Anti-species Antibodies Research Articles

Comparative Characteristics of Immunochromatographic Test Systems for Tylosin Antibiotic in Meat Products

Tylosin (TYL) is a macrolide antibiotic widely used in animal husbandry. Due to associated health risks, there is a demand for sensitive methods for mass screening of TYL in products of animal origin. This article describes the development of lateral flow immunoassays (LFIAs) for TYL detection using direct (anti-TYL antibodies conjugated with nanoparticles) and indirect antibody labeling (anti-species antibodies conjugated with nanoparticles and combined with native anti-TYL antibodies). The choice of LFIA conditions, such as concentrations of hapten–protein conjugates, specific antibodies, and gold nanoparticle (GNP) conjugates with antibodies, as well as incubation time of reagents and the concentration of detergent in the sample buffer, is presented. The achieved limits of TYL detection using LFIAs with indirect labeling were 0.8 ng/mL (visual) and 0.07 ng/mL (instrumental), compared to 4 ng/mL (visual) and 0.4 ng/mL (instrumental) for the case of direct labeling. The sensitivity of the LFIA using the indirect format was up to seven times higher, allowing the determination of the target analyte at low concentrations. TYL detection in ground meat using LFIA with indirect antibody labeling ranged from 76–119%.

Comparison of competitive and sandwich immunochromatographic analysis in the authentication of chicken in meat products

Cheap chicken meat is often used as an undeclared substitute in meat products. In this study, two formats of the immunochromatographic assay (ICA) of immunoglobulins of class Y (IgY) as a biomarker for chicken authentication were developed. In both competitive ICA (cICA) and sandwich ICA (sICA), gold nanoparticles (GNP) were conjugated with anti-species antibodies. A simple procedure of sample preparation, which took only 30 min, was proposed. Test systems demonstrated high sensitivity and rapidity: visual limits of detection of IgY and assay durations were 12/14 ng/mL and 10/15 min for cICA and sICA, respectively. The absence of cross-reactivity with the mammalian species confirmed the high specificity of the test systems. Good applicability of the assays was confirmed for the detection of chicken in raw meat mixtures: as low as 3% and 0.2% (w/w) of chicken could be revealed in beef and pork by cICA and sICA, respectively. The influence of heat processing of meat-based products on immune recognition and, consequently, the analytical performance of the test systems was revealed. It was shown that sICA is preferable for the detection of IgY even in thermally processed meat. The proposed ICAs can be recommended for rapid on-site control of meat products’ composition.

An immunochromatographic test for the detection of anti-tuberculosis antibodies

Tuberculosis is a dangerous socially significant disease of various animal species. According to Rosselkhoznadzor data for 2022, the situation with bovine tuberculosis is endemic, stable, long-term trends are decreasing, epidemic thresholds for ill health and incidence have not been overcome. It should be noted that in 2022, bovine tuberculosis was not officially detected, but this infection was registered in several pigs and wild boars. Take a point that the reduction in the number of cases of tuberculosis in cattle is decreased (to zero), the overall strategy for combating this disease is successful, but this does not mean that the need to develop new tests for the accelerated diagnosis of this infection has completely disappeared. The safety of service personnel directly depends on minimizing the risk of infection with pathogens common to humans and animals, one of which is tuberculosis. The fastest way to indicate infectious agents is by immunochromatographic analysis. This publication is devoted to the details that it is desirable to focus on when developing a diagnostic test based on immunochromatographic analysis, using the example of bovine tuberculosis. As an antigen in the presented study, native antigens of Mycobacterium bovis grown on a nutrient medium Levenshtein-Jensen with proven antigenic activity were used. Conjugation of antispecies antibodies was carried out with a colloidal solution of gold, (diameter of particle is 25±0,9 nm). In the course of the work, the optimal conditions for the manufacture of a diagnostic test for the detection of anti-tuberculosis antibodies by immunochromatographic analysis were shown.

