Maximizing response to intratumoral immunotherapy in mice by tuning local retention.

Direct injection of therapies into tumors has emerged as an administration route capable of achieving high local drug exposure and strong anti-tumor response. A diverse array of immune agonists ranging in size and target are under development as local immunotherapies. However, due to the relatively recent adoption of intratumoral administration, the pharmacokinetics of locally-injected biologics remains poorly defined, limiting rational design of tumor-localized immunotherapies. Here we define a pharmacokinetic framework for biologics injected intratumorally that can predict tumor exposure and effectiveness. We find empirically and computationally that extending the tumor exposure of locally-injected interleukin-2 by increasing molecular size and/or improving matrix-targeting affinity improves therapeutic efficacy in mice. By tracking the distribution of intratumorally-injected proteins using positron emission tomography, we observe size-dependent enhancement in tumor exposure occurs by slowing the rate of diffusive escape from the tumor and by increasing partitioning to an apparent viscous region of the tumor. In elucidating how molecular weight and matrix binding interplay to determine tumor exposure, our model can aid in the design of intratumoral therapies to exert maximal therapeutic effect.

APB-F1, a long-acting feline granulocyte colony-stimulating factor fusion protein, created by exploiting FL335, a chimeric Fab specific for feline serum albumin.

Off-label use of a human granulocyte colony stimulating factor (hG-CSF) has been allowed to treat dogs and cats with neutropenia. However, repeated administration of hG-CSF induces undesirable anti-drug antibody (ADA) responses, implying the necessity of animal-derived G-CSF as a therapeutic reagent, preferably with a long-acting capability. Herein, we generated a recombinant fusion protein by genetically combining FL335, a chimeric Fab specific for feline serum albumin (FSA), and feline G-CSF (fG-CSF), with the ultimate goal of developing a long-acting therapeutic fG-CSF for cats. The resulting FL335-fG-CSF fusion protein, referred to as APB-F1, was produced well as a functional form in a Chinese hamster ovary (CHO) expression system. In in vitro analyses, APB-F1 bound to FSA at high affinity (KD = 400 pM) and possessed 0.78 × 107 U/mg G-CSF biological activity, clearly proving its biological functionality. Pharmacokinetic (PK) and pharmacodynamic (PD) studies using healthy cats revealed that the serum half-life (t1/2) of APB-F1 was increased five times compared with that of fG-CSF (t1/2 = 13.3 h vs. 2.7 h) in subcutaneous (SC) injections. Additionally, APB-F1 induced a profound and sustained increase in white blood cell (WBC) and actual neutrophil count (ANC) up to 10 days, which was far superior to other G-CSF preparations, including filgrastim (Neupogen™) and even pegfilgrastim (Neulasta™). Conclusively, a long-acting fG-CSF with potent in vivo bioactivity was successfully created by using FL335; thus, we provided evidence that our "anti-serum albumin Fab-associated" (SAFA) technology can be applied reliably in developing valuable long-acting biologics in veterinary medicine.

Combination of lymphocyte count and albumin concentration as a new prognostic biomarker for rectal cancer.

Although numerous studies have highlighted the prognostic values of various inflammation-related markers, clinical significance remains to be elucidated. The prognostic values of inflammation-related biomarkers for rectal cancer were investigated in this study. A total of 448 patients with stage II/III rectal cancer undergoing curative resection were enrolled from the discovery cohort (n = 240) and validation cohort (n = 208). We comprehensively compared the prognostic values of 11 inflammation-related markers-derived from neutrophil, lymphocyte, platelet, monocyte, albumin, and C-reactive protein for overall survival (OS) and recurrence-free survival (RFS). Among 11 inflammation-related markers, only "lymphocyte × albumin (LA)" was significantly associated with both OS and RFS in the discovery cohort (P = 0.007 and 0.015, respectively). Multivariate analysis indicated that low LA was significantly associated with poor OS (hazard ratio [HR] 2.19, 95% confidence interval [CI] 1.09-4.58, P = 0.025), and poor RFS (HR 1.61, 95% CI 1.01-2.80, P = 0.048). Furthermore, using the discovery cohort, we confirmed that low LA was significantly associated with poor OS (HR 2.89, 95% CI 1.42-6.00, P = 0.002), and poor RFS (HR 1.79, 95% CI 1.04-2.95, P = 0.034). LA can be a novel prognostic biomarker for stage II/III rectal cancer.

