PULMONARY HYDROPHOBIC SURFACTANT-ASSOCIATED PROTEIN OF 6,000 DALTONS MOLECULAR WEIGHT AND MULTIMERS THEREOF

Abstract: Novel, isolated, substantially pure, hydrophobic surfactant-associated protein of 6,000 daltons simple molecular weight (SAP-6). When SAP-6 is combined with lipids, its surfactant-like property imparts to the combination significant pulmonary biophysical activity. Such a combination results in enhanced adsorption of the lipids with properties similar to that of natural pulmonary surfactant material. SAP-6 in combination with lipids is highly useful for replacing or supplementing natural pulmonary surfactant material for reducing or maintaining normal surface tension in the lungs, especially in the lungs of patients suffering from hyaline membrane disease (HMD), or other syndromes associated with the lack or insufficient amounts of natural pulmonary surfactant material. A combination of SAP-6 and lipids may be administered as an aerosol spray or in aqueous normal saline with or without calcium chloride for treating or preventing HMD and other surfactant deficiency states. Also disclosed are methods of isolating the SAP-6 from animal tissue.

Description: SPECIFICA ION PULMONARY HYDROPHOBIC SURFACTANT-ASSOCIATED PROTEIN OF 6,000 DALTONS MOLECULAR WEIGHT AND MULTIMERS THEREOF Background Hyaline membrane disease, HMD, is a common disorder of premature infants and is related to diffuse atelectesls, hypoxia and resultant respiratory impairment. More particularly, HMD relates to the lack of vital pulmonary materials necessary for reducing surface tension in the airways of the alveoli. As a result, the alveoli or terminal respiratory sacs of patients suffering from HMD normally collapse. And, because the surface tension at the gas-liquid interface in HMD patients is elevated, their alveoli or terminal respiratory sacs are very difficult to reinflate. Consequently, HMD may be associated with significant morbidity and mortality, especially in premature infants. Present treatments of HMD focus on using high concentrations of oxygen, positive pressure and/or mechanical ventilation to maintain adequate oxygenation. These therapies are complicated by oxygen and pressure related injuries as well as injuries resulting from the need to mechanically access the airway via endotracheal tubes. More recent studies, however, have supported the use of replacement pulmonary surfactant material for therapy of HMD and other syndromes associated with the lack of pulmonary surfactant material. Such therapy has included heretofore the use of aerosolized or liquid synthetic phospholipid mixtures, natural pulmonary surfactant material and various preparations of surfactant material prepared from animal lung. Surface tension lowering ability of the naturally derived preparations is in general better than in the synthetic lipid preparations. Also, preliminary studies using modified bovine surfactants have been promising. Problems with human and animal pulmonary preparations, however, have occurred which include batch-to-batch variability, possible infection and immunologic risks. Obviously, when treating patients for any disease including HMD, it is imperative to include only those active substances necessary to minimize possible immunologic consequences of therapy to the patients. Unfortunately, because the natural pulmonary surfactant material and the preparations available heretofore are in crude form, they are less specific and associated with possibly greater immunologic risks. Natural pulmonary surfactant material is a complex material composed primarily of phospholipids and surfactant-associated proteins or apolipoproteins. The phospholipids, mainly phosphatidycholine (PC), disaturated phosphatidycholine (DSPC) and phosphatidylglycerol (PG), are of paramount importance for the physiological role of natural pulmonary surfactant material in reducing surface tension in the alveoli. Phospholipids, of which DSPC is the principal component, are synthesized in the endoplasmic reticulu of Type II epithelial cells, packaged into lamellar bodies, then secreted into the alveolar space by an exocytotic process. Several of the phospholipids are apparently not catabolized and resynthesized, but rather it is presently believed that they are reutilized primarily as intact molecules and constitute the major components of the naturally existing pulmonary surfactant material. With respect to the surfactant-associated proteins or apolipoproteins, there is considerable disagreement as to their identity and utility. Nonetheless, there is increasing agreement among those with medical expertise in this area that in addition to the lung surfactant phospholipids, at least some of the apolipoproteins are vital for the full biological activity of the natural pulmonary surfactant material in reducing surface tension in the alveoli. Surfactant-associated proteins or apolipoproteins include both serum and lung specific proteins. The major lung specific surfactant-associated protein of Mr«30- 40,000 daltons identified in lung surfactant by King et al. Isolation of Apoproteins from Canine Surfactant Material, Am J Physio1 244:788-795, 1973, is a glycoprotein rich in glycine and containing collagen-like regions rich in hydroxyproline. This protein, herein called SAP-35, is synthesized from Mr»28-30,000 dalton translation products which undergo glycosylation, hydroxylation of proline residues and sulfhydryl-dependent cross-linking to form large oligomers which can be detected in the airway. Proteolytic fragments of SAP-35 have been identified in protein preparations isolated from lavage of patients with alveolar proteinosis and from other mammalian surfactants migrating as proteins of small molecular weight by Whitsett et al, Characteristics of Human Surfactant-Associated Glycoprotein(s) A, Pediatr Res 19:501-508, 1985. While the glycoprotein SAP-35 binds phospholipids and may confer the structural organization of tubular myelin to surfactant lipids, it remains unclear whether SAP-35 is required for the biophysical activity of surfactants. See King et al, Metabolism of the Apoproteins in Pulmonary Surfactant, J Appl Physio1 42:483-491, 1977. Smaller lung specific surfactant-associated proteins have also been identified from a variety of mammalian surfactants. King et al, Am J Physiol 223:715-726, 1972, previously described a 10,000-12,000 daltons protein in pulmonary surfactant material, however, the origin of this protein or its distinction from others was not clarified. This protein described by King et al is now believed to be a fragment