Immunoassay for small cell lung carcinoma

An immunoassay for small cell lung carcinoma is provided which uses small cell lung carcinoma voltage-gated calcium channel antigen (SCC-VGCC Ag) comprising a detectable label and antibodies against SCC-VGCC Ag to detect endogenous SCC-VGCC Ag.

BACKGROUND OF THE INVENTION 
Small cell lung carcinoma (SCC) is the prototype, and most lethal, of a 
group of tumors known as "APUDomas". These tumors express markers that are 
characteristic of neurons, including the .gamma..gamma. isozyme of enolase 
(known as "neuron-specific enolase", or NSE), the BB isozyme of creatine 
kinase and neuronal-type intermediate filaments. In addition, SCC is well 
known for secreting peptide hormones (e.g., ACTH, ADH) and growth factors 
(e.g., gastrin-releasing peptide, also known as "bombesin"). 
Over the years, several of these markers of SCC have been investigated for 
potential clinical applications. For example, products secreted or shed by 
SCC into a patient's serum, or expressed in tumor tissues, could be useful 
for tumor diagnosis, radionuclide imaging or therapeutic targeting of 
tumoricidal therapy. However, none of the SCC markers investigated to date 
has found wide clinical application. Therefore, there is a need to 
identify and isolate SCC factors which can be used in the diagnosis, 
imaging, monitoring and/or treatment of small cell lung carcinoma. 
SUMMARY OF THE INVENTION 
The present invention provides a diagnostic assay for small cell lung 
carcinoma (SCC) which is based on the detection of a new class of SCC 
antigen, hereinafter termed the "voltage-gated calcium channel 
[VGCC]antigen [Ag]of SCC" or "SCC-VGCC Ag". Preferably, the SCC-VGCC Ag in 
a physiological fluid of a human, preferably blood serum, is detected by 
an immunoassay, such as a competitive inhibition radioimmunoassay or a 
solid phase antigen capture assay. 
The SCC-VGCC terminology is derived from the recognition that at least some 
epitopes on VGCC antigen may be common to both cholinergic neurons and the 
small cell carcinomas. 
For example, in the neuromuscular disease, Lambert-Eaton myasthenic 
syndrome (LES), electrophysiologic and ultrastructural studies have 
implicated voltage-gated calcium channels (VGCC) of peripheral cholinergic 
nerve terminals as the target of pathogenic autoantibodies. Approximately 
70% of LES patients have or will develop SCC. Recognition that SCC cells 
in culture exhibit VGCC activity, focussed attention on 
.omega.-conotoxin-GVIA. This toxin, hereafter referred to as 
".omega.-CgTx," is a neurotoxin of the fish-eating snail Conus geographus 
that binds with high affinity to neuronal-type VGCC. It has been discussed 
as a potential tool for identifying an SCC component to which LES IgG 
might bind. H. J. DeAizpurua et al., Trans. Amer. Soc. Neurochem., 20, 221 
(1989) and Cancer Res., 48, 4719 (1988). 
The specific complexing of LES IgG antibodies with an .omega.-CgTx-binding 
molecule extracted from SCC tumors was first demonstrated by V. A. Lennon 
et al., Mayo Clin. Proc., 64, 1498 (1989) and was subsequently confirmed 
by E. Sher et al., J. Autoimmunity, 2, 909 (abstract) (1989) and Cancer 
Res., 50, 3892 (1990). This phenomenon is the basis of a serological test 
that aids the diagnosis of LES (See V. A. Lennon et al., cited above). 
Thus, the SCC-VGCC antigen is a new class of tumor antigen that has not 
been used or considered for use in the diagnosis of cancer. V. A. Lennon 
et al., op. cit., suggested that "if the complete molecular definition of 
the antigens...reveals that some epitopes are SCC-restricted, it should be 
feasible to design synthetic peptide vaccines to enhance a cytotoxic 
anti-tumor response". However, it has not been disclosed or suggested that 
.omega.-CgTx in combination with SCC-VGCC antigen might be applied in an 
assay such as a modified competitive inhibition assay to detect and 
quantitate in human body fluids the SCC-VGCC antigen in a solubilized form 
(secreted or shed) as a diagnostic aid for SCC or other types of cancer. 
