Sensitive Sandwich ELISA Research Articles

A sensitive sandwich ELISA to measure (1→3)-β-d-glucan levels in blood

A highly sensitive (1→3)-β-d-glucan (β-glucan)-specific sandwich ELISA was developed using a fragment of recombinant horseshoe crab factor G protein. The factor G fragment, which was expressed in Escherichia coli, contains a QQWS motif, two β-glucan-binding domains, and an additional N-terminal cysteine residue. The sensitivity of our ELISA was comparable to a conventional (1→3)-β-d-glucan detection method using a horseshoe crab-clotting reaction such as an amebocyte lysate-based assay. In addition, the β-glucan levels measured by our sandwich ELISA in plasma samples showed a good correlation with those measured by the amebocyte lysate-based assay. In the case of our sandwich ELISA, it is not necessary to pre-inactivate interfering substances in plasma samples that is essential for the conventional amebocyte lysate-based assay. Moreover, the assay time of the ELISA method is much shorter than that of the amebocyte lysate-based assay. Because of these advantages, the ELISA system will be more suitable for high-throughput analysis in clinical laboratories using general clinical auto-analyzers.β-glucan is a typical biomarker for fungal infections and the measurements of β-glucan levels by our ELISA could be useful for the diagnosis of fungal infections.

Is it Possible to Accurately Determine Content of Botulinum Neurotoxin Type A in Drug Products?

Botulinum neurotoxin type A (BoNT/A) is the active substance in preparations used both for cosmetic purposes and to treat a variety of neurological disorders. In some preparations, the BoNT/A protein form part of a multi-protein complex. Although the accompanying proteins are considered to play no role in the mechanism of action, their presence has raised concerns regarding the amount of total protein administered, sensitization to the preparation, and the formation of neurotoxin neutralizing antibodies leading to therapeutic failure. Preparations that contain only the BoNT/A protein could potentially limit the development of such an immune response. Hence, it is important to know how much BoNT/A protein is present. The article by Dr Jurgen Frevert published in this issue of Drugs in R&D[1] describes the evaluation of the BoNT/A concentration in three formulations of licensed therapeutic preparations using a sensitive sandwich ELISA assay developed in-house. Only Xeomin®/Bocouture® contains pure BoNT/A protein and is claimed to have the highest purity and specific activity of the formulations tested. The study is relevant for the characterization of therapeutic preparations of botulinum toxin but is potentially controversial. It is therefore essential that the information presented is correct and is adequately scrutinized. The conclusions of the paper appear to be based on rather limited and variable data, particularly for products other than Xeomin®, and should ideally be supported by more focused experimental studies. The claim that the new immunoassay is able to detect only neurotoxin is potentially speculative. It is claimed that antibodies have no ability to detect additional complexing proteins/other clostridial proteins that are known to be present in products other than Xeomin® but no direct evidence of this specificity is directly presented in this paper. The main argument is that not all toxin A products are of the same purity and that a particular brand may be of lowest specific activity, despite the authors own claim of no difference in clinical use and in laboratory mouse bioassays studies. This assertion is not entirely novel as a previous paper by Ekong et al.[2] reached the same conclusion for the licensed therapeutic toxin A products at that time. The conclusion then was based on the observation of differences in the ratio of immunodetected toxin to their biological activity. The capture antibody ELISA methodology described in the 1995 paper is virtually identical to the method published here. Although particular product brands were not specifically identified in the previous publication, those familiar with these products would have been able to calculate that for Dysport®, the toxin A content was approximately 2.0 ng/500 dose that is lethal to 50% of animals tested (LD50) [0.4 ng/100 LD50], which is lower than the content claimed for this brand and slightly lower than, although comparable to, the value reported by Dr Frevert.[1] It is highly likely that further values will be calculated in other laboratories as the results are both method and reagent specific. It may not be possible to be certain that a value determined by Dr Frevert is ‘true’ until it is verified in another laboratory. Furthermore, unlike the previously published study, the biologic activity was not determined by direct comparison but used information provided by the manufacturer. It is not possible to calculate the specific purity of toxin A in proprietary products from LD50 values provided by the individual manufacturers, unless these values are normalized or standardized. Dr Frevert’s conclusions are consistent with other publications on this subject[3–5] that agree the LD50 values indicated on product labels cannot be standardized due to in-house differences in LD50 protocols, which result in differences in the potency and hence bias the calculation of specific activity. Despite these limitations the sandwich ELISA assay is arguably the best available for the detection of toxin but the suitability of this particular version should be confirmed by other laboratories. In addition, estimation of toxin content in other preparations, including pure neurotoxin formulations, such as in samples reported by Sesardic et al.,[5] would be useful, as loss of activity during the production process has been confirmed in bioassays.