of the major glycoprotein SAP-35. Smaller surfactant associated proteins, other than that reported by King et al, have been identified in alveolar lavage material from a number of species and with molecular weights of approximately 10,000 daltons in dog and rabbit, 10,500-14,000 daltons in rat, 11,500-16,500 daltons in pig, and 10,000 daltons in cow. The nature and relationships among these various surfactant associated proteins (SAPs) and the larger protein, SAP-35 or its fragments, have not been established. Nevertheless, the work of Suzuki et al, J Lipid Res 23:53-61, 1982, suggested that a small 15,000 daltons protein in pig alveolar lavage had a greater affinity for lipid than SAP-35. Suzuki et al unfortunately did not distinguish the proteins from the SAP-35 or its fragments or demonstrate if there exists surfactant properties in a purified state. Rather, Suzuki et al only suggested that this 15,000 daltons protein is possibly a physiological regulator for the clearance of alveolar phospholipid. Claypool et al, J Clin Invest 74:677-684, 1984, suggested that a small unidentified protein, isolated from rat alveolar lavage, increased the uptake of liposome by cultured Type II epithelial cells. Work by Wang et al, Amino Acid Composition of Low Molecular Weight Hydrophobic Surfactant Apoproteins, Fed Proc 44:1024 (abstract), 1985, described two distinct small molecular weight proteins in surfactant from rat, one of which is ethanol soluble, the other which is ether/ethanol soluble but, like Suzuki et al, failed to purify and identify surfactant-like activity, or characterize the protein. Instead, Wang et al suggested that these small molecular weight proteins may be involved with surfactant recycling. It also has been suggested that the smaller molecular weight proteins, such as those discussed above, possibly arise as proteolytic fragments of the larger SAP-35 molecule. Heretofore, however, it has been unclear whether SAP-35, one or more of the smaller proteins, or all proteins together are active components imparting biophysical activity to natural mammalian pulmonary surfactant material. Fujiwara, et al, Biochem Biophys. Res. Comm. 135:527-532, 1986, report the isolation of a 5,000 dalton molecular weight proteolipid which, when mixed with phospholipids, was shown to have surface activity based on biophysical testing. However, this . protein, was neither well-characterized nor well-purified. In World Patent Application No. WO86/03408, Schilling, et al describe a human 35,000 dalton protein and nonhuman 10,000 dalton proteins. It was disclosed that both classes of proteins were needed to confer full biophysical activity to surfactant phospholipids. Also, the complete DNA sequence encoding the 35,000 dalton protein and the NH 2 -terminal amino acid sequences for the 10,000 dalton nonhuman proteins were disclosed. However, the 10,000 dalton proteins were unfortunately not purified for use alone or in combination with the 35,000 dalton protein for biophysical assays with phospholipids. Instead, it appears that Schilling et al used a 35,000 dalton protein derived by recombinant DNA methods in combination with naturally-occurring nonhuman 10,000 dalton proteins. Further, Schilling, et al disclose a preliminary (81 bp) partial sequence for a human cDNA clone conjectured to be the 10,000 dalton protein, which was chosen based on its homology to a canine cDNA clone. In view of the present state of the art, there obviously are needs to clarify the nature and role of the surfactant-associated lung specific proteins and to determine the most effective means for treating and preventing^HMD and other syndromes associated with lack or insufficient amounts of pulmonary surfactant material to maximize HMD therapy and to eliminate the disadvantages associated with HMD therapy available heretofore. Summary of the Invention In brief, the present invention alleviates and overcomes the above problems and shortcomings of the present state of the art. This was accomplished through the discovery of novel, isolated, substantially pure, hydrophobic surfactant-associated protein of 6,000 dalton simple molecular weight which, when isolated from animal tissue, comprises two hydrophobic proteins of molecular weights of 6,000 daltons each and may also comprise larger multimers thereof (hereinafter "SAP-6"). SAP-6, when combined with lipids, has significant pulmonary biophysical surfactant activity that can be utilized to effectively treat and prevent HMD and other syndromes associated with lack or insufficient amounts of natural pulmonary surfactant material. Although it is presently believed that SAP-6 is lung specific, its pulmonary biophysical surfactant activity is believed not to be species specific. Therefore, SAP-6 can be purified from animal tissue, specifically pulmonary tissue or amniotic fluid, extracted from a variety of animals, such as dog, cow, human, pig, rabbit, rat and the like, or made by recombinant DNA methods or direct peptide synthesis. The concentration of SAP-6 in pulmonary tissue and lavage is probably greater than that found in amniotic fluid. With respect to other animal tissues or fluid, however, SAP-6 is believed to be present in substantially smaller or undetectable concentrations or completely absent. It is believed that when SAP-6 is combined with lipids, it enhances the surfactant properties of the lipids imparting to the combination significant pulmonary biophysical surfactant activity. As a result of this remarkable property, such a combination is highly useful for replacing or supplementing natural pulmonary surfactant material and for reducing or maintaining normal surface tension in the lungs, especially in the lungs of patients suffering from HMD and other syndromes associated with the lack or insufficient amounts of natural pulmonary surfactant material. And, since SAP-6 can be highly purified from animal tissue or made by recombinant DNA techniques or direct peptide synthesis, the immunologic risks currently associated with the less pure preparations available heretofore for treating or preventing HMD or related syndromes are substantially reduced. It is to be understood that the terms "hydrophobic surfactant-associated protein" and "SAP-6" are used interchangeably herein, and that whenever referenced herein, they are meant to include any small hydrophobic surfactant-associated protein that has surfactant-like activity, that has a simple molecular weight of about 6,000 daltons determined in polyacrylamide gel containing sodium dodecyl sulfate and which is substantially resistant to