Simple ELISA Research Articles

Parallel analysis of mucosally derived B- and T-cell responses to an oral typhoid vaccine using simplified methods

There is a great need for simple methods for analysis of immune responses to mucosal vaccines that can be used on small blood volumes in field trials in both children and adults. We have investigated if mucosally derived B- and T-cell responses can be monitored in parallel by analysis of antibodies and T-cell effector molecules in culture supernatants from circulating blood lymphocytes obtained from orally vaccinated Swedes. Immunization with a live oral model vaccine, i.e. Salmonella enterica serovar Typhi Ty21a, gave rise to secretion of typhoid specific IgA and IgM antibodies from peripheral blood mononuclear cells (PBMCs) and this response could be equally well detected by ELISA and ELISPOT 7 days after vaccination. The ELISA based assay could be further simplified by using buffy coat cells, but not by using whole blood or frozen PBMCs. Vaccine induced T-cell responses appeared later than the B-cell responses, but could be detected by ELISA assessment of IFN-γ and granzymes in supernatants from antigen stimulated PBMCs 21 days after vaccination. Thus, both B- and T-cell responses could be detected using simple ELISA based assays that would be practical to use in large-scale vaccine trials.

A Novel Immunological Assay for Hepcidin Quantification in Human Serum

BackgroundHepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Increased hepcidin concentrations lead to iron sequestration in macrophages, contributing to the pathogenesis of anaemia of chronic disease whereas decreased hepcidin is observed in iron deficiency and primary iron overload diseases such as hereditary hemochromatosis. Hepcidin quantification in human blood or urine may provide further insights for the pathogenesis of disorders of iron homeostasis and might prove a valuable tool for clinicians for the differential diagnosis of anaemia. This study describes a specific and non-operator demanding immunoassay for hepcidin quantification in human sera.Methods and FindingsAn ELISA assay was developed for measuring hepcidin serum concentration using a recombinant hepcidin25-His peptide and a polyclonal antibody against this peptide, which was able to identify native hepcidin. The ELISA assay had a detection range of 10–1500 µg/L and a detection limit of 5.4 µg/L. The intra- and interassay coefficients of variance ranged from 8–15% and 5–16%, respectively. Mean linearity and recovery were 101% and 107%, respectively. Mean hepcidin levels were significantly lower in 7 patients with juvenile hemochromatosis (12.8 µg/L) and 10 patients with iron deficiency anemia (15.7 µg/L) and higher in 7 patients with Hodgkin lymphoma (116.7 µg/L) compared to 32 age-matched healthy controls (42.7 µg/L).ConclusionsWe describe a new simple ELISA assay for measuring hepcidin in human serum with sufficient accuracy and reproducibility.

Quantification of Ferritin from Staple Food Crops

Ferritin-iron has been shown to be as bioavailable as ferrous sulfate in humans. Thus, biofortification to breed crops with high ferritin content is a promising strategy to alleviate the global iron deficiency problem. Although ferritin is present in all food crops, its concentration varies between species and varieties. Therefore, a successful ferritin biofortification strategy requires a method to rapidly measure ferritin concentrations in food crops. The objective of this study was to develop a simple and reliable ELISA using an anti-ferritin polyclonal antibody to detect ferritin in various crops. Crude seed extracts were found to have 10.2 +/- 1.0, 4.38 +/- 0.9, 1.2 +/- 0.3, 0.38 +/- 0.1, and 0.04 +/- 0.01 microg of ferritin/g of dry seed in red beans, white beans, wheat, maize, and brown rice, respectively. Although the measured absolute concentrations of ferritin values were low, the presented method is applicable for rapid screening for the relative ferritin concentrations of large numbers of seeds to identify and breed ferritin-rich crops.

A recombinant vesicular stomatitis virus replicon vaccine protects chickens from highly pathogenic avian influenza virus (H7N1)