Percentage of Natural Killer (NK) Cells in Peripheral Blood Is Associated with Prognosis in Patients with Gastric Cancer: A Retrospective Study from a Single Center.

BACKGROUND Natural killer (NK) cells are important for the prognosis of multiple cancers, but their prognostic value remains to be evaluated in patients with gastric cancer. Thus, this retrospective study was conducted at a single center to investigate the association between percentage of NK cells in the peripheral blood and prognosis in patients with gastric cancer. MATERIAL AND METHODS The data of 180 gastric cancer patients were collected. Univariate and multivariate Cox regression models were applied to screen candidate prognostic factors. A time-dependent receiver operating characteristic curve was employed to evaluate the ability of NK cells as a prognostic marker. Furthermore, we determined the correlation between the NK cells percentage and other parameters and their clinical significance. RESULTS Patients with a higher percentage of NK cells survived longer than those with a lower percentage of NK cells. Cox analysis revealed that NK cells could be used as an independent indicator for patients with gastric cancer. The percentage of NK cells was positively correlated with lymphocyte count and albumin, but was negatively correlated with CA125 and neutrophil-lymphocyte ratio. The area under the curve for NK cells in predicting the 5-year survival rate for gastric cancer was 0.792. This increased to 0.830 upon combining NK cells with neutrophil-lymphocyte ratio. Patients at early T, N, and clinical stages possessed a significantly higher percentage of NK cells compared to those at advanced T, N, and clinical stages of gastric cancer. CONCLUSIONS Our results suggest that a higher percentage of NK cells predicts is associated with longer survival of gastric cancer patients and could serve as an independent prognostic biomarker.

Immune status in sarcoidosis: one size does not fit all.

B-cell immunity and immunoglobulins are less commonly affected in sarcoidosis. We aimed to evaluate immune status in sarcoidosis patients. Retrospective chart review of sarcoidosis patients attending a out-patient clinic over 3 months period. Immunoglobulins levels were recorded (A, M, G, E) along with clinical and serological data. They were divided in group A (normal IgG), group B (increased IgG), group C (decreased IgG) and group D (decreased IgG and IgM and/or IgA). Of 50 subjects, 68% were females and 62% of Caucasian origin. 22 (44%) had normal IgG levels, 16 (32%) had increased IgG levels, 10 (20%) had hypogammaglobulinemia and 2 (4%) had combined hypogammaglobulinemia, diagnosed with combined sarcoidosis and common variable immunodeficiency. Decreased IgA values was found in groups C and D. IgE was high in group B. Globulin was increased in group B and decreased in groups C and D. Decreased neutrophils were found in group D (all statistically significant). Correlation analysis showed significant association of angiotensin converting enzyme with IgA and IgM, inverse correlation of IgG with white blood cells and neutrophils, of IgA with globulin and inverse with albumin and of calcium with albumin. Most sarcoidosis patients have normal or increased immunoglobulin levels, that correlate with serum biomarkers of disease activity. Hypogammaglobulinemia may reflect treatment side effects and accompanied by blood leukocytosis. Combined severe immunodeficiency occurs in sarcoidosis.

Analysis of Sensitization Profiles in Central European Allergy Patients Focused on Animal Allergen Molecules.