OPTIMIZATION OF THE MAIN PARAMETERS OF A LATERAL FLOW ASSAY BASED ON THE RECOMBINANT Р32 ANTIGEN OF LUMPY SKIN DISEASE VIRUS

Lumpy skin disease is a highly contagious infectious transboundary disease of cattle, resulting in significant economic losses. The wide and rapid spread of lumpy skin disease to new territories emphasizes the objective need to improve diagnostic methods. The article outlines the findings of lateral flow assay parameter optimization for lumpy skin disease detection using recombinant P32 antigen. It was found that the optimal amount of protein for immobilization on 20 nm colloidal gold particles was 20 µg/mL. Bovine serum albumin at a final concentration of 0.25% was used to stabilize the conjugate. The optimal dilution of the conjugate was 1:2, and the concentration of recombinant protein and anti-species antibodies for application to the membrane was 500 μg/ml and 250 μg/ml, respectively. The colloidal gold conjugate interacted with antibodies and specifically reacted with the recombinant protein. No cross-reaction with other proteins was noticed at the same time. The created express test allowed for the detection of antibodies from immunized animals when the serum was diluted up to 1:200, however, 1:50 was the ideal ratio. The conjugate remained active after 90 days of storage at room temperature. The findings demonstrate the express method's potential for use in serological research for the prevention and control of the spread of lumpy skin disease.

Modular Set of Reagents in Lateral Flow Immunoassay: Application for Antibiotic Neomycin Detection in Honey

A scheme of modular competitive immunochromatography with an analyte-independent test strip and changeable specific immunoreactants has been proposed. Native (detected) and biotinylated antigens interact with specific antibodies during their preincubation in solution, that is, without the immobilization of reagents. After this, the detectable complexes on the test strip are formed by the use of streptavidin (which binds biotin with high affinity), anti-species antibodies, and immunoglobulin-binding streptococcal protein G. The technique was successfully applied for the detection of neomycin in honey. The visual and instrumental detection limits were 0.3 and 0.014 mg/kg, respectively, and the degree of neomycin revealed in honey samples varied from 85% to 113%. The efficiency of the modular technique with the use of the same test strip for different analytes was confirmed for streptomycin detection. The proposed approach excludes the necessity of finding the condition of immobilization for each new specific immunoreactant and transferring the assay to other analytes by a simple choice of concentrations for preincubated specific antibodies and the hapten-biotin conjugate.

Highly sensitive immunochromatographic assay for simultaneous determination of azaperone and azaperol in pork

In this study, a sensitive immunochromatographic assay (ICA) was developed for simultaneously detecting azaperone (AZN) and its metabolite azaperol (AZL) based on the high-affinity monoclonal antibody (mAb). Herein, the hapten AZL-SA was synthesized by succinic anhydride method, and then the conjugates AZL-SA-OVA and AZL-SA-KLH were prepared by EDC/NHS method. Subsequently, mAb was produced for targets monitoring through two detection modes. In direct ICA (using gold nanoparticles labeled specific antibodies), the visual LOD of AZN was 80 ng/g. For indirect ICA (using gold nanoparticles labeled anti-species antibodies), the visual and instrumental LODs of AZN were 8 and 0.14 ng/g, and AZL were 8 and 0.12 ng/g, respectively. The results indicated that the visual detection limit (LOD) of indirect format was tenfold lower than that of direct format. The established analytical method obtains the results within 15 min and provides a sensitive and simple tool for on-site detection of AZN and AZL.

Novel immunochromatographic estimation of lamb content in meat products using IgG as biomarker

The aim of this study was to develop a novel immunochromatographic analysis (ICA) for the determination of lamb additives in raw meat and meat products. Sheep immunoglobulins of class G (IgG) served as the molecular identifier. The ICA was implemented in a sandwich format using anti-species antibodies labeled with gold nanoparticles. Sheep IgG was detected with the instrumental detection limit of 60 ng/mL and the cutoff of 10 ng/mL within 13 min. The high specificity of the ICA towards only sheep IgG was confirmed within the determination of immunoglobulins of other mammals and birds and analysis of extracts from the corresponding sorts of meat. It was shown that the test system can reliably determine 1% lamb additive in chicken meat. The test system was successfully used for lamb identification in dumplings of various compositions. The proposed ICA is a promising approach for the determination of species-specific immunoglobulins as biomarkers of composition and adulteration of meat products.

