Parallel reaction monitoring with multiplex immunoprecipitation of N-glycoproteins in human serum for detection of hepatocellular carcinoma.

The N-glycosylation of proteins is one of the most important post-translational modifications relevant to various biological functions. The identification and quantification of N-glycoproteins in liquid chromatography-mass spectrometry (LC-MS) is challenging because of their low analytical sensitivity and selectivity. This is due to their microheterogeneity and the difficulty of synthesizing N-glycopeptides as an internal standard. Parallel reaction monitoring (PRM) is widely used in targeted LC-MS. The key advantage of LC-PRM is that it can identify N-glycopeptides using tandem mass spectrometry (MS/MS) fragmentation, even without an internal standard. We investigated the feasibility of analyzing N-glycoproteins using multiplex immunoprecipitation to improve sensitivity and selectivity. We targeted N-glycoproteins [α-fetoprotein (AFP), vitronectin (VTN), and α-1-antichymotrypsin (AACT)] that are abnormally glycosylated in hepatocellular carcinoma (HCC). Their tryptic N-glycopeptides were selected to determine the percentages of fucosylated N-glycopeptides using Y ions, which include glycopeptide fragments with amino acid sequences. Finally, we confirmed that the area under the receiver operating characteristic curve (AUC = 0.944) for the combination of AFP and VTN increased more so than for a single glycopeptide (AUC = 0.889 for AFP and 0.792 for VTN) with respect to discriminating between HCC and cirrhosis serum. This study shows that an LC-PRM method using multiplex N-glycoproteins immunoprecipitated from serum could be applied to develop and verify cancer biomarkers. Graphical abstract.

Quality control of hospital preparations: Establishment of a simple and rapid method for quantifying ulinastatin in vaginal suppositories.

Ulinastatin vaginal suppositories, used to prevent threatened premature delivery, are frequently used in hospitals. However, there is no established method for quantifying ulinastatin contained in suppositories. Therefore, we investigated a simple and efficient method for quantifying ulinastatin contained in suppositories. Our analytical method involved removal of the base; optimising the enzyme inhibition reaction time and enzyme reaction time; and measuring the absorbance. The modified method was reproducible, operation time was significantly shortened, and cost was reduced to approximately 1/17 of that of the previously reported method. This simple and rapid quantitative method could contribute to the improvement of quality control of ulinastatin vaginal suppositories as an extemporaneous hospital preparation.

Disposable bioreactors: maturation into pharmaceutical glycoprotein manufacturing.

Modern biopharmaceutical development is characterised by deep understanding of the structure activity relationship of biological drugs. Therefore, the production process has to be tailored more to the product requirements than to the existing equipment in a certain facility. In addition, the major challenges for the industry are to lower the high production costs of biologics and to shorten the overall development time. The flexibility for providing different modes of operation using disposable bioreactors in the same facility can fulfil these demands and support tailor-made processes.Over the last 10 years, a huge and still increasing number of disposable bioreactors have entered the market. Bioreactor volumes of up to 2,000 L can be handled by using disposable bag systems. Each individual technology has been made available for different purposes up to the GMP compliant production of therapeutic drugs, even for market supply. This chapter summarises disposable technology development over the last decade by comparing the different technologies and showing trends and concepts for the future.

Serum YKL-40 as a marker for cervical adenocarcinoma.

BACKGROUND: The current study examined the clinical usefulness of YKL-40 in detection and prognosis of uterine cervical cancer. PATIENTS AND METHODS: Serum levels of YKL-40, cancer antigen 125 (CA 125), carbohydrate antigen 19-9 (CA19-9), and squamous cell carcinoma (SCC) antigen were determined by enzyme-linked immunosorbent assay in women with benign gynecologic disease (n=24), cervical malignancy (SCC, n=104; adenocarcinoma, n=37), and age-matched healthy controls (n=45). Immunohistochemical analysis for local YKL-40 expression was carried out on 28 adenocarcinomas. RESULTS: Receiver operating characteristic curve analysis showed that YKL-40 [area under the curve (AUC)=0.882] was significantly better at discriminating adenocarcinoma from healthy control than SCC antigen, CA 125, and CA19-9. For SCC, YKL-40 (AUC=0.898) carried out similarly to SCC antigen and was better than CA 125 and CA19-9. Using a cut-off YKL-40 value of 92.2 ng/ml, sensitivity of YKL-40 in stage I adenocarcinoma (68%) was higher than that of the other three markers (11%-21%). Tumor-associated macrophages showed immunoreactivity for YKL-40 in 2 of 28 adenocarcinoma tissue samples, but adenocarcinoma cells themselves were nonimmunoreactive in all samples. Multivariate Cox regression analysis revealed that elevated pretreatment YKL-40 levels predicted unfavorable prognosis, independent of International Federation of Gynecology and Obstetrics stage and age at diagnosis. CONCLUSIONS: Pretreatment serum YKL-40 level is a possible prognosticator of cervical adenocarcinoma.

