Production of antibody and development of enzyme-linked immunosorbent assay for therapeutic drug monitoring of eravacycline.

Eravacycline (ERC) was approved for clinical use in 2018. It is more potent than other tetracyclines and can overcome resistance, making it an attractive option for combating multidrug-resistant bacterial infections. Intensive pharmacokinetic (PK) studies are currently being conducted to ensure the effectiveness and safety of ERC in various groups of patients, including those undergoing extracorporeal therapies. This study is the first attempt to develop a simple, efficient, and high-throughput immunoassay for quantifying ERC in human or animal serum. BSA-ERC conjugate as immunogen elicited antibody production in rabbits. Monitoring of the immune response and comparison of homologous and heterologous coating antigens allowed selection of immunoreagents and development of an assay that was selective for ERC possessing sensitivity (IC50), dynamic range (IC20-IC80) and detection limit equal to 3.3 ng/mL, 0.27-54 ng/mL and 0.09 ng/mL, respectively. The developed ELISA showed acceptable recovery of ERC (85-105 %) from rabbit and human serum in the clinically relevant concentration range of 0.1-3.0 mg/L. The method was used to quantify serum ERC concentration in the pilot PK study in Soviet chinchilla rabbits. The results were confirmed by HPLC-MS/MS.

Immunotechniques for the Group Determination of Macrolide Antibiotics Traces in the Environment Using a Volume-Mediated Sensitivity Enhancement Strategy.

Macrolide antibiotics, which are effective antimicrobial agents, are intensively used in human and veterinary medicine, as well as in agriculture. Consequently, they are found all over the world as environmental pollutants, causing harm to sensitive ecological communities and provoking a selection of resistant forms. A novel azithromycin derivative, which was used as hapten conjugate, ensured the group immunorecognition of six major macrolide representatives (105-41%), namely erythromycin, erythromycin ethylsuccinate, clarithromycin, roxithromycin, azithromycin, and dirithromycin in a competitive immunoassay based on anti-clarithromycin antibodies. The heterologous hapten-based ELISA format resulted in a 5-fold increase in sensitivity, with an IC50 value of 0.04 ng/mL for erythromycin. In this study, we proposed an underexploited strategy in an immunoassay field to significantly improve the detectability of analytes in environmental samples. Unlike most approaches, it does not require special enhancers/amplifiers or additional concentration/extraction procedures; instead, it involves analyzing a larger volume of test samples. A gradual volume increase in the samples (from 0.025 to 10 mL) analyzed using a direct competitive ELISA, immunobeads, and immunofiltration assay formats based on the same reagents resulted in a significant improvement (more than 50-fold) in assay sensitivity and detection limit up to 5 and 1 pg/mL, respectively. The suitability of the test for detecting the macrolide contamination of natural water was confirmed by the recovery of macrolides from spiked blank samples (71.7-141.3%). During 2022-2023, a series of natural water samples from Lake Onega and its influents near Petrozavodsk were analyzed, using both the developed immunoassay and HPLC-MS/MS. The results revealed no contamination of macrolide antibiotic.

Tigecycline Immunodetection Using Developed Group-Specific and Selective Antibodies for Drug Monitoring Purposes.

Tigecycline (TGC), a third-generation tetracycline, is characterized by a more potent and broad antibacterial activity, and the ability to overcome different mechanisms of tetracycline resistance. TGC has proven to be of value in treatment of multidrug-resistant infections, but therapy can be complicated by multiple dangerous side effects, including direct drug toxicity. Given that, a TGC immunodetection method has been developed for therapeutic drug monitoring to improve the safety and efficacy of therapy. The developed indirect competitive ELISA utilized TGC selective antibodies and group-specific antibodies interacting with selected coating TGC conjugates. Both assay systems showed high sensitivity (IC50) of 0.23 and 1.59 ng/mL, and LOD of 0.02 and 0.05 ng/mL, respectively. Satisfactory TGC recovery from the spiked blood serum of healthy volunteers was obtained in both assays and laid in the range of 81-102%. TGC concentrations measured in sera from COVID-19 patients with secondary bacterial infections were mutually confirmed by ELISA based on the other antibody-antigen interaction and showed good agreement (R2 = 0.966). A TGC pharmacokinetic (PK) study conducted in three critically ill patients proved the suitability of the test to analyze the therapeutic concentrations of TGC. Significant inter-individual PK variability revealed in this limited group supports therapeutic monitoring of TGC in individual patients and application of the test for population pharmacokinetic modelling.