INTRODUCTION: Frequently observed multiple sensitizations to several animals highlights the importance of a molecular diagnosis, distinguishing between sensitizations specific to single species and sensitizations due to cross-reactivity. OBJECTIVE: The aim of our study was to assess the usefulness of a molecular diagnosis in the description of sensitization profiles in allergy patients living in Central Europe, with a particular focus on animal-derived molecules. METHODS: The molecular diagnosis was performed using the ImmunoCAP ISAC microarray. Results of 1,255 allergy patients were subjected to statistical analysis. RESULTS: The highest sensitization rates were observed for uteroglobin Fel d 1 (31.8%) and kallikrein Can f 5 (16.4%), followed by animal lipocalins Can f 1 (13.9%), Equ c 1 (6.2%), Fel d 4 (5.3%), Can f 2 (4.2%), and Mus m 1 (4.1%). Sensitization rates to serum albumins Fel d 2, Can f 3, Equ c 3, and Bos d 6 were very low, with the highest being 3.2% to Fel d 2. Detailed subanalysis confirmed the dominant role of Fel d 1 or Can f 5 and/or Can f 1 in cat- or dog-sensitized patients, respectively. Further analysis focused on lipocalins and albumins confirmed a high rate of cosensitizations within both groups. CONCLUSION: The sensitization to animal allergen molecules is very frequent in Central Europe. The most common is sensitization to species-specific cat uteroglobin Fel d 1 and dog kallikrein Can f 5, followed by sensitizations to animal lipocalins. Our data suggest that commonly observed multiple sensitizations detected by extract approach can be explained not only by true cosensitization, but also by cross-reactivity, mainly in the frame of lipocalins. Cross-reactive serum albumins are minor sensitizers and are probably not important from this point of view.

Self-Albumin Camouflage of Carrier Protein Prevents Nontarget Antibody Production for Enhanced LDL-C Immunotherapy.

Elevated low-density lipoprotein cholesterol (LDL-C) increases the risk of atherosclerotic cardiovascular disease. Peptide-based PCSK9 vaccines have shown a promising prospect of reducing LDL-C. In peptide vaccine (pVax) design, the peptide antigens need to conjugate with carrier protein (CP). However, CP incorporation can induce undesirable anti-CP antibodies, which sterically mask peptide epitopes from being recognized by specific B cells and impair subsequent therapeutically antibody production. This epitopic suppression has posed a barrier in clinical translation of conjugate vaccines all along. A model CP (keyhole limpet hemocyanin, KLH) is herein camouflaged with serum albumin (SA) into hybrid nanocarriers (SA@N), with PCSK9 peptide being anchored onto the surface to form nanovaccine (SA@NVax). Such camouflage of KLH via high "self" SA coverage is able to inhibit KLH from extracellular immune recognition and prevent detectable anti-KLH antibody production. Furthermore, the nanovaccine around 70 nm stabilized by intermolecular disulfide network is ideal for internalization and biodegradation by antigen presenting cells as well as better retention in draining lymph nodes and spleen. As expected, the SA@NVax efficiently primes higher anti-PCSK9 IgG antibody titer than PCSK9 pVax.

Milk and Meat Allergens from Bos taurus ß-Lactoglobulin, α-Casein, and Bovine Serum Albumin: An In-Vivo Study of the Immune Response in Mice.

The mechanism of food allergy may vary. This study aimed to compare the effects of milk, yogurt, or beef meat supplementation on humoral and cellular immune responses in a mice model. Mice were divided into four groups: The "Milk group" was sensitized with a ß-lactoglobulin (ß-lg)/α-casein (α-CN) mixture and supplemented cow milk; the "Yogurt group" was sensitized with ß-lg/α-CN and supplemented yogurt; the "Beef group" was immunized with bovine serum albumin (BSA) and supplemented beef meat; and the "PBS group" received PBS in all procedures. ELISA was used to measure humoral response, including: Total IgE, specific IgG, and IgA. Cellular response was determined by phenotyping lymphocyte from lymphoid tissue and measuring the Th1/Th2 cytokine concentration with flow cytometry. The qPCR method was used for quantification of the fecal microbiota. The results obtained revealed a lower IgE level for the Yogurt group than for the Milk one. In the Yogurt group, the contribution of regulatory T cells to MLN and PP was higher compared to the other groups. We confirmed that diet supplementation with yogurt modulates the immune response to the prime allergen, and changes the activity of serum antibodies to milk proteins and BSA. Based on a specific antibodies level, we cannot exclude the possibility of CMA mice reaction against BSA.

Red meat allergy in children and adults.

