ABSTRACT
The selection and characterization of a set of mouse mAbs against high-risk human papillomavirus (HPV) E7 oncoprotein and the development of protocols for immunocytochemistry (ICC) are described here. A large number of antibodies raised towards HPV16 and 18 E7 were tested for high-risk specificity by ELISA using a panel of HPV E7 proteins. Antibodies detecting low-risk E7 were discarded, resulting in 38 high-risk HPV E7-specific antibodies. The corresponding epitopes were mapped using overlapping HPV E7 fragments displayed on phage particles. Functionality in ICC against formalin-fixed cervical cancer cell lines was demonstrated for ten mAbs; their high-risk specificity was confirmed by Western blot analysis and ICC on transiently transformed cells expressing high- or low-risk HPV E7. These mAbs were specific for one or several of the high-risk strains HPV16, 18, 31, 35 and 45. Specific E7 staining of liquid-based cytology (LBC) samples was demonstrated for seven mAbs and optimized protocols were established. The E716-41 and E718-79 mAbs demonstrated particularly strong and specific staining of cells stored in LBC fluid for at least 6 months. It is proposed that the high-risk HPV E7 staining protocols established in this study may have the potential to be included in a complementary test for the detection and identification of malignantly transformed cells, in for example atypical squamous cells of undetermined significance samples.
Subject(s)
Antibodies, Monoclonal , Antibodies, Viral , Immunohistochemistry/methods , Papillomavirus E7 Proteins/analysis , Papillomavirus Infections/diagnosis , Animals , Blotting, Western , Early Detection of Cancer/methods , Epitope Mapping , Female , Humans , Mice , Mice, Inbred BALB C , Sensitivity and SpecificityABSTRACT
Fourteen monoclonal antibodies with specificity against native or recombinant antigens within the S100 family were investigated with regard to immunoreactivity. The specificities of the antibodies were studied using ELISA tests, Western blotting epitope mapping using competitive assays, and QCM technology. The mimotopes of antibodies against S100A4 were determined by random peptide phage display libraries. Antibody specificity was also tested by IHC and pair combinations evaluated for construction of immunoradiometric assays for S100B. Out of the 14 antibodies included in this report eight demonstrated specificity to S100B, namely MAbs 4E3, 4D2, S23, S53, 6G1, S21, S36, and 8B10. This reactivity could be classified into four different epitope groups using competing studies. Several of these MAbs did display minor reactivity to other S100 proteins when they were presented in denatured form. Only one of the antibodies, MAb 3B10, displayed preferential reactivity to S100A1; however, it also showed partial cross-reactivity with S100A10 and S100A13. Three antibodies, MAbs 20.1, 22.3, and S195, were specific for recombinant S100A4 in solution. Western blot revealed that MAb 20.1 and 22.3 recognized linear epitopes of S100A4, while MAb S195 reacted with a conformational dependent epitope. Surprisingly, MAb 14B3 did not demonstrate any reactivity to the panel of antigens used in this study.
Subject(s)
Antibodies, Monoclonal/immunology , Recombinant Proteins/immunology , S100 Proteins/immunology , Animals , Antibody Specificity , Binding Sites, Antibody , Blotting, Western , Cattle , Enzyme-Linked Immunosorbent Assay , Epitope Mapping , Humans , Immunoassay , Immunoenzyme Techniques , Peptide Library , Radioimmunoassay , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , S100 Proteins/chemistry , S100 Proteins/metabolismABSTRACT
Squamous cell carcinoma antigen (SCCA) is a serological marker of squamous cell carcinomas (SCC). To study whether any of the SCCA isoforms would provide additional and more specific/sensitive clinical information than total SCCA, immunoassays specific for the different forms of SCCA (free SCCA2, total SCCA2, total SCCA1 and total SCCA) were developed. SCCA isoforms were determined before therapy and in follow-up samples from patients with cervical cancer and in serum samples from healthy females. Serum samples from patients with benign skin diseases (psoriasis and eczema) were also selected based on elevated SCCA levels. Rising levels of SCCA1 and SCCA2 were seen in patients with recurrence or progressive disease at the end of the study. The rise in SCCA2 was usually more prominent than that in SCCA1. The dominating serological form of SCCA was free SCCA2 both in healthy controls and in patients with cervical cancer. Both SCCA1 and SCCA2 were elevated in serum from cervical cancer patients and followed the clinical course of the disease during therapy monitoring. SCCA2 did not show improved tumor specificity as compared to SCCA1. A larger study is however necessary to make firm conclusions.