protease enzymes, endoglycosidase F and collagenase. The present invention further resides in a method of separating SAP-6 from animal tissue which involves separating the animal tissue into a particulate fraction and a liquid fraction, and extracting from the liquid fraction the SAP-6 in a substantially pure state. The methods of this invention are further concerned with separating SAP-6 from the larger, novel hydrophobic multimers thereof. As already mentioned above, this can be accomplished by, for instance, gel electrophoresis migration or other suitable techniques. The present invention still further contemplates novel medicaments, preparations and methods employed to treat animals, including human infants, suffering from HMD and other syndromes related to the lack or insufficient amounts of natural pulmonary surfactant material. Also contemplated by the present invention are novel antibodies and antisera directed against SAP-6. Accordingly, it can be appreciated that the present invention provides a solution to the art that has long sought to understand natural pulmonary surfactant material and effective means to treat or prevent HMD and other syndromes associated with the lack or insufficient amounts of natural pulmonary surfactant material. The above features and advantages will be better understood with reference to the Figs., Detailed Description and Example set out hereinbelow. It will also be understood that the compositions and methods of this invention are exemplary only and are not be regarded as limitations of this invention. Brief Description of the Figs. Reference is made to the accompanying Figs, in which is shown isolated SAP-6 and multimers thereof. Fig. 1. SAP-6 from bovine. SAP-6 was purified and delipidated as described herein and applied to 10-20% sodium dodecyl sulfate- polyacrylamide gel electrophoresis gels in the absence of beta-mercaptoethanol. Lane B represents approximately 2 micrograms of protein detected by silver staining. SAP-6 from bovine, Lane B, migrated at Mr=6,000, 14,000, 20,000 and 26,000. Fig. 2. SAP-6 from human, canine and bovine. SAP-6 was purified and delipidated as described herein and applied to 10-20% sodium dodecyl sulfate-polyacryl mide gel electrophoresis gels in the presence of beta-mercaptoethanol. Each lane represents approximately 2 micrograms of each protein detected by silver staining. SAP-6 from each species, H * human, D = canine, and C = bovine, migrated at Mr=6,000 and 14,000. The beta-mercaptoethanol artifact is also observed at Mr=65-70,000. Standard molecular weight markers are seen on the right. Detailed Description of the Invention By way of illustrating and providing a more complete appreciation of the present invention and many of the attendant advantages thereof, the following detailed description is provided concerning SAP-6, novel medicaments based on SAP-6, novel antibodies and antisera and methods of preparation and utilization thereof. The present invention provides SAP-6 isolated from animal tissue, specifically from pulmonary tissue and amniotic fluid or made by recombinant DNA methods or direct peptide synthesis. SAP-6, when isolated from animal tissue, comprises two small hydrophobic surfactant-associated proteins with simple molecular weights of about 6,000 daltons each (hereinafter referred to as "SAP-6 monomers"), and may also comprise larger, hydrophobic protein multimers which may be covalent or noncovalent aggregates of SAP-6 monomers having molecular weights of about 14,000, about 20,000 and about 26,000 daltons based on size estimation using SDS-PAGE gel electrophoresis (hereinafter referred to as "SAP-6 multimers"). It should be realized that other SAP-6 multimers may exist but in smaller undetectable concentrations. As used herein, "simple molecular weight" refers to the molecular mass of what is thought to be the smallest polypeptide chain after sulfhydryl reduction which serves as a repeating building block for a SAP-6 multimer. To determine the molecular weights and separate the SAP-6 monomers from the larger SAP-6 multimers, for instance, a gel with about 3-27% polyacrylamide, in particular about 15% polyacrylamide (PAGE), and containing about 2% of sodium dodecyl sulfate (SDS) can be used to separate and determine the molecular weights of SAP-6 monomers and multimers in SDS-PAGE gel. See Fig. 1. For comparison, low molecular weight protein markers such as trypsin inhibitor (6,200), lysozyme (14,000), beta- lactalbumin (18,400), alpha-chymotrypsin (25,700) and ovalbumin (43,000) can be used. These can be obtained from BRL Inc., of Bethesda, MD. When gel separation is performed on SAP-6 in the absence of sulfhydryl reducing agents, such as beta-mercaptoethanol, dithiothreitol (DTT) , or after reduction and alkylation, an increase is observed in the larger, SAP-6 multimers over SAP-6 monomers. Presently, it is believed that sulfhydryl bonding, non-sulfhydryl aggregation and/or interpeptide bonding of a fraction of SAP-6 account for the larger, hydrophobic SAP-6 multimers. It should be understood that the molecular weights are only estimates based upon relative migrations and that other suitable techniques may be employed to make more accurate molecular weight determinations. As used herein, "hydrophobic," refers to solubility in non-polar solvents, such as 3:1 ether/ethanol, chloroform, chloroform/methanol in various ratios, such as 3:1, and having an abundance of hydrophobic non-charged a ino acids. The term "surfactaηf-associated protein," as used herein, refers to proteins associated with binding to or co-purifying with the phospholipid components of mammalian surfactants during centrifugation in isotonic solution. SAP-6 can be further characterized as being substantially resistant to protease enzymes (trypsin, chymotrypsin and staph V-8), endoglycosidase F, and collagenase. It has been discovered that SAP-6 is not degraded nor is its size heterogeneity significantly altered by these enzymes. In further characterizing SAP-6 it is localized in pulmonary type II cells and lung surfactant in a variety of mammalian species, as well as in human amniotic fluid near term gestation. With respect to the determination of the amino acid composition of SAP-6, purified samples are hydrolyzed by two different methods. In the first method, the samples are hydrolyzed in constantly boiling 5.7N HC1 containing 0.3% phenol and 0.1% beta- mercaptoethanol at 110°C under vacuum for about 24 and 48 hours. In a second method, these same samples are hydrolyzed in 12N HCl/trifluoroacetic acid (2:1) containing 0.3% phenol at 150°C