Setting of methods for analysis of mucosal antibodies in seminal and vaginal fluids of HIV seropositive subjects from Cambodian and Italian cohorts.

BackgroundGenital mucosae play a key role in protection from STD and HIV infection, due to their involvement in both horizontal and vertical disease transmission. High variability of published observations concerning IgA isolation and quantification underlies the strong requirement of specific methods able to maximize investigation on HIV-specific IgA.MethodologyGenital fluids from 109 subjects, including male and female cohorts from Italy and Cambodia, were collected, aliquoted and processed with different techniques, to assess optimal conditions maximizing mucosal antibody recovery. Three sampling techniques, up to sixteen preservation conditions, six ELISA methods and four purifications protocols were compared.Principal FindingsThe optimal method here described took advantage of Weck-Cel sampling of female mucosal fluids. Immediate processing of genital fluids, with the addition of antibiotics and EDTA, improved recovery of vaginal IgA, while the triple addition of EDTA, antibiotics and protease inhibitors provided the highest amount of seminal IgA. Due to low amount of IgA in mucosal fluids, a high sensitive sandwich ELISA assay was set; sensitivity was enhanced by milk-based overcoating buffer and by a two-step biotin-streptavidin signal amplification. Indeed, commercial antisera to detect human immunoglobulins showed weak cross-reactivity to different antibody types. Three-step affinity purification provided reproducible immunoglobulin recovery from genital specimens, while conventional immuno-affinity IgA purification was found poorly manageable. Affinity columns were suitable to isolate mucosal IgA, which are ten-fold less concentrated than IgG in genital specimens, and provided effective separation of IgA monomers, dimers, and J-chains. Jacalin-bound resin successfully separated IgA1 from IgA2 subfraction.Conclusions/SignificanceSpecific, effective and reliable methods to study local immunity are key items in understanding host mucosal response. The sequence of methods here described is effective and reliable in analysing humoral local responses, and may provide a solid advance to identify and measure the effective mucosal responses to HIV.

Production, characterization, and applications of mouse monoclonal antibodies against gonadotropin, somatolactin, and prolactin from common carp ( Cyprinus carpio)

The gonadotropin α subunit (cGTHα), gonadotropinIIβ subunit (cGTHIIβ), somatolactin (cSL), and prolactin (cPRL) were isolated from the pituitaries of common carps, purified by traditional chromatographic analysis, identified by mass-chromatographic analysis, and used as immunogens in the B-lymphocyte hybridoma technique. Totally, 7, 11, 17, and 8 hybridoma cell lines were established, which were able to stably secrete monoclonal antibodies (mAbs) against cGTHα, cGTHIIβ, cSL, and cPRL, and designated as FMU-cGTHα1–7, FMU-cGTHIIβ1–11, FMU-cSL 1–17, and FMU-cPRL 1–8, respectively. The isotype, titer, and specificity were identified by enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemical staining, respectively, and application of these mAbs in the aforementioned tests has been proved. Furthermore, sensitive sandwich-ELISA systems for quantitative detection of the hormones mentioned above were also developed.

