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Justin Lengfeld,1 Hongtao Zhang,2 Steven Stoesz,1 Ramachandran Murali,3 Franklin Pass,1 Mark I Greene,2 Peeyush N Goel,2 Payal Grover2 1Martell Diagnostic Laboratories, Inc., Roseville, MN, 55113, USA; 2Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA; 3Department of Biomedical Sciences, Research Division of Immunology; Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USACorrespondence: Justin LengfeldMartell Diagnostic Laboratories, Inc., Roseville, MN, 55113, USAEmail JLengfeld@martelldiagnostic.comPayal GroverDepartment of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USAEmail Payalgrover13@gmail.com; pgrover@pennmedicine.upenn.eduAbstract: Breast cancer is a highly prevalent malignancy that shows improved outcomes with earlier diagnosis. Current screening and monitoring methods have improved survival rates, but the limitations of these approaches have led to the investigation of biomarker evaluation to improve early diagnosis and treatment monitoring. The enzyme-linked immunosorbent assay (ELISA) is a specific and robust technique ideally suited for the quantification of protein biomarkers from blood or its constituents. The continued clinical relevancy of this assay format will require overcoming specific technical challenges, including the ultra-sensitive detection of trace biomarkers and the circumventing of potential assay interference due to the expanding use of monoclonal antibody (mAb) therapeutics. Approaches to increasing the sensitivity of ELISA have been numerous and include employing more sensitive substrates, combining ELISA with the polymerase chain reaction (PCR), and incorporating nanoparticles as shuttles for detection antibodies and enzymes. These modifications have resulted in substantial boosts in the ability to detect extremely low levels of protein biomarkers, with some systems reliably detecting antigen at sub-femtomolar concentrations. Extensive utilization of mAb therapies in oncology has presented an additional contemporary challenge for ELISA, particularly when both therapeutic and assay antibodies target the same protein antigen. Resolution of issues such as epitope overlap and steric hindrance requires a rational approach to the design of diagnostic antibodies that takes advantage of modern antibody generation pipelines, epitope binning techniques and computational methods to strategically target biomarker epitopes. This review discusses technical strategies in ELISA implemented to date and their feasibility to address current constraints on sensitivity and problems with interference in the clinical setting. The impact of these recent advancements will depend upon their transformation from research laboratory protocols into facile, reliable detection systems that can ideally be replicated in point-of-care devices to maximize utilization and transform both the diagnostic and therapeutic monitoring landscape.Keywords: breast cancer, diagnosis, ELISA, detection, plasmonics, sensitivity

Medi-Ject Corporation (now Antares Pharma, Inc.) has been providing delivery devices for the needle-free administration of insulin for over 25 years. This study was one of the final steps in the development and premarket evaluation of Medi-Ject's newest needle-free system, the Medi-Jector Vision. This study was conducted to evaluate the performance of this device in the hands of experienced jet injection users in a home environment. Diabetic subjects currently using a needle-free device for the administration of their insulin were studied. Subjects used the new Medi-Jector Vision for all of their insulin administration during the course of the study. Insulin was injected on schedule and at doses consistent with their standard of practice as directed by their health care provider. All subjects were required to document each injection in a daily diary. Study subjects utilized all common insulin types (rapid, regular, intermediate, and long acting), and injections were administered in all of the common injection sites (arms, thighs, abdomen, and buttocks). Once subjects optimized the system to the most appropriate orifice size based on completeness of injection, average completeness percentages were greater than 94% for all orifice sizes. Most patients using the new Medi-Jector Vision in the home were able to manage their insulin therapy without significant complication. We conclude that the jet delivery of insulin with the new Medi-Jector Vision is well accepted by people with diabetes and offers a reliable alternative to the use of needles.

Immunosuppressed patients such as renal transplant recipients are prone to increased incidence of wart disease. We examined 48 tissue specimens from 36 renal transplant recipients using human papillomaviruses (HPVs) 1, 2, 3, 4, 5, and 6 in filter hybridization under stringent conditions. The results showed that 90% of the samples contained HPV DNA. Of these 43 positive samples, we found HPV-1 in 2%, HPV-2 in 56%, HPV-3 in 19%, HPV-4 in 47%, HPV-5 in 9%, and HPV-6 in 5%. In several cases, more than one type of HPV DNA was observed. In a few of these cases, the clinical appearance of the lesions differed from what might have been expected, such as those lesions containing HPV-3- or HPV-5-related DNAs.