Highly pathogenic avian influenza viruses (HPAIV) of subtypes H5 and H7 cause fatal disease in poultry (fowl plague) but also have zoonotic potential. Currently commercially available vaccines often do not provide sufficient protection and do not allow easy discrimination between vaccinated and infected birds. Therefore, vaccination of domestic poultry against H5 and H7 HPAIV is not allowed in many countries, or is only possible after special permission has been provided. We generated a recombinant marker vaccine based on non-transmissible vesicular stomatitis virus (VSV) expressing the HA antigen of HPAIV A/FPV/Rostock/34 (H7N1) in place of the VSV G gene. This virus, VSV*ΔG(HA), was propagated on a helper cell line providing VSV G in trans. Since no progeny virus was produced after infection of non-complementing cells, the vector was classified as biosafety level 1 organism (“safe”). Chickens were immunized via the intramuscular route. Following booster vaccination with the same replicons high titers of serum antibodies were induced, which neutralized avian influenza viruses of subtypes H7N1 and H7N7 but not H5N2. Vaccinated chickens were protected against a lethal dose of heterologous HPAIV A/chicken/Italy/445/99 (H7N1). Secretion of challenge virus was short-term and significantly reduced. Finally, it was possible to discriminate vaccinated chickens from infected ones by a simple ELISA assay. We propose that VSV replicons have the potential to be developed to high-quality vaccines for protection of poultry against different subtypes of avian influenza viruses.

Serum Antigen and Antibody Detection in Echinococcosis: Application in Serodiagnosis of Human Hydatidosis

Diagnosis of hydatidosis is based on immunodiagnostic methods along with radiological and ultrasound examinations. The objectives of the present study were to develop a specific and simple antigen-based ELISA method for diagnosis of hydatidosis and compare it with antibody detection method. The subjects in this study included 89 patients in the following groups: surgically confirmed hydatidosis patients (35 cases), control with other parasitic diseases (29 cases), and healthy controls (25 cases). Hyperimmune serum was raised against hydatid cyst fluid in rabbits. Anti-hydatid cyst IgG was purified by affinity chromatography using protein A column and labeled with horseradish peroxidase. Collected sera were assessed for hydatid cyst antigens and antibody by ELISA. Circulating hydatid antigen was found in 9 out of 35 patients with surgically confirmed hydatidosis. A sensitivity of 25.7% and a specificity of 98.0% were calculated for the antigen detection assay. Antibody detection by indirect ELISA, using antigen B, showed that 94.2% of patients (33 cases) have anti-hydatid cyst antibodies in their serum while cross reaction was noted in a few of non-hydatidosis patients. A sensitivity of 94.2% and specificity of 81.6% were found for the antibody detection assay. Findings of this study indicated that antibody detection assay is a sensitive approach for diagnosis of hydatid cyst while antigen detection assay might be a useful approach for assessment of the efficacy of treatment especially after removal of the cyst.

Stable lyophilized reagents for the serum ferritin assay

Two monoclonal antibodies to human ferritin, including one that was coupled to horseradish peroxidase, were lyophilized. These reagents show little loss of activity on reconstitution and demonstrate acceptable stability in the accelerated degradation test. When applied in a simple ELISA for the assay of serum ferritin along with the WHO standard for serum ferritin (80/602) they provide a robust assay with standardized reagents which is potentially suitable for use as a reference assay.

Generation and Characterization of Monoclonal Antibodies AgainstKlebsiella pneumoniae

The present work was undertaken with the aim to generate highly specific monoclonal antibodies (MAb) targeted against Klebsiella pneumoniae that could be incorporated into a highly sensitive and simple ELISA (plate and dot) based system for the rapid identification of K. pneumoniae strains. Four murine monoclonal antibodies (KP-1, KP-2, KP-3, and KP-4) reactive to K. pneumoniae were generated and stabilized. Monoclonal antibodies KP-1 and KP-2 were of IgM type and KP-3 and KP-4 were of IgG type. All of the MAbs were found to be specific to K. pneumoniae strain, as they did not show any cross reactivity when tested with 22 different species and stains of members of the Enterobacteriaceae group. In Western blotting with whole cell lysate antigens of K. pneumoniae, KP-3 and KP-4 were found to react with 70 to 110 kDa regions. The nature of the epitope recognized by all four monoclonal antibodies was carbohydrate as determined by periodate and trypsin treatment of antigens. The monoclonal antibodies were further evaluated on 20 biochemical identified K. pneumoniae strains. The KP-3 and KP-4 monoclonal antibodies produced strong reactions with 19 and 18 strains, respectively, by dot-ELISA. KP-1 and KP-2 (IgM) did not show reaction in dot-ELISA but by agglutination test these could detect 11 and 13 strains, respectively.

