A NOVEL GENERATION OF CLONED HORSESHOE CRAB RECOMBINANT FACTOR C FOR DETECTION AND REMOVAL OF ENDOTOXIN |
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| 申请号 | EP98945748.6 | 申请日 | 1998-09-18 | 公开(公告)号 | EP1015609B1 | 公开(公告)日 | 2004-09-01 |
| 申请人 | NATIONAL UNIVERSITY OF SINGAPORE; | 发明人 | DING, Jeak, Ling, Ind. Techn. Relations Office; HO, Bow, Ind. & Techn. Relations Office; | ||||
| 摘要 | |||||||
| 权利要求 | |||||||
| 说明书全文 | The present invention relates to recombinant Factor C (rFC) of a horseshoe crab, produced in an insect cell system. The invention also relates to vectors for producing the protein by recombinant DNA methods and to methods for using the recombinant Factor C to detect endotoxins in a sample or for removal of endotoxins from a sample by affinity methods. The amoebocytes of horseshoe crabs contain an efficient coagulation cascade system which is activated by endotoxin, also known as lipopolysaccharide (LPS) from Gram negative bacteria. The enzymatic components of the coagulation cascade and the molecular events responsible for the subsequent gelation of the amoebocyte lysate have been characterized in Tachypleus tridentatus1 and Carcinoscorpius rotundicauda2,3,4. Factor C has been shown to be the intracellular endotoxin-sensitive serine protease that initiates the coagulation cascade system5. By spiking, the LAL test detects femtogram levels of LPS6. Owing to its extreme sensitivity, the amoebocyte lysate, in particular, the Limulus amoebocyte lysate (LAL) has been developed into a commercial assay for widespread use in the detection of pyrogenic LPS in drugs and other pharmaceutical products7,8. This assay is based on the LPS-induced coagulation reaction of the lysate, culminating in formation of a gel clot. However, (a) the possible lack of specificity due to 1-3 β-D glucan and (b) the batch-to-batch variation in the sensitivity of commercial lysate to LPS, due to seasonal and geographical differences in the starting material9 has prompted our laboratory to employ recombinant DNA technology to genetically-engineer Factor C as an alternative source of novel "limulus lysate" for endotoxin detection. cDNAs encoding Factor C have been Cloned1,10,15. There are six potential glycosylation sites in the amino acid sequence of the Factor C from Carcinoscorpius rotundicauda (CrFC)10,15. Cloned cDNA encoding CrFC has been expressed in E. coli11 and also in yeast expression systems12,24. The rFC obtained from yeast was found to be immunoreactive and capable of binding LPS, although only limited amounts of rFC produced in yeast were soluble13,14. Also, it was found that LPS could not activate the enzymatic activity of yeast rFC, thus, a direct enzyme-based LPS detection is not possible using rFC produced in yeast14. SG 42456 A discloses isolated Carcinoscorpius rotundicauda Factor C proteins with defined sequences of 1083 and 1019 amino acids. Further disclosed are an isolated and purified DMA encoding a protein having Factor C enzymatic activity useful in assays for Gram-negative bacterial endotoxin. The present inventors believed that expression in insect cells rather than in a prokaryotic or simple eukaryotic expression system is suitable for producing rFC with full biological activity. Furthermore, horseshoe crabs and insects belong to the same phylum, Arthropoda, and so insect cells might more closely resemble the cells of the horseshoe crab than yeast cells in their physiology and biochemistry. Thus, rFC produced in insect cells might more closely resemble the protein as purified from the horseshoe crab and retain the bioactivity of having a serine protease activity activated by LPS. The present invention relates to genetic engineering of a bioactive rFC, which unequivocally exhibits full biological functionality. It is capable of specifically recognizing and binding LPS and Lipid A in both free and immobilized forms. Interference from 1-3 β-D-glucan, which switches on the alternate pathway in the coagulation cascade in conventional LAL, is not anticipated in assays of the present invention that use only Factor C as the LPS-binding, serine protease enzyme. Both the LPS-activated enzymatic assays of rFC and the ELISA lipid A binding assay could be formulated into a rapid high throughput mass screening test for LPS. Thus, a novel generation of "limulus amoebocyte lysate" has been invented, being capable of rapid and sensitive diagnosis and removal of subpiogram levels of endotoxin. The invention provides a standardized and convenient source of enzyme-based diagnostic reagent for detection of