Subject(s)
Antigens, Neoplasm/blood , Carcinoma, Squamous Cell/diagnosis , Enzyme-Linked Immunosorbent Assay , Serpins/blood , Uterine Cervical Neoplasms/diagnosis , Antibodies, Monoclonal/immunology , Antigens, Neoplasm/immunology , Female , Humans , Hybridomas/immunology , Protein Isoforms/blood , Protein Isoforms/immunology , Serpins/immunologyABSTRACT
Squamous cell carcinoma antigen (SCCA), a member of the serine protease inhibitor (serpin) family, is a tumor-associated antigen and a serological marker for squamous cell carcinomas (SCC). Elevated serum levels are correlated with the clinical stage of the disease. Gene cloning has previously revealed two tandemly arrayed genes, SCCA1 and SCCA2. Using RT-PCR, an SCCA1/A2 transcript was identified in 6 out of 8 cell lines and the reciprocal SCCA2/A1 transcript was identified in 6 out of 8 analysed cell lines. Southern blot analysis showed an aberrant band pattern in 3 out of 5 cell lines. The cell lines demonstrating a normal band pattern corresponded to the cell lines where no fusion transcript was detected using PCR. Complex binding studies show that SCCA1/A2 binds to cathepsin G but not to cathepsin L, indicating that the SCCA1/A2 fusion protein has the specificity of SCCA2, but the transcription may be regulated as that of SCCA1. SCCA2/A1 reacts in the opposite way. The clinical relevance of these fusion transcripts is not yet known. Studies using primary tumours are underway to elucidate if these fusion transcripts are tumour specific and if they might be used as a diagnostic and prognostic marker for SCC.
Subject(s)
Antigens, Neoplasm/genetics , Serpins , Adenocarcinoma/genetics , Base Sequence , Binding Sites , Breast Neoplasms/genetics , Cloning, Molecular , Oncogene Proteins, Fusion/geneticsABSTRACT
Carcinoma ex pleomorphic adenoma (CexPA) is a carcinoma developing within a pre-existing benign pleomorphic adenoma (PA). Here we describe the identification and characterization of a series of genetic events leading to translocation, deletion/amplification, and overexpression of the HMGIC and MDM2 genes in a CexPA at an early stage of development. The tumor had a pseudodiploid stemline karyotype with a del(5)(q22-23q32-33) and a t(10;12)(p15;q14-15). In addition, there were several sidelines with double minute chromosomes (dmin) or homogeneously staining regions (hsr). Fluorescence in situ hybridization (FISH) mapping revealed that the 12q14-15 breakpoint was located centromeric to HMGIC and that the entire gene was juxtaposed to the der(10) chromosome. Detailed analysis of cells with dmin and hsr revealed that HMGIC and MDM2 were deleted from the der(10) and that the dmin and hsr were strongly positive for both genes. Southern blot analysis confirmed that both HMGIC and MDM2 were amplified and that no gross rearrangements of the genes had occurred. Immunostaining revealed that the HMGIC protein was highly overexpressed particularly in the large polymorphic cells within the carcinomatous part of the tumor. These findings suggest that amplification and overexpression of HMGIC and possibly MDM2 might be important genetic events that may contribute to malignant transformation of benign PA.