An enzyme immunoassay for determining 17α-hydroxyprogesterone in dried blood spots on filter paper using an ultramicroanalytical system

Background 17α-hydroxyprogesterone has been used for the diagnosis of congenital adrenal hyperplasia (CAH) in the newborn period. Methods A simple and rapid competitive ultramicro ELISA assay based on competition between 17-OHP-alkaline phosphatase conjugate and 17-OHP in blood specimens for a limited number of binding sites on specific polyclonal rabbit anti-17-OHP antibodies, has been developed for the measurement of 17-OHP in dried blood spots on filter paper. The assay buffer contains danazol to displace 17-OHP from steroid-binding proteins. Results The 17-OHP assay was completed in 3 h, with a measuring range of 10–250 nmol/l. The intra- and inter-assay CV were 5.5–8.2% and 6.4–9.1%, respectively, depending on the 17-OHP concentrations. The recovery ranged from 98–103%. Of 3750 newborn samples collected on filter paper, 903 from the national neonatal screening program were analyzed, and the mean 17-OHP concentration was 12.2 nmol/l. Our assay showed high Pearson and concordance correlations with the commercially available ICN Neoscreen ELISA 17α-hydroxyprogesterone kit. Conclusions The analytical performance characteristics of our 17-OHP Neonatal UMELISA suggest that it can be used for the neonatal screening of CAH.

Retrospective Analysis of Monkeypox Infection

Serologic cross-reactivity between orthopoxviruses is a substantial barrier to laboratory diagnosis of specific orthopoxvirus infections and epidemiologic characterization of disease outbreaks. Historically, time-consuming and labor-intensive strategies such as cross-adsorbed neutralization assays, immunofluorescence assays, and hemagglutination-inhibition assays have been used to identify orthopoxvirus infections. We used cross-adsorption to develop a simple and quantitative postadsorption ELISA for distinguishing between monkeypox and vaccinia infections. Despite the difficulty of diagnosing clinically inapparent monkeypox in previously vaccinated persons, this technique exhibited 100% sensitivity and 100% specificity for identifying clinically overt monkeypox infection irrespective of vaccination history. We also describe a Western blot technique in which up to 3 diagnostic bands may be used to distinguish between vaccinia and monkeypox infection. The techniques described provide independent diagnostic tests suitable for retrospective analysis of monkeypox outbreaks.

Prediction of clonal specificity frequencies for hybridomas using affinity partitioning analysis of test sera

The development of monoclonal antibodies (Mabs) that can differentiate between closely related organic compounds can be exceptionally difficult. Recent studies attempting to produce high affinity antibodies that can differentiate between DNT and TNT have met with success by first screening antisera from candidate mice using an affinity partitioning technique. Although simple ELISA procedures may yield equivalent titers to such closely related molecules, affinity partitioning of the same parent antisera have demonstrated up to a 150‐fold higher affinity for DNT over TNT within a subset of the entire antibody pool. Splenic fusion correspondingly demonstrated that >30% of the hybridomas were specific for DNT. Thus, this procedure enables the investigator to predict the frequency and specificity of future hybridomas, even when no differences can be distinguished simply by ELISA titers. This research was supported by the Office of Naval Research and the Virginia Institute of Marine Science.

PART 3: ELEVATED LEVELS OF THE SALIVARY PROTEIN IMMUNOGLOBULIN E, NERVE GROWTH FACTOR, MYOGLOBIN, AND INSULIN IN PATIENTS WITH TYPE 2 DIABETES OR WITH AUTOIMMUNITY CAN BE PREVENTED BY LT-10-TREATMENT

It is known that serum from allergy and asthma patients contains elevated levels of immunoglobulin E. This research reports use of a simple ELISA test for measuring levels of endogenous proteins, including immunoglobulin E, nerve growth factor, myoglobin, and insulin in non-invasively collected saliva. The elevated immunoglobulin E is believed to play a role in mediating symptoms of allergy/asthma, type 2 diabetes, and autoimmune diseases. High levels of immunoglobulin E were associated with increased glucose concentration and disruption of the regulation of levels of other endogenous proteins. Saliva from normal people and patients suffering from type 2 diabetes and various other disorders, such as autoimmune diseases, were assayed for immunoglobulin E, nerve growth factor, myoglobin, and insulin using a simple ELISA test employing the respective anti-sera. It was observed that saliva specimens from individuals with type 2 diabetes and various autoimmune diseases contained elevated levels of immunoglobulin E, nerve growth factor, myoglobin, and insulin in comparison with saliva samples from normal control individuals.

