Sandwich Enzyme-linked Immunosorbent Assay Method Research Articles

Low serum brain derived neurotrophic factor in non-suicidal out-patients with depression: Relation to depression scores

Context:Low brain-derived neurotrophic factor (BDNF) has been implicated in the pathophysiology of depression. The relation between BDNF and severity of depression has been investigated.Aims:In this study, we aimed to measured serum BDNF levels in never-treated non-suicidal out-patients with depression and relate this to the severity of depression.Settings and Design:This study was conducted in an out-patient setting in a tertiary care psychiatric hospital on consenting depressed patients.Materials and Methods:Forty three (19 females) antidepressant-naive out-patients with depression, aged between 18 and 55 years and 24 (13 females) age-matched healthy volunteers gave consent for the study. Serum BDNF levels were assayed by using the sandwich enzyme-linked immunosorbent assay method on morning serum samples before starting treatment. These were compared between patients and controls using independent sample t-test. Pearson's correlation coefficient was used to assess the association between baseline BDNF and Hamilton depression rating scale (HDRS).Results:Serum BDNF was significantly lower in patients with depression (mean±standard deviation (SD)=18.59±4.9 ng/ml) than in healthy volunteers (mean±SD=23.6±5.6 ng/ml; P=0.001). There was a significant negative correlation between the HDRS total scores and BDNF levels (P=0.04), indicating that more severely depressed patients had lower BDNF scores.Conclusions:Serum BDNF level is lower in non-suicidal out-patients with depression. The inverse correlation between ratings of depression and BDNF levels suggests possible relationship between depression, (role of illness on) BDNF levels and neuroplasticity thereof.

Increased circulating soluble LR11 in patients with acute coronary syndrome

BackgroundLR11 (also so called SorLA or SORL1) is a novel marker of intimal smooth muscle cell (SMC) proliferation. Vascular SMCs play important roles in the development of atherosclerosis interacting with macrophages in a vulnerable plaque of patients with acute coronary syndrome (ACS).The present study determines whether soluble LR11 (sLR11) is associated with ACS. MethodsWe studied 100 patients with coronary artery disease (CAD) comprising 50 consecutive patients with acute coronary syndrome (ACS; mean age 62.3±13.0years; male 78.0%) who were successfully treated with percutaneous coronary intervention and 50 age- and sex-matched stable angina pectoris (SAP) patients as control. Concentration of sLR11 was measured by sandwich enzyme-linked immunosorbent assay method. ResultsCirculating sLR11 was significantly increased in patients with ACS compared with SAP (9.88±2.78 vs. 8.18±1.11ng/ml, p<0.01). Multivariate logistic regression analysis indicated that sLR11 was independently associated with ACS (odds ratio (OR), sLR11 quartile increment, 2.18, 95% confidence interval (CI) 1.21–4.19, p<0.01). Among various biomarkers of acute coronary syndrome, hsCRP were significantly correlated with LR11 (r=0.480, p<0.01). ConclusionsThere is a statistical significant association between LR11 and ACS and may be a useful biomarker for the development of acute coronary syndrome.

Two novel sandwich ELISAs identify PAD4 levels and PAD4 autoantibodies in patients with rheumatoid arthritis

The peptidylarginine deiminase 4 (PAD4) gene and PAD4 autoantibodies have been associated with rheumatoid arthritis (RA) and its pathogenesis. Therefore, methods for accurately determining their levels in the peripheral blood of these patients would be a diagnostic asset. The objective of our study was to adapt the enzyme-linked immunosorbent assay (ELISA) method for evaluating PAD4 levels in human blood. We prepared recombinant human (h)PAD1, -2, -3, and -4 proteins to develop mouse monoclonal antibodies specific to hPAD4. We then generated six monoclonal antibodies against hPAD4 and developed two new sandwich ELISA methods for evaluating hPAD4 and PAD4 autoantibodies in the peripheral blood from 32 patients with RA, ten patients with osteoarthrosis, and 20 healthy individuals. The distribution of hPAD4 in the patients' plasma was determined. Two populations were identified: one group with high hPAD4 levels (>0.57 ng/mL) and a second group with near-zero levels (<0.1 ng/mL). Most patients approximating zero hPAD4 levels had PAD4 autoantibodies. In contrast, most of those with higher plasma hPAD4 levels did not have detectable PAD4 autoantibodies. The combination of these sandwich ELISA methods may be a potentially beneficial clinical tool for diagnosing RA.