Comparative polymyxin B pharmacokinetics in critically ill patients with renal insufficiency and in continuous veno-venous hemodialysis.

PURPOSE: The aim of this study was to assess polymyxin B pharmacokinetics (PK) in patients with varying degrees of renal dysfunction and in patients who require continuous veno-venous hemodialysis (CVVHD). METHODS: The study enrolled 37 patients with sepsis, including 13 patients with glomerular filtration rate (GFR) below 80 mL/min and 11 patients on CVVHD. Each patient received a loading dose of polymyxin B (200-300 mg) and at least 3 subsequent doses of 100-150 mg every 12 h. For every patient, 6-8 blood samples were collected between doses. Polymyxin B (PMB) serum concentration was determined using enzyme-linked immunosorbent assay. RESULTS: In sepsis, patients with preserved renal function mean area under the curve over 24 h (AUC0-24 h) value reached 67.8 ± 9.8 mg*h/L, while in patients with GFR below 80 mL/min, mean AUC0-24 h was 87 ± 5.8 mg*h/L. PMB PK in patients with renal insufficiency was characterized by significantly lower clearance (CL) compared to the normal renal function group (2.1 ± 0.1 L/h vs 3.9 ± 0.4 L/h respectively). In patients on CVVHD, mean AUC0-24 h was 110.4 ± 10.3 mg*h/L, while CL reached 2 ± 0.23 L/h. The median recovery rate from dialysate constituted 22%. Simulation of different dosage regimens that indicate a fixed maintenance dose of 100 mg q12h with a loading dose of 200 mg is optimal for patients on CVVHD, and no dosage increase is required. CONCLUSION: This study demonstrates decreased clearance of PMB in patients with renal insufficiency, which puts them at risk of toxicity. Therefore, patients with extremes of renal function might benefit from therapeutic drug monitoring. For patients with anuria, who require CVVHD, we suggest a fixed dose of 100 mg q12h.

Unbound serum polymyxin B in patients with sepsis: Detection approaches and limited sampling strategy for clinical practice and research.

Polymyxins are increasingly used to treat multidrug resistant bacteria in critically ill patients, however these drugs have a narrow therapeutic window. Highly variable antibiotic pharmacokinetics in critically ill patients puts them at high risks of toxicity and underdosing, therefore robust tools suitable for therapeutic drug monitoring and pharmacokinetic studies are in great demand. It is now recognized that only antibiotics not bound to serum proteins possess antimicrobial effects and free drug concentration is the most appropriate pharmacokinetic target. However, data on unbound polymyxin B (PMB) determination is very limited, especially in different cohorts of critically ill patients. Performance of rapid equilibrium dialysis and ultrafiltration in unbound PMB measurement, as well as different approaches to predict free PMB exposure based on a single measurement were explored in present study. In comparison to ultrafiltration rapid equilibrium dialysis demonstrated a lower degree of non-specific binding, which makes the latter a more reliable tool. Overall, unbound drug determination is a costly and labor-intensive procedure, hence different limited sampling strategies were proposed for further clinical application. Although pooled sample analysis is commonly used for this purpose, the results showed inconsistency between observed and predicted values. An alternative approach using 1-3 sampling points and Bayesian estimation is proposed.

Immunoassay for Natamycin Trace Screening: Bread, Wine and Other Edibles Analysis.

The antifungal drug natamycin (NAT) is widely used in medicine and in the food industry as preservative E235 for a wide variety of foods. The risk of the development of resistance to NAT and its spread in relation to other polyene antibiotics is fraught with the emergence of incurable infections. This work is devoted to the development of an immunoassay to investigate the prevalence of NAT use for food preservation. Two immunogen designs based on tetanus toxoid, conjugated to NAT through different sites of hapten molecules, were compared in antibody generation. Assay formats using heterologous coating antigens were superior for both antibodies. The ELISA variant demonstrated the highest sensitivity (IC50 = 0.12 ng/mL), and a limit of detection of 0.02 ng/mL was selected for NAT determination. The optimized extraction procedure provided a recovery rate of 72-106% for various food matrixes with variations below 12%. Cyclodextrins, as well as NAT-cyclodextrin complex formulations, showed no interference with the quantification of NAT. One hundred and six food product brands, including baked goods, wines, beers, drinks, sauces, and yogurts, were tested to assess the prevalence of the undeclared use of NAT as a preservative. The screening examination revealed three positive yogurts with an undeclared NAT incorporation of 1.1-9.3 mg/kg.