the ubiquitously contaminating endotoxin in pharmaceutical products. This inexhaustible supply of genetically-engineered Factor C can be easily standardized to circumvent batch-to-batch variations in sensitivity to LPS, a problem faced by the conventional LAL industry. Furthermore, the ability of the rFC of the invention to protect mice from endotoxemia, as well as its bacteriostatic activity, adds to its value in in vivo applications. Furthermore, the availability of rFC obviates the need for routine harvesting of the horseshoe crab for procurement of their amoebocyte lysate, and therefore, conserves this endangered "living fossil". The present inventors have succeeded in expressing biologically active rFC using recombinant baculoviruses and insect host cells. The rFC obtained is enzymatically active. Thus, expression of rFC in insect cells is a convenient and economical source of rFC protein for use in rapid, sensitive, specific and quantitative determination of LPS in pharmaceutical products and other biological fluids. Thus, the present invention comprises purified rFC that is enzymatically active. The phrase "enzymatically active" means that the Factor C protein has the biological activity of binding LPS or lipid A, being activated as to its serine protease activity upon LPS or lipid A binding. Enzymatically active rFC will induce coagulation of an amoebocyte lysate and will also cleave synthetic substrates such as, but not limited to, Boc-Val-Pro-Arg-MCA, Mu-Val-Pro-Arg-AFC and Boc-Val-Pro-Arg-pNA. The present invention is also embodied in a method for producing substantially purified, enzymatically active rFC. The method comprises expressing DNA encoding a Factor C protein having the enzymatic activity described above in a culture of insect cells, then isolating the enzymatically active Factor C protein. The isolation preferably includes an ultrafiltration step. The purification preferably also includes a step of gel-filtration chromatography on a matrix having an exclusion limit of 100 kilodaltons. The gel filtration is preferably applied after the ultrafiltration. The exclusion limit of the gel filtration matrix can vary substantially; an effective matrix will provide at least about a 4-fold increase in the serine protease activity of an ultrafiltered crude preparation as measured by the fluorometric assay described herein. The present invention also encompasses host-vector systems for expressing enzymatically active rFC. The host cells in these embodiments of the invention are insect cells, preferably leptidopteran cells. The vectors in these embodiments support replication of inserted DNA in insect cells and expression of heterologous DNA in insect cells. The vectors are preferably baculovirus or plasmid vectors. The heterologous DNA is sufficient to encode a Factor C enzyme of a horseshoe crab, preferably of the genus Carcinoscorpius, Tachypleus or Limulus. Preferred heterologous DNA is a polynucleotide having the sequence shown in SEQ ID NO: 1 or SEQ ID NO:3. The present invention is also embodied in assays for endotoxin comprising contacting a sample to be assayed for the presence of endotoxin or LPS or Lipid A with enzymatically active rFC according to the invention and measuring the serine protease activity of the rFC. The amount of serine protease activity of the rFC will reflect its activation due to binding of LPS or Lipid A or of another endotoxin known in the art to bind to Factor C of a horseshoe crab. The serine protease activity is conveniently measured by any method known in the art but is preferably measured by a chromogenic or fluorogenic method. In such a method formation of a product from a substrate by cleavage of the substrate by the serine protease activity of the rFC, resulting in a change in color or in fluorescence emission, is measured. Preferred substrates for such a chromogenic or fluorogenic assay are N-t-BOC-Val-Pro-Arg-MCA, Mu-Val-Pro-Arg-AFC and Boc-Val-Pro-Arg-pNA. Additional embodiments of the invention include immunologic methods for assaying the presence of Lipid A or LPS or endotoxin in a sample. These methods of the invention rely upon binding of antibody that specifically binds to Factor C and subsequent detection or quantitation of the amount of the Factor C-antibody complex. In a preferred embodiment, the sample to be assayed is contacted with immobilized antibody that specifically binds to Lipid A or LPS or endotoxin as the ligand to form immobilized ligand. The immobilized ligand is then contacted with rFC according to the present invention to form immobilized