Double Immunochromatographic Test System for Sensitive Detection of Phycotoxins Domoic Acid and Okadaic Acid in Seawater and Seafood.

In this investigation, a double immunochromatographic analysis (ICA) of two relevant phycotoxins, domoic acid (DA) and okadaic acid (OA), was developed for the first time. The ICA was performed in the indirect competitive format using gold nanoparticles conjugated with anti-species antibodies. Under optimal conditions, the instrumental detection limits/cutoffs for simultaneous detection of DA and OA were 1.2/100 and 0.1/2.5 ng/mL, respectively. The time of the assay was 18 min. The ICA was applied to test seawater and a large panel of seafood, including mussels, tiger shrimps, octopuses, whelks, crabs, and scallops. The proposed simple sample preparation method for seafood takes only 20 min. For seawater, a dilution by buffer was implemented. The assay recoveries varied from 80.8% to 124.5%. The competitive potential of the proposed technique as a tool to control natural water and seafood samples is determined by its simplicity, rapidity, and sensitivity.

Cascade-Enhanced Lateral Flow Immunoassay for Sensitive Detection of Okadaic Acid in Seawater, Fish, and Seafood.

In this investigation, a new approach for developing a sensitive lateral flow immunoassay (LFIA) was proposed for the detection of the hazardous marine toxin okadaic acid (OA). It is based on the indirect format with anti-species antibodies labeled by gold nanoparticles (AuNPs) and cascade signal amplification. The latter is performed by first passing a mixture of anti-OA antibodies and a tested sample along the immunochromatographic test strip and then performing several cycles of the interaction of anti-species antibodies conjugated with AuNPs with free antibodies, which bind to anti-species antibodies but are not specific to the target analyte. As a result, branched aggregates are formed, due to which the colorimetric signal intensification occurs. The developed test system enabled the detection of OA with an instrumental detection limit of 30 pg/mL and a cutoff of 1 ng/mL, which exceeds these characteristics in the LFIA without amplification by 7 and 2 times, respectively. The OA recoveries from seawater, fish, and seafood varied from 76.9% to 126%. The test system may be required for point-of-care monitoring of samples for phycotoxin contamination; the developed principle of signal amplification can be used in cases where highly sensitive detection of trace amounts of a contaminant is required.

Development of a rapid lateral flow immunochromatography assay for the detection of group-specific antibodies against VP7 protein of bluetongue virus in multiple species

Bluetongue virus (BTV), the causative agent of bluetongue disease infects many domestic and wild ruminants. In the present study, colloidal gold nanoparticle-based lateral flow immunochromatography assay (LFIA) was developed to detect the group-specific antibodies to BTV in serum samples of sheep, goats, cattle, and camel. The recombinant VP7 protein of BTV conjugated to colloidal gold nanoparticles (GNPs) was used as a detector reagent. Recombinant streptococcal protein G and monoclonal antibody to BTV group-specific antigen were immobilized as the test and the control line, respectively on a nitrocellulose membrane. The protein G could capture the specific antibodies to BTV present in the serum of multiple ruminant species susceptible to BTV in a common test format and could eliminate the requirement of multiple anti-species antibodies. Upon addition of serum sample, GNP-rVP7 protein-serum complex migrated laterally onto the strip via capillary action and results were analyzed based on appearance of red colour band at test and control line. Serum samples (n = 481) of sheep, goats, cattle, and camel segregated as positive and negative by the commercial competitive-ELISA (c-ELISA) kit were tested in the fabricated LFIA strips to analyze the performance of the assay. In comparison with c-ELISA, the relative diagnostic sensitivity (DSn) of 95.2% with 91.6–97.6 (95%)) confidence interval and relative diagnostic specificity (DSp) of 99.6% 97.8–100.0 (95%) confidence interval were obtained for the optimized LFIA. The agreement between the LFIA and the c-ELISA was excellent as indicated by the kappa coefficient value of 0.949 (SE = 0.0142) with 0.9219 to 0.9779 (95%) confidence interval. The recombinant protein G based LFIA is a sensitive, specific, rapid, one-step test that can be used in the field or poorly equipped laboratories for serological diagnosis and serosurveillance of bluetongue in multiple susceptible species.