Development and application of amphotericin B immunoassay for pharmacokinetic studies and therapeutic drug monitoring in critically ill patients.

Amphotericin B (ATB) is a broad spectrum antibiotic used to combat severe systemic fungal and protozoan infections. Existing and new ATB formulations designed to address the problem of poor solubility and side effects of ATB require pharmacokinetic (PK) studies and dosing controls, especially in critically ill patients. Given that, the present study was devoted to development of competitive immunoassay of ATB and its testing on real human serum samples. A novel immunogen design was based on alternative ATB carboxyl-mediated conjugation to tetanus toxoid (TTd). The resulting conjugates retained antifungal (C.albicans) activity, which indicates the preservation and spatial availability of the ergosterol-binding site, bioactive polyene epitope. Antibody generated against click reaction product, TTd-ATB(cuaac), was able to recognize a group of polyenes ATB, nystatin, natamycin and deoxycholate ATB in heterologous ELISA as 100%, 255%, 99% and 70%, respectively. The sensitivity (IC50), detection limit (IC10) and dynamic range of assay (IC20-IC80) were 6.0, 0.1 and 0.6-46 ng/mL, respectively, and made it possible to quantify total and unbound ATB in the therapeutic range of concentrations in serum. ATB recovery from spiked serum samples was in the range of 95-106% and unbound ATB fractions in ultrafiltrates were about 12%. PK parameters were estimated in single COVID-19 patient with secondary lung Rhizopus microspores infection who was treated with ATB and received veno-venous extracorporeal membrane oxygenation.

Comparative polymyxin B pharmacokinetics in patients receiving extracorporeal membrane oxygenation.

OBJECTIVES: To describe polymyxin B pharmacokinetics in patients receiving veno-venous extracorporeal membrane oxygenation (ECMO) in comparison with critically ill patients without ECMO support and to explore potential covariates that could affect the pharmacokinetics in this group of patients. PATIENTS AND METHODS: In 13 critically ill patients on ECMO and in 21 critically ill patients without ECMO support, 6-8 blood samples were collected during 12 h intervals after reaching steady state. Polymyxin B concentration in serum was determined using a previously developed ELISA. Protein binding was assessed by rapid equilibrium dialysis. RESULTS: In 13 critically ill patients on ECMO who received polymyxin B, the median area under the concentration-time curve over 12 h (AUC0-12h) was 48.38 mg/h/L for the total drug and 14.08 mg/h/L for the free drug. The unbound fraction was 0.35. Total body clearance was 1.16 L/h. In non-ECMO patients, the median AUC0-12h was 34.7 mg/h/L and the median CL was 1.76 L/h. The volume of distribution was significantly lower in ECMO patients (19.7 versus 30.4 L, respectively). We found a moderate negative correlation between the ECMO blood flow rate and AUC0-12h, a strong negative correlation between SOFA score and polymyxin B clearance and a moderate correlation between polymyxin B clearance and renal function in ECMO patients. CONCLUSIONS: Currently recommended polymyxin B dosage regimens are sufficient for patients receiving ECMO and no dosage increase is required. In our study, polymyxin B exposure was higher in ECMO patients compared with the control group.

Influence of Endogenous Factors of Food Matrices on Avidin-Biotin Immunoassays for the Detection of Bacitracin and Colistin in Food.