rFC. Then the immobilized rFC is contacted with a second antibody that specifically binds the rFC. Finally, the presence or preferably the amount of the rFC-second antibody complex is determined. This determination can be performed by any method typical in the art such as a third antibody that binds the second antibody, perhaps through its Fc portion, or the like. In an alternate embodiment of this aspect of the invention, the second antibody is omitted and the enzymatic activity of the immobilized rFC is measured. In another embodiment of the invention, the specific binding of LPS or lipid A to rFC is employed in a BIACORE™ assay (Pharmacia Biotech). By immobilizing the rFC on the substrate plate of the BIACORE™ apparatus, the presence of LPS or lipid A in a sample can be detected. Optimization of the amount of the rFC to be immobilized for a given load of LPS in a sample is considered within the skill of the ordinary practitioner. The BIACORE™ apparatus is operated in accord with the manufacturer's instructions. Also, the present invention is embodied in methods for removal of endotoxin from a sample, wherein immobilized rFC is contacted with the sample, under conditions such that endotoxin in the sample binds the immobilized rFC, then the bound endotoxin is separated from the sample. According to a first aspect of the present invention there is provided a recombinant DNA vector comprising a baculovirus-derived vector and a cDNA encoding an enzymatically active Factor C of a horseshoe crab. According to a second aspect of the present invention there is provided an assay for endotoxin comprising:
According to a third aspect of the present invention there is provided an assay for endotoxin comprising:
According to a fourth aspect of the present invention there is provided a recombinant DNA vector comprising (i) a baculovirus-derived vector or a vector for expressing heterologous DNA in an insect cell line and (ii) a nucleic acid encoding a polypeptide having 75% or greater sequence identity to an amino acid sequence selected from the group consisting of SEQ. ID. NO. 2, SEQ. ID. NO. 4 and the amino acid sequence of Factor C of Tachypleus tridentatus. According to a specific method of the present invention there is provided a method for removing endotoxin or lipid A from a sample comprising:
The present application relates to rFC of the horseshoe crab. A preferred horseshoe crab that can serve as a source of DNA or mRNA for producing the rFC of the invention is Carcinoscorpius rotundicauda (CrFC). The present invention relates especially to expression of rFC by means of baculovirus host-vector systems. The present application also relates to a fluorometric assay for endotoxin that makes use of the rFC expressed by recombinant DNA methods. cDNAs encoding Factor C proteins from Carcinoscorpius rotundicauda have been previously described10,15. rFC from Carcinoscorpius rotundicauda (rCrFC) has been produced in vitro by coupled transcription/translation systems10,15. However, the present invention resides partly in the development of in vivo systems, especially using insect cells as the host cell, for efficient production of rFC by expression of cloned DNA. Also, the protection of rFC from activation and subsequent self-proteolysis by binding of endotoxin which may be present in solutions used in isolation of the protein is described in reference 15. Basically, dimethylsulfoxide (Me2SO, DMSO) is added to solutions which are used during the purification process. Even greater protection of the rFactor C is achieved by also adding an agent effective for chelating divalent metal ions to the purification solutions. cDNAs appropriate for expression in the presently-described system can be cDNAs encoding Factor C of any horseshoe crab. Two representative nucleotide sequences are presented as SEQ ID NO:1 and SEQ ID NO:3 (encoding the amino acid sequences of SEQ ID NOs:2 and 4). A composite DNA sequence, assembled from incomplete cDNA fragments, encoding the Factor C of Tachypleus tridentatus is disclosed by Muta et al.1 For use in the LPS binding assays and LPS removal according to the invention, the Factor C can be produced by any method typical in the art, but is preferably made in a eukaryotic host cell. Production of rFC in yeast host-vector systems is described in reference 16. As it has been the Inventors' recent experience that Factor C produced in yeast lacked serine protease activity, rFC for use in enzymatic activity-based assays is preferably produced by a baculovirus host-vector system. "Stringent conditions" for