Lateral flow immunoassay for sensitive detection of undeclared chicken meat in meat products

This study presents the development of an immunochromatographic test system aimed at the detection of chicken additives in meat products. It is based on sandwich-format lateral flow immunoassay (LFIA) of immunoglobulins as a biomarker for species identification. The LFIA based on gold nanoparticles as a label for anti-species antibodies was used to determine chicken immunoglobulins and, accordingly, chicken meat in food products. Absence of cross-reactivity with mammalian species tested in the study confirmed high specificity of the determination. The test system showed good sensitivity and rapidity, allowing for the detection of as low as 0.063% (w/w) chicken meat in raw meat mixtures within 20 min. As a result of the testing of raw and cooked meat products, it was shown that the test system can reliably recognize specific immunoglobulins even after heat processing. The proposed assay can be recommended for rapid on-site screening control of the composition and quality of meat products.

Sensitive lateral flow immunoassay of an antibiotic neomycin in foodstuffs.

Aminoglycosides belong to a class of antibiotics now widely used in agriculture and veterinary medicine and expected to contaminate food products. In this study, a sensitive lateral flow immunoassay (LFIA) of an aminoglycoside neomycin (NEO) was developed. Two methods of immunochromatographic detection based on various techniques of gold nanoparticles (AuNPs) introduction as a label were compared. It was demonstrated that the indirect labeling (a conjugation of anti-species antibodies with a marker) allowed for an increase in assay sensitivity by 80 times. The test system was characterized by an instrumental limit of detection of 0.1ng/mL and the cutoff level of 10ng/mL; the assay duration was 15min. Specificity only toward NEO was demonstrated. The developed LFIA has been tested to detect NEO in different foodstuffs. It has been demonstrated that 70-119% of NEO (coefficients of variations < 10%) can be determined in milk, turkey meat, honey, and eggs using simple procedures of preliminary sample preparation. Testing the samples showed the coincidence of the results for the developed lateral flow assay and for commercial ELISA kit.

ОПТИМИЗАЦИЯ ПРОТОКОЛА ИММУНОФЕРМЕНТНОГО АНАЛИЗА ДЛЯ ИНДИКАЦИИ Т-2 ТОКСИНА

T-2 toxin is one of the most common and toxic trichothecene mycotoxins – secondary metabolites of molds that develop on cereals and some other crops. This article discusses the development stage of a test system for the determination of T-2 toxin based on enzyme-linked immunosorbent assay (ELISA), which uses an enzyme label – a conjugate of anti-rabbit goat antibodies with peroxidase. An important step in the creation of any ELISA-based test system is the preliminary titration of reaction components. The aim of the work was to determine the optimal concentration of the conjugate of antispecies antibodies in an indirectly competitive enzyme-linked immunosorbent enzyme immunoassay with the indication of T-2 toxin. A number of dilutions of the antivirus conjugate were examined: 1 : 1000; 1 : 2000; 1 : 3000; 1 : 4000; 1 : 5000; 1 : 7500; 1 : 10000; 1 : 12 500; 1 : 15,000; 1 : 17,500; 1 : 20,000; 1 : 30 000. In the ELISA staging protocol, calibration solutions of T-2 toxin at concentrations of 0.0 were used; 2.5; 5, 10, 20, 40, 80, 160 ng/ml and specific polyclonal rabbit antibodies to the T-2-BSA conjugate. Based on the average optical density values, calibration plots were constructed using the percentage of signal absorption from the zero standard. When evaluating the results of the study, the criterion for choosing the dilution of the conjugate of anti-species antibodies was considered the greatest, at which the best linearity of the grading plot is achieved and the level of its non-specific reaction with the zero standard would be the lowest. It was established that the optimal variants of dilutions of the conjugate of anti-species antibodies under the same experimental conditions tested were 1: 4000, 1 : 5000 and 1 : 12 500. Dose-dependent signal absorption was observed in all concentrations of anti-species antibodies. Dilutions of the conjugate of anti-species antibodies 1 : 1000–1 : 3000 and 1 : 17 500–1 : 30 000 were not taken into account, since the optical density of most wells was higher than the optimal boundaries in the first case (&gt; 3.9) and lower in the second (&lt; 0.4). Based on the foregoing, optimal dilution of the conjugate of anti-species antibodies was selected 1 : 12 500.