The stability of house dust mite allergens in glycerinated extracts.

BACKGROUND: Mite allergen vaccines are important diagnostic and immunotherapeutic reagents. Previous studies on mite allergen stability under different storage conditions have yielded contradictory results. OBJECTIVE: We sought to compare, over a 12-month period, the stability of mite allergens reconstituted in 50% glycerol and stored at different temperatures and to examine the role of protease inhibitors in enhancing allergen stability. METHODS: Lyophilized allergen extracts were reconstituted in 50% glycerol, with and without protease inhibitors, and stored at -70 degrees C, -20 degrees C, 4 degrees C, or 37 degrees C for 12 months. At 6 and 12 months, the extracts were compared with freshly dissolved extracts by competition ELISA with pooled allergic sera, 2-site ELISA with mite-specific mAbs, and immunoblot analyses. RESULTS: The overall potencies of the stored extracts measured by competition ELISA were stable at -20 degrees C and 4 degrees C. As determined by means of the immunoblot and 2-site ELISA, Der f 1 levels decreased at 4 degrees C. Levels of Der f 2, Der p 1, and Der p 2 decreased in at least one of the allergen-specific assays. Storage at 37 degrees C led to overall loss of potency and allergen content, whereas storage at -70 degrees C was associated with a moderate loss of potency that increased with multiple freeze-thaw cycles. Protease inhibitors had no effect on allergen stability. CONCLUSION: Although overall potency of the extracts, as measured by competition ELISA, was preserved at -20 degrees C and 4 degrees C, allergen-specific assays indicated loss of allergens. These findings suggest that the competition ELISA is insensitive to decreases in the concentrations of individual allergens.

Monoclonal antibodies to recombinant Der p 2, a major house dust mite allergen: specificity, epitope analysis and development of two-site capture ELISA.

House dust mite allergens have been well established as sensitizing agents that are important in the induction of allergic diseases. In order to analyze epitopes of the allergen and to develop a quantitative method of the allergen exposure, monoclonal antibodies against a recombinant Der p 2 (rDer p 2), one of the major allergens of Dermatophagoides pteronyssinus, were produced. Four monoclonal antibodies produced were species-specific and did not cross-react to the D. farinae crude extract. Two of the monoclonal antibodies were found to be IgG1 and the others were IgM. For the analysis of epitopes, a Der p 2 cDNA encoding 126 amino acids (aa) was dissected into three fragments with several overlapping peptides. A (aa residues 1-49), B (44-93), and C fragment (84-126). Three monoclonal antibodies showed reactivities to the recombinant B fragment and to the full-length rDer p 2, but one monoclonal antibody reacted only with the full-length rDer p 2. Two-site capture ELISA was developed using two different monoclonal antibodies for quantitating Der p 2 in house dust. The sensitivity limit was 4 ng/ml with rDer p 2 and 8 micrograms/ml with the D. pteronyssinus crude extract. The result suggested that the assay using monoclonal antibodies against rDer p 2 could be useful for the environmental studies and for the standardization of mite allergen extracts.

Recombinant glycoprotein product quality in proliferation-controlled BHK-21 cells.

We analyzed product quality to determine the applicability of proliferation-controlled mammalian cells for recombinant pharmaceutical protein production. Baby hamster kidney (BHK)-21 cells were engineered to express a dicistronic, stabilized, self-selecting growth control system consisting of a beta-estradiol-activatable transcription factor IRF-1 fusion protein. IRF-1 activity led to a reduced growth rate, whereas productivity, protein integrity, and glycosylation pattern of the industrially relevant secreted pharmaceutical glycoprotein erythropoietin remained consistent, showing that this technique has the potential for improving the consistency of high-quality pharmaceutical products and thus warrants further development.