hybridization are those that provide for hybridization of sequences having less than 15% mismatch, preferably less than 10% mismatch, most preferably 0% to 5% mismatch. Exemplary of such conditions, using probes of 50 bases or longer, are an aqueous solution of 0.9 M NaCl at 65 °C; an aqueous solution of 0.98 M NaCl, 20% formamide at 42-45 °C. The conditions will vary according to the length of the probe, its G+C content and other variables as known to the skilled practitioner17. Exemplary wash conditions following hybridization are an aqueous solution of 0.9 M NaCl at 45-65 °C, preferably 55-65 °C. Lower salt, or addition of an organic solvent such as formamide, in the wash buffer will increase the stringency of the condition as known in the art. A preferred hybridization condition is at 42°C in 50% formamide, 5x SSC, 1x Denhardt's solution, 20 mM phosphate buffer, pH 6.5, 50 µg/ml calf thymus DNA, 0.1% SDS. Salt and temperature conditions equivalent to the hybridization conditions employed can be calculated from the following equation18: A preferred washing condition is in 1x SSC, 0.1% SDS washing solution at room temperature, followed by washing at high stringency with 0.1x SSC, 0.1% SDS at 42°C and 2x with 0.1x SSC/0.1% SDS for 15 min. each at 42°C. Sf9 insect cells were maintained as a monolayer culture in serum-free SF 900 II SFM medium supplemented with 50 U/ml penicillin and 50 µg/ml streptomycin (Life Technologies, Inc.) in a humidified incubator (Forma, USA) at 27°C. The plasmid pFastBac I™ and the competent DH10Bac E. coli were from Life Technologies, Inc., USA. The strategy for cloning CrFC21 into the pFastBac I™ (Life Technologies, Inc.) expression shuttle vector is shown in Fig 1. The recombinant plasmids were verified by restriction enzyme digestion. The 5' cloning sites were further confirmed by dideoxynucleotide sequencing using the forward primer designed from the -44 position of the polyhedrin promoter region, before they were used for transfection in insect cells. PCR and Southern analyses of the pFastBac/CrFC21 DNA confirmed the authenticity of recombinant baculoviruses. The CrFC21 cDNA10 from pGEM11Zf+/CrFC2111 was recloned in two steps into pBluescript II SK+ (pBSSK), to yield pBSSK/CrFC 21. Further manipulations using pBBSK/CrFC21 and the baculoviral expression vector, pFastBac I™ were carried out using standard methods to clone full-length CrFC21, thus, yielding the recombinant construct, pFastBac/CrFC21 (Fig. 1). pFastBac/CrFC21 was transformed into competent E. coli, DH10Bac, and cultured in LB agar containing 50 µg/ml kanamycin, 7 µg/ml gentamycin, 10 µg/ml tetracycline, 30 µl of 2% X-gal and 40 µg/ml of IPTG. Screening19 for positive clones involved the use of the 2.3 kb 32P-CrFC21/EE fragment as probe10. The recombinant bacmid DNA was isolated and transfected into Sf9 cells. Early log phase recombinant Sf9 cells were seeded at 6.5 x 104 cells per well. The culture was incubated in a sealed bag at 27 °C for 1 h. Meanwhile, the virus stock was serially diluted 10-fold with SFM containing 10% FBS to give final dilutions of 10-2 to 10-4. The BacPak™ Baculovirus Rapid Titer Kit (InVitrogen) was used for plaque assay. It is an immunoassay which uses a primary monoclonal antibody raised to an AcMNPV envelope glycoprotein (gp64). A secondary goat anti-mouse HRP-conjugated antibody enables visualization of the infected cells as blue-stained viral plaques or foci seen under the light microscope. The virus titer (pfu/ml) was calculated based on the following formula : (Average no. of foci per well x dilution factor x 40) x 2 where 40 represents the inoculum volume normalization factor. The culture supernatant from the 6-well plates was harvested and the viral stock was amplified by re-infection of Sf9 cells grown in 25 cm2 flasks, using a multiplicity of infection (MOI) of 0.1 - 1.0. In such cultures, the viral stock reached a titer of 2 x 107 pfu/ml. Aliquots of this viral stock were re-inoculated at a MOI of 5-10 into Sf9 cells grown in 15 ml SFM medium in 75 cm2 flasks. The volume of the viral inoculum was determined using the formula: Subsequently, Sf9 cells were passaged twice and conditioned to grow in suspension in 100 ml SFM medium, in spinner flasks (Bellco, USA). At the mid log phase of growth, the viral stock from the 75 cm2 flask cells was inoculated at a MOI of 5-10. In the same manner, the cell culture volume was scaled up further in increasingly larger spinner flasks of 250, 500 and 1000 ml, infected with proportionally increasing volumes of viral stock at the same MOI.