PURPOSE OF REVIEW: To highlight recent advances in our understanding of the clinical features, prevalence, and pathophysiology of red meat allergy. RECENT FINDINGS: Allergic reactions to red (i.e. mammalian) meat have historically been considered rare and described primarily in young atopic children. It is now clear that red meat allergy is not uncommon in some parts of the world in other age groups. Strikingly, the majority of these cases relate to specific IgE to galactose-α-1,3-galactose, an oligosaccharide of nonprimate mammals. The mechanism of sensitization in this syndrome relates to bites of certain hard ticks and the clinical reactions often have a delay of 3 to 6 h. An additional form of red meat allergy relates to inhalant sensitization to mammalian proteins. The best characterized example involves cat-sensitized patients with specific IgE to cat serum albumin who can react to ingested pork because of cross-sensitization to pork serum albumin. SUMMARY: Red meat allergy is more common than previously appreciated and relates to at least three different forms that are distinguished by mechanisms of sensitization and have characteristic clinical and immunologic features.

Intact bioactivities and improved pharmacokinetic of the SL335-IFN-ß-1a fusion protein that created by genetic fusion of SL335, a human anti-serum albumin fab, and human interferon-ß.

Recombinant human interferon beta (rIFN-ß) has long been used as a first-line treatment for multiple sclerosis (MS), and any attempt to develop a long-acting rIFN-ß is desirable since only one pegylated version of long-acting rIFN-ß-1a (Plegridy) is currently available in clinics. Previously, we reported that SL335, a human Fab molecule specific to serum albumin, exhibits an extended serum half-life via utilizing the FcRn recycling mechanism. With the ultimate goal of developing a long-acting rIFN-®, we generated a fusion construct by linking human IFN-ß cDNA to the C-terminus of the SL335 H chain at the DNA level followed by expression of the fusion protein, referred to as SL335-IFN-ß-1a, in Chinese hamster ovary-S (CHO-S) cells. In its N-linked glycosylated form, the resulting fusion protein was easily purified from the culture supernatant via a three-step chromatography process. In vitro functional assays revealed that the fusion protein retained its intrinsic binding capabilities to human serum albumin (HSA) and interferon α/ß receptor (IFNAR) that were almost identical to those of parental SL335 and rIFN-ß-1a (Rebif). In addition, the fusion protein possessed an antiviral potency and anti-proliferation activity comparable to those of Rebif. In pharmacokinetic (PK) analyses using Lewis rats and cynomolgus monkeys, SL335-IFN-ß-1a exhibited at least a two-fold longer serum half-life and a significantly reduced renal clearance rate compared to those of Rebif. Finally, a four-week repeated dose toxicity study revealed no abnormal toxicological signs. In conclusion, our results clearly demonstrated that SL335-IFN-ß-1a is worthy of further development as an alternative long-acting IFN-ß therapeutic.

Selective and sensitive fluorescence detection method for pig IgG based on competitive immunosensing strategy and magnetic bioseparation.

A novel fluorescence detection method based on competitive immunoassay and magnetic bioseparation technique was developed and applied to the determination of pig immunoglobulin G (IgG) in serum samples. Core-shell structured Fe3O4@SiO2 nanoparticles were synthesized by chemical coprecipitation, followed by functionalization with amino groups and immobilization of pig IgG antibodies. The synthesized Fe3O4@SiO2-antibody nanoparticles were employed as the probe for the competitive immune recognition of the target antigens in samples and the antigens labeled with fluorescein isothiocyanate (FITC). After the magnetic separation of probes binding with these two types of antigens, fluorescence of the free FITC-labeled antigens was measured for the quantification of the target antigens, since the ratio of the FITC-labeled antigens in supernatant before and after the competitive immune recognition depends on the amount of the target antigens in sample, due to the competitive nature of the binding of the antibody for these two types of antigens. Under the optimal conditions, a linear relationship was obtained between the change of fluorescence intensity and the concentration of pig IgG in a range from 0.75 to 23.50 µg L-1, with a detection limit (LOD) of 0.031 µg L-1. With the facile-prepared probes, this fluorescence competitive method can provide a rapid, specific and highly sensitive immunoassay protocol for the determination of target proteins in complex matrix samples.

Effect of early feed restriction programs and genetic strain on humoral immune response production in broiler chickens.