Allergen source materials and quality control of allergenic extracts.

Allergen extracts are prepared from a wide variety of source materials including pollens, fungi, arthropods, animal danders, foods, and dusts. The composition of allergen extracts can vary depending on the allergen source, manufacturing process, and storage conditions. Allergen-specific immunoglobulin E (IgE) assays and skin tests employ a variety of allergen-containing reagents that confer specificity on the test. Given that the allergen source materials are heterogenous mixtures of proteins, glycoproteins, carbohydrates, and other substances that are not allergenic, it is not unexpected that variability exists between test results obtained with different allergen-containing reagents. Variability within a single manufacturer's allergen product can be controlled by using reproducible extraction and processing procedures, single large lots of allergen source materials, and solid-phase supports. These controls do not, however, ensure the consistency of products between manufacturers or laboratories because allergen source materials, manufacturing procedures, and acceptance criteria for allergen reagents may vary.

Quality parameters for the production of mite extracts.

Mites present in house dust are of great etiological importance in type I hypersensitivity, with those belonging to the Dermatophagoides genus (D. pteronyssinus and D. farinae), of the Pyroglyphidae family, being the most frequent and principal source of allergens. For the production of allergenic extracts destined for specific diagnostic and treatment purposes of allergic diseases, the culture of such mites is absolutely necessary. In accordance with studies carried out in our laboratories to obtain adequate extracts, one must bear in mind the culture mite phase. Three growth phases have been distinguished for both species: latency phase (F1), growth phase (F2) in which the allergenic proteins are expressed with greater intensity, and death phase of the culture (F3). In the same study, the biological standardization of the extracts demonstrated that those produced from the maximum growth phase gave both in vitro and in vivo results, at least three times more sensitive than those from the other phases. We checked the reproducibility of the production method, obtaining different batches in similar conditions with a high homogeneity regarding allergenic activity. The sensitivity and specificity of the allergenic extracts depends just as much upon the production method as the standardization method. During the biological cycle of Dermatophagoides in culture, it is only from the maximum growth phase (F2), that allergenic extracts with an excellent diagnostic value, high sensitivity and specificity, can be obtained.

[Ulinastatin Reference Standard (Control 971) of the National Institute of Health Sciences].

The "Ulinastatin Reference Standard (control 971)" of National Institute of Health Sciences was prepared. The standard material was evaluated in collaboration with one domestic laboratory, and the potency of trypsin inhibiting activity was determined to be 3, 100 units/vial by relative assay method against the Ulinastatin Reference Standard (control 942). Other analytical data obtained were as follows: UV maximum absorption was observed at 277 nm, and the molecular weight was estimated to be about 66,300 by gel filtration method. Maximum variance of material contents in 10 vials was 2.29%. Based on the above results, this standard material was authorized to be the "Ulinastatin Reference Standard (control 971)" of the National Institute of Health Sciences.

Measurement of airborne mite allergen exposure in individual subjects.

To evaluate the extent of personal exposure to airborne mite allergens, subjects were asked to carry a personal air sampler when in their houses. The level of Der 1 allergen trapped by the sampler was measured with a highly sensitive immunoassay. There were great variations in airborne Der 1 exposure in each subject. When used bedding was replaced with new allergen-free bedding, we detected a decrease in the allergen level. The use of new bedding seems to be an effective measure for reducing airborne mite allergen exposure.

An in-house ELISA method for measuring surfactant protein A.

An in-house enzyme-linked immunoabsorbant assay (ELISA) for SP-A was successfully developed using in-house polyclonal anti SP-A and a commercial polyclonal anti-rabbit immunoglobulin horseradish peroxidase conjugate system. The standard curve, generated by using 50 ng of SP-A to coat the plate and 1:500 dilution of polyclonal anti SP-A as a primary antibody, was linear for concentrations of SP-A ranging from 4 micrograms/l to 4000 micrograms/l and reproducible. Results of recovery study of SP-A from a known sample of tracheal aspirate ranged from 94%-114%. Intra- and inter-assay coefficients of variations were 2.7% and 5.6% respectively for a known sample of tracheal aspirate. Interference study showed that tracheal aspirate did not interfere with the assay. The assay developed was intended to be used for SP-A measurement in tracheal aspirates obtained from neonates with and without respiratory distress syndrome.