Therefore, polyclonal or monoclonal antibodies which specifically bind to 
SCC-VGCC Ag can be used alone, or in combination with labelled SCC-VGCC 
Ag, to detect tumor antigens (or fragments thereof) in body fluids (e.g., 
serum, cerebrospinal fluid, ascites, pleural effusions and possibly urine) 
obtained from patients afflicted with small cell lung carcinoma and 
possibly, with related tumors (e.g., APUDomas, neuroblastoma). Testing of 
body fluids would be indicated clinically: 1) as a diagnostic test to 
initially screen for tumor antigens in persons at high risk for SCC (e.g., 
smokers with a family history of cancer) or in patients already suspected 
of having lung cancer; 2) to serially monitor, in the course of treatment 
(e.g., chemotherapy, X-irradiation), the disappearance of tumor antigen 
from serum; 3) as a periodic follow-up test, to detect tumor recurrence 
early.

DETAILED DESCRIPTION OF THE INVENTION 
In its broadest aspect, the present invention comprises using SCC-VGCC 
antigen which can incorporate a detectable label, or a binding site for a 
detectable label, in an immunoassay to detect endogenous SCC-VCGCC Ag in a 
physiological fluid, e.g., of a human such as a patient afflicted with 
SCC, or a human suspected of having SCC or related cancer. 
SCC-VGCC Ag of a useful purity can be obtained from well-characterized SCC 
tumor lines by detergent extraction. The lines are derived from Mayo 
Clinic patients and established as continuous cell lines in culture and in 
athymic nude mice, in the Neuroimmunology Laboratory of the Mayo Clinic, 
Rochester, MN. The murine source of tumor can provide the quantities of 
antigen that are currently required for serologic testing. Recent studies 
indicate that normal human brain gray matter may be a satisfactory and 
more readily available alternative source of SCC-VGCC antigen. See C. L. 
Williams et al., Soc. Neurosci. Abstr., 14, 66 (1988) and V. A. Lennon, 
Ann. Neurol., 28, 281 (abstract) (1990). 
To detect endogenous SCC-VGCC Ag in a sample by a competitive inhibition 
immunoassay, a known amount of anti-SCC-VGCC Ag antibody is added to a 
sample containing an unknown amount of endogenous SCC-VGCC Ag. The known 
amount is selected to be less than the amount required to complex all of 
the SCC-VGCC Ag suspected to be present, e.g., that would be present in a 
sample of the same amount of physiological fluid obtained from a patient 
known to have SCC. Next, a known amount of SCC-VGCC Ag comprising a 
detectable label is added. If endogenous SCC-VGCC Ag is present in the 
sample, no antibodies will be available to bind the labelled SCC-VGCC 
antigen, and it will remain free in solution. If no endogenous SCC-VGCC Ag 
is present, the added labelled SCC-VGCC antigen will complex with the 
added anti-SCC-VGCC antibodies to form binary complexes. Next, the binary 
antibody-antigen complexes are precipitated by an anti-human IgG antibody. 
The amount of radioactivity in the precipitate (a ternary complex) is 
inversely proportional to the amount of endogenous SCC-VGCC Ag that is 
present in the sample, e.g., a pellet free of radioactivity is indicative 
of the presence of endogenous SCC-VGCC Ag. 
Presently, the most suitable source of the exogenous anti-SCC-VGCC 
antibodies is serum derived from selected LES patients (preferably 
non-smokers without evidence of cancer). Murine monoclonal anti-SCC-VGCC 
antibodies may also be useful in this assay. 
Solid phase assays provide alternative systems for detecting SCC-VGCC 
antigens. For example, immobilized monoclonal anti-SCC-VGCC antibodies 
(rodent or human) e.g., bound to polystyrene plates or particles, can be 
used as tools for capturing soluble SCC-VGCC Ag. In a feasibility study, 
the capture of detergent-solubilized SCC-VGCC Ag by an immobilized rat 
anti-SCC-VGCC monoclonal antibody was demonstrated by the subsequent 
binding of .sup.125 I-.omega.-CgTX. An immobilized irrelevant control rat 
monoclonal antibody did not capture solubilized SCC-VGCC Ag as judged by 
the insignificant binding of subsequently added .sup.125 I-.omega.-CgTX. 
