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1. (WO1991018290) IMMUNOASSAY OF ELASTIN PEPTIDES IN URINE
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IM UNOASSAY OF ELASTIN PEPTIDES IN URINE
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Field of the Invention
This invention relates generally to a method for th immunological identification of elastin-derived peptides (EDP) i urine to assess the existence of lung tissue damage and/or chronic obstructive pulmonary disease (COPD) .
Background Art
Chronic obstructive pulmonary disease usually develops over many years, and it is not until lung structure and function have been significantly compromised that COPD can be diagnosed with certainty by radiological and pulmonary function tests.
The connective tissue protein, elastin, is largely responsible for maintaining the elasticity of major blood vessels and lung tissue. In experimental animal models of emphysema, the major emphasis in research has been the study of the destruction of mature elastin fibers by administration of selective proteases by aerosol or intratracheal instillation. In these systems, there is a strong correlation between the production of emphysema and the digestion of insoluble amorphous lung elastin. Similarly, destruction of elastin fibers in humans is a likely prerequisite for the development of COPD and/or emphysema.
An early study involving the pathogenesis of destructive lung disease involved the immunologic identification of peptides derived from lung elastin appearing in blood or urine. See Harel, S., Janoff, A., Yu, S.U., Hurewitz, A., Bergofsky, E.H., Measurement of Elastin in Degradation ja vivo by Desmosine Radioimmunoassay, Am.Rev.Respir.Dis. 122:769-73 (1980), which describes the use of a radioimmunoassay to quantify the characteristic elastin cross-link, desmosine, in human urine and found significantly higher levels in emphysematous patients compared to normal controls. However, more recent studies failed to detect any difference in urine desmosine content between individuals with normal lung function and those with obstructive lung disease. See Davies, S.F., Offord, K.P., Brown, M.G., Campe, H. , Niewoehner, D. , Am.Rev.Respir.Dis. 128:473-75 (1983), which found that urine desmosine is unrelated to cigarette smoking or to spirometric measurements of lung function.
In Kucich, U. , Christner, P., Weinbau , G., Rosenbloo , J. (hereinafter Kucich, I), Immunologic Identification of Elastin-Derived Peptides in the Serums of Dogs with Experimental Emphysema, Am.Rev.Respir.Dis. 122:461-65 (1980), the present inventors and others used antibodies against elastin-derived peptides (EDP) to detect elastin peptides in the sera of animals with experimental emphysema. See also Darnule, T.V. , Osman, M. , Darnule, A.T., Mandl, I., Turino, G.M. , Immunologic Detection of Lung Elastin Peptides in the Serum of Rats with Elastin Induced Emphysema, Am.Rev.Respir.Dis. 121:331 (1980).
In an analysis of human plasma by an enzyme-linked immunosorbent assay (ELISA) , the three present inventors with others demonstrated that significantly higher values of elastin peptides were found in emphysema patients than in individuals with normal lung function. Kucich, U. , Christner, P. , Lippmann, M. , Kimbel, P., Williams, G. , Rosenbloom, J. , Weinbaum G. , Utilization of a Peroxidase-Antiperoxidase Complex in an Enzyme-Linked Immunosorbent Assay of Elastin-Derived Peptides in Human Plasma, Am.Rev.Respir.Dis. 131:709-13 (1985) , (hereinafter Kucich II). See also Am.Rev.Respir.Dis. 127:S28-30 (1983).
The analysis of human sera by radioimmunoassay has previously demonstrated that significantly higher values of elastin peptides were found in emphysema patients than in individuals with normal lung functions. Darnule, T.V. , McKee, M. , Darnule, A.T., Turin, G.M., Mandl, I., Anal .Biochem. 122:302- 07 (1982) .
While the Kucich II and the Darnule studies suggest the possible utility of such tests, their value in monitoring the pathogenesis of destructive lung disease remains to be proven. In the case of Kucich II, the patients with COPD had elevated peptide levels (127 ± 47 ng/ml) as compared to normal non-smokers (58 ± 17 ng/ml) , while normal smokers had intermediate values (76 ± 42 ng/ml) . With a small sample number, PiZZ individuals als appeared to have elevated levels (93 ± 18 ng/ml) . These peptid levels however, lack sensitivity, particularly with the larg variation of values in patients with COPD.
