US 5,516,643 A · Immunochemical assays for cancer-associated SCM-recognition factor

Abstract

Disclosed are antibodies to peptides active in the structuredness of the cytoplasmic matrix test (SCM-factor peptides) and to fragments of the peptides by immunization of antibody-producing animals with the peptides or fragments. Both polyclonal and monoclonal antibodies can be prepared. Particularly useful are antibodies specifically binding the peptides M-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-V-P-L-F-M-G-K (SEQ ID NO: 2) and F-L-M-I-D-Q-N-T-K (SEQ ID NO: 3). The antibodies can be labeled and are suitable for performing immunoassays to detect the presence of SCM cancer-recognition factors in cell cultures or body fluids. One particularly useful immunoassay can distinguish SCM factor from partially homologous peptide sequences, and comprises: (a) incubating a first aliquot of the sample with a first antibody specific for the cancer-recognition factor to bind the first antibody to the cancer-recognition factor in the first aliquot; (b) reacting a second aliquot of the sample with a second antibody specific for the amino-terminal portion of the partially homologous peptide sequence to bind the second antibody to the partially homologous peptide sequence in the second aliquot; and (c) comparing the quantity of the first antibody bound to the first aliquot with the quantity of the second antibody bound to the second aliquot to detect the cancer recognition factor.

Claims67

Dependent claims

A method for detecting a selected peptide factor active in the structuredness of the cytoplasmic matrix (SCM) test in a sample that may also contain a second peptide sequence that is partially homologous to the selected peptide factor, comprising the steps of:

(a) incubating a first aliquot of the sample with a first antibody capable of forming an antigen-antibody complex with a peptide factor active in the SCM test, the factor being a peptide of nine amino acid residues to 35 amino acid residues including a core sequence of nine amino acid residues having an amphipathicity profile substantially equivalent to that of the sequence F-L-M-I-D-Q-N-T-K, wherein the sixth amino acid of the core sequence is selected from the group consisting of Q and N, the seventh amino acid of the core sequence is selected from the group consisting of N and Q, and the ninth amino acid of the core sequence is selected from the group consisting of Q and R, the factor producing at least a 10% decrease in the intracellular fluorescence polarization value of lymphocytes capable of responding in the SCM test as isolated from donors afflicted with cancer to bind the first antibody to the peptide factor and to the partially homologous peptide sequence in the first aliquot;
(b) incubating a second aliquot of the sample with a second antibody specific for a portion of the partially homologous sequence lacking any substantial homology with any portion of the peptide factor active in the SCM test to bind the second antibody only to the partially homologous peptide sequence in the second aliquot;
and
(c) detecting a peptide factor active in the SCM test by an immunochemical reaction by comparing the quantity of the first antibody bound to the first aliquot with the quantity of the second antibody bound to the second aliquot.

1 more element

A method for detecting a peptide factor active in the structuredness of the cytoplasmic matrix (SCM) test in the presence of .alpha..sub.1 -antitrypsin, comprising the steps of:

(a) incubating a first aliquot of cells with a first rabbit IgG antibody capable of forming an antigen-antibody complex with the peptide factor active in the SCM test to bind the first antibody to the cells of the first aliquot, the first antibody being selected from the group consisting of:
(i) an antibody produced by immunization with a peptide of the sequence M-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-V-P-L-M-G-K-C (SEQ ID NO:
1) conjugated at its carboxy-terminal cysteine residue to a carrier protein;
and (ii) an antibody produced by immunization with a peptide of the sequence M-I-P-P-E-V-K-F-N-K-P-F-V-F-L-M-I-D-Q-N-T-K-V-P-L-F-M-G-K (SEQ ID NO:
2), the first antibody binding both to the peptide factor active in the SCM test and the .alpha..sub.1 -antitrypsin in the first aliquot;
(b) incubating a second aliquot of the cells with a second rabbit IgG antibody specific for the amino-terminal 19-amino acid sequence of .alpha..sub.1 -antitrypsin, the amino-terminal 19-amino acid sequence of .alpha..sub.1 -antitrypsin lacking any substantial homology with any portion of the sequence of the peptide factor active in the SCM test, the second antibody binding only to the .alpha..sub.1 -antitrypsin in the second aliquot;
(c) reacting the incubated aliquots of steps (a) and (b) separately, each incubated aliquot being reacted with an antibody specific for rabbit IgG antibody coupled to an enzyme label to label the first antibody bound to the first aliquot and the second antibody bound to the second aliquot;
(d) incubating the bound enzyme-labeled antibodies of the first and second aliquots separately, each with an indicator that yields a detectable product in response to enzymatic activity of the enzyme;
and
(e) detecting the peptide factor by an immunochemical reaction by comparing the quantity of detectable product produced by the first and second aliquot.

7 more elements

Dependencies are inferred from the claim text.

Immunochemical assays for cancer-associated SCM-recognition factor

Life of this patent

Immunochemical assays for cancer-associated SCM-recognition factor

Life of this patent

Abstract

Claims67