Immunosensor Based on the ZnO Nanorod Networks for the Detection of H1N1 Swine Influenza Virus

This paper presents an immunosensor fabricated on patterned zinc oxide nanorod networks (ZNNs) for detecting the H1N1 swine influenza virus (H1N1 SIV). Nanostructured ZnO with a high isoelectric point (IEP, approximately 9.5) possesses good absorbability for proteins with low IEPs. Hydrothermally grown ZNNs were fabricated on a patterned Au electrode (0.02 cm2) through a lift-off process. To detect the H1N1 SIV, the sandwich enzyme-linked immunosorbent assay (ELISA) method was employed in the immunosensor. The immunosensor was evaluated in an acetate buffer solution containing 3,3',5,5'-tetramethylbenzidine (TMB) via cyclic voltammetry at various H1N1 SIV concentrations (1 pg/mL-5 ng/mL). The measurement results of the fabricated immunosensor showed that the reduction currents of TMB at 0.25 V logarithmically increased from 259.37 to 577.98 nA as the H1N1 SIV concentration changed from 1 pg/mL to 5 ng/mL. An H1N1 SIV immunosensor, based on the patterned ZNNs, was successfully realized for detecting 1 pg/mL-5 ng/mL H1N1 SIV concentrations, with a detection limit of 1 pg/mL for H1N1 SIV.

A study on Haemophilus influenzae type b growth rate and capsule production in different media

In the present study, seven isolates of Haemophilus influenzae type b were cultured in four different media to compare growth rate and capsule production. Four liquid media namely brain heart infusion broth (BHI), trypticase soy broth (TSB), Mueller Hinton Broth (MHB) and Gonococci broth (GC) with added supplements (1% hemoglobin, 1% Isovitalex) were used. The growth was measured by colony counting (CFU/ml) using serial dilution. Four of the isolates showed the highest growth rate with the average of 10 13 CFU/ml on BHI broth while TSB had the second highest growth of more than 10 10 CFU/ml in an 18hour culture at 37 oC culture. In the next step, the amount of capsular polysaccharide (CPS-b) antigen which is made of Polyribosyl ribitol phosphate (PRP) was assessed all isolates by two methods: modified Indirect Sandwich Enzyme-linked immunosorbent assay (ELISA) and Bial method to select the isolate producing the highest amount of PRP. The antisera used in sandwich ELISA were prepared by immunization of Rabbit and Rat. The maximal amount of PRP was produced by isolate Hib (5s) with the amount of 321 mg/lit.

Purification of HRSV F protein from a eukaryotic expression vector and establishment of a sandwich ELISA method

In order to increase the expression of the fusion (F) protein and lay a foundation for the construction of a genetically engineered vaccine and rapid clinical detection, the F protein of the human respiratory syncytial virus (HRSV) was expressed and purified, and a sandwich enzyme-linked immunosorbent assay (ELISA) method was established. The F1 fragment of the HRSV F protein was amplified following reverse transcription, and was then combined with the vector and transformed into eukaryotic cells. The recombinant protein was induced and purified. The purified protein was used to immunize mice to produce antiserum and establish indirect ELISA. The established method was tested and verified by analyzing 100 samples using gold immunochromatography (GICA). The F1 fragment of the F gene was successfully amplified, the DNA (+) recombinant was selected, and a protein of molecular weight approximately 45,000 was obtained after the induction. The optimal reaction conditions and working concentration of ELISA were determined. The optimal concentration of mice anti-F1 IgG is 3.2 µg/ml, the best reaction time of the samples is 70 min at 37 ˚C, and the working concentration of the rabbit anti‑mouse IgG is 1:6,000. Compared with the GICA method, the sample's positive co-efficient of variation was 3.2-8.6%, and the negative co-efficient of variation was 5.1-8.3%. These were <10%, indicating that the ELISA method was reproducible. The F1 protein can be greatly expressed in transfected eukaryotic cells, and the purified F1 protein has good immunogenicity. The antiserum produced by the purified recombinant protein can be precisely detected using the ELISA detection method described in this study.