Analytical Validation and Biological Evaluation of a High–Molecular-Weight Adiponectin ELISA

Of the 3 circulating multimeric forms of adiponectin, the high-molecular-weight (HMW) form, as measured by size-exclusion and/or immunoblotting techniques, is a better index of insulin sensitivity for monitoring health and disease than is total adiponectin. We aimed to develop a simple ELISA to measure HMW adiponectin. We pretreated serum or plasma samples with digestion solution containing proteinase K (Millipore, ESDS). HMW (Millipore, EZHMWA-64K) and total adiponectin (Millipore, EZHADP-61K) concentrations were measured in treated and untreated samples, respectively, from 108 individuals and from 20 morbidly obese patients before and at 1, 3, 6, and 12 months after gastric-bypass surgery. The ELISA has a dynamic range of 3-200 microg/L and a detection limit of 0.8 microg/L. Intraassay and interassay CVs were <4% and <10%, respectively. Sample-dilution curves paralleled the calibration curves. Fast protein liquid chromatography profiles of the proteinase K-treated samples revealed predominantly HMW adiponectin. Values for HMW adiponectin produced with this method are comparable with those obtained with Western blot analysis (y = 0.77x - 0.15; r = 0.96; n = 56). Body mass index (BMI)- and sex-related changes were more pronounced for HMW adiponectin and percentage of HMW adiponectin than for total adiponectin. HMW and total adiponectin increased after bypass surgery, but changes in HMW adiponectin were more pronounced and preceded changes in total adiponectin. This simple, rapid ELISA for HMW adiponectin recognizes the HMW isoform, produces results closely correlated with those obtained with Western blotting, and appears to better distinguish BMI-, sex-, and weight loss-associated differences than assays for total adiponectin.

Simultaneous direct detection of toxigenic and non-toxigenic Vibrio cholerae from rectal swabs and environmental samples by sandwich ELISA

A mAb-based simple, specific and rapid two-tip dipstick ELISA was developed for simultaneous detection of toxin- and non-toxin-producing strains of Vibrio cholerae, and for direct detection of V. cholerae from rectal swabs of patients and from environmental water samples. Rabbit polyclonal antibodies and murine mAbs were raised against recombinant protein (r-protein) antigens of cholera toxin B (CtxB) and outer membrane protein W (OmpW). Rabbit polyclonal antibodies to both r-proteins were coated individually onto the tips of nitrocellulose (NC) membranes of a two-tipped NC dipstick as capture antibodies and a mixture of two mAbs was used for the detecting antibodies. The test was found to be specific for V. cholerae strains O1, O139, non-O1 and non-O139, and did not show any cross-reaction to closely related bacterial strains. The test was evaluated on rectal swabs collected at the bedside of 75 hospitalized diarrhoeal patients and on 50 environmental water samples after enrichment for 4 h in alkaline peptone water. The mAb two-tip dipstick ELISA detected V. cholerae in 52/75 rectal swabs and 2/50 environmental water samples for CtxB antigen, and in 1/50 environmental water samples for the non-toxin OmpW antigen of V. cholerae within 1.5 h. These findings were identical to those observed using PCR and conventional culture methods. Thus, this mAb-based two-tip dipstick ELISA could be used for early and reliable simultaneous detection of toxigenic and non-toxigenic strains of V. cholerae from clinical and environmental water samples.

Development of a Novel Immunoassay for the Assessment of Plasma Gas6 Concentrations and Their Variation with Hormonal Status

Gas6 is a vitamin K-dependent antiapoptotic protein that has been implicated in cardiovascular pathophysiology. We report the development and validation of an ELISA for Gas6, and the variation of plasma Gas6 with hormonal status in a study designed to evaluate the effect of oral contraception on plasma markers. After validation of the main stages of the ELISA assay, we measured plasma Gas6 concentrations in 94 male and 88 female healthy volunteers ages 18 to 38 years. Forty-five of the women then received an oral contraceptive, which contained ethinylestradiol and levonorgestrel, for 3 months before a new measurement was performed at the same time point in their menstrual cycles. Interassay imprecision was 5.8%-11.8%, and the detection limit was 5.9 microg/L. Mean Gas6 plasma concentrations were significantly lower in men (52.0 microg/L) than in women not receiving oral contraceptives (63.8 microg/L, P <0.001). In the women who received oral contraceptives, Gas6 concentrations decreased after 3 months of therapy from 63.6 microg/L to 51.9 microg/L (P <0.001). We have developed a simple and reproducible ELISA assay for measuring plasma Gas6 concentrations, which vary with sex and are decreased by oral contraceptive use. These results suggest regulation of plasma Gas6 concentrations by sex hormones. Future clinical studies may require participants to be stratified by sex.