under vacuum for 2 hours, 6 hours, 24 hours and 48 hours. The cysteine composition of these samples was determined separately as cysteic acid after performic acid oxidation. Hydrolysis was then performed on the oxidized samples in 12N HCl/trifluoroacetic acid (2:1) containing 0.3% phenol at 150°C for three hours. The amino acids are determined using a Beckman 6300 amino acid analyzer with a SICA 7000A Integrator. Table I illustrates the amino acid compositions that have been determined for bovine and canine SAP-6. It should be understood to those skilled in the art that since the amino acid compositions have been determined using a Beckman 6300 analyzer, the amino acid residue compositions per protein molecule are only estimates based upon this analytical technique. TABLE I BOVINE SAP-6 HUMAN SAP-6 CANINE SAP-6 ASX 2.1 1 0.5 THR 0.2 1 0.3 SER 0.6 1 4.0 GLX 0.3 k 0.4 PRO 4.3 5 4.6 GLY 4.1 A 5.1 ALA 2.3 7 2.3 CYS 3.6 5 1.6 VAL 23.1* 7 19.2* MET 1.8 1 1.7 ILE 4.7* 6 5.9* LEU 13.8* 10 13.9* TYR 0.2 2 — PHE 0.3 1 1.9 HIS 1 LYS 2.0 2 2.0 TRP ARG 1.9 2.0 ESTIMATED MOL. WT. 6588.0 6620.0 'HIGHEST VALUE OBTAINED FROM HYDROLYSIS As previously mentioned, SAP-6 comprises two 6,000 molecular weight proteins or SAP-6 monomers. In order to characterize the SAP-6 monomers herein, each protein will be separately referred to as SAP-6-Val and SAP-6-Phe. Presently, the sequences for the first 25, the first 23, and the first 19 NH 2 ~terminal amino acid residues, respectively, for bovine, canine and human SAP-6-Val have been determined and are shown in Table II. Presently, the sequences for the first 6, the first 7, and the first 12 NH 2 -terminal amino acid residues, respectively, for the bovine, canine and human SAP-6-Phe have been determined and are shown in Table III, TABLE II SAP-6-Val NH2-Terminal Amino Acid Sequences Bovine : Leu-Ile-Pro-Cys-Cys-Pro-Val-Asn-Ile-Lys-Arg- Leu-Leu-I le-Val-Val-Val-Val-Val-Val-Leu- (Val) Leu-Val-Val-Val (Val ) Canine : Ile-Pro-Cys-Phe-Pro-Ser-Ser-Leu-Lys-Arg-Leu- Leu-Ile-I le-Val-Val-Val-Val -Val-Val-Val- Human : Leu -He-Pro-Cys-Cys-Pro-Val-Asn-Leu-Lys-Arg- (lle) Leu-Leu-I le-Val-Val-Val-Val-Val Amino acids having alternate amino acids in parenthesis underneath have not been confirmed. TABLE III SAP-6-Phe NH2~Terminal Amino Acid Sequences Bovine: Phe-Pro-Ile-Pro-Ile-Pro Canine: Ile-Pro-Ile-Pro-Ile-Pro-Tyr Human; Phe-Pro-Ile-Pro-Leu-Pro-Tyr-Cys-Trp-Leu-Cys Arg-Ala-Leu In sequencing, the SAP-6 samples are prepared by Ed an sequence analysis by first dialyzing the samples against water. The samples are taken up in methanol and applied to a glass fiber filter. The analysis is performed on an Applied BioSystems model 470A Gas Phase Protein Sequencer. Analysis of the resulting phenylthiohydantoins (PTH) is accomplished by high pressure liquid chromatography (HPLC) at 50°C on an Altex reversed-phase PTH-C,g column. A binary buffer system consisting of ammonium acetate buffered acetonitrile, pH of 4.5, is used. The chromatography procedure is conducted utilizing buffer A which contains about 10% acetonitrile and buffer B which contains about 90% acetonitrile. The initial chromatography conditions are 70% buffer A and 30% buffer B with a flowrate of 1.0 ml per minute. One minute after injection, the concentration of buffer B is linearly increased to 50% over a 3 minute period and held at this level for 8 minutes. The concentration of Buffer B then is reduced to 30% over one minute. After re- equilibration, which takes approximately 10 minutes, another PTH sample is injected. A discussion of this separation procedure appears in greater detail in Jospeph L. Meuth and J. Lawrence Fox, Separation of Amino Acid Phenylthiohydantoin Derivatives by High-Pressure Liquid Chromatography, Analytical Biochem, Vol. 154, pp. 478-484, 1986 which is incorporated herein by reference in its entirety. As already indicated, SAP-6 comprises two 6,000 dalton hydrophobic surfactant-associated proteins. With respect to these SAP-6 monomers, the following has been observed. First, the NH 2 -terminal amino acid residue sequences of SAP-6-Val for canine, bovine and human, although similar to one another as indicated above, appear to be substantially different from the NH -terminal amino acid residue sequences of SAP-6-Phe for all three species. Similarly the NH 2 ~terminal amino acid residue sequences of SAP-6-Phe for all three species are similar to one another but substantially different from the SAP-6-Val sequences. Secondly, when SAP-6 is subjected to gel electrophoresis migration in the absence of beta-mercaptoethanol, a sulfhydryl reducing agent, major and minor amounts of protein appear. This can be better understood with reference to Fig. 1 wherein major amounts of silver stained protein exist at the 6 , 000 and 14 ,000 molecular weight migratory regions , whereas only minor amounts of silver stained protein exist at the 20,000 and 26 , 000 molecular weight migratory regions . Thirdly, when SAP-6 is subjected to gel electrophoresis migration in the presence of beta-mercaptoethanol, silver stained proteins appear only at the 6 , 000 and 14 , 000 molecular weight migratory regions as shown in Fig. 2. Therefore, two distinct 6 , 000 dalton SAP-6 monomers exist that are believed to co-elute together , to co-purify together via gel electrophoresis migration, to have similar molecular weights of about 6 , 000 daltons determined in a polyacrylamide gel containing sodium dodecyl sulfate, to have similar biophysical surfactant-like activity and to have similar enzyme resistance . Further , it is believed that the larger SAP-6 multimers , i . e . , Mr=20 , 000 and 26 , 000 , resulting from one or more of the 6, 000 dalton SAP-6 monomers are possibly bonded together via sulfhydryl bonds in view of their migration patterns in the absence and presence of beta-mercaptoethanol , as illustrated in Figs . 