While it has been demonstrated that the monospecific antibody used in the plasma assay performs satisfactorily, it is believed that the use of a monospecific antibody in a urine assay has yet to be successfully employed. In prior work performed by the present inventors in developing a plasma test for detecting and monitoring COPD, they successfully demonstrated a significant elevation in antigenic reactivity using a monospecific antibody in samples from subjects with COPD (average of 432 ng/ml equivalents) compared to controls (108 ng/ml equivalents) . In contrast however, the present inventors have not been able to successfully develop the parallel use of a monospecific antibody in a comparable urine test for COPD and lung damage testing.
For' an extended time period, many researchers believed that a similar immunological test for urine was impractical, if not impossible, as reinforced by the failure of the urine desmosine studies noted herein. This theory was held, in part, because scientists believed that the peptides degraded from elastin and circulating in the plasma, would be further degraded by kidney filtration and other bodily processes, so that they would be effectively undetectable in urine, thereby preventing the development of a reliable quantitative test.
There has therefore been a long-felt need for a urine test which can quickly, easily and inexpensively determine the presence of lung tissue damage, emphysema, and/or COPD. Using urine as a test fluid is less intrusive to the patient and permits a larger sample to be collected and used, and is therefore preferable to using plasma and other more complex methods.
Objects of the Invention
Accordingly, it is a general object of the instant invention to provide a non-intrusive, quantitative test of improved sensitivity that is capable of identifying the presence of lung damage at an early stage, in asymptomatic individuals. This test would be useful in identifying individuals who are at risk of developing emphysema and COPD, and in monitoring disease progression.
Summary of the Invention
These and other objects of this invention are achieved by providing a non-intrusive, quantitative urine test of improved sensitivity that is capable of identifying the presence of lung damage at an early stage, before symptoms develop. Such a quantitative test of improved sensitivity is useful in identifying individuals who are at risk of developing emphysema. Such a test may also be used in monitoring the progression of the disease. The method comprises the steps of preparing a synthetic peptide or providing elastin-derived peptides and using either the synthetic peptide or the elastin-derived peptides to generate antibodies. The antibodies are then incubated with increasing amounts of either a synthetic peptide or the elastase-digested elastin-derived peptides in an indirect ELISA.
A mixture of antibodies and peptides is then applied to microtiter plates coated with the elastase-digested elastin-derived peptides or synthetic peptides so that the antibodies and peptides combine to form a first complex. The microtiter plates containing the first complex are washed to remove excess unbound antibodies. A known amount of a conjugate of enzyme-antibody that is very specific and will cling only to bound antibodies is then used. A standardization curve based on the foregoing steps is then prepared.
A sample of urine containing an unknown amount of elastin-derived peptides is obtained and analyzed in accordance with the above steps. The results of such analysis are corrected for creatinine excretion and compared to the standardization curve. From these results one can determine whether the amount of elastin-derived peptides in the unknown urine sample is sufficiently in excess of a standard value in the urine of a group of control individuals not having lung damage or chronic obstructive pulmonary disease, to establish the existence of lung disease or chronic obstructive pulmonary disease.
The kit of the above invention for carrying out a method for immunologic detection of elastin-derived peptides in urine comprises a synthetic peptide or natural peptide, and is employable with elastin-derived peptides prepared from the amorphous component of human lung elastin. The kit includes a method to use the synthetic peptide in an indirect ELISA to quantify the elastin-derived peptides in a urine sample, and to compare the results obtained with an established standard.
Description of the Drawings
Fig. 1 is a plot of the molecular sieving of a urine sample which illustrates the elution of elastase-derived elastin- derived peptides in a solution containing known molecular weight standards .
Fig. 2 is a bar graph showing the quantity of elastin-derived peptides determined in a group of individuals comprising a non-smoker control group, a smoker group and a group of COPD patients, in accordance with the method of the instant invention.
Detailed Description of the Preferred Embodiment
Materials and Methods
Goat anti-rabbit (GAR) serum may be obtained from