Paraffin sections of condylomata acuminata removed from the lower genital canal were stained for papillomavirus antigen by th peroxidase-antiperoxidase test using a broadly cross-reactive antiserum. The antiserum was prepared by immunization of a rabbit with disrupted capsids of papillomavirus purified from a pool of plantar warts. Specific staining was seen as a brown granular reaction in the nuclei of the epithelial cells; this reaction occurred most consistently in the more superficial cells. Papillomavirus antigen was demonstrable in about half of the 50 specimens examined. The antigen was found in both flat and papillary lesions from the vulva, vagina, and cervix. The distribution of the antigen was widely variable and ranged from abundant in some specimens to patchy and sparse in others. In papillary lesions, antigen-positive cells were found characteristically at the tips of the epithelial fronds. The ability to detect the viral antigen in genital condylomas may help in understanding the pathogenesis of these lesions and in evaluating the role of papillomaviruses in th etiology of lower genital tract cancer.

Great progress has been made over the last five years in our understanding of papillomavirus (PV) biology. New technology has enabled investigators to understand the relationship between the PV and its host. The PV cannot be cultured in vitro, and this has led to limitations for those wishing to study the biology of this virus. However, utilizing recombinant DNA technology, investigators now have abundant quantities of human papillomavirus (HPV) DNA for study. Such HPV genomes may be labeled with a radioisotope such as P32 and used as a "probe" in hybridization studies to see if a given tissue contains HPV DNA. No longer are we limited to electron microscopy and immune studies in our efforts to identify HPV within benign or malignant tissues. Ultimately, we hope to understand the relationship between the virus and its host. This paper will concentrate on one aspect of this relationship--the immunology of HPV.

We have recently identified two unusual human papillomavirus (HPV) isolates while engaged in an ongoing study of wart disease in meat handlers and veterinarians. The papillomas from which these two viruses were isolated clinically resembled verruca vulgaris rather than either flat warts or epidermodysplasia verruciformis (EV). These two previously uncharacterized HPVs were molecularly cloned and characterized with respect to known HPVs. The genomes of the two viruses exhibited dramatically different restriction endonuclease cleavage patterns but were found to have significant sequence homology to each other, as well as to HPV-3 and a new virus isolated from a patient with EV. Neither of the two new HPV isolates exhibit detectable sequence homology under stringent conditions of hybridization or share similar restriction endonuclease cleavage patterns with previously characterized HPV types 1,2,4,5,6b, or a previously isolated HPV from meat handlers.

Five antigenically distinct serotypes of human papillomavirus (HPV) cause various cutaneous verrucae with distinct clinical features. Laryngeal and other mucosal papillomas, also suspected to have a viral etiology, were examined for the presence of a genus-specific (common) antigen of HPV using a peroxidase-antiperoxidase (PAP) technique. The primary antiserum used was reactive against laryngeal papillomas from 26 of 35 randomly selected patients (74%); overall, 49 of the 102 squamous papillomas stained were positive for HPV (48%). HPV antigen was not demonstrated within frankly dysplastic squamous cells. Nuclear localization of virus was confirmed by electron microscopy in 3 cases positive for HPV. PAP localization of HPV is rapid and more efficient than routine electron microscopy. Our data indicate a common pathogenesis for some papillomas and cutaneous verrucae and suggest a future role for immunohistochemistry in evaluating the epidemiology and pathobiology of laryngeal papillomatosis.