This is a prototype example of an alternative system that could be used to 
detect secreted or shed SCC-VGCC Ag in a patient's body fluids. The amount 
of SCC-VGCC Ag detected in a patient's body fluid sample in this assay is 
expressed in terms of moles of labelled probe, e.g., .sup.125 
I-.omega.-CgTX that specifically bind to the captured SCC-VGCC Ag, after 
the immobilized monoclonal anti-SCC-VGCC antibody is exposed to the 
patient's body fluid. 
A second monoclonal anti-SCC-VGCC antibody can be used as an alternative 
probe to .sup.125 I-.omega.-CgTX for detecting SCC-VGCC Ag captured by the 
immobilized monoclonal antibody. The second antibody can be labelled 
radioisotopically (e.g., by .sup.125 I) or conjugated directly to a 
detector enzyme (e.g., alkaline phosphatase or horse radish peroxidase), 
or can be labelled indirectly with a binding site for a detectable label, 
e.g., via biotinylation. The biotinylated antibody can then be detected by 
its ability to bind to an avidin-linked enzyme. If the second antibody is 
biotinylated, a detector enzyme conjugated to avidin will be subsequently 
added. The final step for detecting enzymes conjugated to monoclonal 
antibody or to avidin is the addition of a substrate appropriate for the 
enzyme to allow quantitative colorimetric detection of reaction product. 
The value (read in optical density units) is converted to fmol of SCC-VGCC 
Ag by reference to a standard curve generated in a control assay in which 
a standard extract of detergent-solubilized SCC-VGCC Ag is added in graded 
concentrations to the immobilized anti-SCC-VGCC monoclonal antibody. 
A preferred method to label SCC-VGCC Ag is to react it with 
.omega.-conotoxin-GVIA or ".omega.-CgTx", which is available in a 
radiolabelled form that retains its high affinity for SCC-VGCC Ag (for 
example, .sup.125 I-labelled .omega.-CgTx is available from Amersham 
Corp., Arlington Heights, Ill.). Complexes formed between radiolabelled 
.omega.-CgTx and SCC-VGCC Ag retain their ability to bind to anti-SCC-VGCC 
Ag antibodies. 
The invention will be further described by reference to the following 
detailed examples. 
EXAMPLE 1 
Preparation of SCC-VGCC Ag 
SCC tumors (excised from athymic nude mice) or fresh human brain gray 
matter is used as a source of "detector" antigen, and for establishing a 
quantitative standard curve of immunoreactive units (fmol/ml). All steps 
are performed at 4.degree. C. Tissues are chopped finely and dissociated 
in physiological phosphate-buffered saline by pushing the tissue through a 
stainless steel sieve (0.010 inch diameter wire, 40 mesh). After 
centrifugation at 850.times.g, the pelleted cells are transferred to a 
Teflonglass homogenizer in an equal volume of extraction buffer (20 mM 
Tris with 2% CHAPS, 20% glycerol, 0.02% NaN.sub.3 and solid HEPES buffer 
to pH 7.5), with fresh protease inhibitors (PMSF, 10 .mu.M; Pepstatin, 0.1 
.mu.g/ml; and aprotinin, 1 KIU/ml). After 5 manual strokes, the homogenate 
is shaken intermittently for 2 hr, and then ultracentrifuged at 
100,000.times.g. Aliquots of the clear supernate are stored in plastic 
vials at -70.degree. C. for use as SCC-VGCC antigen. 
EXAMPLE 2 
Labelling of Standard SCC-VGCC Extract With .sup.125 I-.omega.-CgTX 
The .sup.125 I-labelled .omega.-CgTx (Amersham) is reconstituted in 1% 
acetic acid according to the supplier's instructions, and stored at 
-70.degree. C. in polypropylene microcentrifuge tubes (Sarstedt, 
Princeton, N.J.) that are pretreated with 10% bovine serum albumen for 16 
hr at 4.degree. C. Immediately before use, the frozen toxin is thawed 
rapidly, neutralized (pH 7.4) with 1M NaOH, and added (221 fmol/ml) to a 
standard preparation of SCC-VGCC Ag prepared in accord with Example 1. 