1 and 2. Therefore, it should be understood that any 6 , 000 dalton hydrophobic surfactant-associated protein, SAP-6, is well within the contemplation of this invention. There is the possibility that SAP-6*-Phe is migrating between 6000 and 14000 m.w. regions^ which, mayarise as a result ot proteolysis of the peptide. Thus there is noted a slight heterogeneity of the forms between 6000 and 14000 which may reflect various proteolytic processing of SAP-6-Pheprecursors. The present invention further relates to a novel method for isolating SAP-6. SAP-6 can be purified from, for example, animal tissue and fluids, cells, cultivated cells, suitable pulmonary surfactant replacement preparations available, such as CLSE or Surfactant-TA, and the like. It should be appreciated therefore that the expression "animal tissue" as used herein is meant in a broad sense to encompass all appropriate sources for SAP-6, including but not limited to animal tissue and fluid, cells, cultivated cells, pulmonary surfactant replacement preparations, such as CLSE and Surfactant-TA, and the like. It is preferred, however, to isolate SAP-6 from animal pulmonary tissue or amniotic fluid, and more preferably human pulmonary tissue. Solutions containing SAP-6 are obtained by extraction of animal tissue in which SAP-6 occurs. For instance, mammalian lung is excised, the trachea preferably should be cannulated and washed repeatedly with several volumes of an iced, buffered solution, such as 0.9% NaCl (pH 7.0-7.4), to remove surfactant from the alveoli. The wash is centrifuged at low speed, for example, 1,000 x g for ten minutes, and should be repeated if necessary to remove contaminating blood and white cells or macrophages. All procedures preferably should be performed at about 2-4°C. This surfactant material should then be centrifuged at high speed from the alveoli wash to sediment particulate materials at about 10,000 x g for about 30 minutes or longer. This pellet preferably should be resuspended and washed by repeated centrifugation and sonicated or freeze thawed, several times if necessary, in a suitable buffer, such as the one above, to uniformly resuspend the materials. The resulting white surfactant is pelleted by centrifugation, the aqueous material is removed and is extracted with about 10-100 volumes of approximately 2:1 ether/ethanol, 3:1 chloroform/methanol or other suitable organic solvents at about -30°C to produce a supernatant containing SAP-6. Extractions should proceed overnight. The material should then be centrifuged again at about 10,000 x g for about 20 minutes or longer. The resulting liquid fraction will contain lipid and include the SAP-6 of this invention. The liquid fraction thereafter can be dried (concentrated) with, for instance, N 2 stream, resuspended in an organic solvent, such as chloroform or other suitable organic solvents, and applied to a silicic acid column or LH-20 column, approximately 50cm x 2.5cm in size which is pre-equilibrated with chloroform or other suitable organic solvents at room temperature. The column is then eluted with, for example, 50 to 200 mis of a mixture of chloroform and alcohol, such as methanol or the like, increasing the alcohol concentration from 0% to about 100% in stepwise or gradient fashion. SAP-6 is eluted in the solvents containing alcohol having concentrations of greater than about 40% up to about 100% and more particularly about 40% to about 80%. Most of the lipids present in the SAP-6 will be separated from the proteins at this stage. It should be appreciated to those skilled in the art that the silicic acid column can also be eluted with any other suitable solvents capable of extracting therefrom the SAP-6 of this invention. An example of isolating SAP-6 is disclosed in the following example: EXAMPLE I. Isolating Surfactant Material Surfactant associated proteins are isolated from lung lavage material which is obtained from adult dogs and cows after sacrifice. Proteins are also isolated from human surfactant which are obtained from human cadavers after autopsy or from lung lavage of consenting adults. In animal studies, the trachea is cannulated and the lung is lavaged three times with several volumes of iced 0.9% NaCl, 50 mM Na 2 H 0 4 , and 5 mM EDTA, pH 7.2. Cells and debris are removed by centrifugation at 80 x g for about 10 minutes (twice) and a particular fraction is collected by centrifugation at 40,000 x g for about 30 minutes at 4°C. The pelleted material is then resuspended in the above buffer containing 1 mM phenylmethylsulfonylfluoride and sonicated for 10 seconds with a Branson sonifier. Surfactant is pelleted by repeated centrifugation at about 40,000 x g for about 30 minutes at 4°C. II. Purification of Hydrophobic SAP-6 Proteins From Surfactant Material The surfactant pellet is further processed by extraction in about 3:1 ether/ethanol or chloroform/ methanol at about -30°C for about 16 hours. Ether/ ethanol or chloroform/methanol extracts containing SAP-6 are evaporated to near dryness with N 2 gas and are redissolved in chloroform. The redissolved residue in chloroform is applied to a BioSil-HA column (silicic column dimensioned 2.5 cm by 40 cm that is obtained from Bio-Rad, Richmond, California) and is equilibrated in chloroform. Lipid-protein fractions are recovered by stepwise elution with chloroform-methanol mixtures (200 ml each) with successive increases in concentration of methanol (10% intervals) in chloroform. The SAP-6 proteins are eluted between 3:2 and 1:4 chloroform/ methanol solvent elution or about 40% to about 80% methanol in chloroform. Fractions containing the SAP-6 of this invention now can be assessed by, for example, fluorescamine assay and by gel electrophoresis migration followed by staining with silver stain or Coomassie brilliant blue staining. The SAP-6 isolated at this point is considered to be substantially pure and homogeneous. By "substantially pure" it is meant herein that the SAP-6 proteins are substantially free of contaminants, such as cells, cellular debris, DNA, RNA, and major surfactant glycoproteins or fragments thereof. It should be appreciated, however, that some lipids still remain in the now substantially pure SAP-6. In the most preferred form, the substantially pure SAP-6 contains on the order of about 10% or less of contaminants. If desired, to further delipidate the now isolated, substantially pure SAP-6, consisting essentially of the SAP-6 monomers and the larger multimers thereof, the fractions containing the eluted SAP-6 preferably are pooled, evaporated to near dryness and subjected to repeated dialysis in, for example, a cellulose dialysis bag with about 100-500 volumes of about 2:1 of chloroform/ methanol or other suitable mixtures or acidified chloroform/methanol mixtures acidified with HC1 or other appropriate acids at about room temperature. This procedure^should proceed until the SAP-6 is virtually free of contaminating lipids. This substantially purified SAP-6 can again be assessed by fluorescamine assay and gel electrophoresis migration. It should be realized that other suitable delipidating steps may also be employed to accomplish the above delipidating objective. The approximate amino acid composition also can now be determined and the proteins identified after PAGE with Coomassie brilliant blue, silver