IgA anti-actin antibodies ELISA in coeliac disease: A multicentre study

Background Previous studies have demonstrated that serum anti-actin antibodies are a reliable marker of intestinal damage severity in coeliac disease. Aims To validate in a multicentre study the clinical usefulness of serum IgA anti-actin antibody ELISA and its possible use in monitoring intestinal mucosa lesions during gluten-free diet. Patients and methods Four centres recruited 205 newly diagnosed coeliac disease patients with villous atrophy, 80 healthy controls and 81 “disease” controls. Twelve coeliac disease patients on gluten-free diet but with persistent symptoms underwent serum IgA anti-actin antibody assay and intestinal histology evaluation. IgA anti-actin antibody ELISA was performed with a commercial kit. All coeliac disease patients underwent intestinal histology study. Results IgA anti-actin antibodies showed a sensitivity of 80% and a specificity of 85% in the diagnosis of coeliac disease patients with villous atrophy. The area under the receiving operator curve for anti-actin antibodies was 0.873 [95% C.I. 0.805–0.899]. Serum anti-actin antibodies values were significantly higher in coeliac disease patients than in healthy or “disease” controls ( P < 0.0001). Serum anti-actin antibodies were positive in 41 of the 60 coeliac disease patients with mild intestinal histology lesions (69%) and in 123 of the 145 with severe lesions (85.3%) ( P < 0.05). There was a significant inverse correlation between anti-actin antibody values and the villi/crypts ratio ( r = −0.423; P < 0.0001). In the 12 coeliac disease patients on gluten-free diet who underwent re-evaluation as they were persistently symptomatic, intestinal histology showed three cases with persistent villous atrophy: all of these were positive for serum anti-actin antibodies ELISA, whereas both serum anti-tTG and EmAs were negative. The other nine patients showed normal intestinal villi and were negative for serum anti-actin antibodies. Conclusions Anti-actin antibodies are a reliable marker of severe intestinal mucosa damage in coeliac disease patients and a simple ELISA technique offers an accurate method for their determination. These antibodies seem to be a very reliable marker of persistent intestinal damage in coeliac disease patients.

Clinical validation of combined serological biomarkers for improved hepatocellular carcinoma diagnosis in 961 patients

BackgroundAlpha-fetoprotein (AFP), the only serological marker currently available for the detection of hepatocellular carcinoma (HCC), is unsatisfactory because of its poor sensitivity, as are other recently proposed markers. Therefore new biomarkers are badly needed. Squamous cell carcinoma antigen (SCCA), a serine protease inhibitor physiologically present in the skin, has recently been reported to be present in HCC patients, as also the immunocomplexed (IC) forms of SCCA and AFP: SCCAIC and AFPIC, respectively. MethodsTo determine the diagnostic accuracy of new serum biomarkers for the diagnosis of HCC a rapid, simple ELISA test was applied in 961 patients. Sensitivity and specificity were determined for each marker and for all the markers combined in detecting smaller and larger HCC versus liver cirrhosis. ResultsIn smaller HCC, receiver operating characteristics analysis yielded the following AUC: AFP 0.714 (CI 95% 0.679–0.748), AFPIC 0.691 (CI95% 0.655–0.748), SCCA 0.703 (CI95% 0.667–0.736), SCCAIC 0.694 (CI 95% 0.659–0.728). SCCA was inversely correlated with size. The combined use of AFPIC, SCCA and SCCAIC in patients displaying low levels of AFP (<20 IU/mL) identified 25.6% HCC (186/725). ConclusionThis study suggests that the use of a combination of all these markers in clinical practice provides a non invasive and simple test that could increase the accuracy of HCC diagnosis.

Papel de los marcadores biológicos en la enfermedad inflamatoria intestinal

The role played by the distinct biological markers in chronic inflammatory bowel disease (IBD) remains insufficiently characterized. C-reactive protein (CRP) has a short half-life and consequently it is elevated early after the onset of the inflammatory process and rapidly decreases after its resolution, making it an attractive marker of disease activity. Moreover, this test is inexpensive and easy to perform and is unaffected by medication. While Crohn's disease is associated with a marked CRP response, there is little or no elevation in the synthesis of this protein in ulcerative colitis. Erythrocyte sedimentation rate provides some advantages such as its ease of determination, availability, and reduced cost. Nevertheless, it also has several disadvantages, notably the fact that its concentration depends on age, the presence of anemia, smoking, and the use of certain drugs. Moreover, its utility is limited by its long half life and consequent prolonged latency period after changes in chronic IBD activity. In theory, fecal markers have the advantages of showing greater specificity in the diagnosis of chronic IBD. Several gastrointestinal diseases, including chronic IBD, show greater leukocyte elimination in feces and a close correlation has been described between fecal calprotectin concentration and leukocyte excretion quantified by 111indium. Advantages of this fecal marker are that it can be detected through a simple and inexpensive technique and also shows excellent stability in feces for prolonged periods. Like calprotectin, fecal lactoferrin is also quantified by a simple and inexpensive ELISA method, although there is considerably less experience with this latter marker.