After holding the mixture at 4.degree. C. for 20 hr, unlabelled 
.omega.-CgTx is added (20,000-fold molar excess) to prevent further 
binding of .sup.125 I-.omega.-CgTX. SCC-VGCC Ag complexed with .sup.125 
I-.omega.-CgTX is now ready to use. 
To determine nonspecific binding, a control extract of SCC tumor prepared 
as in Example 1, is preexposed for 1 hr to 20,000 fold molar excess of 
unlabelled .omega.-CgTx before adding .sup.125 I-.omega.-CgTx. Specific 
binding sites for .sup.125 I-.omega.-CgTx are enumerated as the difference 
in radioactive (.gamma.) counts in samples with and without unlabelled 
.omega.-CgTx, determined by vacuum filtration on glass microtiter filters 
(Whatman, England) pretreated with 1% polyethylenimine in accord with the 
methodology of R. F. Bruns et al., Anal. Biochem., 132, 74 (1983). 
EXAMPLE 3 
Establishment of an Antigen (SCC-VGCC Ag)-Binding Curve 
Serial dilutions of a standard source of high affinity anti-SCC-VGCC Ag 
antibodies (currently 5.mu.l or less of serum from a selected LES patient) 
are dispensed in duplicate siliconized glass tubes (10 by 75 mm; Baxter 
Scientific Products, McGraw Park, Ill.) containing 0.1 ml of assay buffer 
(40 mM sodium phosphate, 10 mM sodium chloride, 0.1% Tween 20, and 0.02% 
NaN.sub.3). Normal human serum is added to make 5 .mu.l the final volume 
of human serum. 
The standard SCC-VGCC Ag extract complexed with .sup.125 I-.omega.-CgTX is 
admixed (17-40 pmol/l) with the antibody dilutions, and held at 4.degree. 
C. for 16 hr. Anti-human IgG antibodies (e.g., from a goat hyperimmunized 
with human IgG and adjuvants) are added. After 30 min at 22.degree. C., 
polyethylene glycol (PEG) is then added to a final concentration of 0.7% 
(to enhance the precipitation of immune complexes which, after 2 hr at 
4.degree. C., are pelleted by centrifuging for 5 min at 1750.times.g). The 
pelleted complexes are washed twice by repeated resuspension and 
recentrifugation in assay buffer containing 0.7% PEG. The pellets are 
counted for .gamma.-emission with a gamma detector. The mean value for 
precipitates obtained from three tubes containing 5 .mu.l of the normal 
human serum is subtracted from the value of each dilution of the anti-VGCC 
antiserum. The latter values are converted to moles of bound .sup.125 
I-.omega.-CgTx by reference to the counts per minute for a daily standard 
of .sup.125 I-.omega.-CgTx (allowing for radioactive decay). 
The dilution of anti-SCC-VGCC antiserum that precipitates 50% of the 
radioactive counts precipitated by undiluted (i.e., 5 .mu.l) anti-SCC-VGCC 
antibodies is selected as the limiting dilution to be used in testing body 
fluids of patients with suspected cancer for the presence of 
immunoreactive SCC-VGCC Ag. A representative graph derived from these data 
is shown in FIG. 1. 