Increased levels of stromelysin-1 and tissue inhibitor of metalloproteinases-1 in sera from patients with rheumatoid arthritis.

OBJECTIVE: To evaluate the efficacy of stromelysin-1 (matrix metalloproteinase-3 [MMP-3]) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in serum as markers for joint inflammation in rheumatoid arthritis (RA). METHODS: Levels of both macromolecules in sera from 97 healthy controls, 109 patients with RA, and 47 patients with osteoarthritis (OA) were measured by respective 1-step sandwich enzyme immunoassays. In the patients with RA, serum levels of MMP-3 and TIMP-1 were investigated in relation to laboratory and clinical measures of disease activity. In addition, the relationships between serum and synovial fluid (SF) levels in paired samples from individual patients were examined. RESULTS: Serum levels of both MMP-3 and TIMP-1 in RA patients were significantly higher than those in OA patients and in healthy controls (P < 0.001), and were shown to correlate with traditional systemic markers of inflammation including the erythrocyte sedimentation rate and C-reactive protein level, and with the Lansbury articular index. In addition, it was noted that serum levels of MMP-3 correlated with the corresponding values in paired SF samples obtained concurrently from patients with RA (rs = 0.588, P < 0.001), while such correlations were not found for TIMP-1 levels. CONCLUSION: Our results support the notion that levels of both MMP-3 and TIMP-1 in RA patient sera are increased in association with inflammation. Furthermore, the level of MMP-3 in serum provides a particularly useful marker of inflammatory activity in the joints of patients with RA.

[Ulinastatin reference standard (Control 941) of the National Insutitute of Health Sciences].

The raw material of ulinastatin was examined for preparation of the "Ulinastatin Reference standard". The candidate material was evaluated in collaboration with one domestic laboratory, and the potency of trypsin inhibiting activity was determined to be 3500 unit/vial. Other analytical data obtained were as follows: UV maximum absorption was observed at 276 nm, the molecular weight was estimated to be about 66000 +/- 5000 by gel filtration method. Maximum variance of material contents in 10 vials was 6.52% by means of the weight variation test in JP XII. Based on the above results, this raw material was authorized to be the first "Ulinastatin Reference Standard" of the National Institute of Health Sciences.

Quantitative skin prick tests and specific IgE (CAP System) for D. pteronissynus--correlation of results in a paediatric population.

The CAP System compared with RAST has a greater sensitivity, with the same specificity, as well as an excellent correlation with skin prick tests (SPT), as documented by others. Standardization of SPT is essential for routine and investigational purposes. We used a standardized lancet (DHS), and the methodology of reading the SPT was a computerized digitalized graphics tablet coupled to an IBM-PC AT using CAD software, expressing the result as wheal areas with cut off +/- at 7 mm2. The method is precise and reproducible with a mean coefficient of variation (cv) intratester--T1: 3.1%, T2: 3.5% and a cv intertester: 2.93%. We compare the results of SPT of two different allergen extracts for Dermatophagoides Pteronissynus (DPt) (Bencard; Merck/Allergo-Pharma, standardized in SBU) and correlate them with specific IgE, in 122 patients, 72 males and 50 females, aged 3 to 19 years. The results between the extracts and the results of specific IgE obtained by CAP System with each of the extracts, were correlated. 34 patients were considered to be non atopic. 88 patients showed at least one positive result with one test, being those used to correlate the positive results.... The results showed good correlations for quantitative SPT between Bencard and Merck/Allergo-Pharma, as well as for the capacity of eliciting positive results. When SPT were compared to specific IgE as +/- and as quantitative results, there were good correlation for Bencard/CAP and Merck/CAP. The results obtained suggest that CAP can be used to validate allergen extracts of DPt and that studies with other extract are advisable.

Proposal of standardized methods and reference for assaying recombinant human tumor necrosis factor.

Two assay methods for recombinant human tumor necrosis factor (rH-TNF) were developed, one a biological L-cell assay and the other an enzyme-linked immunosorbent assay. The accuracy and reproducibility of each and the correlation between the two were studied. As a result of this investigation, the two assay methods were found appropriate for standardization of rH-TNF. A freeze-dried reference was prepared, and examination of its potency and stability showed it to be suitable for use as a reference standard for rH-TNF assays.