staining or other standard staining techniques. It should be appreciated that the above methods are described in generality and can be utilized with other suitable solvents and methods to separate and duplicate the SAP-6 from appropriate sources. A discussion of SAP-6 proteins appears in Jeffrey A. Whitsett et al, Hydrophobic Surfactant- Associated Protein (SAP6-14) In Whole Lung Surfactant and Its Importance for Biophysical Activity in Lung Surfactant Extracts Used for Replacement Therapy, Pediatr Res 20:460-467, 1986, and Jeffrey A. Whitsett et al, Immunologic Identification of a Pulmonary Surfactant- Associated Protein of Mr=6,000 Daltons, Pediatr Res 20:744-749, 1986, in which these articles are incorporated herein by reference in their entireties. It is believed that the SAP-6 of this invention has useful "surfactant-like activity". As used herein, "surfactant-like activity", refers to the ability to interact with phospholipids or other lipids to reduce surface tension spread on a surface and/or reduce surface tension at an air/liquid interface. And, when SAP-6 i.e., SAP-6 monomers, or the monomers in combination with multimers thereof, are mixed with lipids, their surfactant-like activity is believed to impart to the mixture significant pulmonary biophysical surfactant activity. Such a mixture with this activity is highly useful for replacing or supplementing natural pulmonary surfactant material for reducing or maintaining normal surface tension in lungs, especially in the lungs of animals suffering from HMD and other syndromes associated with the lack or insufficient amounts of natural pulmonary surfactant material. To make a preparation for medicinal application comprising SAP-6 and lipids, isolated SAP-6 can be stored in chloroform under nitrogen or air at about -30°C prior to reconstitution with lipids. Preparations may also be made with alcohols. Those lipids, including neutral or other lipids as well as phospholipids, that may be employed can be derived from a number of suitable phospholipids, such as phosphatidylcholine, disaturated phosphatidylcholine, phosphatidylglycerol, dipalmitoylphosphatidylcholine, phosphatidylinositol and mixtures thereof, cholesterol or cholesterol esters and mixtures thereof, and the like. For example, purified SAP-6 can be mixed in an amount of about 0.1 to about 2.0% with synthetic phospholipids, such as about 65% dipalmitoylphosphatidylcholine, about 20% egg-phosphatidylcholine, about 7.5% of soy-phospha¬ tidylinositol, and about 7.5% egg-phosphatidylglycerol. If desired, in preparing such a medicament, the mixture may further contain a physiological buffer comprising 0.9% sodium chloride with or without about 0.5 to about 1.5 mM calcium chloride or any other suitable pharmaceutically accepted carrier. After reconstitution or mixing, the material should be sonicated or vortexed and tested for surface tension lowering capacity and adsorption on a Wilhelmy balance or other surface tension measuring devices. Adsorption studies can be conducted by the methodology disclosed by Notter et al, Chem. Phys . Lipids 33: 67-80, 1983 and Ross et al, Phospholipid Binding and Biophysical Activity of Pulmonary Surfactant-Associated Protein SAP-35 and Its Non-Collagenous C-Terminal Domains, In press, 1986 in which they are incorporated herein by reference in their entirety. A phospholipid mixture (SM) containing about 65% dipalmitoylphosphatidylcholine (DDPC), about 20% egg- phosphatidylcholine (PC), about 7.5% egg-phosphatidyl¬ glycerol (PG) and about 7.5% soy-phosphatidylinositol (PI) is combined with delipidated SAP-6 and tested in a Wilhelmy balance and nitrogen bubble surfactometer. The SAP-6 phospholipid mixture is absorbed rapidly, lowers surface tension by about 30-47 dynes/cm and is found to be more active than the phospholipid mixture or SAP-35, the major surfactant-associated glycoprotein, mixed with the same phospholipid tested in identical fashion. See Tables IV-VI. The methods employed to generate Tables IV-VI can be found in detail in Notter et al and Ross et al referenced hereinabove. TABLE IV ADSORPTION FACILITY OF A SYNTHETIC LIPID MIXTURE (SM)*, A SYNTHETIC LIPID MIXTURE (SM) COMBINED WITH SAP-35, AND A SYNTHETIC LIPID MIXTURE (SM) COMBINED WITH SAP-6 Lipid Adsorption Surface Pressure at time Concentration (mg lipid/ml Mixture subphase) t=0 min 5 min 10 min 15 min SM 0.6 mg/ml 1 dyne/cm 1 1 3 SM + SAP-35** 0.6 mg/ml 20 dynes/cm 22 22 22 (canine) SM + SAP-6** 0.6 mg/ml 43 dynes/cm 45 45 45 (human) *SM is a synthetic lipid mixture composed of DPPC:egg-PC: egg-PG:soy-PI 65:20:7.5:7.5. **SAP-35 and SAP-6 proteins have been added in similar molar ratios . TABLE V ADSORPTION OF DELIPIDATED SAP-35 AND VARIOUS SEGMENTS AND SAP-6 COMBINED WITH A SYNTHETIC PHOSPHOLIPID MIXTURE **Surface Pressure (dynes/cm) Surfactant Mixture* 0 min 5 min 10 min 15 min 20 min 30 min 1. SM 1 1 1 3 3 2. SM + Ovalbumin 1 2 4 5 5 3. SM + SAP-35 9 13 14 15 18 4. SM + SAP-18 8 16 17 17 18 5. SM + SAP-21 2 4 6 7 9 6. Natural LS 17 46 46 47 47 7. SM + Canine 19 26 29 31 33 37 SAP-6 (25ug) 8. SM + Canine 37 47-48 47-48 47-48 47-4 SAP-6 (50ug) 9. SM + Canine 46 47 47 47 47 SAP-6 (lOOug) *Delipidated proteins studied are glycoproteins SAP-35, SAP-21 and SAP-18 and canine SAP-6. Each of these proteins are combined with a synthetic phospholipid mixture (SM) composed of DPPC:egg-PC:egg-PG:soy-PI 65:20:7.5:7.5. Natural LS (lipid-surfactant) represents optimally active surfactants; ovalbumin is used as a non-specific protein control. Phospholipid concentration for all adsorption experiments is about 0.063 mg/ml 0.15 M NaCl with about 1.4 mM CaCl2,' temperature = 35 _ 2°C. For cases 2-5, the lipid to protein ration is (wt/wt) 99/1 to 98/2. All mixtures are dispersed by vortexing at room temperature except natural LS (resuspended) . **Surface pressure is the amount of surface measured within 10 seconds after addition of bolus of surfactant dispersion to the stirred subphase at time zero. Values given for 1-6 are the means of 4-10 experiments with SEM (standard error of measure) always less than 3 dynes/cm, except for SAP-18 data which are the. mean of 2 experiments with a deviation of _ 0.5 dynes/cm about the mean. TABLE VI DYNAMIC SURFACE TENSION LOWERING DURING DYNAMIC COMPRESSION ON AN OSCILLATING BUBBLE Minimum Surface Tension After Cycling Surfactant D:ispersion Cone (dynes, /cm) For Dispersion* (mq/lipid/ml) 0.5 min 2 min 5 min 10 min 11 min 1. SM lmg/ml 55 21 21 21 2. Egg-PC lmg/ml 53 42 27 22 3. SM + SAP-35 lmg/ml 36 36 36 36 4. SM + SAP-35 2mg/ml 35 23 21 21 5. SM + SAP-21 lmg/ml 