Screening for co-existence of α-thalassemia in β-thalassemia and in HbE heterozygotes via an enzyme-linked immunosorbent assay for Hb Bart’s and embryonic ζ-globin chain

We sought to demonstrate the ability of levels of Hb Bart's and ζ-globin chain quantified by enzyme-linked immunosorbent assay (ELISA) in detecting α-thalassemia in β-thalassemia and HbE heterozygotes. We developed an in-house sandwich ELISA method using monoclonal antibodies (mAbs) to Hb Bart's and ζ-globin chain, and quantified levels of Hb Bart's and ζ-globin chain in 172 and 223 anonymous blood samples of β-thalassemia and HbE heterozygotes, respectively. Genotypes of α-thalassemia 1, β-thalassemia were identified, and HbE allele was confirmed using a newly developed multiplex allele-specific PCR. The in-house sandwich ELISA method detected Hb Bart's in 6.4% of β-thalassemia heterozygotes, of which 5.2% showed detectable amounts of the ζ-globin chain. 15.2% of individuals heterozygous for HbE showed a detectable amount of Hb Bart's, and the ζ-globin chain was detected in 11.2% of this cohort. All samples having detectable amounts of Hb Bart's and the ζ-globin chain were verified to be SEA-type α-thalassemia 1. ELISA-quantified Hb Bart's and ζ-globin chain levels can be used to detect double heterozygosity of α- and β-thalassemia and of α-thalassemia and HbE. This strategy may be useful in screening for co-existence of α-thalassemia in β-thalassemia and in HbE heterozygotes, particularly in countries where α-, β-thalassemia and HbE are endemic.

Daily variance of urinary excretion of AQP2 determined by sandwich ELISA method

Urinary excretion of aquaporin 2 (AQP2) is a useful marker of kidney collecting duct function. A specific and convenient method to measure AQP2 in human urine would help to treat water balance disorders. It is unknown whether urinary excretion of AQP2 shows any daily variance. A sandwich enzyme-linked immunosorbent assay (ELISA) method for AQP2 was established using two different kinds of antibodies, and its sensitivity and specificity were examined. Daily variance of urinary excretion of AQP2 and responses to acute water load were examined. The established ELISA specifically detected urine AQP2 with high sensitivity (detected as low as 0.34 pmol/mL). Urinary excretion of AQP2 did not show daily variance between 9 a.m. and 9 p.m. in healthy subjects. The developed ELISA method using two different antibodies is convenient and highly sensitive, and could be useful in clinical practice. Urinary excretion of AQP2 is relatively constant from morning to night, and spot urine sampling at any time during this time period does not affect the results.

High Sensitive Detection of Cry1Ab Protein Using a Quantum Dot-Based Fluorescence-Linked Immunosorbent Assay

Protein-based detection methods, enzyme-linked immunosorbent assay (ELISA) and lateral flow strip, have been widely used for rapid, spot, and sensitive detection of genetically modified organisms (GMOs). Herein, one novel quantum dot-based fluorescence-linked immunosorbent assay (QD-FLISA) was developed employing quantum dots (QDs) as the fluorescent marker for the detection of the Cry1Ab protein in MON810 maize. The end-point fluorescent detection system was carried out using QDs conjugated with goat anti-rabbit secondary antibody. The newly developed Cry1Ab QD-FLISA assay was highly specific to the Cry1Ab protein and had no cross-reactivity with other target proteins, such as Cry2Ab, Cry1F, and Cry3Bb. The quantified linearity was achieved in the value range of 0.05-5% (w/w). The limits of detection (LOD) and quantification (LOQ) of the QD-FLISA were 2.956 and 9.854 pg/mL, respectively, which were more sensitive than the conventional sandwich ELISA method. All of the results indicated that QD-FLISA was a highly specific and sensitive method for the monitoring of Cry1Ab in GMOs.