Development of monoclonal antibodies and quantitative ELISAs targeting insulin-degrading enzyme

BackgroundInsulin-degrading enzyme (IDE) is a widely studied zinc-metalloprotease implicated in the pathogenesis of type 2 diabetes mellitus, Alzheimer disease (AD) and varicella zoster virus infection. Despite more than six decades of research on IDE, progress has been hampered by the lack of well-characterized reagents targeting this biomedically important protease. To address this important need, we generated and characterized new mouse monoclonal antibodies (mAbs) targeting natively folded human and rodent IDE.ResultsEight monoclonal hybridoma cell lines were derived in house from mice immunized with full-length, natively folded, recombinant human IDE. The mAbs derived from these lines were shown to detect IDE selectively and sensitively by a wide range of methods. Two mAbs in particular—designated 6A1 and 6H9—proved especially selective for IDE in immunocytochemical and immunohistochemical applications. Using a variety of methods, we show that 6A1 selectively detects both human and rodent IDE, while 6H9 selectively detects human, but not rodent, IDE, with both mAbs showing essentially no cross reactivity with other proteins in these applications. Using these novel anti-IDE mAbs, we also developed sensitive and quantitative sandwich ELISAs capable of quantifying IDE levels present in human brain extracts.ConclusionWe succeeded in developing novel mAbs that selectively detect rodent and/or human IDE, which we have shown to be suitable for a wide range of applications, including western blotting, immunoprecipitation, immunocytochemistry, immunohistochemistry, and quantitative sandwich ELISAs. These novel anti-IDE mAbs and the assays derived from them constitute important new tools for addressing many unresolved questions about the basic biology of IDE and its role in multiple highly prevalent human diseases.

Development of Animal Models and Sandwich-ELISA Tests to Detect the Allergenicity and Antigenicity of Fining Agent Residues in Wines

Food allergy can cause food-related anaphylaxis. Food allergen labeling is the principal means of protecting sensitized individuals. This motivated European Directive 2003/89 on the labeling of ingredients or additives that could trigger adverse reactions, which has been in effect since 2005. During this study, we developed animal models with allergy to ovalbumin, caseinate, and isinglass in order to be able to detect fining agent residues that could induce anaphylactic reactions in sensitized mice. The second aim of the study was to design sandwich ELISA tests specific to each fining agent in order to detect their residue antigenicity, both during wine processing and in commercially available bottled wines. Sensitized mice and sandwich ELISA methods were established to test a vast panel of wines. The results showed that although they were positive to our highly sensitive sandwich-ELISA tests, some commercially available wines are not allergenic in sensitized mice. Commercially available bottled wines made using standardized processes, fining, maturation, and filtration, do not therefore represent any risk of anaphylactic reactions in sensitized mice.

Extracts of wheat gluten activate complement via the alternative pathway

We studied the ability of wheat gluten and its subfractions to activate complement directly. A sensitive sandwich ELISA employing a monoclonal antibody (MoAb) to a C9 neoepitope exposed in the terminal complement complex (TCC), a functional haemolytic assay for C5b6 generation, and Laurell's electrophoretic method of estimating C3 conversion to C3bi were used. On a weight-for-weight basis, enzyme solubilized Frazer's fraction three of gluten (FIII) produced approximately 75% of the complement activation seen with the potent activator zymosan. By contrast, activation with whole insoluble undigested gluten was very weak and similar to that seen with ovalbumin or beta-lactoglobulin. The results were the same using normal human serum or sera from patients with coeliac disease, dermatitis herpetiformis, or hypogammaglobulinaemia as the complement source. Activation by both zymosan and FIII was blocked in 0.01 M EDTA, but not in 0.01 M EGTA with 0.0025 M magnesium chloride. Zymosan and FIII activated complement in a serum from a patient with an intact alternative pathway but classical pathway haemolytic activity (CH50) of zero. Preferential heat inactivation of the alternative pathway inhibited both zymosan- and FIII-induced activation. Our results confirm that FIII is a strong activator of the alternative pathway. We discuss how gluten enteropathy might be initiated by complement.

Measurement of IL-4 in tears of patients with seasonal allergic conjunctivitis and vernal keratoconjunctivitis.

