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Indirect enzyme-linked immunosorbent assay (ELISA) is an important diagnostic method as it enables the quantification of the presence of autoantibodies in human blood sera. However, unspecific binding of antibodies to the solid phase causes considerable serum-specific background noise (SSBN), involving the risk of false positive diagnosis. Therefore, we present a simple and concise, yet obvious proof-of-principle of a recently suggested normalization method. The method is based on subtracting SSBN by using non-coated ELISA wells as a control for each serum-of-interest. We performed ELISA to quantify anti-fibroblast growth factor receptor 3 (FGFR3) antibody levels in three positive controls (two anti-FGFR3-positive patients and a rabbit antiserum against FGFR3) and 58 negative controls (healthy blood donors). In all subjects, we found considerable unspecific reactivity which strongly varied among subjects. The conventional normalization method was not able to balance this strong SSBN, as demonstrated by 2/58 false positive healthy controls and one FGFR3-positive patient that was hidden in the noise (false negative). SSBN normalization reduced the frequency of false-positives to 0/58. Further, all three anti-FGFR3-positive sera were successfully detected and even doubled their z-score used to determine positivity. Albeit occupying more space on the ELISA plate, we strongly recommend considering this normalization method when working with blood sera. To better put the idea across to the community, we depict the SSBN issue and its solution in a graphic scheme. We conclude that SSBN normalization increases the sensitivity and specificity of indirect ELISA and thereby reduces the risk of false positive and false negative diagnosis. © 2019. Licensed under the Creative Commons [CC BY-NC 4.0 licence, https://doi.org/10.1016/j.jim.2019.01.004 ].

Birch (Betula) pollen is a major cause of allergy in northern and central Europe. The allergenic potency of products for diagnosis and therapy of birch pollen allergy is adjusted nearly exclusively to the major birch pollen allergen Bet v 1. Although every fifth patient is additionally sensitized to Bet v 4, both content and variability of this minor allergen in birch allergen products remain unclear due to a lack of simple and cost-effective quantitative methods. This study aimed to develop and in-house validate the first Bet v 4-specific sandwich enzyme-linked immunosorbent assay (ELISA). Based on a murine monoclonal antibody in combination with a polyclonal rabbit antiserum, the ELISA proved to be highly sensitive, with a lower limit of quantification of 30 pg/ml Bet v 4. After confirmation of satisfactory accuracy, reproducibility, and robustness, the ELISA was utilized to quantify Bet v 4 in 30 authorized birch allergen products. The allergen was detected in all samples tested, ranging from 0.2 to 4.4 μg/ml. No significant correlation of Bet v 4 was found with the respective amount of Bet v 1. In contrast to Bet v 1, also no correlation of Bet v 4 with total protein content or total allergenic activity could be observed. Thus, it seems presently unfeasible to base birch allergen product standardization additionally on Bet v 4. In light of these results, the continuous monitoring of Bet v 4 in birch allergen products with the presented ELISA will provide a basis for the understanding of the clinical relevance of minor allergens.

Sulfamethazine was used as target analyte to produce IgY from chicken aiming to compare the performance of these two antibodies in term of yield, titer, sensitivity, selectivity and matrix effect under parallel conditions. The results showed that the total yield of IgY produced by one chicken during 43 weeks' experimental period was about 15 g. This output is much more than IgG (150 mg from one rabbit). Besides, IgY titers increased during 10 weeks and remained stable over 30 weeks. The peak titer of IgY was 1:218. As the antibody titer rose, the sensitivity was increased. For IgG, the maximum titer was observed to be 1:223. In addition, both IgY and IgG were highly sensitive. The limits of detection were 0.54 ng/mL for IgY and 0.24 ng/mL for IgG. These results indicated that the IgY potentially provides a practical and ethical alternative to IgG in veterinary drug residue immunoanalysis.