Two experiments were conducted to compare the immunocompetence of Cobb high performance and rustic Label Rouge broilers and the influence of reduced growth rates subsequent to feed restriction on the IgY anti-bovine serum albumin (BSA) response. In the first experiment (EXP), 360 broilers were assigned to 36 cages from 1 to 42 days of age. A completely randomized design was applied in a 3 × 2 factorial arrangement, with 3 groups (Label Rouge, Cobb ad libitum, and Cobb Restricted Intake), and 2 levels of energy (3,100 and 2,800 kcal/kg); there were 6 replicates per treatment. In the second EXP, 384 Cobb 500 male broilers were randomly assigned to the following feed restriction programs from day 8 to 16: Control, fed ad libitum; Quantitative (80% of the control amount); By Time (fed for 8 h/d), and Qualitative (80% limiting nutrients) restriction. Blood samples were collected on days 35 and 42 (EXP 1) and weekly from day 7 to 42 (EXP 2) for IgY anti-BSA quantification. In EXP 1, the production of IgY anti-BSA was lower in the Cobb groups (P < 0.0001) than in the Label Rouge group, and higher in the Cobb Restricted Intake group (P < 0.0001) compared with the same genetic strain fed ad libitum. Birds fed the low energy diet presented lower (P ≤ 0.06) IgY anti-BSA, independent of genetics. In EXP 2, no difference (P > 0.05) was observed 1 wk after the first BSA inoculation. However, at day 28, birds in all feed restriction programs had higher (P < 0.05) IgY anti-BSA than the Control group fed ad libitum. At day 35, the greatest residual effect of IgY anti-BSA was observed in the Quantitative restriction group. No differences (P > 0.05) were observed between groups after 42 d. The 3 early feed restriction programs had beneficial effects on the humoral immune response. Overall, Quantitative restriction promoted a longer lasting IgY anti-BSA response. Lower growth rate, due to feed restriction or genetic potential, improves humoral immunity in broiler chickens.

Urinary albumin measurement methods.

The quantification of urine albumin is a common practice in Medical Biology laboratories. It allows the assessment of renal injury in common pathologies and many studies have confirmed its role in the diagnosis and prognosis of these disorders. The physicochemical characteristics of albumin in the urine, very different from those in the blood, do not allow the use of the same standardized assay techniques for the blood albumin determination and make it difficult its quantification. Indeed, because of a physiological fragmentation phenomenon, urinary albumin is present in the urine as various small specific peptides. We will present here the main methods of determination of albumin in the urine, which are immuno-turbidimetric and immuno-nephelometric methods, high performance liquid chromatography with steric exclusion and liquid chromatography coupled with mass spectrometry. Currently, immunoanalysis techniques are the most used and are not standardized; large bias can be found between the different kits. This observation calls for a standardization of its determination in the urine.

A case of monoclonal gammopathy of undetermined significance with abnormal low levels of plasma glycated albumin by M protein.

BACKGROUND: It is known that an immunoglobulin abnormality affects various clinical laboratory measurements and leads to abnormal values. We experienced a case of monoclonal gammopathy of undetermined significance (MGUS) showing a falsely low plasma glycated albumin (GA) level. CASE REPORT: The patient was a 75-y-old male who visited our hospital for thrombocytosis identified during a medical checkup. Based on further examinations, he was diagnosed with MGUS (IgM-κ type). Laboratory examinations revealed that the plasma GA level was significantly low at -1.3% but the serum GA level was reasonable at 15.5%. We investigated the cause of the falsely low plasma GA level. RESULTS: The patient's plasma became turbid after mixing with the first reagent for GA measurement. The plasma GA level was increased by dilution of the plasma. The plasma GA level was falsely decreased only at the time of measurement on a sample collected using a blood-collecting tube with heparin sodium. The GA level was decreased by adding heparin sodium to the patient's serum, whereas the GA level was increased by neutralization of the patient's plasma with protamine sulfate. The GA level was increased after adding polyethylene glycol to the patient's plasma. Serum GA levels in healthy controls were decreased by adding purified M protein from the patient's serum. CONCLUSIONS: We report a patient with MGUS whose plasma GA concentration was falsely decreased by M protein when blood was drawn in a heparin sodium-containing tube.