To elucidate the mechanism of ocular surface allergic disease, we focused on IL-4, which is one of the key factors in regulating IgE production, and thus determined the concentration of IL-4 in tears. IL-4 concentration was determined in the tears of 15 patients with seasonal allergic conjunctivitis, 15 vernal keratoconjunctivitis (VKC), 10 giant papillary conjunctivitis (GPC), 10 patients with non-allergic conjunctivitis and post-cataract surgical conjunctivitis as intermediate conjunctivitis, and 10 normal subjects using a highly sensitive sandwich ELISA. The mean level of IL-4 in normal controls was low, and seasonal allergic conjunctivitis, VKC and GPC showed a significant elevation (P < 0.05), respectively. IL-4 of VKC and GPC were also significantly higher than allergic conjunctivitis, and non-allergic conjunctivitis and post-cataract surgical conjunctivitis were not higher than normal. These results raise the possibility that the increased level of IL-4 in tears could play a role in allergic disease and its severity in patients.

Functionally active complement proteins C6 and C7 detected in C6- and C7-deficient individuals

Two sensitive sandwich ELISAs based on monoclonal antibodies directed to native C6 and C7 allowed the detection and quantitation of these complement proteins in 20 out of 37 serum samples from individuals who had previously been classified as deficient in these proteins as assessed by immunochemical and/or functional assays. Furthermore, serum from four C6-deficient and one combined C6-/C7-deficient individual showed an increase in the terminal complement complex (TCC) and a decrease in native C6 and C7 after complement activation as assayed by specific ELISAs. Despite their (incomplete) deficiencies, these individuals therefore possess functionally active terminal complement proteins with respect to their ability to generate the TCC. As these individuals have no history of a susceptibility to neisserial infections, even low concentrations of functionally active C6 and C7 may provide sufficient protection against those micro-organisms whose destruction requires TCC formation.

A Sandwich ELISA for Phosphoglycerate Kinase

Phosphoglycerate kinase (PGK1) is a key enzyme in glycolysis that can also be released from certain cells. In the extracellular milieu, PGK1 reportedly acts as a disulphide reductase to activate plasmin, resulting in the production of angiostatin, a potent angiogenesis inhibitor. Certain cancer cell lines secrete unusually large amounts of PGK1, raising the possibility that serum PGK1 levels can be used to screen for cancer. To facilitate the characterization of the PGK1 secretory pathway and to monitor serum levels of PGK1, we have developed a sensitive sandwich ELISA using an immuno-affinity-purified chicken polyclonal antibody for capturing PGK1 and an immuno-affinity-purified rabbit polyclonal antibody for detecting it. The assay is about 10-fold more sensitive than other reported PGK1 ELISAs. We used the ELISA to quantify the amount of PGK1 released from HeLa cells and PGK1 serum levels in cancer patients. Of 10 cancer patients whose serum was tested, 3 of 4 with pancreatic cancer had 65–900% higher levels of PGK1 than that found in normal serum.

Development and partial characterization of high-affinity monoclonal antibodies for botulinum toxin type A and their use in analysis of milk by sandwich ELISA

Botulinum neurotoxins (BoNT), produced by the anaerobic bacterium Clostridium botulinum, cause severe neuroparalytic disease and are considered the most toxic biological agents known. While botulism is rare in the U.S. it often is fatal if not treated quickly, and recovery is long, requiring intensive treatment. BoNT is synthesized as a 150 kDa precursor protein (holotoxin), which is then enzymatically cleaved to form two subunit chains linked by a single disulfide bond. The ‘gold standard’ for BoNT detection relies on a mouse bioassay. This is a time consuming (up to 4 days) assay and it lacks specificity, however, it gives a sensitivity (mouse LD 50) of approximately 10 pg mL − 1 . Most BoNT immunoassays are much less sensitive. In this study we describe the development of four high-affinity (dissociation constants (Kd's) in the low pM range) monoclonal antibodies (mAbs) that specifically bind BoNT serotype A (BoNT/A). These antibodies, designated F1-2, F1-5, F1-40, and F2-43 are IgG1 subclass mAbs with kappa light chains and they specifically bind BoNT serotype A. Western blot analyses following SDS-PAGE demonstrate that mAbs F1-2 and F1-5 bind the 100 kDa heavy chain subunit and that mAb F1-40 binds the 50 kDa light chain. The fourth antibody demonstrated strong binding to the 150 kDa holotoxin in the ELISA and on Western blots following electrophoresis on native gels. However binding in Western blot studies was not observed for mAb F2-43 following SDS-PAGE. A highly sensitive sandwich ELISA, capable of detecting as little as 2 pg/mL BoNT/A was developed using mAbs F1-2 and F1-40. Such an assay represents a realistic, high sensitivity alternative to the mouse bioassay.