EXAMPLE 4 
Establishment of an Antigen (SCC-VGCC Ag)-Inhibition Curve 
Graded quantities of SCC-VGCC Ag extract (currently 0.1 through 10.0 
fmoles) complexed with an excess of unlabelled .omega.-CgTx (442 pmol/ml 
for 1 hr at 4.degree. C. before use) are dispensed into duplicate 
siliconized glass tubes. Extraction buffer containing bovine serum albumin 
(30 mg/ml) is added to make the final volume 200 .mu.l. A limiting 
dilution of the standard anti-SCC-VGCC Ag antiserum (with normal human 
serum to make a final solution of 5 .mu.l human serum) is added in a final 
volume of 100 .mu.l with assay buffer. The samples are held for 4 hr at 
4.degree. C. (to allow interaction of anti-SCC-VGCC Ag antibodies with 
immunoreactive fragments in the .omega.-CgTx-SCC-VGCC Ag extract). Next 
the standard SCC-VGCC Ag complexed with .sup.125 I-.omega.-CgTx is added 
for 16 hr at 4.degree. C. Anti-human IgG antibodies (prepared in a goat) 
are added for 30 min at 22.degree. C. Polyethylene glycol is then added to 
a final concentration of 0.7%. 
After 2 hr at 4.degree. C., antigen-antibody complexes are pelleted by 
centrifugation and washed twice by repeated resuspension and 
recentrifugation in assay buffer containing 0.7% PEG. The pellets are 
counted for .gamma.-emission with a gamma detector. The mean value for 
precipitates obtained from three tubes containing 5 .mu.l of normal human 
serum is subtracted from the value of each sample tested. The latter 
values are used to establish a reference inhibition curve by plotting 
counts per minute of .sup.125 I-.omega.-CgTx-SCC-VGCC Ag precipitated vs. 
moles of .omega.-CgTx-SCC-VGCC Ag [unlabelled] added per tube. A 
representative graph derived from these data is shown in FIG. 2. 
EXAMPLE 5 
Competitive Inhibition Radioimmunoassay For Detecting SCC-VGCC Ag in Serum 
Fresh or deep frozen human blood serum is the most useful specimen to test. 
In a patient with a large burden of SCC tumor, soluble immunoreactive 
SCC-VGCC Ag can be detected in 10 .mu.l of serum. However, with unknown 
samples it is preferred to use about 0.5 ml of serum. Protease inhibitors 
are added to the sample of serum (final concentration 0.01 mM PMSF, 0.1 
.mu.l ml Pepstatin A, 1 KIU/ml Aprotinin), which is then depleted of IgG 
by adding Sepharose beads conjugated with staphylococcal protein A and 
precoated with rabbit anti-human IgG. After 1 hr at 22.degree. C. the 
beads are removed by centrifugation, and a limiting dilution of standard 
anti-SCC-VGCC Ag antiserum is added (e.g., 1 .mu.l), with normal human 
serum added to make a final solution containing the equivalent of 5 .mu.l 
of human serum. The samples are held for 4 hr at 4.degree. C. (to allow 
interaction of anti-SCC-VGCC Ag antibodies with any immunoreactive 
fragments in the patient's IgG-depleted serum). 
Next, the standard SCC-VGCC Ag complexed with .sup.125 I-.omega.-CgTx is 
added for 16 hr at 4.degree. C. (together with 20,000 fold molar xs of 
nonradioactive .omega.-CgTx to prevent the .sup.125 I-.omega.-CgTx binding 
to putative SCC-VGCC Ag present in the patient's body fluid sample). Next, 
anti-human IgG antibodies are added for 30 min at 22.degree. C. 
Polyethylene glycol is then added to a final concentration of 0.7%. After 
2 hr at 4.degree. C., antigen-antibody complexes are pelleted by 
centrifugation and washed twice by repeated resuspension and 
recentrifugation in assay buffer containing 0.7% PEG. The pellets are 
counted for .gamma.-emission with a gamma detector. The mean value for 
precipitates obtained from three tubes containing 5 .mu.l of normal human 
serum is subtracted from the value of each sample tested. The latter 
values are converted to moles per liter of SCC-VGCC Ag by reference to an 
inhibition curve such as that shown in FIG. 2. 