Robot-assisted laparoscopic cystectomy with intracorporeal urinary diversion vs. open mini-laparotomy cystectomy: evaluation of surgical inflammatory response and immunosuppressive ability of CO2-pneumoperitoneum in an experimental porcine study.

OBJECTS: To compare surgical inflammatory response (SIR) after radical cystectomy (RC) in a porcine model using minimal invasive techniques. Additionally we aimed to investigate the potential immunosuppressive ability of preoperative CO2-pneumoperitoneum (CO2P). MATERIALS AND METHODS: Forty female landrace pigs were randomized to five groups: Three intervention groups all having a cystectomy and an ileal conduit either done by robot-assisted laparoscopic technique with intracorporeal urinary diversion (RALC) or an open mini-laparotomy with or without prior CO2P (OMC ± CO2P). Two control sham groups with or without prior CO2P (S ± CO2P). Serum samples were obtained preoperatively, immediately postoperative, 24, 48 and 72 hours postoperatively, and the inflammatory mediators CRP, Haptoglobin, Ceruloplasmin, Albumin, Cortisol, IL-4, IL-6, IL-12 and IFN-α were measured. RESULTS: Operative time was significantly longer in RALC compared to open groups (OMC ± CO2P) (p's < .0001). CRP and Haptoglobin levels were significantly higher for surgical intervention groups (SIG) compared to controls 24, 48 and 72 hours postoperatively (p's < .001). At 48 hours, CRP was higher for RALC vs OMC + CO2P (p = .029). At 72 hours, Haptoglobin was higher for RALC vs open groups (p's < .024). Ceruloplasmin, cortisol, albumin, IL-4, IL-6, IL-12 and IFN-α, revealed no significant differences between SIG. CONCLUSIONS: No major differences were found between RALC and OMC regarding the degree of tissue trauma quantified by inflammatory markers. Thirty minutes of CO2-insufflation preoperative appears to have a transient immunosuppressive effect of the innate postoperative SIR, whereas prolonged CO2P apparently diminishes this effect.

Relative efficiencies of peptidylarginine deiminase 2 and 4 in generating target sites for anti-citrullinated protein antibodies in fibrinogen, alpha-enolase and histone H3.

OBJECTIVE: Peptidylarginine deiminase 2 (PAD2) and PAD4 are expressed in the synovium of rheumatoid arthritis (RA) patients and catalyze citrullination of arginine residues in proteins targeted by anti-citrullinated protein antibodies (ACPAs). Little is known about the relative importance of PAD2 and PAD4 in generating citrullinated self-antigens. Here we investigate the ability of PAD2 and PAD4 to generate citrullinated targets for ACPAs in four human proteins. METHODS: Synovial fluid (SF) and plasma were collected from 42 RA patients. Human fibrinogen, human alpha-enolase (ENO1), human histone H3, and human serum albumin (HSA) were citrullinated in vitro by PAD2 or PAD4. The total degree of citrullination was determined using the anti-modified citrulline approach. Antibody binding to native and citrullinated proteins was measured by ELISA. RESULTS: ACPAs within pooled SF from multiple RA patients reacted equally well with, and cross-reacted with, PAD2- and PAD4-citrullinated fibrinogen. ACPAs from most individual patient SF and plasma samples bound equally well to PAD2- and PAD4-citrullinated fibrinogen or ENO1. When histone H3 was used as target, PAD4 was generally superior in generating epitopes recognized by ACPAs. No binding to citrullinated HSA was observed. CONCLUSION: In most patients, PAD2 and PAD4 are equally efficient in generating citrullinated target sites for ACPAs in fibrinogen and ENO1. The binding of autoantibodies to histone H3 was generally higher after citrullination with PAD4 than with PAD2. Citrullinated HSA is not a target for ACPAs.

Immunological Comparison of Native and Recombinant Hen's Egg Yolk Allergen, Chicken Serum Albumin (Gal d 5), Produced in Kluveromyces lactis.