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect the venom of Indian cobra ( Naja naja naja) in various tissues (brain, heart, lungs, liver, spleen, blood, kidneys, and tissue at the site of injection) of mice after cobra venom injected at different time intervals (0, 2, 4, 6, 8, and 12 h intervals up to 24 h). Whole venom antiserum or individual venom protein antiserum (14, 29, 65, 72, and 99 kDa) could recognize N. n. naja venom by Western blotting and ELISA, and antibody titer was also assayed by ELISA. Antiserum raised against cobra venom in rabbit significantly neutralized the toxicity of venom-injected mice at different time intervals after treatment. The assay could detect N. n. naja venom levels up to 2.5 ng/ml of tissue homogenate, and the venom was detected up to 24 h after venom injection. Venom was detected in brain, heart, lungs, liver, spleen, kidneys, tissue at the bite area, and blood. As observed in mice, tissue at the site of bite area showed the highest concentration of venom and the brain showed the least. Moderate amounts of venoms were found in liver, spleen, kidneys, heart, and lungs. Development of a simple, rapid, and species-specific diagnostic kit based on this ELISA technique useful to clinicians is discussed.

A polyclonal antibody specific to an egg protein of the Suminoe oyster, Crassostrea ariakensis, was developed to assess reproductive effort. After 2 mo of immunization, rabbit antiserum showed strong specificity to the egg protein in an enzyme-linked immunosorbent assay (ELISA). The rabbit antioyster egg immunoglobulin G detected as little as 0.2 µg/mL of Suminoe oyster egg protein by ELISA. The quantity of eggs present in an oyster was estimated using ELISA and was expressed as a gonadosomatic index (GSI). Gonadosomatic index values of Suminoe oysters were assessed monthly from January to July 2007 at the Seomjin River estuary off the south coast of Korea. Histology indicated that most oysters were mature and ready to spawn by the middle of July. Mean GSI values for oysters collected in April, when most female oysters are in early developmental stages, varied between 0.6% and 14.0%. In July, most oysters were ready to spawn and GSI values ranged from 17.5–67.0%, with a mean of 47.7%. The potenti...

HMGB1 concentration is currently regarded as an important biological marker in many inflammation-related conditions. Although ELISA has been proposed as a convenient way to quantify HMGB1 in biological fluids, various molecules have been shown to complex with HMGB1 and may interfere with HMGB1 detection by this technique. We describe here a simple technical improvement that dissociates HMGB1 containing complexes and therefore increases ELISA sensitivity. This procedure was validated in sera from patients with septic shock.We prepared in vitro complexes containing HMGB1 protein. Recombinant human HMGB1 (rhHMGB1) was incubated in the presence of molecules that are known to form complexes with HMGB1 such as LPS, IL-1β, or a rabbit antiserum directed against HMGB1. Then we tested the capacity of perchloric acid (PCA) to dissociate these complexes by quantifying rhHMGB1 by ELISA immediately or following PCA treatment.We demonstrated for the first time that incubation of rhHMGB1 with, IL-1β, LPS or specific antibodies significantly reduce the amount of protein detected by conventional ELISA (p0.05). Treating the samples with PCA prior ELISA efficiently reversed this inhibition. As expected, PCA-modified ELISA detected significantly higher amounts of HMGB1 in plasma samples from 40 patients with septic shock compared to conventional ELISA (p=0.0006).We designed a performing assay that allows the detection of masked and unmasked forms of HMGB1 with a high sensitivity and practicability.