Development of a double immunochromatographic test system for simultaneous determination of lincomycin and tylosin antibiotics in foodstuffs

This study is devoted to the development of a sensitive immunochromatographic analysis (ICA) for simultaneous determination of tylosin (TYL) and lincomycin (LIN) as antibiotics of the macrolide and lincosamide classes, widely used in animal husbandry and implicated in the contamination of foodstuffs. The ICA was implemented in an indirect competitive format, using antispecies antibodies conjugated with gold nanoparticles (GNPs) as a label. After the multistep optimization, the developed double ICA allowed for antibiotics detection with instrumental limits of detection/cutoff levels of 0.09/2 ng/mL and 0.008/0.8 ng/mL for TYL and LIN, respectively, within 10 min. The cross-reactivity was 40% to lincosamide clindamycin and negligible to other antibiotics tested. The test system allowed for the detection of TYL and LIN in milk, honey, and eggs. The recoveries of antibiotics from foodstuffs were 87.5–112.5%. The results demonstrate that the developed double ICA is an effective approach for the detection of other food contaminants.

An immunochromatographic test system for the determination of lincomycin in foodstuffs of animal origin

The aim of this study was to develop a rapid and sensitive immunochromatographic test system for the detection of lincomycin (LIN), which belongs to the lincosamide group of antibiotics and contaminates food products of animal origin. Two formats of immunochromatographic analysis (ICA) based on different approaches of introducing gold nanoparticles (GNPs) as a label were compared. It was demonstrated that an indirect ICA method where GNPs were conjugated with anti-species antibodies allowed the achievement of both instrumental and visual detection limits of LIN almost two orders of magnitude lower than those achieved in the standard direct ICA format. In the optimized conditions, the developed indirect ICA allowed for the detection of LIN within 15 min, with instrumental and visual detection limits of 8 pg/mL and 0.8 ng/mL. The assay showed 40% cross-reactivity to clindamycin (CLIN) as a structural analogue of LIN, with no interaction with antibiotics from other classes. The developed ICA was applied for LIN detection in a panel of food products. No treatment of cow milk was necessary before the analysis. For chicken eggs and honey, a simple procedure of preliminary sample preparation was developed, which fully prevented a matrix influence on the assay results. It was demonstrated that ICA could detect LIN in food products while preserving the same analytical characteristics as in the buffer. The analytical recoveries of LIN in foodstuffs were 93.8–125% with coefficients of variations of 5.3–14.0%.

Highly sensitive lateral flow test with indirect labelling for zearalenone in baby food

ABSTRACT Maximum permissible levels of mycotoxins in baby food may be 1% of those in ordinary food. Therefore, highly sensitive methods of mycotoxin control are in demand. To detect such low amounts, expensive instrumental methods are commonly used. Advantages of immunochromatographic analyses are their low cost and simple sample preparation; however, their sensitivity needs to be increased to contend with instrumental methods. A scheme for competitive immunochromatography with indirect labelling was implemented and developed for the detection of mycotoxin zearalenone (ZEA). Two separate reagents were used for the assay, namely free specific antibodies and antispecies antibodies conjugated with gold nanoparticles. This made it possible to simultaneously increase the sensitivity of the assay and the reliability of measurements. The instrumental detection limit of ZEA in baby food was 5 pg/mL (100 pg/g). Thus, the sensitivity attained is comparable with liquid chromatography characteristics. The duration of the analysis was 17 min.

Development of A Lateral Flow Highway: Ultra-Rapid Multitracking Immunosensor for Cardiac Markers.