Rational screening of antibodies and design of sandwich enzyme linked immunosorbant assay on the basis of a kinetic model

A rational strategy for the rapid establishment of a sensitive sandwich enzyme linked immunosorbant assay was developed. The kinetic properties required for the solid-phase and enzyme-conjugated antibodies of sandwich ELISA were determined rationally on the basis of a kinetic model describing antibody-antigen interaction. Some antibodies possessing the required kinetic properties against a model antigen, C-reactive protein (CRP), were successfully isolated from a phage antibody library under the screening conditions that were designed on the basis of simulation results. The best combination of solid-phase and enzyme-conjugated antibodies that gives the most sensitive sandwich ELISA was determined by simulation on the basis of the apparent association and dissociation rate constants of the isolated antibodies. It was confirmed by experiment that the sandwich ELISA using the best combination of antibodies was actually the most sensitive one. Our strategy would be useful for the rapid establishment of sensitive sandwich ELISAs compared with the traditional hybridoma method in which the best condition is determined by trial and error.

Tenascin‐W, a new marker of cancer stroma, is elevated in sera of colon and breast cancer patients

Tenascins are extracellular matrix proteins present during the development of organisms as well as in pathological conditions. Tenascin-W, the fourth and last member of the tenascin family remains the least well-characterized one. Our study aimed to evaluate the potential significance of tenascin-W as cancer biomarker by monitoring its presence in the serum of colorectal and breast cancer patients and its expression in colorectal tumor tissues. To measure serum tenascin-W levels, a sensitive sandwich-ELISA was established. Mean tenascin-W concentration in sera of patients with nonmetastatic colorectal cancer at time of diagnosis was highly increased compared to that of healthy volunteers. A similar tendency was observed for tenascin-C in the same patient cohort. However, the increase was much more striking for tenascin-W. We also detected elevated tenascin-W levels in sera of breast cancer patients. Furthermore, we could show a prominent expression of tenascin-W in extracts from colorectal tumor tissues by immunoblot analysis, whereas tenascin-W was not detectable in the corresponding normal colon mucosa. To confirm the western blot results, we performed immunohistochemistry of frozen sections of the same patients as well as of an additional, independently chosen collection of colorectal cancer tissues. In all cases, similarly to tenascin-C, tenascin-W was detected in the tumor stroma. Our results reveal a clear association between elevated levels of tenascin-W and the presence of cancer. These results warrant further studies to evaluate the potential value of serum and tissue tenascin-W levels as diagnostic, prognostic or monitoring biomarker in colorectal, breast and possibly other solid cancers.

Validated sandwich ELISA for the quantification of tissue transglutaminase in tissue homogenates and cell lysates of multiple species

Tissue transglutaminase (tTG) is a calcium dependent enzyme that displays diverse functions in various physiological processes. In addition to these physiological functions, there is strong evidence for the implication of tTG in a number of pathologies, including celiac disease, cancer and neurodegeneration. To explore the expression and function of tTG during (patho)physiological conditions, it is of utmost importance to have an assay that specifically measures tTG protein levels in various species and matrices. Therefore, we have developed a sensitive sandwich ELISA to measure tTG protein levels in tissue homogenates and cell lysates of human, rat and mouse origin. The ELISA uses commercially available antibodies, and human recombinant tTG as the standard protein. The limit of detection is 100 pg/ml; the coefficients of intra- and inter-assay variation range from 2.4% to 6.6% and from 12.7% to 15.1%, respectively. Clear detectable levels of tTG protein were measured in human and rat liver and cerebral cortex, as well as in brain-derived neuronal and glial cells. tTG levels in mouse tissues were much lower than observed in human and rat tissues. No cross-reactivity against keratinocyte TG (TG1), epidermal TG (TG3) or blood coagulation factor XIII was observed. The tTG specific sandwich ELISA presented in this paper is a sensitive and reliable tool to accurately measure tTG protein levels in different matrices (cell/tissue) of rat, mouse and human origin. It provides a better alternative for the widely used transglutaminase activity assay with respect to sensitivity and specificity, and may serve as a valuable tool to investigate protein expression levels as part of the approach to unravel the contribution of tTG to health and disease.

