Monitoring an antibody affinity chromatography with a label-free optical biosensor technique.

In the production of polyclonal antibody, a purification step is necessary which is often done by affinity chromatography. We present a biosensor system based on reflectometric interference spectroscopy (RIfS) to monitor the quantity and quality in terms of affinity and kinetic constants of the antibody during this procedure. Biosensors are rapid compared with ELISA, which is done in practice and can work fully automated. They provide additional information about the active antibody to protein concentration ratio and the affinity of the antibody. We show how to determine these values very accurate. In addition, we describe a new rapid method to monitor the affinity chromatography in process. This gives the possibility to select antibody fractions with best properties in respect to the application.

Detection of squamous cell carcinoma of the oral cavity by imaging 5-aminolevulinic acid-induced protoporphyrin IX fluorescence.

OBJECTIVES: Early cancer detection is the best way to improve the prognosis of patients with oral cancer. Therefore this study presents quantitative fluorescence measurements and results in the visualization of cancerous oral mucosa with 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PPIX). METHODS: Time progression and type of porphyrin accumulation were analyzed in neoplastic and surrounding healthy tissue of 58 patients with a suspected cancer of the oral cavity by measuring emission spectra of 5-ALA-induced PPIX fluorescence. Fluorescence images in the red and green spectral range from the tumor tissue were recorded with a charge-coupled device camera. RESULTS: After topical application of 0.4% 5-ALA and incubation for 1 to 2.5 hours, all patients revealed higher intensities of red fluorescence in neoplastic tissue compared with the surrounding normal tissue. Maximum contrast was reached after 1.5 hours of incubation. In 13.8% (n = 8) of the patients, additional findings like dysplasia, carcinoma in situ, primary tumor, secondary carcinomas, and tumor branches were found by means of fluorescence marking in contrast to white light examination. An evaluation of the biopsy specimens resulted in a specificity of 60% and a sensitivity of 99%. CONCLUSIONS: As a fluorescent marker, PPIX could represent a possible new diagnostic tool to detect early malignant and secondary lesions in the oral cavity. In addition, 5-ALA-induced PPIX fluorescence is promising as a useful intraoperative tool for determining adequate surgical margins of resection. Further investigations aim to assess this diagnostic procedure as a sensitive and clinically reliable method for patients with oral cancer.

Fluorescence staining of oral cancer using a topical application of 5-aminolevulinic acid: fluorescence microscopic studies.

INTRODUCTION: Topical application of 5-aminolevulinic acid (5-ALA) by means of a rinsing solution has been shown to be a promising new procedure in the diagnosis of oral malignancies. However, for assessing the reliability of this method regarding fluorescence-guided tumor resections and photodynamic therapy, further information on the distribution and penetration depth of 5-ALA-induced protoporphyrin IX (PPIX) in the tissue is needed. METHODS: 24 patients suffering from oral cancer were included in this investigation. Biopsies were taken immediately after fluorescence examination and either used as native sections for immediate fluorescence microscopic examination (n = 3) or shock frozen in liquid nitrogen and prepared as frozen sections (n = 46). Fluorescence imaging and digital image processing were utilized in order to determine the presence of PPIX in regions of various histologies as well as the penetration depth of PPIX into solid tumor. RESULTS: PPIX fluorescence in the tissue was limited to the epithelium. Both normal and dysplastic epithelium showed PPIX fluorescence. In the stroma, no PPIX fluorescence was found. In some cases (n = 3/4) invasive carcinomas did not show PPIX fluorescence, while the adjacent or overlying normal epithelium was strongly fluorescent. The penetration depth of PPIX after topical application of 5-ALA was found to be limited to less than 1 mm. CONCLUSION: PPIX fluorescence induced by topical application of 5-ALA can be very useful in the determination of superficial tumor margins. However, due to the limited penetration depth there is a risk of not accurately recognizing the infiltration depth of solid tumors. The aim of further investigations will be to assess the tissue distribution and depth of penetration of PPIX following systemic application of 5-ALA.

Autofluorescence imaging and spectroscopy of normal and malignant mucosa in patients with head and neck cancer.

BACKGROUND AND OBJECTIVE: An early detection of oral cancer might improve the patient's prognosis. We present preliminary results of autofluorescence photodetection of cancerous oral mucosa. MATERIALS AND METHODS: 49 patients were investigated altogether. In 30 patients, malignant and healthy oral mucosa were excited with violet light (lambda = 375 to 440 nm). Images were recorded by a sensitive CCD camera. Spectrophotometric analysis in the green spectral range was performed on tumorous and innocuous mucosa in 36 patients. RESULTS: In 13 patients (43.3%), tumors were subjectively better distinguishable from their surroundings through a reduction of green autofluorescence than by ordinary inspection. Tumor detection abilities varied for different locations and tumor morphologies. Spectral analysis showed contrasts in autofluorescence intensities between tumor and normal tissues in 34 patients (94.4%). Autofluorescence spectra of normal mucosa varied both inter- and intraindividually. CONCLUSIONS: Using violet excitation light, camera-based autofluorescence photodetection in the green spectral range presented a highly promising tool for the diagnosis of oral malignomas in almost half of the cases examined. The possible ways on how the obtained results could serve to find a more advanced method for a precise tumor detection in the oral cavity are being discussed.

Fluorescence staining of laryngeal neoplasms after topical application of 5-aminolevulinic acid: preliminary results.

BACKGROUND AND OBJECTIVE: The prognosis of patients suffering from laryngeal carcinomas can be improved by early diagnosis. Exact demarcation of tumor margins could contribute to an optimum preservation of the larynx. Therefore, the aim of the present study was the evaluation of 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PPIX) fluorescence as a new diagnostic procedure for the detection of laryngeal cancer. STUDY DESIGN/MATERIALS AND METHODS: Sixteen patients with suspected malignancies of the larynx received 0.6 wt% 5-ALA-NaCl solution by means of a medical nebulizer. After a period of 1-2 hours, the patients underwent microlaryngoscopy under white light and fluorescence illumination (lambda(ex) = 375-440 nm). A quantitative analysis of the fluorescence contrast between neoplastic and surrounding tissue was performed using an optical multichannel analyzer. RESULTS: Carcinoma, carcinoma in situ, and dysplasia showed red fluorescence that could be attributed to the 5-ALA-induced formation of PPIX. The surrounding normal tissue exhibited autofluorescence in the green spectral range, which was greatly reduced within the tumor. The results of macroscopic red fluorescence staining were correlated with the histologic diagnosis. CONCLUSION: According to these preliminary results, the presented method seems to be a promising adjunct diagnostic procedure for the early identification of malignant neoplasms in the larynx. The aim of further investigations is the assessment of sensitivity and specificity and an evaluation of fluorescence-guided laser resections of laryngeal cancer. Lasers Med. Surg. 25:414-420, 1999.

Optical biosensors. Monitoring studies of glycopeptide antibiotic fermentation using white light interference.

This paper describes the design, characterization, and use of an optical biosensor suited for the process control of biotechnological processes. The detector principle is based on reflectometric interference spectroscopy (RIfS). RIfS enables a label-free, product-specific monitoring, with a future outline for on-line process control. The potential of the RIfS biosensor is exemplified by the qualitative and quantitative monitoring of the microbial production of vancomycin-type glycopeptide antibiotics.