EXAMPLE 6 
Solid Phase Capture Assay for Detection and Quantification of SCC-VGCC 
Antigen in a Patient's Body Fluid 
Monoclonal IgG anti-SCC-VGCC antibodies (of rodent, human or chimeric 
murine-human hybridoma origin) are purified (e.g., by affinity 
chromatography on protein A-Sepharose) from tissue culture medium or from 
ascites fluids of athymic nude mice inoculated intraperitoneally with 
cells secreting the anti-SCC-VGCC antibodies. An anti-SCC-VGCC monoclonal 
IgG is coated directly onto a support substrate such as polystyrene beads 
or the wells of a plastic microtiter plate by conventional methodology. To 
increase the number of SCC-VGCC Ag capture sites, the monoclonal IgG can 
be biotinylated (by standard methodology). The support substrate in this 
case is precoated with a biotinylated linker protein (e.g., bovine serum 
albumin). After adding avidin to the plate coated with biotinylated linker 
protein, and washing, the biotinylated anti-SCC-VGCC monoclonal IgG is 
added. After 3 hr at 37.degree. C. the monoclonal IgG solution is replaced 
by phosphate-buffered saline containing 0.05% Tween-20 and 10% normal goat 
serum ("wash buffer") for 1 hr at room temperature (to block residual 
non-specific protein-binding sites). The support substrate coated with 
anti-SCC-VGCC IgG (directly or via biotin-avidin-biotin linkage) is now 
ready to specifically capture SCC-VGCC antigen that might be present in a 
patient's body fluid. 
The patient's body fluid sample (e.g., 200 .mu.l of serum) is brought into 
contact with the anti-SCC-VGCC antibody-coated solid phase substrate (with 
protease inhibitors) for 1 hr at 22.degree. C. followed by 16 hr at 
4.degree. C. Next the substrate is washed three times with "wash buffer". 
At this stage, a labelled probe is added to detect (and quantitate) any 
SCC-VGCC antigen complexed by the immobilized monoclonal IgG (in a binary 
complex). This can be done by adding a predetermined excess amount of 
.sup.125 I-.omega.-CgTx at pH 7.4 in phosphate-buffered saline containing 
0.05% Tween-20, 2% normal goat serum and 200 .mu.g/ml lysozyme (to prevent 
non-specific binding of the basically charged .sup.125 I-.omega.-CgTx). 
After 120 min at 22.degree. C., and 16 hr at 4.degree. C., the ternary 
complex bound to the plate is washed three times with phosphate-buffered 
saline containing 0.05% Tween-20, and the cpm of .sup.125 I-.omega.-CgTx 
bound to each well is counted by a gamma detector. After subtracting the 
means value for cpm of .sup.125 I-.omega.-CgTx bound to duplicate wells to 
which a control normal human serum sample has been added instead of a 
patient's serum, the corrected value (i.e, specific cpm) bound by the 
patient's sample is converted to the moles of bound .sup.125 
I-.omega.-CgTx by reference to the cpm for a daily standard of .sup.125 
I-.omega.-CgTx (allowing for radioactive decay). 
An alternative to using .sup.125 I-.omega.-CgTx as the probe for detecting 
SCC-VGCC antigen captured from the patient's serum by the immobilized 
monoclonal anti-SCC-VGCC IgG, is to use a second monoclonal anti-SCC-VGCC 
antibody directed at a different epitope of the SCC-VGCC antigen. The 
second 
monoclonal antibody, labelled radioisotopically (e.g., .sup.125 I) or 
conjugated directly with enzyme (e.g., alkaline phosphatase or horse 
radish peroxidase), is allowed to bind for 45 min at 37.degree. C. to form 
a ternary complex. After washing three times in phosphate buffered-saline 
containing 0.05% Tween-20, bound radioactivity is counted by a gamma 
detector. In the case of an enzyme-conjugated second antibody, an 
appropriate substrate is added to the ternary complex for 30 min at 
37.degree. C., and the reaction product is measured colorimetrically in 
terms of optical density units. This value is converted to fmol of 
SCC-VGCC antigen by reference to a standard curve generated in a control 
plate, in which, instead of a patient's body fluid, a standard extract of 
detergent-solubilized SCC-VGCC antigen was added in graded concentrations 
to the immobilized anti-VGCC monoclonal IgG. 
All publications cited herein are incorporated by reference herein. The 
invention has been described with reference to various specific and 
preferred embodiments and techniques. However, it should be understood 
that many variations and modifications may be made while remaining within 
the spirit and scope of the invention.