The integration of several controlled parameters within a single test system is experiencing increased demand. However, multiplexed test systems typically require complex manufacturing. Here, we describe a multiplexed immunochromatographic assay that incorporates a conventional nitrocellulose membrane, which is used together with microspot printing, to construct adjacent microfluidic “tracks” for multiplexed detection. The 1 mm distance between tracks allows for the detection of up to four different analytes. The following reagents are applied in separate zones: (a) gold nanoparticle conjugates with antibodies against each analyte, (b) other antibodies against each analyte, and (c) antispecies antibodies. The immersion of the test strip in the sample initiates the lateral flow, during which reagents of different specificities move along their tracks without track erosion or reagent mixing. An essential advantage of the proposed assay is its extreme rapidity (1–1.5 min compared with 10 min for common test strips). This assay format was applied to the detection of cardiac and inflammatory markers (myoglobin, D-dimer, and C-reactive protein) in human blood, and was characterized by high reproducibility (8%–15% coefficient of variation) with stored working ranges of conventional tests. The universal character of the proposed approach will facilitate its use for various analytes.

Indirect Labeling of Antibodies as a Universal Approach to Increase Sensitivity of Lateral Flow Tests: A Case Study for Mycotoxins Detection

Objective: The study aimed at increasing the sensitivity of immunochromatographic tests for the control of toxic contaminants (on the examples of aflatoxin B1 and T-2 toxin) in agricultural products. Methods: For reliable immunochromatographic detection of low concentrations of analytes, a replacement of the (specific antibodies – gold nanoparticle) conjugate by a combination of native specific antibodies and anti-species antibodies conjugated with gold nanoparticles was proposed. Different variants of test systems based on the principle of indirect labeling were realized and compared. Results: Immunochromatographic assays with indirect labeling for aflatoxin B1 and T-2 toxin were implemented experimentally. A reduction in the detection limit by one to two orders of magnitude was demonstrated. Conclusion: The presented results confirm that indirect labeling of specific antibodies overcomes the limitations of the competitive immunochromatographic analysis and can be used to detect analytes of different chemical nature.

Development of Immunochromatographic Assay for Determination of Tetracycline in Human Serum.

Determining antibiotic concentration in human blood provides useful pharmacokinetic information. Commonly used methods such as ELISA require a long time to obtain results and thus cannot be applied when information is needed immediately. In this study, a novel antibody-based lateral flow technique was developed for tetracycline detection in human serum. Contrary to tests developed to analyze food samples, the features of work with serum as analyzed probe were studied for the first time here. The application of labeled and unlabeled specific antibodies was compared. For this purpose, specific and anti-species antibodies were labeled with gold nanoparticles and used for antigen–antibody interaction on the membrane surface with observed staining in the test zone. For both schemes, optimal conditions were established to provide the best sensitivity. The developed assay has a limit of visual detection as low as 35 and 11 ng/mL for the direct and indirect labeled antibodies, respectively. The limit of instrumental detection is from 0.4 to 3.5 ng/mL for diluted and undiluted sera. The use of indirect antibody labeling showed a small increase in sensitivity compared to traditional direct antibody labeling. The developed method showed no cross-reactivity with antibiotics of other classes. The method was used to test samples of serum. The results showed high correlation with the data obtained by ELISA (R2 = 0.98968). The assay provides a quick assessment of the amount of antibiotics in the blood and keeps them under control throughout the duration of therapy.

Complexes of Gold Nanoparticles with Antibodies in Immunochromatography: Comparison of Direct and Indirect Immobilization of Antibodies for the Detection of Antibiotics

Complexes of spherical gold nanoparticles with antibodies, obtained by direct adsorption and indirect (nanoparticle–anti-species antibody–specific antibody) interaction, were studied. The binding processes for nanoparticle-labeled antibodies in immunochromatographic analysis (ICA) and competitive detection of low molecular weight analytes are considered. Based on the developed mathematical model, the advantages of indirect labeling of antibodies are shown; the parameters determining the gain in the detection limit between two kinds of systems are characterized. The obtained complexes of gold nanoparticles with antibodies were used for the development of ICA for antibiotics. The reached detection limit of ampicillin ICA is 100 ng/mL for direct labeling and 16 ng/mL for indirect; in the case of tetracycline, these values are equal to 4.2 and 1.3 ng/mL, respectively.