Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-02-21
2004-05-18
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007800, C435S068100, C435S173300, C435S810000, C435S091500, C436S536000, C436S513000, C530S381000, C530S388150
Reexamination Certificate
active
06737243
ABSTRACT:
TECHNICAL FIELD
The invention relates to methods for determining the pulmonary surfactant protein SP-C, to SP-C-specific antibodies and to the provision of a reagent kit for carrying out the methods.
PRIOR ART
The lungs of all vertebrates contain a substance mixture called “pulmonary surfactant”. It has surface-active properties and reduces surface tension in the alveolar region of the lungs. In addition to phos-pholipids such as dipalmitoylphosphatidylcholine (DPPC) and phosphatidylglycerol (PG), the pulmonary surfactant contains proteins as further essential components. To date, four different surfactant proteins have been described, which are designated SP-A, SP-B, SP-C and SP-D, corresponding to the order in which they were discovered (Possmayer, F., A Proposed Nomenclature for Pulmonary Surfactant-associated Proteins. Am. Rev. Respir. Dis. 1988, 138, 990-998). The term SP stands for surfactant protein (surfactant-associated protein, SP).
SP-B and in particular SP-C are strongly hydrophobic proteins. The total proportion of hydrophobic proteins in the pulmonary surfactant is approximately 1% (Curstedt, T., Jörnvall, H., Robertson, B., Bergmann, T., Bergren, P.: Two Hydrophobic Low-Molecular-Mass Protein Fractions of Pulmonary Surfactant. Characterization and Biophysical Activity. Eur. J. Biochem. 1987, 168, 255-262).
The determination (detection and in particular quantification) of these hydrophobic proteins in samples of pulmonary surfactant (for example from lung lavage) frequently gives unsatisfactory results when the techniques employed for hydrophilic proteins are used, if it is possible at all. The methods which are customarily used for separating and determining hydrophilic proteins, such as, for example, “Western blotting” or ELISA (Enzyme-Linked Immunosorbent Assay) techniques can be applied to hydrophobic proteins only under certain conditions, since on the one hand the “Western blot” per se is semiquantitative and, on the other hand, the use of an ELISA is very problematic, since it can generally only be carried out with proteins which are soluble in aqueous systems. In most cases, only traces of the analytes to be quantified are present. Furthermore, in the samples to be analyzed, SP-B and SP-C are frequently associated with other hydrophobic substances (for example lipids), which renders quantification by customary methods even more difficult. In the ELISA, the substance to be examined is usually not separated prior to the determination from the other components which are contained in the mixture. Van Eijk and co-workers (Van Eijk, M., De Haas, C. G. M. and Haagsman, H. P.: Quantitative Analysis of Pulmonary Surfactant Proteins B and C. Analytical Biochemistry 1995, 232, 231-237) describe a process for quantifying SP-C and SP-B in samples of pulmonary surfactant. Extracts containing surfactant proteins are separated by high-pressure liquid chromatography, and SP-B or SP-C are detected and quantified by absorption at 228 nm. (The detection limits are at 1 &mgr;g of SP-B and 4 &mgr;g of SP-C.)
For SP-B, a quantitative detection by ELISA technique is described (Krämer, H. J., Schmidt, R., Günther, A., Becher, G., Suzuki, Y., Seeger, W.: ELISA Technique for Quantification of Surfactant Protein B (SP-B) in Bronchoalveolar Lavage Fluid. Am. J. Respir. Crit. Care Med. 1995, 152, 1540-1544). An immunoassay for the determination of SP-C has hitherto not been described, apparently because the preparation of SP-C-specific antibodies has not been successful (Beers, M. F., Wali, A., Eckenhoff, M. F., Feinstein, S. I., Fisher, J. H. and Fisher, A. B.: Am. J. Respir. Cell Mol. Biol. 1992, 7, 368-378).