Benign papillomas from a patient with a family history of epidermodysplasia verruciformis were examined for the presence of human papillomavirus (HPV) DNA. Employing stringent hybridization conditions that allow identification of a single type of HPV and radioactively labeled HPV-5 DNA as a probe, we have detected HPV DNA exhibiting sequence homology to HPV-5 in these tumors. Restriction endonuclease analysis of this HPV DNA confirmed its identity as HPV type 5. However, when hybridization was performed under less stringent conditions that allow all of the known types of HPV to react with the radioactively labeled HPV-5 DNA probe, two additional species of HPV DNA unrelated to HPV-5 were identified. As these two HPV types do not hybridize with HPV 1, 2, 3, or 4 under stringent conditions, they appear unique and have, as yet, not been reported to be associated with patients exhibiting epidermodysplasia verruciformis. Thus we have observed three distinct HPV species in benign papillomas from a single patient. These observations have important implications when attempting to correlate the type of HPV present in the various wart disease syndromes that have been described to date and further suggest that extreme care must be taken when analyzing carcinomas, occupying similar anatomic sites and suspected to have arisen from papillomas, for HPV species.

Inflammation was induced on the forearms of volunteers by twenty-four closed patch tests to either the irritant 10% sodium lauryl sulfate (SLS) or Toxicodendron extract. Each chemical was tested at eight sites on the ventral forearms of each volunteer in order to assess the variability of response among test sites in individual subjects. Inflammation was assessed about 10 minutes after patch tests were removed. The degree of inflammation elicited by both Toxicodendron and SLS was variable among subjects, but variation among individual test sites was much more marked in subjects tested with SLS (p less than 0.002). The marked variability of responses to irritation that occur in any single subject may explain why irritant patch test responses do not reliably identify the irritation-prone individual.

There are two traditional ways to modify a virus for immunization: (1) kill the virus or (2) use a live, attenuated virus. There are three modern ways to prepare vaccines: (1) extract and purify a part of the virus that is immunogenic, (2) synthesize a polypeptide immunogen piece of the virus, or (3) use recombinant deoxyribonucleic acid or gene splicing to prepare an immunogenic portion of the virus. The last three produce subunit vaccines that can be made to contain no deoxyribonucleic acid. They are not infectious and are likely to be nononcogenic. Using recombinant deoxyribonucleic acid techniques, a vaccine for bovine papillomavirus has been prepared. This is in clinical trials and probably will be licensed for use in cattle in 1988. A vaccine for herpes simplex virus has been prepared using glycoprotein D from the surface of the virus. This immunizes animals but it has not reached clinical trials in humans.

Human papillomavirus (HPV) genomes were identified in two cases of verrucous carcinoma of the vagina, using Southern blot DNA hybridization under low-stringency conditions. Type (group) 6 HPV DNA (HPV-6) was identified, using molecularly cloned HPV-1 through HPV-6 DNA probes under high-stringency conditions in both cases. In addition, DNA extract in one case hybridized with HPV-1, HPV-3, and HPV-4 DNA probes. No HPV structural proteins were demonstrated in either case by immunocytochemical tests, using HPV antibodies. In one case viruslike intranuclear particles were observed by transmission electron microscopy. These two cases suggest a strong associative relationship between HPV and verrucous carcinoma (Ackerman) of the lower part of the genital tract.

An antiserum prepared by immunization of a rabbit with sodium dodecyl sulfate-disrupted virions from a pool of plantar warts was cross-reactive with virus-positive papillomas of other animal species by both indirect immunofluorescence tests on frozen sections of wart tissues and peroxidase-antiperoxidase tests of sections of Formalin-fixed tissues. The antiserum stained plantar warts, common warts, and skin lesions of epidermodysplasia verruciformis, all from humans; bovine fibropapilloma, experimentally produced with bovine types 1 and 2; and transmissible canine oral papillomas. The staining was localized to nuclei of the upper granular layers of the peithelium and was similar in distribution to the pattern produced by antiserum specifically prepared against that papillomavirus. The antiserum did not stain virus-negative warts, or cells infected with simlan virus 40, human polyomavirus BK, and murine polyomavirus. These data suggested that papillomaviruses share a common internal antigen unrelated to a similar antigen described previously for the polyomaviruses (which include simian virus 40 and polyomavirus subgroups).

The formation of human skin warts may depend on transformation by human papilloma virus or result from a productive virus infection. To explore this problem, methods were developed for the primary tissue culture of epidermal cells (keratinocytes) derived from wart tissues and normal skin. Wart-derived keratinocytes continued to proliferate for periods exceeding 3 months, but the patterns of cell growth were identical to normal keratinocytes. Electron microscopic and immunohistologic examination of wart-derived cultures did not evidence virion assembly.