Five µg of each cell lysate, or culture supernatant, harvested from 24, 48 and 72 h p.i. was analyzed on 10% SDS-PAGE gels, under denaturing conditions21. The electrophoretically-resolved bands were then transferred onto Immobilon™ PVDF membrane (Millipore, USA). The membrane was washed in PBS for 30 min, and blocked in 1% skimmed milk-PBS for 1 h followed by overnight incubation with rabbit anti-Factor C antibody diluted 1:500 in 0.2% Tween-20-PBS containing 1% BSA. Horseradish peroxidase-conjugated secondary goat anti-rabbit antibody, diluted 1:10000, was subsequently incubated with the membrane. For visualization of protein bands, the membrane was treated with SUPERSIGNAL™ chemiluminescent substrate (Pierce, USA) for 5-10 min, followed by 3 min exposure of the membrane to an X-ray film. The Western analysis revealed 3 bands of immunoreactive rFC proteins of 132, 88 and 44 kDa, expressed by pFastBac/CrFC21 recombinant baculoviruses at 24, 48 and 72 h post infection, p.i. (Fig. 2A). At 24 h p.i., rFC was observed in the culture supernatant, but not in the cell lysate. The 48 h and 72 h p.i. culture supernatant showed increasing amounts of rFC. The rFC in the cell lysate started to appear as a faint 132 kDa band only at 48 h p.i., and reached a substantial level at 72 h p.i. (Fig. 2B). The immunoblot thus showed that the bulk of the rFC produced was released from the infected Sf9 cells into the medium. This is probably due to lysis of the infected cells, which released the recombinant protein. On ultracentrifugation at 100,000 xg for 1 h at 4 °C, rFC was found to be in the soluble fraction. The results show that rFC protein was expressed correctly under the direction of the viral late promoter from the polyhedrin gene using the native translation start site from the CrFC cDNA. As there are six potential glycosylation sites in the CrFC cDNA sequence10; the protein band of 132 kDa represents the intact glycosylated form of Factor C. The 88 and 44 kDa proteins are likely the activated products of rFC whose molecular sizes correspond closely to the heavy and light chains, respectively, of double-chain Factor C3. Autoactivation could have occurred in the presence of picogram levels of ubiquitous endotoxin during the preparation of the protein sample for SDS-PAGE. Figure 2B shows a comparison of rFC from 72 h p.i., electrophoresed under reducing and non-reducing conditions of SDS-PAGE. The 88 and 44 kDa bands became more prominent under reducing conditions. Under non-reducing conditions, LPS-activated rFC still retained its 132 kDa band, thus indicating the double-chain form of rFC3. The presence of a double chain form of rFC was further proven when the BIOMAX™-purified rFC was pre-incubated with LPS before Western blotting. Under reducing condition, the LPS-treated rFC showed activated products of 88 and 44 kDa which were absent in the untreated rFC sample (Fig. 2C). However, under non-reducing conditions (Fig. 2D), the 132 kDa band was intact for both the LPS-treated and -untreated rFC. In order to visualize and test the ability of rFC to specifically bind the biologically potent component of LPS, diphosphoryl lipid A (E. coli K12, D31M4, List Biologicals, Inc., USA) ranging from 0.01 to 100 ng in 100 µl volumes was immobilized onto 96-well Nunc IMMUNO-PLATES™ (PolySorp). The immobilization was carried out overnight at room temperature. Unbound lipid A was removed and the plates were washed 6 times with wash buffer containing 0.01% Tween 20 and 0.01% thimerosal in PBS. The excess sites were blocked for 1 h at room temperature with the same buffer containing 0.2% BSA, after which the wells were again washed 6 times. Aliquots of 100 µl of BIOMAX™ 50-treated rFC from culture supernatant containing 20 µg total protein was then added to the wells and incubated overnight at room temperature. Unbound rFC was removed, and the wells were washed 6 times in wash buffer. This was followed by addition of aliquots of 100 µl of 1:500 diluted rabbit anti-Factor C antibody and incubation was continued for 2 h at 37°C. Subsequently, the wells were washed 6 times in