A highly sensitive immunoassay for the detection of prion‐infected material in whole human blood without the use of proteinase K

The causal association of variant Creutzfeldt-Jakob disease (vCJD) with bovine spongiform encephalopathy has raised significant concerns for public health. Assays for vCJD infection are vital for the application of therapeutics, for the screening of organ donations, and to maintain a safe blood supply. Currently the best diagnostic tools for vCJD depend upon the detection of disease-associated prion protein (PrP(Sc) ), which is distinguished from normal background PrP (PrP(C) ) by proteinase K (PK) digestion, which can also degrade up to 90% of the target antigen. A sandwich enzyme-linked immunosorbent assay method was developed using unique antibodies for the detection of disease-associated PrP in the absence of PK treatment. In combination with immunoprecipitation the assay was optimized for the detection of pathogenic PrP in large volumes of whole blood. Optimization of the assay allowed detection of 2×10(4) LD(50) units/mL spiked in whole blood. Application of the assay to clinically relevant volumes enabled the detection of 750 LD(50) units/mL in 8mL of whole blood. By combining the use of a unique antibody that selectively immunoprecipitates PrP(Sc) with glycoform-restrictive antibodies we have developed a rapid assay for vCJD infection that does not require any PK treatment to achieve high levels of specificity in whole human blood, the most challenging potential analyte. The sensitivity of detection of vCJD infection is greater than the equivalent of a more than 2.5 million-fold dilution of infected brain, providing a highly sensitive immunoassay compatible with blood screening.

Monoclonal antibody-based triple antibody sandwich-enzyme-linked immunosorbent assay and immunocapture reverse transcription-polymerase chain reaction for Odontoglossum ringspot virus detection

Odontoglossum ringspot virus (ORSV) infects numerous commercially important orchids and causes significant losses worldwide. The coat protein ( CP) gene of ORSV was cloned and expressed in Escherichia coli by using the pET-32a expression vector, and the expression of recombinant protein was confirmed by Western blotting using anti-ORSV antibodies. The recombinant protein was purified using Ni-NTA agarose, and the purified protein was used as an immunogen to produce monoclonal antibodies (MAbs) and polyclonal antibodies (PAbs). Five murine MAbs against ORSV CP were obtained. Among them, two MAbs (6B4 and 1D1) also reacted with TMV CP. The triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) and immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) methods using the MAb (8A5) were then developed for sensitive, specific, and rapid detection of ORSV. TAS-ELISA and IC-RT-PCR could detect ORSV in the infected leaf saps with dilutions of 1:10,240 and 1:81,920 (w/v, g mL −1), respectively. TAS-ELISA and IC-RT-PCR detections indicated that ORSV was prevalent in orchids in the Zhejiang Province of China.

Immunomodulating property of Ocimum sanctum by regulating the IL-2 production and its mRNA expression using rat's splenocytes

Objective To investigate the regulatory effect of aqueous extract of leaves of Ocimum sanctum on IL-2 cytokine production in vivo and in vitro, and the effect of leaves extract on general blood picture including T& B lymphocytes. Methods For in vivo studies albino Wistar rats were treated with aqueous crude leaves extract of Ocimum sanctum for 20 consecutive days. Spleen cells were harvested and assayed for IL-2 production by using sandwich enzyme-linked immunosorbent assay (ELISA) and mRNA expression methods. For in vitro study aqueous Ocimum sanctum leaves extract = in different concentrations (25–500 μg/mL) was added into culture plates containing ConA stimulated splenocytes. To study the overall effect on blood picture, density gradient purified lymphocytes analysis and conventional methodology for total and differential leukocyte count and hemoglobin level were also done. Results It indicated that the rats treated with Ocimum sanctum leaves extract had significantly enhanced ( P < 0.001) ability of spleen cells to secrete IL-2. Investigation in vitro also showed regulation of IL-2 production. Blood study exhibited leucocytosis and augmentation of T& B lymphocytes by 25% approximately. 4–5% increase in Hemoglobin value was also noticed. Conclusion Aqueous Ocimum sanctum leaves extract may have stimulatory effect on T & B lymphocytes particularly on Th1 subset of lymphocytes as shown by enhancement in IL-2 production.