Chicken serum albumin (CSA) is a hen's egg yolk allergen causing IgE-mediated allergy. The objective of this study was to produce a recombinant version of CSA and compare its IgE reactivity to natural CSA (nCSA). CSA was cloned and expressed as a soluble fraction in the yeast Kluyveromyces lactis (K. lactis) protein expression system. The gene encoding CSA was amplified with a C-terminal hemagglutinin epitope tag by polymerase chain reaction (PCR) and cloned into the pKLAC2 expression vector prior to transforming into K. lactis. Recombinant CSA (rCSA) was purified by immunoprecipitation. Twenty-one patients allergic to hen's egg white were examined for sensitisation against nCSA. 38% of patients were found to be sensitised to CSA based on Western immunoassay. Immunoglobulin E (IgE) binding capacity of rCSA and nCSA was analysed by ELISA using sera from patients sensitised to CSA. Levels of IgE-binding were similar for both the recombinant and the natural CSA, indicating the existence of similar epitopes. rCSA produced in this study is a potential candidate to be used in component-resolved diagnosis (CRD) of egg yolk allergy. The usefulness of rCSA in CRD of egg yolk allergy warrants further characterisation using sera from patients with allergy to hen's egg yolk in future studies.

Development and Validation of a Surface Plasmon Resonance Biosensor for Specific Detection of Porcine Serum Albumin in Food.

Food allergies are a potential food safety and public health concern worldwide. To assure the safety of people who experience allergic reactions, it is necessary to establish effective and reliable methods for rapid detection of food allergens. This paper reports an innovative method for the rapid detection and analysis of porcine serum albumin (PSA), known as a major allergen in pork, based on a surface plasmon resonance (SPR) biosensor. The antibodies known to have a high bioactivity against PSA were verified by competitive indirect-ELISA and then immobilized on the SPR sensor surface, thus allowing them to capture PSA. The developed SPR demonstrated a linear range from 1.0 to 450 ng/mL for the measurement of PSA with a detection limit of 19.81 ng/mL. Within-day RSD (1.97-4.02%) and between-day RSD (1.88-4.15%) were no more than 5%. The SPR was evaluated for analysis of six commercial food samples and showed almost perfect agreement between the results obtained by ELISA test kits without significant differences (P > 0.05). Therefore, this assay permits accurate, specific, and sensitive detection of PSA in pork and pork products.

Influence of Gamma Irradiation on Porcine Serum Albumin Structural Properties and Allergenicity.

Pork provides an ideal source of food energy; however, pork can elicit an allergic reaction, and porcine serum albumin (PSA) has been identified as a major allergen. This study examined the impact of gamma irradiation on the allergenicity and structural qualities of PSA; the PSA solution was gamma-irradiated at 1, 2, 4, 6, and 8 kGy. Allergenicity was investigated by immunoblotting and competitive indirect ELISA using serum from patients who are allergic to pork, and conformational changes in irradiated PSA were measured by circular dichroism, sulfhydryl group detection, and fluorescence emission spectra. The secondary and tertiary structures of gamma-irradiated PSA were altered, and the allergenicity of PSA was lowered by boosting the amount of irradiation. In addition, there is high correlation between depletion in the α-helix and immunoglobulin E-binding capability of PSA. The results show a new possibility in using gamma irradiation to reduce the allergenicity of pork products.

An overview of in vitro and in vivo glycation of albumin: a potential disease marker in diabetes mellitus.

Non-enzymatic glycation of macromolecules, especially proteins leading to their oxidation is increased in diabetes mellitus due to hyperglycaemia and play an important role in associated complications of the disease. Protein glycation mostly occurs in intra chain lysine residues resulting in the formation of early stage Amadori products which are finally converted to advance glycation end products (AGEs). This review deals with the structural studies of in vitro and in vivo glycated human serum albumin (HSA). The aim of this review is to explain the disturbance in secondary and tertiary structure of albumin upon glucosylation and the immunogenic potential of modified albumin. Amadori-albumin may have enough potential to provoke the immunoregulatry cells and generate autoantibodies in diabetic patients. Role of Amadori-albumin in the induction of autoantibodies in type2 diabetes especially in chronic kidney disease (CKD) patients has been discussed. This review also considers various studies that investigate the effects of glycation on the structural and immunological properties of HSA. The use of glycated albumin (GA) as a short to intermediate term marker for glycaemic control in diabetes is also focused.