the wash buffer before addition of 100 µl aliquots of 1:2000 diluted goat anti-rabbit antibody conjugated with horseradish peroxidase. After washing the wells 6 times, 1 mg/ml of substrate ABTS, 2,2'-azino-di-[3-ethylbenzthiazoline sulfonate (6)] diammonium salt (Boehringer Mannheim) was added in 200 µl aliquots and incubated at room temperature for 15 min. The formation of a green product was quantified by reading its absorbance at 405 nm. rFC binding the immobilized lipid A results in positive color formation via the ELISA test. The ELISA test for lipid A-binding indicates that rFC is capable of specifically recognizing and binding to immobilized lipid A (Fig. 3) and hence, it could be used in the detection of endotoxin. With increasing amounts of lipid A in the wells, there is an increasing intensity of color development in the ABTS product (Fig. 3A). Compared to the cell lysate, the 72 h p.i. culture supernatant consistently yielded more efficacious rFC for detection of lipid A (Fig. 3B). Furthermore, it was observed that blocking of excess sites in the wells with 0.2% BSA removed non-specific background binding and drastically improved the specificity of lipid A binding (Fig. 3C). This assay indicates that rFC can be used for mass screening of pharmaceutical products for LPS contamination, with the capability of quantifying LPS. This efficacy is comparable to the commercially available natural lysate derived from the Limulus or Tachypleus amoebocyte lysate. Two hundred µl samples containing either control wild-type supernatant (w/t Sf9, uninfected Sf9 cell supernatant) or partially-purified rFC samples (obtained by BIOMAX™-ultrafiltration), diluted in PBS to 10, 25 or 50 µg total protein per 200 µl were coated/immobilized onto each of the wells of a 96-well microtiter plate (NUNC, USA). The plates were left overnight at 4°C. Unbound protein was removed from the wells, and 200 µl of 0.2% BSA (depyrogenized by ultrafiltration) dissolved in PBS was added to the wells for 1 h at 37°C, to block unoccupied sites. The wells were washed 3 times with PBS. This was followed by addition of 200 µl FITC-conjugated LPS (E. coli 055:5B, List Biological Labs, USA) to the wells. The plate was incubated at 37°C for 1 h, after which each well was washed 6x with PBS. The fluorescence was read at Ex495nm and Em525um using LS-50B Spectrofluorimeter (Perkin Elmer). Wells coated with 10, 25 or 50 µg of partially purified proteins containing rFC showed increasing efficiency of binding LPS-FITC. Blocking of the wells with 0.2% BSA reduced the background fluorescence reading, indicating improvement in the specificity of binding of LPS to the immobilized rFC. Immobilization of negative control proteins (w/tSf9: wild-type Sf9 cell culture supernatant of Sf9 cells infected with AcNMPV DNA alone, and rFCSN: yeast rFC derived from the truncated recombinant rFC devoid of LPS-binding domain described in ref. 16, to the wells did not capture or bind LPS, thus indicating the specificity of recognition of LPS by the immobilized rFC. As a proenzyme, Factor C becomes catalytically activated by trace levels of LPS. Thus, conversion of its enzymatic substrate to product indicates the presence of LPS. rFC samples present in the crude cell lysate and culture supernatant were used for analysis of LPS-activated Factor C enzyme activity by using two different substrates. The first, in a conventional tube assay format, is based on a modification of the fluorimetric assay of Iwanaga et al.22 Using rFC obtained from a 72 h p.i. culture supernatant, 10 µg total protein in a volume of 0.1 ml was mixed with 1.9 ml of 50 mM Tris-HCl, pH 8.0, containing 0.1 M NaCl and 0.05 M CaCl2. The mixtures were preincubated with 0.01 to 100 pg of LPS (E. coli 055:B5, Sigma) at 37 °C for 1 h before addition of 15 µl of 2 mM fluorimetric substrate, Boc-Val-Pro-Arg-MCA (Sigma). Incubation was continued for 30 min and the reaction was terminated with 0.1 ml glacial acetic acid. The product AMC was read in Fluorescence Units (FU) at Ex380nm (slit 10 nm) and Em460nm (slit 5 nm) using a Perkin Elmer Luminescence