DESCRIPTION OF THE INVENTION
It is an object of the invention to provide processes permitting the determination of pulmonary surfactant protein SP-C in a sample in a simple manner and with high sensitivity.
Surprisingly, we have succeeded in providing antibodies which are specific for SP-C, and thus allow SP-C to be determined by an immunological method.
Accordingly, the invention provides processes for determining SP-C in a sample, where the determination is carried out by an immunological method.
Further subject matters follow from the subclaims.
In the context of the invention, the determination of SP-C is to be understood as the detection and, in particular, the quantification of SP-C.
In the context of the invention, the term “SP-C” is to be understood, in analogy to the nomenclature proposed by Possmayer (Possmayer, F.: A Proposed Nomenclature for Pulmonary Surfactant-associated Proteins. Am. Rev. Respir. Dis. 1988, 138, 990-998), as the “family” of surfactant proteins which is present in natural pulmonary surfactant or the amnionic fluid of mammals designated SP-C.
Furthermore, the term “SP-C” also includes chemically synthesized or recombinantly prepared SP-C and modifications of SP-C, for example those modifications where one or more amino acids are missing or have been replaced by other amino acids. Chemically synthesized or recombinantly prepared SP-C and modifications of SP-C are described, for example, in WO91/18015, WO91/00871, WO89/04326, WO93/21225 and also in WO95/32992.
SP-C is preferably understood as meaning the surfactant protein SP-C which is present in human pulmonary surfactant or in human amnionic fluid.
In the context of the present invention, “immunological methods” are understood as meaning analytical methods based on immunochemistry, in particular on an antigen-antibody reaction. Examples of immunological methods include immunoassays such as radioimmunoassay (RIA), enzyme immunoassay (EIA, combined with solid-phase technique: ELISA) or else immunofluorescence assays. The immunoassay is carried out by exposing the sample to be investigated to an SP-C-binding antibody and detecting and quantifying the amount of antibody which binds to SP-C. In these assays, detection and quantification is carried out directly or indirectly in a known manner. Thus, detection and quantification of the antigen-antibody complexes is made possible by using suitable labels which may be carried by the antibody directed against SP-C and/or by a secondary antibody directed against the primary antibody. Depending on the type of the abovementioned immunoassays, the labels are, for example, radioactive labels, fluorescent dyes or else enzymes, such as phosphatase or peroxidase, which can be detected and quantified with the aid of a suitable substrate.
In one embodiment of the invention, the immunological method is carried out with the aid of a suitable solid phase. Suitable solid phases which may be mentioned include the customary commercial microtiter plates made of polystyrene or membranes (for example made of polyvinylidene difluoride, PVDF) which are customarily used for the ELISA technique. Surprisingly, it has been found that even chromatography plates are suitable for use as solid phase in the process according to the invention. The implementation of the process according to the invention using chromatography plates is hereinbelow also referred to as immuno-TLC.
To carry out the process according to the invention, the sample is applied to the solid phase. The sample is preferably a solution of SP-C in a suitable solvent, and the solvent is evaporated after the sample has been applied to the solid phase. To prepare a solution of the sample to be investigated in a suitable solvent, it is advantageous to use organic solvents or solvent mixtures which have proved to be suitable for solubilizing hydrophobic proteins. Examples which may be mentioned are short-chain alcohols, in particular methanol, ethanol, 2-propanol (isopropanol) or n-propanol. Furthermore, in connection with immuno-TLC, mixtures of non-polar and polar solvents were found to be useful, suitable non-polar solvents being, in particular, chloroform, methylene chloride and toluene, and suitable polar solvents being short-chain alcohols. Chloroform/methanol mixtures may be mentioned as being particularly preferred.
If desired, small amounts of water and/or acid or base may be added to the abovementioned solvents o
Günther Andreas
Ise Wolfgang
Schmidt Reinhold
Steinhilber Wolfram
Altana Pharma AG
Cheu Changhwa J.
Jacobson & Holman PLLC
Le Long V.
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