Fifty-nine (24 plantar, 35 common) warts surgically excised from 44 patients (15 female, 29 male), 18 to 32 years old, were examined by electron microscopy (EM) for papillomavirus particles and by fluorescent antibody (FA) and peroxidase-antiperoxidase (PAP) tests for human papillomavirus type 1 (HPV-1)-specific antigens and papillomavirus genus-specific (common) antigens. Fifty per cent of plantar warts and 52 per cent of common warts were positive for HPV by EM. When examined by FA and PAP for genus-specific antigens, 58 per cent of plantar warts and 68 per cent of common warts were positive. Fifty per cent of plantar warts and 11 per cent of common warts were caused by HPV-1 as determined by reactivity with HPV-1 type-specific antiserum. All warts positive by EM were positive by FA and PAP. Sampling error accounted for warts positive by FA and PAP but not by EM. Forty-two per cent of plantar warts and 31 per cent of common warts were negative by both EM and FA and PAP. The warts caused by HPV-1 contained more virus and were more clinically aggressive than warts caused by other HPV. This study shows that the results obtained by the PAP technique using papillomavirus genus- and type-specific antisera on formalin-fixed, paraffin-embedded tissue available to the diagnostic pathologist are concordant with the results obtained by methodology usually available only to the experimental pathologist.

Allergic contact dermatitis was elicited with Toxicodendron antigen and the patch test site examined at various time intervals up to one week. The degree of inflammation was rather constant during the observation period. The mean erythema score at 168 hours was not significantly different from the score at 24 hours. These data support the use of a delayed (96-hour) patch test reading as a guide to discriminating between allergic and irritant patch test reactions.

Epidermodysplasia verruciformis (EV) is a rare disease characterized by the early onset and unremitting progression of wart-like lesions and frequent association of cutaneous carcinomas. We report two siblings with EV. Immunologic study of both patients demonstrated normal immunoglobulin levels, normal numbers of T-lymphocytes and B-lymphocytes, but markedly depressed in vitro blastogenic reactivity to mitogens and antigens. Cutaneous anergy to a variety of common skin test antigens was noted. These observations may reflect an inherited abnormality in immune function, or the depressed immune function may result from the viral infection of EV.

The presence of papillomaviruses in epithelial-derived cancers from several animal species has led to the speculation that these viruses may also have a pathogenic role in the development of certain human carcinomas, particularly those associated with the anogenital tract. Recently, human papillomavirus (HPV) DNA has been detected in epithelial-derived cancers, both cutaneous and metastatic, from patients exhibiting the rare, chronic flat wart disease, epidermodysplasia verruciformis (EV). Except for patients exhibiting this chronic wart syndrome, the association of HPV genomes with human epithelial cancers has not been demonstrated. In an attempt to delineate the association and possible involvement of papillomaviruses with human anogenital carcinomas, we have begun an analysis of these cancers for the presence of HPV-specific nucleotide sequences by using highly sensitive hybridization procedures capable of detecting distantly related papillomaviruses at low copy number. Here we demonstrate the presence of HPV DNA in several types of anogenital tumours: Bowenoid papulosis, carcinoma in situ, and verrucous carcinoma. These data indicate that HPV can be detected in several types of premalignant and malignant tumours, supporting the contention that this group of viruses may be involved in the development of certain types of human epithelial-derived cancers.

Virions isolated from warts of 2 siblings with epidermodysplasia verruciformis (EV), a rare disease characterized by the lifelong growth of warty skin tumors containing papova-like virions, were compared to isolates of human papillomavirus (HPV) from 3 pools of plantar and common hand warts. The length of relaxed, circular (form II) molecules of EV virion DNA approximated the length of HPV DNA but was 3.3% longer. Antisera prepared in rabbits against the 3 HPV pools coated and aggregated HPV in immune electron microscopy (IEM) tests but did not react with EV virions. These antisera reacted at high titers in complement fixation (CF) tests with HPV and reacted only weakly in CF tests with EV virions. Rabbit antisera to EV virions coated and aggregated EV virions but reacted only weakly or not at all with HPV virions in IEM tests. These sera reacted in CF with EV virions only. The data indicated that virions from the EV patients represent an immunologically distinct papillomavirus.