Diagnostic role of serum glypican‐3 in differentiating hepatocellular carcinoma from non‐malignant chronic liver disease and other liver cancers

The role of glypican-3 (GPC3), a novel serum marker, in differentiating hepatocellular carcinoma (HCC) from non-malignant chronic liver disease and other malignant space-occupying lesions in the liver is largely unknown. The aims of this study were to evaluate its diagnostic role and clinical correlations in patients with HCC. Six groups were studied which included 40 healthy subjects, 50 patients with chronic hepatitis (CH), 50 patients with liver cirrhosis (LC), 100 patients with HCC, 50 patients with intrahepatic cholangiocarcinoma (ICC) and 50 patients with metastatic carcinoma (MCA). Serum GPC3 levels were measured by using a sandwich enzyme-linked immunosorbent assay method. Fifty-three percent of HCC patients had elevated serum GPC3 levels with values ranging 35.5-7826.6 ng/mL. The serum marker was undetectable in other groups except one patient (2%) with LC and another patient (2%) with MCA. In most cases of HCC, elevated GPC3 values did not correlate with alpha-fetoprotein (AFP) levels. Detectable GPC3 was significantly correlated with the presence of viral hepatitis markers but was not correlated with tumor size and stage of HCC. Serum GPC3 was superior to AFP in detecting small HCC (56.3% and 31.3%, respectively). A combination of serum GPC3 and AFP yielded an improved sensitivity for detecting small HCC to 75%. Serum GPC3 is highly specific for detecting HCC. The combined use of serum GPC3 and AFP provides a potentially promising tool to better differentiate HCC from benign liver disorders, as well as from other liver cancers.

Effect of Maternal Egg Consumption on Breast Milk Ovalbumin Concentration

Palmer D, Gold M, Makridess M. Clin Exp Allergy. 2008;38(7):1186–1191PURPOSE OF THE STUDY. To assess human milk ovalbumin concentrations after daily maternal ingestion of 1 cooked egg for a 3-week period.STUDY POPULATION. There were 32 mothers of singleton, breastfed, egg-sensitive infants with moderate-to-severe eczema. Egg sensitivity was identified by a positive skin-prick test result. Eczema was evaluated by using a standardized scoring system.METHODS. Families had an initial home visit by an experienced dietitian, which involved collection of demographic and dietary information. All women and children were asked to follow an egg-free diet from day 1 through the duration of the trial. Adherence to the egg-free diet was assessed via detailed dietary intake records for both mothers and children on days 1 to 3, 10 to 12, and 21 to 23. Mothers were randomly allocated to receive identical-appearing egg-free muffins or muffins containing 1 (55 g) whole egg. Each mother was given a 3-week supply of frozen muffins corresponding to her randomization group and consumed 1 muffin per day on days 3 through 23. Atopic dermatitis assessments were performed for each child at the commencement and completion of the trial. The mothers completed the Infant's Dermatitis Quality of Life Index 3 times during the trial. On days 3, 12, and 23, the mothers manually expressed 5 mL of breast milk into sterile containers before and 2, 4, and 6 hours after eating the test muffin. Breast milk samples were stored in the home freezer and collected on day 24. The breast milk samples were queried for ovalbumin concentration by using a sandwich enzyme-linked immunosorbent assay method. Breast milk ovalbumin concentrations (nanograms per milliliter) were plotted against time, and the resulting curve was used to determine peak ovalbumin concentrations and total ovalbumin excretions (nanograms per milliliter per hour). Independent-sample t tests, Mann-Whitney U tests, and Pearson's χ2 tests were used to investigate differences between the diet groups.RESULTS. Women in the egg group had higher ovalbumin concentrations in breast milk than did the control group at all time points. Within each dietary group, the frequency of ovalbumin detection, peak ovalbumin concentration, and total ovalbumin excretion did not differ at days 3, 12, and 23. Ovalbumin was not detected in the breast milk of 25% of the women in the egg group. Infant eczema symptom scores were significantly reduced with time for both groups.CONCLUSIONS. Human milk ovalbumin is related to maternal dietary egg intake. Comparable detection of ovalbumin across time suggests that ovalbumin does not accumulate in human milk. One quarter of the women had no ovalbumin detected in their breast milk on any of the study days, which suggests that some women either do not excrete ovalbumin in their breast milk when challenged with 1 egg or have delayed excretion beyond 6 hours. Maternal dietary avoidance of well-cooked egg may not be necessary for all breastfed infants with egg sensitivity and eczema.REVIEWERS COMMENTS. For breastfed infants with food allergy, strict avoidance of the offending food proteins for both mother and child is frequently recommended. Total dietary avoidance of egg is difficult for patients to achieve. Additional study is needed to substantiate or to refute the preliminary observation that regular maternal ingestion of a small quantity of well-cooked egg did not markedly exacerbate eczema symptoms in egg-sensitive breastfed infants.