Even though carrot allergy is common in Europe, the amount of different allergens in carrots is still unknown due to a lack of methods for quantitative allergen measurements. The current study aimed at the development of quantitative ELISA tests for the known carrot allergens, namely Dau c 1.01, Dau c 1.02, and Dau c 4 in pure carrot extracts. Monoclonal antibodies targeting the major carrot allergen isoforms Dau c 1.01 and Dau c 1.02 were generated and combined in sandwich ELISA with rabbit antisera against Api g 1, the celery homologue of Dau c 1. A competitive ELISA for the carrot profilin Dau c 4 was based on a polyclonal rabbit antiserum. The three ELISA tests were allergen-specific and displayed detection limits between 0.4 and 6 ng allergen/ml of carrot extract. The mean coefficient of variation (CV) as a means of intraassay variability of the Dau c 1.01, Dau c 1.02 and Dau c 4 ELISA tests was 8.1%, 6.9%, and 11.9%, and the mean interassay CV 13.3%, 37.1% and 15.6%, respectively. Target recovery ranged between 93 and 113%. In conclusion, the specific, accurate and reproducible quantification of three important carrot allergens may help to identify less allergenic carrot varieties, as well as to standardize the amount of allergens in extracts used for carrot allergy diagnosis.

Various strains of E. coli were used to produce experimental haematogenous pyelonephritis in rats. The persistence in the renal tissue of the common enterobacterial antigen (CA) shared by these micro-organisms was then studied by means of indirect immunofluorescent technique with rabbit antiserum against CA. In fresh infections, CA could regularly be demonstrated in and around abscesses, in macrophages, in dilated tubules and in pelvic exudate. However, as early as 2 weeks in the course of the infection, CA was hardly detectable in macrophages. By means of specific antiserum against one of the strains (E. coli 04), it could, however, be shown that other antigens from this strain were still present in the macrophages in animals infected with this strain. In renal tissue with chronic inflammatory changes, examined more than 4 weeks after the bacterial injection, bacterial antigen could no longer be detected with antiserum against CA, whereas antiserum against E. coli 04 still revealed positive reactions on renal tissue from the rats infected with this strain.

The stability of a polyvalent Pseudomonas aeruginosa antigen (St-Ag) was analysed under different experimental conditions by quantitative immunoelectrophoretic methods. St-Ag did not undergo proteolytic degradation by plasmin which is present in the corresponding polyvalent rabbit antiserum (St-Ab) against St-Ag. Storage at -30 degrees C for up to 4 years caused no qualitative changes in St-Ag and, after 1 year, only minor quantitative changes could be revealed. Repeated thawing and freezing caused no changes in St-Ag whereas storage at 4 degrees C, or at room temperature, caused gradual degradation of most of the antigens of St-Ag, beginning after 1 week. Heating up to 40 degrees C for 2 min did not change St-Ag, but heating for longer periods of time or at higher temperatures gradually destroyed most of the antigens of St-Ag. The most thermostable antigen in St-Ag was number 37 which is related to the group specific O antigens.

Cross-reactions between antigens from S. typhi and 24 other bacterial species were studied by quantitative immunoelectrophoretic methods. A sonicated S. typhi antigen preparation and a corresponding pooled rabbit antiserum regularly presenting 86 immunoprecipitates were used as a reference system. Antigens from Enterobacteriaceae cross-reacted intensively with the S. typhi antigens. No cross-reactions between S. typhi and antigens from gram-positive bacteria were found, and only a few cross-reactions with antigens from gram-negative bacteria outside the Enterobacteriaceae. The taxonomic perspectives of the immunoelectrophoretic approach are stressed by results showing that the number of cross-reacting antigens between S. typhi and other Enterobacteriaceae was positively correlated to the number of identical reactions in a collection of 28 biochemical tests.

Human primary and secondary malignant, and primary benign brain tumours were short time cultured in order to detect a coating with a protein, immunologically indistinguishable from complement component Cl inactivator. The investigations were done by cytophotometry of immunoflurescence on individual cells from human brains. The cell cultures were incubated with specific rabbit antiserum against human Cl inactivator, conjugated with fluorescein isothiocyanate. The majority of the malignant brain tumours, and a few of the benign brain tumours, were shown to carry the Cl inactivator—resembling protein. The primary malignant brain tumour cells seemed to lose this coating during prolonged culturing. Metastatic brain carcinomas retained their inactivator coating.