Development of an ELISA-based kit for the on-farm determination of progesterone in milk

Effective reproductive management is a crucial concern in the dairy industry and missed oestrus and late recognition of unsuccessful insemination can lead to substantial financial loss. Progesterone levels change in a predictable manner with progress of the oestrus cycle and with the onset of pregnancy. We report here the development of a simple ELISA test for the measurement of progesterone levels in bovine milk. The method is based on the use of polyclonal antibodies raised against 11&amp;alpha;-hydroxyprogesterone-hemisuccinate and competition for antibody binding sites between free progesterone and horseradish peroxidase-labelled progesterone. Binding is quantified by means of the colour reaction between horseradish peroxidase and 3,3&amp;prime;,5,5&amp;prime;-tetramethylbenzidine. The method has proved to be a cost effective, sensitive and easy to perform alternative to currently available methods of oestrus and pregnancy detection. The practical feasibility of the method has been proven under dairy farm conditions.

Development and evaluation of an ELISA for quantification of human alpha-1-proteinase inhibitor in complex biological mixtures

Human alpha-1-proteinase inhibitor 1 Also known as antitrypsin. 1 (α 1-PI) is the most abundant serine protease inhibitor in plasma. Its major function is inhibition of neutrophil elastase in lungs. α 1-PI deficiency may result in severe, ultimately fatal emphysema. Three plasma-derived (pd-) α 1-PI products are licensed in the US for replacement therapy of deficient patients. The recombinant versions (r-α 1-PI), proposed as alternatives to pd-α 1-PI products, have been under intensive investigation. For accurate determination of α 1-PI from different sources and in various forms, there is an obvious need for reliable standardized assays for α 1-PI quantification and potency measurements. As a part of our multi-step research focused on α 1-PI structure-function investigation, we have established a simple and reproducible double-sandwich ELISA based on commercially available polyclonal antibodies. The developed ELISA allows the quantification of both pd-α 1-PI and r-α 1-PI in various complex matrices. A validation of the ELISA was performed with the working range of the assay (3.1–50 ng/ml) established on the bases of the following parameters: linearity (3–100 ng/ml, r 2 = 0.995); accuracy (87.3–114.6% recovery); intra-assay precision (%CV, 2.8%); inter-assay plate-to-plate precision (3.9% per day and 4.1% day-to-day); detection limit (1.10 ng/ml); and quantification limit (3.34 ng/ml). The analytical performance of the α 1-PI ELISA indicates that this assay can be used for monitoring concentration levels of α 1-PI in multi-component biological matrices, based on the following: (a) quantification of r -α 1-PI in various fermentation mixtures ( E. coli and A. niger); (b) investigation of α 1-PI enzymatically digested in the conditions of harsh fungal proteolysis; (c) evaluation of thermally polymerized α 1-PI; (d) quantification of α 1-PI in human serum; and (e) comparative quantification of α 1-PI in commercially available products.

Detection of mutations in the 7A allele of wheat ( Triticum aestivum) granule-bound starch synthase (Wx-7A) with a monoclonal antibody produced by targeted peptide immunisation

A peptide sequence unique for the granule-bound starch synthase (GBSS1) of hexaploid wheat ( Triticum aestivum) was identified by sequence alignment of the three isoforms. Multiple antigenic peptides (MAP) of different length (16 and 20 AA) were synthesised against the selected sequence. The assessment of the immune response in mice against differently presented forms (resin-bound, free) and two types of adjuvants indicated that the free MAP with the longer peptide is more immunogenic. A 20AA MAP that elicited a strong immune response was used to produce a monoclonal antibody for the 7A isoform of GBSS 1. Epitope mapping of the selected Mab (F5-1F2) revealed that a 12mer partial sequence of the immunising peptide was specifically detected. Based on this Mab, a simple high throughput ELISA was developed that allows the quick identification of wheat lines carrying the 7A allele of GBSS 1 with minute amounts of sample.