Level of Vascular Endothelial Growth Factor in Aqueous Humor and Surgical Results of Ahmed Glaucoma Valve Implantation in Patients With Neovascular Glaucoma

To determine the associations between the levels of growth factors in aqueous humor and the surgical outcomes of Ahmed glaucoma valve implantation in patients with neovascular glaucoma (NVG). From 19 NVG eyes of 19 patients, a sample of aqueous humor was taken just before Ahmed glaucoma valve implantation. Levels of vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-beta1, and TGF-beta2 in aqueous humor aspirates were measured using a sandwich enzyme-linked immunosorbent assay method. When the postoperative intraocular pressures were >or=21 mm Hg with or without antiglaucoma medications at 2 successive visits or when any other surgical interventions were needed to lower intraocular pressure, that surgery was considered a failure. After patients were classified into success versus failure groups, the levels of the growth factors between the 2 groups was compared. The cumulative probability of success according to Kaplan-Meier analysis was also determined. Mean postoperative follow-up period was 40.9+/-19.6 months and cumulative probability of success was 43% at 57 months after surgery. Mean VEGF level in the failure group was higher than that of the success group (P=0.014). However, there was no statistical difference in the levels of TGF-beta1, TGF-beta2, and protein between 2 study groups (all P>0.05). The level of VEGF in aqueous humor was significantly higher in the failure group after the Ahmed glaucoma implantation compared with the success group. Our results imply that VEGF may play a role in determining surgical success after Ahmed valve implantation in patients with NVG.

A Reliable Enzyme Linked Immunosorbent Assay for the Determination of Bovine and Porcine Gelatin in Processed Foods

Since gelatin-containing foods pose a risk for eliciting allergic reactions in sensitized individuals, a novel sandwich enzyme linked-immunosorbent assay (ELISA) for the detection and quantification of bovine and porcine gelatin in processed foods was developed. Rabbits and goats were immunized with bovine gelatin, and three antisera (pAb1 and pAb2 from rabbits, and pAb3 from goats) were obtained. We established a sandwich ELISA method based on a combination of these antibodies. In this study, two sandwich ELISA methods, rabbit pAb2-pAb1 and goat pAb3-pAb3, were evaluated for sensitivity, specificity, cross-reactivity, and applicability. Both ELISA methods were highly specific for bovine and porcine gelatin but had little reactivity with fish gelatin. The detection and quantification limits for porcine gelatin were found to be 0.78 ng/mL and 1.56 ng/mL, respectively. The established sandwich ELISA methods produced no false-positives, except for heated meat products or false negatives when various commercial foods were analyzed for their gelatin content. The rabbit pAb2-pAb1 ELISA cross-reacted with boiled squid, while the goat pAb3-pAb3 ELISA did not. Thus, the proposed goat pAb3-pAb3 ELISA method is a reliable tool for the detection of gelatin contaminants present in processed foods.