Spectrophotometer (LS-50B). For multiple samples, this assay was routinely scaled down to 96-well microtiter plate assay. Briefly, the microassay involved 1 h preincubation of LPS with rFC in a volume of 100 µl, followed by addition of 1.5 µl of 2 mM fluorimetric substrate and 100 µl of 100 mM Tris-HCl, pH 8.0, containing 0.2 M NaCl and 0.1 M CaCl2 and further incubation for 30 min at 37 °C before termination of the reaction with 10 µl of glacial acetic acid. The fluorescence was read in a 96-well microtiter plate reader module. The second enzymatic assay for LPS involved a modification of the colorimetric test23 where preincubation of culture supernatant proteins with LPS ranging from 0.01 to 10 pg was carried out at 37 °C for 1 h. The reaction volume was scaled down to 200 µl in 0.1 M Tris-HCl (pH 8.0) containing 5 mM MgCl2. This was followed by addition of 50 µl of 2 mM of a colorimetric substrate, Boc-Val-Pro-Arg-p-nitroanilide (Seikagaku, Japan). Incubation at 37 °C was resumed for 1 h before termination of the reaction with 28 µl of glacial acetic acid. This substrate is hydrolyzed by rFC to produce pNA that was measured colorimetrically at OD405nm. From 24 to 48 to 72 h p.i., there was progressively increasing trend in the enzymatic activity of rFC in supernatants of cultures of insect cells transformed with the construct of Example 1, as indicated by the increase in fluorimetric units of the AMC product hydrolyzed from Boc-Val-Pro-Arg-MCA substrate. A comparison of the amount of total proteins present in the cell lysate (Lysate: 50 µg) and culture supernatant (Sup: 5 µg) illustrates that the culture supernatant from 72 h p.i. contained rFC that is >5-10 fold more effective in LPS detection. Twenty µg of BIOMAX™-purified rFC was able to detect 0.01 ng LPS. Using 40 to 80 µg of this protein, the detection limit could be easily extended to LPS levels below 0.01 pg or 0.001 ng/ml (Fig. 4). Purification of rFC by chromatography through SEPHADEX™ G-100 yielded enzymatic activity of even higher sensitivity to LPS (Fig. 5). It is envisaged that more elaborate purification of rFC following the methods covered in reference 24 would vastly improve the efficacy of the rFC for endotoxin detection. Furthermore, when the fluorimetric assay was modified to ∼ 200 µl, using a 96-well microtiter plate, the sensitivity to LPS was improved by 10-fold (Fig. 6). This was directly attributable to the removal of background fluorescence by gel filtration. Furthermore, the LPS-activated rFC enzyme assay was also conveniently quantifiable by a colorimetric assay with the Boc-Val-Pro-Arg-pNA substrate. The sensitivity to LPS was 0.1 pg (0.01 ng/ml) with 100 µg of BIOMAX™-50-treated culture supernatant when 2 mM of the pNA substrate was employed (Fig. 7). Similar to the fluorimetric assay, the colorimetric test also showed that the SEPHADEX™ G-100-purified rFC exhibited improved sensitivity to LPS, where 40 µg of purified rFC (instead of 100 µg of BIOMAX™ rFC) was sufficient to detect subpicogram levels of LPS (Fig. 8). Use of gel filtration-purified rFC resulted in a 4-fold increase in sensitivity to LPS. A direct comparison of the 2 microassays revealed that with gel filtration-purified rFC, the colorimetric assay achieved sensitivity to LPS comparable to the fluorimetric assay. Thus, using the scaled down, yet improved sensitivity assay for LPS detection, high throughput screening of samples can be conveniently achieved by either the colorimetric or fluorimetric assay using the 96-well microtiter plate assays. This enables rapid and mass screening of samples with limited volumes. The invention being thus described, modification of the invention with respect to various materials and methods will be apparent to one of ordinary skill in the art. Such modifications are to be considered as falling within the scope of the invention, which is defined by the claims hereinbelow. Articles of the scientific and patent literature referred to herein are incorporated by reference in their entirety by citation thereto.
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