54 47 47 42 6. LS lmg/ml 16 10 2 1 7. SM + Canine lmg/ml 15 15 14 3 1 SAP-6 (25ug) 8. SM + Canine 0.5mg/ml 17 16 16 16 16 SAP-6 (25ug) 9. SM + Canine 2mg/ml 16 9 4 3 1 SAP-6 (25ug) 10. SM + Canine 0.5mg/ml 16 15 1 1 etc. SAP-6 (50ug) SM + Canine lmg/ml 15 0 0 0 etc. SAP-6 (50ug) SM + Canine 2mg/ml 15 13 1 etc. etc. SAP-6 (50ug) ♦ Surfactant dispersions are studied on the oscillating bubble at about 37°C, about 100% humidity, cycling rate about 20 cpm and area compression about 50%. The concentration is 1 or 2 mg lipid/ml bubble subphase as noted. Natural LS (lipid-surfactant) represents active lung surfactant preparation against which to compare the lipid-protein combined mixtures of cases 3-5 and 7-10. In treating HMD and other related disorders, mixtures containing SAP-6 and naturally occurring or synthetic phospholipids can be instilled intratracheally, for instance, in liquid form or as an aerosol spray in doses of about 0.1% to about 2.0% SAP-6 by weight, with about 20 to about 100 mg phospholipid per kilogram for treatment or prevention of HMD and other related disorders. Another utility for SAP-6 is in the preparation of SAP-6 antibodies or antisera. These antibodies or antisera can then be used in immunochemical methods for detecting, determining or purifying SAP-6. For example, SAP-6 antibodies and antisera can be used for Immunoblot and ELISA analysis for immunological detection and quantification or other immunological assays of SAP-6. SAP-6 antibodies and antisera which can be used for this purpose can be obtained as follows: Albino rabbits are immunized with repeated injections of delipidated SAP-6 and Freund's complete adjuvant. Usable antisera should be obtainable after four injections with approximately 100 micrograms of the delipidated SAP-6 per injection. The SAP-6 used for injection should contain SAP-6 monomers and a smaller amount of the SAP-6 multimers assessed by silver staining analysis after one dimensional SDS-PAGE. Polyclonal antibodies may be isolated from the antisera by affinity chromatography.. With respect to the production of monoclonal antibodies against SAP-6 proteins, standard known techniques for producing such antibodies are certainly within the contemplation of this invention. As one example, lymphocytes from mice immunized with isolated SAP-6 may be fused with mouse myeloma cells in the presence of, for example, polyethylene glycol, generally according to the technique of Kohler and Milstein, 256:495-497 (1975) which is incorporated herein by reference in it entirety. The surviving hybrids would then be screened by selective cloning to obtain only cells which produce the anti-SAP-6 which, when separated from contaminating myeloma antibody by immuno-absorption, may be employed in immunoassay techniques for detection of SAP-6 in, for instance, a tissue sample. For example, such antibodies can bind to or precipitate SAP-6 from a mixture containing SAP-6, increase the sedimentation rate of SAP-6, can modify elution characteristics of SAP-6 on gel filtration, and can identify or quantify SAP-6 from a sample. The detection and determination of SAP-6 by immunological methods is of diagnostic importance. In normal situations, since SAP-6 occurs in some tissues of the human body, for example, lung tissue, lung lavage and amniotic fluid, SAP-6 can be detected. In illnesses associated with SAP-6, the proteins will not appear or will appear in altered concentrations in the serum, tissue, such as the lungs, or other body fluids. The detection or lack of detection of these proteins can be useful for diagnostic determination of an illness or for monitoring the course of a disease and for closely controlling the therapy of such an illness. SAP-6 has been detected in human amniotic fluid near term gestation by immunoblot analysis using anti-SAP-6 antibodies. The present invention may, of course, be carried out in other specific ways than those herein set forth without departing from the spirit and essential characteristics of the invention. The present embodiments are, therefore to be considered in all respects as illustrative and not restrictive and all changes coming within the meaning and equivalency range of the appended claims are intended to be embraced herein. 30:1801o

Claims: WHAT IS CLAIMED IS: 1. Isolated and substantially pure hydrophobic surfactant-associated protein (SAP-6), said protein enhancing surfactant-like activity of lipids, and having a simple molecular weight of about 6,000 daltons determined in a polyacrylamide gel containing sodium dodecyl sulfate and which is substantially resistant to protease enzyme, endoglycosidase F, and collagenase. 2. Isolated SAP-6 of claim 1 wherein said protein is obtained from animal tissue. 3. Isolated SAP-6 of claim 1 further comprising hydrophobic multimers thereof. 4. SAP-6 of claim 3 wherein said multimers are selected from a class of hydrophobic multimers having molecular weights determined in a polyacrylamide gel containing sodium dodecyl sulfate of about 14,000 daltons, about 20,000 daltons, about 26,000 daltons and mixtures thereof. 5. SAP-6 of claim 3 wherein said SAP-6 is extracted from animal tissue. 6. Isolated and substantially pure human SAP-6, said human SAP-6 enhancing surfactant-like activity of lipids, having a simple molecular weight of about 6,000 daltons determined in a polyacrylamide gel containing sodium dodecyl sulfate and wherein the sequence of the NH 2 -terminal amino acid residues of said human SAP-6 is selected from the group consisting of: Phe-Pro-Ile-Leu-Pro-Tyr-Cys-Trp-Leu-Cys-Arg-Ala-Leu and Leu-Ile-Pro-Cys-Cys-Pro-Val-Asn-Leu-Lys-Arg-Leu-Leu ( He) I le-Val-Val-Val-Val-Val 7. Isolated and substantially pure canine SAP-6, said canine SAP-6 enhancing surfactant-like activity of lipids, having a simple molecular weight of about 6,000 daltons determined in a polyacrylamide gel containing sodium dodecyl sulfate and wherein the sequence of the NH 2 -terminal amino acid residues of said canine SAP-6 is selected from the group consisting of: Ile-Pro-Ile-Pro-Ile-Pro-Tyr and Ile-Pro-Cys-Phe-Pro-Ser-Ser- Leu-Lys-Arg-Leu-Leu-Ile- Ile-Val-Val-Val-Val-Val (lleMIle) Val-Val 8. Isolated and substantially pure bovine SAP-6, said bovine SAP-6 enhancing surfactant-like activity of lipids, having a simple molecular weight of about 6,000 daltons determined in a polyacrylamide gel containing sodium dodecyl sulfate and wherein the sequence of the NH 2 -terminal amino acid residues of said bovine SAP-6 is selected from the group consisting of: Phe-Pro-Ile-Pro-Ile-Pro and Leu-Ile-Pro-Cys-Cys-Pro-Val-Asn-Ile-Lys-Arg-Leu- Leu-I