Association of the mutation for the human carboxypeptidase E gene exon 4 with the severity of coronary artery atherosclerosis

To test the hypothesis that the identification of mutation in the carboxypeptidase E (CPE) gene which leads to marked hyperproinsulinaemia is consistent with a possible role for mutations in CPE in the development of coronary atherosclerosis. The study subjects consisted of 1084 consecutive patients (812 males and 272 females) who will undergo coronary angiography. The severity of coronary atherosclerosis was defined by the Gensini's score system. The proinsulin level was measured using highly sensitive two-site sandwich ELISA methods. Screening for mutations of the 4th exon and exon-intron junctional region of the CPE gene was performed by polymerase chain reaction followed by bidirectional sequencing. Sequencing of the CPE gene exon 4 in 1084 consecutive patients revealed 2 genetic variants, the G-to-A substitution at nucleotide 2855 in exon 4 (synonymous mutation) and A-to-G substitution at nucleotide 2925 in intron 4. Although the proinsulin level was not influenced by the presence of the two point mutation, the Gensini score was significantly influenced by the presence of the A2925G mutant (P = 0.023). Furthermore, we found a higher prevalence of subjects with the A2925G heterozygous mutant among higher Gensini score subjects than it among lower Gensini score subjects, and this difference reached statistical significance (P = 0.006, OR 1.465, 95%CI 1.116-1.924). In addition, the frequency distribution of the G2855A mutant was differed in the higher Gensini score subjects than it in the lower Gensini score subjects belonging to high-risk category as smokers, and the statistical significance was reached (P = 0.043, OR 2.075, 95%CI 1.024-4.207). In the present study, the severity of the coronary atherosclerosis estimated by Gensini score was significantly influenced by the presence of the A2925G mutant and G2855A mutant of the CPE gene, and the exactly mechanism underlying the association needs further study.

Molecular scanning of the human carboxypeptidase E gene for mutations in Chinese subjects with coronary atherosclerosis

To test the hypothesis that the identification of mutation in the carboxypeptidase E (CPE) gene which leads to marked hyperproinsulinaemia is consistent with a possible role for mutations in CPE in the development of coronary heart disease. The study subjects consisted of 51 consecutive patients (34 males and 17 females) who will undergo coronary angiography for suspected or known coronary atherosclerosis. Coronary heart disease (CHD) was defined as having a luminal diameter stenosis > or =50% in at least one of three major coronary arteries by coronary angiography or based on the Rose Questionnaire. The insulin and proinsulin level were measured using highly sensitive two-site sandwich ELISA methods. Screening for mutations of the eight exons of the CPE gene was performed by polymerase chain reaction followed by bidirectional sequencing. We scanned eight exons and exon-intron junctional region. Overall, we found 12 distinct variants in the intron region and three variants in the exon region. Among the 15 variants, 10 mutations were rare. The further explored study reveal that the above five non-rare variants would not affect the level of glucose, insulin, and proinsulin. However, the results suggest that the prevalence of the coronary heart disease was significant difference between the wild type group and mutant type group according to the A4545G (P = 0.020). The results from the logistic regression reveal that the subjects with the CPE mutation of A4545G, the odds ratio for the coronary heart disease was 0.196 (95% CI: 0.046 to 0.830, P = 0.027). In the present study, the mutation of CPE gene would not affect the level of glucose, insulin, and proinsulin. The hypothesis of a possible role for mutations in CPE in the development of coronary heart disease needs further study.

