplicate to a 96-well cell culture tray and processed according to the manufacturer's instructions. The mean rate of change in optical density (OD) at 405 nm over time was measured using a microplate spectrophotometer (SYNERGY™ HT; Biotek Instruments) and unknowns were interpolated from a standard curve. Serum galactomannan was measured using a commercially available kit (PLATELIA™ Aspergillus EIA, Bio-Rad Laboratories). Serum was heat-treated following the addition of an EDTA acid solution. Treated supernatant was added to microwells containing conjugate and the rat monoclonal antibody EB-A2. Following incubation, microwells were washed and the substrate solution added forming a complex with the monoclonal antibody. The OD values of each sample, positive control, negative control, and cut-off control were measured using a microplate spectrophotometer at 450 and 630 nm, and the galactomannan index (OMI) was calculated as the OD of each sample divided by the mean cut-off of the control. The lateral-flow assay and the (1.fwdarw.3)-β-D-glucan and galactomannan assays were performed in separate laboratories by different investigators blinded to the results of the other.

(62) For each biomarker, the time to positivity was defined as the first time point at which three serum samples became positive. Time to positivity was plotted by Kaplan-Meier analysis, and differences in median time at which the assays became positive were analyzed by the log-rank test. Differences in the number of positive samples per time point between the assays were determined by Fisher's exact test. The overall specificity of each assay was also measured in uninfected controls. All statistical tests were performed using Prism 5.0 (GraphPad Software, Inc.).

(63) The assays were negative 1 hour post-inoculation prior to the onset of invasive disease with the exception of a galactomannan test result (Table 3), which likely represents a false positive result, as invasive disease is not yet established. Each biomarker became positive early with more than three samples positive for each assay by day 5 post-inoculation. In serial samples from the same animals, each biomarker continued to increase throughout the study (FIGS. 32A, B, and C). When the weakly positive lateral-flow device results were considered positive, this assay became positive on day 3, which was significantly shorter compared to the galactomannan (day 5, p=0.03) and (1.fwdarw.3)-β-D-glucan assays (day 7, p<0.001). When the weakly positive lateral-flow results were considered negative and only the moderately and strongly positive results positive, the time to positivity for each biomarker assay occurred at the day 5 time point.

(64) The sensitivity of each biomarker increased throughout the study period (Table 3). Similar to the time to positivity results, when the weakly positive results were considered positive, the sensitivity of the lateral-flow device on day 3 (48%) was greater than the galactomannan (4%, p<0.001) and (1.fwdarw.3)-β-D-glucan glucan assays (0%, p<0.001). The sensitivity of the lateral-flow device also remained higher than the (1.fwdarw.3)-β-D-glucan assay on day 5 (82% vs. 23%, respectively; p<0.001), but was not significantly different than the galactomannan assay (59%). When the weakly positive lateral-flow device results were considered negative and only the moderately to strongly positive results positive, the sensitivity of this biomarker was similar to that of the galactomannan and (1.fwdarw.3)-β-D-glucan assays (35%, 59%, and 23%, respectively; p>0.05). Each biomarker was 100% sensitive at the day 7 time point. Excellent specificity was also observed for each biomarker with only two false positives observed in uninfected animals with the (1.fwdarw.3)-β-D-glucan assay (Table 3).

(65) TABLE-US-00003 TABLE 3 Comparison of the lateral flow device and galactomannan and (1.fwdarw.3)-β-D-glucan assays Lateral-Flow Beta-glucan Galactomannan Index Time Point Device (+) (≥80 pg/mL) (≥0.5) 1 hour Number positive 0/5 0/5  1/5 Day 3 Number positive 12/25 0/25  1/25 Sensitivity  48%  0%  4% Day 5 Number positive 14/17 4/17 10/17 Sensitivity  82% 23%  59% Day 7 Number positive 6/6 6/6  6/6 Sensitivity 100% 100%  100% Uninfected  0/10 2/10  0/16 Specificity 100% 80% 100%

(66) The table below discloses the “DNA Sequences” as SEQ ID NOS 1, 3, 5, 38, 7, 9, 12, 14, and 16 and the “Amino Acid Sequences” as SEQ ID NOS 26-34, 36, and 37, all respectively, in order of appearance.