Cappel Laboratories of Cochranville, PA. Rabbit peroxidase-antiperoxidase (PAP) complex may be obtained from Sternberger

Meyer Immunocytochemicals, Inc. of Jarrettsville, MD. Immulon #2

Microtiter plates may be obtained from Scientific Accessories of

Andalusia, PA. Other chemicals should be of reagent grade.
I. Preparing the Synthetic
Peptide for Antibody Production
To generate antibodies for use in the present invention, it is necessary to prepare either a synthetic peptide (perhaps with the amino acid sequence GFPGGACLGKACGRKRK) or elastin-derived peptides from human lung elastin (to be described below) . The following table sets forth the amino acid which corresponds to each one-letter symbol in the preceding amino acid sequence:
One-Letter S mbol Amino Acid



To prepare the antibodies against the synthetic peptides, a synthetic amino acid sequence, such as GFPGGACLGKACG- RKRK may be coupled to keyhole limpet hemocyanin (KLH) using glutaraldehyde. This complex may then be used to produce antibodies for use in the method of the present invention as described below. Baron, M.H., and Baltimore, D. , Antibodies

Against a Chemically Synthesized Genome-Linked Protein of Polio

Virus React With Native Virus-Specified Proteins, Cell, (1982).
II. In Vitro Preparation of Elastin-Derived
Peptides Using Elastase for the Production
of Antibodies
To prepare the elastin-derived peptides (EDP) for the production of antibodies as described below and as previously described in Kucich I, the amorphous component of human lung elastin may be digested with, human neutrophil elastase at a 1:500 ratio of enzyme to elastin (w/w) for 24 hours at 37 °C.
III. Preparing Elastin Peptides from Urine in
Vitro for the Production of Antibodies
In accordance with established practices and methods for producing antibodies, it is believed that the antibodies to elastin-derived peptides isolated from urine may be prepared by the following procedure, in accordance with the preferred method of the present invention. Preparation and use of antibodies against the antigens isolated from urine should result in increased sensitivity of the present invention, as these anti- bodies are believed to be more specific to the EDP antigen in an junknown sample of urine.
A 50-100 ml sample of human urine containing elastin- derived peptides can be concentrated 10-fold by ultrafiltration in an Amicon cell Model 52, and should result in an approximate recovery of 60-70% of the elastin-derived peptides present in the urine sample. The urine sample can then be centrifuged in a Sorvall refrigerated centrifuge, DuPont Company, RC-5B rotor, at 12,000 rpm for 10 minutes to remove solids. The supernatant should then be removed and frozen at -20 °C as the elastin-derived peptides may be unstable at 4°C. The urine sample will then be fractionated on a molecular sieve P-10 column, as shown in Fig. 1, and should result in a more than 90% recovery of elastin derived peptides.
Fig. 1 demonstrates the fractionation of a human urine sample by molecular sieving on the P-10 column, using an eluting solution of 0.2 M phosphate buffer, 0.2% sodium azide, pH = 7.2, along with the conventional known molecular weight standards of bovine serum albumin (BSA) , soy trypsin inhibitor (SBTI) and cytochrome C (CYTO C) . Fractions of 5 mis were collected and absorbance was read at 280 nm to provide EDP concentrations. It is believed that ninety percent of the elastin immunoreactivity should elute between 4-20,000 daltons with little contaminating protein.
The desired fractions can then be collected and the sample may then be placed on a Superose 12 liquid chromatography column (FPLC) which separates materials according to molecular weight. It is expected that desired column effluent fractions will contain peptides having a molecular weight in the range of 13,000 daltons. Peptides having a molecular weight in this range exhibit the desired activity when subjected to peptide detection analysis. These purified EDP from urine can then be used to generate antibodies in accordance with the method of Section IV below.