le-Val-Val-Val-Val-Val-Val-Leu -Leu - Val-Val-Val (Val ) (Val) 9. Isolated and substantially pure SAP-6 with an amino acid residue composition comprising the following: Approximate Amino Acid Residues Amino Acid Residues Per Protein Molecule Cys about 3 to about 6 ASX about 1 to about 3 Thr 0 to about 1 Ser about 1 to about 6 Glx above 1 to about 4 * Pro about 4 to about 6 Gly about 4 to about 7 Ala about 2 to about 7 Val about 7 to about 25 Met about 1 to about 2 He about 4 to about 7 Leu about 10 to about 16 Tyr 0 to about 2 Phe 0 to about 3 His 0 to about 1 Lys about 2 to about 3 Trp 0 to about 2 Arg about 2 to about 5 10. The SAP-6 of claim 9 wherein said SAP-6 is human and has amino acid residue composition comprising the following: Approximate Amino Acid Residues Amino Acid Residues Per Protein Molecule .. Cys about 5 Asx about 1 Thr about 1 Ser about 1 Glx about 4 Pro about 5 Gly about 4 Ala about 7 Val about 7 Met about 1 He about 6 Leu about 10 Tyr about 2 Phe about 1 His about 1 Lys about 2 Arg about 5 11. The SAP-6 of claim 9 wherein said SAP-6 is canine and has amino acid residue composition comprising the following: Approximate Amino Acid Residues Amino Acid Residues Per Protein Molecule Cys about 2 Asx about 1 Thr about 1 Ser about 4 Glx about 1 Pro about 5 Gly about 5 Ala about 3 Val about 20 Met about 1 to about 2 He about 6 Leu about 14 Tyr about 1 Phe about 2 Lys about 2 Arg about 2 12. The SAP-6 of claim 9 wherein said SAP-6 is bovine and has amino acid residue composition comprising the following: Approximate Amino Acid Residues Amino Acid Residues Per Protein Molecule Cys about 4 ASX about 3 Ser about 1 Glx about 0 to about 1 Pro about 5 Gly about 5 Ala about 3 Val about 24 Met about 2 He about 5 Leu about 14 Phe about 0 to about 1 Lys about 2 Arg about 2 13. A method of isolating substantially pure SAP-6 from animal tissue which comprises the steps of: separating the animal tissue into a particulate fraction and a liquid fraction; and extracting from the liquid fraction the substantially pure SAP-6. 14. A method of claim 13 wherein said separation step comprises: centrifuging the animal tissue to generate a cell fraction and a liquid supernatant; centriguing the liquid supernatant to generate a pellet fraction containing SAP-6; and obtaining from the pellet fraction with an organic solvent the liquid fraction wich contains SAP-6. 15. A method of claim 13 wherein said extraction step comprises: concentrating the liquid fraction, thereby obtaining a material containing SAP-6; dispersing the material in chloroform to form a dispersion; applying the chloroform dispersion to a silicic acid column; and eluting the silicic acid column in stepwise increments with selected solvents in series wherein the selected solvents comprise an alcohol and chloroform, wherein the alcohol concentration in each selected solvent of the series is continuously increased from 0% up to about 100% with each of the stepwise increments. 16. A method of claim 13 which after said extraction step further comprises the step of delipidating the substantially pure SAP-6 to further remove lipids thereform. 17. A method of claim 16 wherein said delipidating step is carried out with dialysis. 18. A substantially pure SAP-6 produced in accordance with the method of claim 13. 19. A substantially pure SAP-6 produced in accordance with the method of claim 16. 20. Substantially pure SAP-6 produced by a process of claim 13 wherein the process further comprises the step of delipidating the substantially pure SAP-6 to further remove lipids therefrom, the delipidating step being carried out with dialysis. 21. A medicament for reducing or maintaining normal pulmonary surface tension in the alveoli of an animal comprising substantially pure SAP-6, a lipid and pharmaceutically acceptable carrier, said SAP-6 and said lipid being present in a effective amount to reduce or maintain normal pulmonary surface tension in the alveoli. 22. A medicament of claim 21 further including SAP-6 multimers. 23. A medicament of claim 21 wherein said lipid is a synthetic phospholipid, a naturally occurring phospholipid or mixture thereof. 24. A medicament of claim 21 wherein said lipid is selected from a class consisting of phospholipids, neutral lipids, cholesterol, cholesterol esters and mixtures thereof, phosphatidylcholine, disaturated phosphatidylcholine, phosphatidylglycerol, dipalmitoylphosphatidylcholine, phosphatidylinositol and mixtures thereof. 25. A preparation for replacing pulmonary surfactant material of an animal comprising an isolated SAP-6 mixed with a lipid. 26. A preparation of claim 25 further including SAP-6 multimers. 27. A preparation of claim 25 wherein said lipid is a synthetic phospholipid, a naturally occurring phospholipid or mixtures thereof. 28. A preparation of claim 25 wherein said lipid is selected from a class of phospholipids consisting of phospholipids, neutral lipids, cholesterol, cholesterol esters and mixtures thereof, phosphatidylcholine, disaturated phosphatidylcholine, phosphatidylglycerol, dipalmitoylphosphatidylcholine, phosphatidylinositol and mixtures thereof. 29. A method of treating ' or preventing hyaline membrane disease or other syndromes associated with insufficient pulmonary surfactant material in animals comprised of administering to the animal in an effective dose, isolated SAP-6 and a lipid to reduce or maintain normal pulmonary surface tension. 30. A method of claim 29 wherein the protein and lipid are administered to the animal as a liquid or as an aerosol spray. 31. A method of claim 29 wherein said administration step further includes administrating SAP-6 multimers. 32. A specific, immunologically active anti-SAP-6 antibody obtained as a serum immunoglobulin component from an animal immunized with SAP-6 and characterized by its donation to the immunoglublin of the following properties: a.) the ability of said immunoglobulin to bind to or precipitate SAP-6 from a mixture containing SAP-6; b.) the ability of said immunoglobulin to increase the sedimentation rate of SAP-6; c.) the ability of said immunoglobulin to modify elution characteristics of SAP-6; and d.) the ability of said immunoglobulin to identify or quantify SAP-6 from a sample. 33. The anti-SAP-6 antibody of claim 32 wherein the antibody is a monclonal or polyclonal antibody. 30:1801o

Inventors: WHITSETT JEFFREY A, WHITSETT, JEFFREY, A.
Applicants: CHILDRENS HOSP MEDICAL CENTER, CHILDREN'S HOSPITAL MEDICAL CENTER
Application year: 1986
Main classification: C07K15
IPC codes: A61K38, C07K1, C07K14, C07K16, G01N33