(67) TABLE-US-00004 Reference DNA Sequenc Amino Acid Sequence VH3-1 AGCTTCTCGAGTCTGGAGGTGCCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTG MDFGLIFFIVALLKGVQCEVKLLESGGGLVQ CAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGA PGGSLKLSCAASGFDFSRYWMSWVRQAPGK AAGGGCTAGAATGGATTGGAGAAATTAATCCAGATAGCAGTAAGATAAACTATATGCCA GLEWIGEINPDSSKINYMPSLKDKFIISRDNA TCTCTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAA KNTLYLQMSKVRSEDTALYYCARPRGYYA ATGAGCAAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGACCTCGGGGTTAC MDFWGQGTSVTVSSATTTAPSVFPLA TACGCTATGGACTTCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCTACAACAACA GCCCCATCCGTCTTCCCCCTGGCAC VH3-2 AGCTTCTCGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTG MDFGLIFFIVALLKGVQCEVKLLESGGGLVQ CAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGA PGGSLKLSCAASGFDFSRYWMSWVRQAPGK AAGGGCTAGAATGGATTGGAGAAATTAATCCAGATAGCAGTAAGATAAACTATATGCCA GLEWIGEINPDSSKINYMPSLKDKFIISRDNA TCTCTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAA KNTLYLQMSKVRSEDTALYYCARPRGYYA ATGAGCAAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGACCTCGGGGTTAC MDFWGQGTSVTVSSATTTAPSVFPLA TACGCTATGGACTTCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCTACAACAACA GCCCCATCCGTCTTCCCCCTGGCAC VH3-4 ATGGATTTTGGGCTGATTTTTTTTATTGTTGCTCTTTTAAAAGGGGTCCAGTGTGAGGTGA MDFGLIFFIVALLKGVQCEVKLLESGGGLVQ AGCTTCTCGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTG PGGSLKLSCAASGFDFSRYWMSWVRQAPGK CAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGA GLEWIGEINPDSSKINYMPSLKDKFIISRDNA AAGGGCTAGAATGGATTGGAGAAATTAATCCAGATAGCAGTAAGATAAACTATATGCCA KNTLYLQMSKVRSEDTALYYCARPRGYYA TCTCTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAA MDFWGQGTSVTVSSATTTAPSVFPLA ATGAGCAAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGACCTCGGGGTTAC TACGCTATGGACTTCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCTACAACAACA GCCCCATCCGTCTTCCCCCTGGCAC VH3-8 ATGGATTTTGGGCTGATTTTTTTTATTGTTGCTCTTTTAAAAGGGGTCCAGTGTGAGGTGA MDFGLIFFIVALLKGVQCEVKLLESGGGLVQ AGCTTCTCGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTG PGGSLKLSCAASGFDFSRYWMSWVRQAPGK CAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGA GLEWIGEINPDSSKINYMPSLKDKFIISRDNA AAGGGCTAGAATGGATTGGAGAAATTAATCCAGATAGCAGTAAGATAAACTATATGCCA KNTLYLQMSKVRSEDTALYYCARPRGYYA TCTCTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAA MDFWGQGTSVTVSSATTTAPSVSPWR ATGAGCAAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGACCTCGAGGTTAC TACGCTATGGACTTCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCTACAACAACA GCCCCATCGGTCTCCCCCTGGCGC VH5-1 ATGGATTTTGGGCTGATTTTTTTTATTGTTGCTCTTTTAAAAGGGGTCCAGTGTGAGGTGA MDFGLIFFIVALLKGVQCEVKLLESGGGLVQ AGCTTCTCGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTG PGGSLKLSCAASGFDFSRYWMSWVRQAPGK CAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGA GLEWIGEINPDGSKINYMPSLKDKFIISRDNA AAGGGCTAGAATGGATTGGAGAAATTAATCCAGATGGCAGTAAGATAAACTATATGCCA KNTLYLQMSKVRSEDTALYYCARPRGYYA TCTCTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAA MDFWGQGTSVTVSSATTTAPPVYPLVPEAW ATGAGCAAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTGCAAGACCTCGGGGTTAC TACGCTATGGACTTCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCTACAACAACA GCCCCACCCGTCTATCCACTGGTCCCTGAAGCTTGGG VH5-2 ATGGATTTTGGGCTGATTTTTTTTATTGTTGCTCTTTTAAAAGGGGTCCAGTGTGAGGTGA MDFGLIFFIVALLKGVQCEVKLLESGGGLVQ AGCTTCTCGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTG PGGSLKLSCAASGFDFSRYWMSWVRQAPGK CAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGA GLEWIGEINPDSSKINYMPSLKDKFIISRDNA AAGGGCTAGAATGGATTGGAGAAATTAATCCAGATAGCAGTAAGATAAACTATATGCCA KNTLYLQMSKVRSEDTALYYCARPRGYYA TCTCTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAA MDFWGQGTSVTVSSATTTAPPVYPLAP ATGAGCAAAGTGAGATCTGAGGACACAGCCCTTTATTACTGTCCAAGACCTCGGGGTTAC TACGCTATGGACTTCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCTACAACAACA GCCCCACCCGTCTATCCCCTGGCCCCTGG JF5VH MDFGLIFFIVALLKGVQCEVKLLESGGGLVQ Consensus PGGSLKLSCAASGFDFSRYWMSWVRQAPGK Sequence GLEWIGEINPDSSKINYMPSLKDKFIISRDNA KNTLYLQMSKVRSEDTALYYCARPRGYYA MDFWGQGTSVTVSSATTTAPSVFPLA VL4-1 ATGGAGTCACATACCCAGGTCTTTATATTCGTGTTTCTCTGGTTGTCTGGTGTTGACGGAG MESHTQVFIFVFLWLSGVDGDIVMTQSHKV ACATTGTGATGACCCAGTCTCACAAAGTCATGTCCACATCAGTAGGAGACAGGGTCAGCA MSTSVGDRVSITCKASQDVSTAVAWHQQKP TCACCTGCAAGGCCAGTCAGGATGTGAGTACTGCTGTAGCCTGGCATCAACAGAAACCA GQSPKPLIYSASYQYTGVPDRFTGSGSGTDFT GGACAATCTCCTAAACCACTGATTTACTCGGCATCCTACCAGTACACTGGAGTCCCTGAT FTISSVQAEDLAVYYCQQHYSIPWTFGGGTK CGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGCAGGCT LEIKRADAAPTVSIFPPSSKLG GAAGACCTGGCAGTTTATTACTGTCAGCAACATTACAGTATTCCGTGGACGTTCGGTGGA GGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCA TCCAGTAAGCTTGGG VL4-8 ATGGAGACACAGTCTCAGGTCTTTGTATTCGTGTTTCTCTGGTTGTCTGGTGTTGACGGAG METQSQVFVFVFLWLSGVDGDIVMTQSHKV ACATTGTGATGACCCAGTCTCACAAAGTCATGTCCACATCAGTAGGAGACAGGGTCAGCA MSTSVGDRVSITCKASQDVSTAVAWHQQKP TCACCTGCAAGGCCAGTCAGGATGTGAGTACTGCTGTAGCCTGGCATCAACAGAAACCA GQSPKPLIYSASYQYTGVPDRFTGSGSGTDFT GGACAATCTCCTAAACCACTGATTTACTCGGCATCCTACCAGTACACTGGAGTCCCTGAT FTISSVQAEDLAVYYCQQHYSIPWTFGGGTK CGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGCAGGCT LEIKRADAAPTVSIFPPSSKLG GAAGACCTGGCAGTTTATTACTGTCAGCAACATTACAGTATTCCGTGGACGTTCGGTGGA GGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCA TCCAGTAAGCTTGGG VL4-18 CCCAGGTCTTTGTATTGGTGTTTCTCTGGTTGTCTGGTGTTGACGGAGACATTGTGATGAC QVFVLVFLWLSGVDGDIVMTQSHKVMSTSV CCAGTCTCACAAAGTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGG GDRVSITCKASQDVSTAVAWHQQKPGQSPK CCAGTCAGGATGTGAGTACTGCTGTAGCCTGGCATCAACAGAAACCAGGACAATCTCCTA PLIYSASYQYTGVPDRFTGSGSGTDFTFTISSV AACCACTGATTTACTCGGCATCCTACCAGTACACTGGAGTCCCTGATCGCTTCACTGGCA QAEDLAVYYCQQHYSIPWTFGGGTKLEIKR GTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAG ADAAPTVSIFPPSSKLG TTTATTACTGTCAGCAACATTACAGTATTCCGTGGACGTTCGGTGGAGGCACCAAGCTGG AAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTAAGCTTG GG JF5VL MESXSQVFVFVFLWLSGVDGDIVMTQSHKV Consensus MSTSVGDRVSITCKASQDVSTAVAWHQQKP Sequence GQSPKPLIYSASYQYTGVPDRFTGSGSGTDFT FTISSVQAEDLAVYYCQQHYSIPWTFGGGTK LEIKRADAAPTVSIFPPSSKLG