Evaluation of the novel serum markers B7-H4, Spondin 2, and DcR3 for diagnosis and early detection of ovarian cancer

Objective Early detection through regular screening could significantly reduce mortality from ovarian cancer. Advances in biomarkers and imaging continue to improve the sensitivity and specificity of cancer detection, but further improvements are still needed. In this study, we identified and evaluated three new serum biomarkers that may be used to improve detection of ovarian cancer. Methods Through genomic analysis, we identified B7-H4, Spondin 2, and DcR3 as over-expressed genes in ovarian cancer tissues. Sensitive sandwich ELISAs were developed to analyze the level of these novel markers in 68 serum samples from patients with ovarian cancer (16 early stage, 52 late stage) and 108 control samples, and 20 healthy women from which two serum samples were collected 1 year apart. CA125 levels were measured in all samples. Results Markers were evaluated for their ability to identify clinical disease. The three novel markers and CA125 were elevated in serum of ovarian cancer patients as compared to normal controls. B7-H4 showed the highest specificity, with the lowest frequency of elevation in all control groups. When all cases were compared against all controls, CA125, Spondin 2, B7-H4, and DcR3 showed areas under the ROC curve of 0.87, 0.78, 0.74, and 0.71, respectively. CA125 and B7-H4 showed the best diagnostic performance for early-stage, with AUCs of 0.90 and 0.80, respectively. Conclusion This study demonstrates that B7-H4, Spondin 2, and DcR3 are promising new ovarian cancer markers that may improve early detection of cancer when used in combination with traditional diagnostic tests.

A Sensitive Sandwich ELISA for Buffalo Prolactin

Two murine monoclonal antibodies have been developed against the buffalo prolactin (buPRL). These were designated as 1501 and 1504. Using two MAbs and anti‐buPRL rabbit serum, an analysis was performed for the development of a sandwich ELISA (sELISA). The 1504/buPRL/anti‐buPRL rabbit serum system was found feasible for sELISA. The sELISA had a sensitivity of 156 pg/mL. The buffalo serum sample showed a parallelism with the standard curve. The intra‐ and inter‐assay coefficients of variance were 8.4% and 9.06%, respectively. These data proved the validity of the assay.

Tau in cerebrospinal fluid: A sensitive sandwich enzyme-linked immunosorbent assay using tyramide signal amplification

Tau in cerebrospinal fluid (CSF) has been proposed as a diagnostic marker for Alzheimer's disease (AD). This paper presents a new sensitive sandwich ELISA allowing quantitation of tau from 8 μl CSF/well. A human specific monoclonal tau antibody HT7 was used as a capture antibody and a mixture of polyclonal tau antibodies, 92e and R134d was used as reporter antibodies. Tyramide signal amplification (TSA) technology was used in the last step to increase the sensitivity. With this TSA–ELISA, the lowest detection limit for tau was 14.3 pg/ml. Tau levels in CSF were found to be increased in AD patients (807 ± 304 pg/ml, p < 0.001) compared with controls (252 ± 94 pg/ml). Thirty-five of 38 AD cases (92% sensitivity) yielded signals greater than cutoff, while only 1 of 38 control cases (97% specificity) was greater. A highly significant correlation was found between this assay and a commonly used kit, INNOTEST hTAU Antigen.

Preparation and Identification of Human Soluble sPD-L1 and Its Antibodies

This study reports the preparation and identification of soluble programmed death-1 (PD-1) ligand-1 (sPD-L1) and its antibodies of mouse origin. Immobilized metal ion affinity chromatography was used to perform on-column refolding with simultaneous purification of denatured sPD-L1, and soluble sPD-L1 with purity of 95 % was obtained. The purified sPD-L1 was verified by immunoblotting using a commercial goat-anti-human PD-L1 antibody. An ELISA-based assay showed that it also had high binding activity for its cognate receptor PD-1. Furthermore, mouse anti-sPD-L1 antiserum of high titer was raised using the purified sPD-L1 as an immunogen, and the specific IgG antibodies were purified using sPD-L1-HiTrap affinity chromatography. In addition, a sensitive sandwich ELISA was established using the purified IgG antibodies together with the commercial goat antibodies. In conclusion, the preparation of soluble sPD-L1 and its antibodies provide the basis for detection of the potential anti-PD-L1 antibodies and soluble PD-L1 in humans as well as for further investigation of its in vivo bioactivities and characterization of its potential receptors.