Reliable Enzyme-Linked Immunosorbent Assay for the Determination of Soybean Proteins in Processed Foods

Among allergenic foods, soybean is known as a food causing adverse reactions in allergenic patients. To clarify the validity of labeling, the specific and sensitive detection method for the analysis of the soybean protein would be necessary. The p34 protein, originally characterized to be p34 as an oil-body associated protein in soybean, has been identified as one of the major allergenic proteins and named Gly m Bd 30K. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of the soybean protein in processed foods was developed using polyclonal antibodies raised against p34 as a soybean marker protein and the specific extraction buffer for extract. The developed sandwich ELISA method was highly specific for the soybean protein. The limit of detection (LOD) and the limit of quantification (LOQ) of the developed ELISA were 0.47 ng/mL (equivalent to 0.19 microg/g in foods) and 0.94 ng/mL (equivalent to 0.38 microg/g in foods), respectively. The recovery ranged from 87.7 to 98.7%, whereas the intra- and interassay coefficients of variation were less than 4.2 and 7.5%, respectively. This study showed that the developed ELISA method is a specific, precise, and reliable tool for the quantitative analysis of the soybean protein in processed foods.

Quantitative Detection of Allergenic Protein Sin a 1 from Yellow Mustard (Sinapis alba L.) Seeds Using Enzyme-Linked Immunosorbent Assay

Allergy to yellow mustard (YM; Sinapis alba L.) seed proteins has been reported and is currently seen as a constraint that hampers expansion of YM protein utilization. The most predominant allergenic protein of YM seed has been recognized as Sin a 1. In this study, Sin a 1 was purified ( S. alba var. Andante), rabbit polyclonal antibodies (pAb) specific to Sin a 1 were generated, and a sandwich enzyme-linked immunosorbent assay (S-ELISA) was developed to detect and quantify Sin a 1 from YM. The S-ELISA method using Sin a 1-pAb and its horseradish peroxidase conjugate resulted in a detection limit of 0.3 microg/mL for purified Sin a 1. The Sin a 1 contents of six YM lines were in the range of 0.82-2.94 mg/g when assayed by the developed S-ELISA method. The results showed that S-ELISA could distinguish Sin a 1 in YM seed-derived extracts rapidly and could be applied in controlling and/or monitoring of YM allergenic proteins.

Determination of peanut allergens in cereal-chocolate-based snacks: metal-tag inductively coupled plasma mass spectrometry immunoassay versus liquid chromatography/electrospray ionization tandem mass spectrometry

A comparison of two methods for the identification and determination of peanut allergens based on europium (Eu)-tagged inductively coupled plasma mass spectrometry (ICP-MS) immunoassay and on liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) with a triple quadrupole mass analyzer was carried out on a complex food matrix like a chocolate rice crispy-based snack. The LC/MS/MS method was based on the determination of four different peptide biomarkers selective for the Ara h2 and Ara h3/4 peanut proteins. The performance of this method was compared with that of a non-competitive sandwich enzyme-linked immunosorbent assay (ELISA) method with ICP-MS detection of the metal used to tag the antibody for the quantitative peanut protein analysis in food. The limit of detection (LOD) and quantitation of the ICP-MS immunoassay were 2.2 and 5 microg peanuts g(-1) matrix, respectively, the recovery ranged from 86 +/- 18% to 110 +/- 4% and linearity was proved in the 5-50 microg g(-1) range. The LC/MS/MS method allowed us to obtain LODs of 1 and 5 microg protein g(-1) matrix for Ara h3/4 and Ara h2, respectively, thus obtaining significantly higher values with respect to the ELISA ICP-MS method, taking into account the different expression for concentrations. Linearity was established in the 10-200 microg g(-1) range of peanut proteins in the food matrix investigated and good precision (RSD <10%) was demonstrated. Both the two approaches, used for screening or confirmative purposes, showed the power of mass spectrometry when used as a very selective detector in difficult matrices even if some limitations still exist, i.e. matrix suppression in the LC/ESI-MS/MS procedure and the change of the Ag/Ab binding with matrix in the ICP-MS method.

A Reliable and Sensitive Immunoassay for the Determination of Crustacean Protein in Processed Foods

Among food allergens, crustacea such as shrimps, crabs, and lobsters are a frequent cause of adverse food reactions in allergic patients. The major allergen has been identified as a muscular protein, tropomyosin. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of crustacean protein in processed foods was developed using the sample dilution buffer that is added to porcine tropomyosin. The sandwich ELISA method was highly specific for the Decapoda group, apart from minor cross-reactivities to other crustacea and mollusks. The recovery ranged from 85 to 141%, while the intra- and interassay coefficients of variation were less than 2.8 and 8.4%, respectively.