IV. Generating Antibodies to the Synthetic
or Elastin-Derived Peptides
After preparing the synthetic or elastin-derived peptides as described above, either type may be used to generate antibodies in New Zealand white rabbits. See Rosenbloom, J. , Kucich, C. , Weinbaum, G., Kimbel, P., and Feierstein, N. , Immunologic Identification of Carboxy Terminal Sequences of Elastin in Human Plasma Using Monospecific Antibodies, Pulmonary Emphysema and Proteolysis, Vol. 2, edited by Taylor, J.C., and Mittman, C. , Academic Press (1986) . The IgG fractions may be purified by ammonium sulfate precipitation followed by DEAE chromatography.
V. The Enzyme-Linked Immunosorbent Assay (ELISA)
Microtiter plates may be coated with 200 μl per well of elastin-derived peptides (250 ng/ml) or synthetic peptide (1000 ng/ml), by incubation at 4°C for 24 hours in 0.1 M carbonate, pH = 9.6, containing 0.02% M sodium carbonate. Standard curves for the indirect ELISA may be generated by incubating 5 μg/ml primary antibody (for the elastin-derived peptides) or 7 μg/ml primary antibody (for the synthetic peptide) with variable concentrations of competing antigen for 16 hours at 4°C. These reaction mixtures (200 μl per well) may be transferred to the coated wells and incubated at 4°C for 1 hour. The wells may be washed with phosphate buffered saline (PBS-Tween 20) , and afterwards 200 μl per well of goat antirabbit serum at a dilution of 1:2000 may be added to the wells and incubated for 1 hour at room temperature. After washing, 200 μl per well of the peroxidase-antiperoxidase complex may be added to the wells at a dilution of 1:2000 in PBS-Tween 20, and incubated for 30 minutes at room temperature. The wells may be washed with PBS-Tween 20 and then 200 μl per well of a 2 mg/ l solution of o-phenylenediamine and 0.006% H2O2 in 0.1 M citrate, pH = 4.5, may be added. After 45-60 minutes, the absorbance values may be determined at 450 nm using an automatic plate reader (Flow Laboratories, McLean, VA) . Unknown urine samples may preferably be analyzed in triplicate at two different concentrations (usually at 30 mis 1:20 and 60 mis 1:10 dilu- tions) . See also the procedure and materials of the ELISA of Kucich II.
VI. Preparing Urine for Analysis
A 50-100 μl sample of human urine containing an unknown amount of elastin-derived peptides is obtained after centrifuga- tion of the collected urine sample in a Sorvall refrigerated centrifuge, DuPont- Company, RC-5B sized rotor, at 12,000 rpm for 10 minutes to remove solids. The supernatant may then be removed and frozen at -20°C, as the elastin-derived peptides may be unstable at 4°C. The urine is then concentrated 10-fold by ultrafiltration in an Amicon cell Model 52, and it is believed that there will be a 60-70% recovering of elastin-derived peptides.
The human elastin-derived peptides or the synthetic peptides may be applied to microtiter plates. The EDP antibodies and peptides (synthetic or urine-derived) should combine to form a complex. The microtiter plates may then be washed to remove any excess unbound antibodies. After binding the enzyme-linked antibodies and the addition of substrate, the absorbance of each unknown is then subjected to the standardization curve previously prepared in order to determine the elastin peptide level in the urine sample. By comparing the concentration of elastin-derived peptides in the urine sample, after correction for creatinine excretion, to those readings obtained from a normal individual, one can establish the existence of chronic obstructive pulmonary disorder, as shown more clearly in Fig. 2.
The results of the above analysis should demonstrate that COPD patients excrete high concentrations of low molecular weight EDP in their urine. In the study of a group of individuals comprising non-smokers, smokers and COPD patients, in accordance with the method of the above invention, the following results were obtained as summarized in Fig. 2 (n = number of individuals) and in Table 1 below (values reported below as ngEDP/mg creatinine) :

COPD
211
20
441
255
275
329
296
123
84
364
350
515
165
222
126
202
324
77
181
17
84
520

Non-smokers n=24 Smokers n=8 COPD n=22

After correction for creatinine excretion, the present inventors determined that EDP levels for COPD patients were 252 ± 162 ng/mg creatinine (n=22) , for non-smokers EDP levels were 88 ± 60 ng/mg (n=24; p<0.001) and for normal smokers 87 ± 57 ng/mg (n=8) . Multiple samples from the same patient revealed that urinary EDP levels varied less than 20-25%. COPD patients appeared to excrete lower molecular weight peptides into the urine, since dialysis through a 3500 M.W. cut-off membrane resulted in the loss of 80% of the immunoreactivity, while the urine of a non-smoker lost less than 35% immunoreactivity. Little EDP antigen may be detected in any urine using an elastin carboxy-terminus antibody, which detects EDP in plasmas of non- smokers, smokers and COPD individuals. Am.Rev.Resp.Dis. 137:A372 (1988) . These data suggest considerable antigen processing o EDP prior to urinary excretion, particularly in COPD patients. The easy sampling and the presence of low molecular weight ED make the urine ideal to use for isolation and characterization o such elastin antigen(s) . Identifying the in vivo - generated elastin antigen(s) may provide tools for developing more sensitive probes of lung destruction and for monitoring therapy.
Without further elaboration the foregoing will so fully illustrate my invention that others may, by applying current or future knowledge, readily adapt the same for use under various conditions of service.