(Strept)avidin-biotin technology is frequently used in immunoassay systems to improve their analytical properties. It is known from clinical practice that many (strept)avidin-biotin-based tests provide false results when analyzing patient samples with a high content of endogenous biotin. No specific investigation has been carried out regarding possible interferences from avidin (AVI) and biotin (B7) contained in food matrices in (strept)avidin-biotin-based immunoanalytical systems for food safety. Two kinds of competitive ELISAs for bacitracin (BT) and colistin (COL) determination in food matrices were developed based on conventional hapten-protein coating conjugates and biotinylated BT and COL bound to immobilized streptavidin (SAV). Coating SAV-B7-BT and SAV-B7-COL complexes-based ELISAs provided 2- and 15-times better sensitivity in BT and COL determination, corresponding to 0.6 and 0.3 ng/mL, respectively. Simultaneously with the determination of the main analytes, these kinds of tests were used as competitive assays for the assessment of AVI or B7 content up to 10 and 1 ng/mL, respectively, in food matrices (egg, infant milk formulas enriched with B7, chicken and beef liver). Matrix-free experiments with AVI/B7-enriched solutions showed distortion of the standard curves, indicating that these ingredients interfere with the adequate quantification of analytes. Summarizing the experience of the present study, it is recommended to avoid immunoassays based on avidin-biotin interactions when analyzing biosamples containing these endogenous factors or enriched with B7.

Development of ELISA formats for polymyxin B monitoring in serum of critically ill patients.

Treating infections in critically ill patients often requires the use of last-line antibacterial drugs such as polymyxins with a narrow therapeutic window and high toxicity. In critically ill patients, the drug pharmacokinetics changes significantly, and as a result, the antibiotic concentrations in blood and infection foci become suboptimal, which leads to therapeutic failures or toxic manifestations. For timely dosage adjustments, a competitive ELISA-based method using antibodies to polymyxin В (PMB) was developed. Among the several considered assays, a direct antibody-coated format was selected for its short duration (1.5 h) and the best agreement with the LC-MS/MS data (R2 = 98 %). The assay dynamic measurement range (IC20-IC80) could be substantially shifted by changing the ratio of immunoreagents. To conveniently measure the therapeutic range of PMB concentrations, it was adjusted to 5.0-192 ng/mL, allowing the samples to be analyzed after a simple 100-fold dilution with the assay buffer. The ELISA sensitivity expressed in half-inhibition concentration (IC50) and the limit of detection were 30.6 and 1.8 ng/mL, respectively. The assay cross-reactivity towards the related analogue colistin (COL) was 95 %, and this compound could also be adequately quantified by the same assay. The PMB and COL recovery from the spiked serum samples was similar and constituted 98-109 %. The trial drug monitoring was carried out in 3 patients with Gram-negative sepsis, and the established pharmacokinetic profiles of PMB revealed the necessity for individual dosage adjustment.

Simultaneous immunodetection of ionophore antibiotics, salinomycin and narasin, in poultry products and milk.

Rabbit polyclonal antibodies were generated against the ionophore antibiotic salinomycin (SAL) as a determinant of the BSA-SAL conjugate. The homologous ELISA format was found to be preferred for similar recognition of SAL and narasin (NAR) with IC50 values of 0.55 and 0.57 ng mL-1, respectively. Both analytes could be determined in the range of 0.1-2.7 ng mL-1 (IC20-IC80) with a detection limit of 0.03 ng mL-1. To analyze matrices, individual pretreatment of samples was required. For chicken muscles, simple buffer extraction was sufficient to recover 87-110% of ionophores. Extraction with acetonitrile followed by evaporation of the solvent was best for recovering 67-108% SAL and NAR from egg homogenate. A feature of the extraction of ionophores from milk was the elimination of fat-mediated interference by organic solvation. It was found that the absence of Na+ and K+ ions during reconstitution of sample extracts was a key factor contributing to the increase in the average recovery of ionophores from 32% to 93%. Thanks to this special pretreatment and improved recovery, the developed immunoassay method was suitable for the analysis of ionophore antibiotics SAL and NAR in a milk matrix, which has not been previously reported.

Development of a latex particles-based lateral flow immunoassay for group determination of macrolide antibiotics in breast milk.

A lateral flow immunoassay (LFIA) using latex particles labeled with antibody to BSA-clarithromycin (CLA) was developed for the rapid simultaneous group determination of six macrolide antibiotics. Optimization of antigen spotting on the membrane and latex probe loading allowed improving visual detectability (vLOD) 100 times, which was 1, 1, 10, 10, 50, and 1000 ng/mL for CLA, roxithromycin, erythromycin, dirithromycin, azithromycin, and oleandomycin in buffer, respectively. The calculated limits of instrumental detection (cLOD) were respectively 0.12, 0.15, 1.4, 2.1, 2.4, and 3.3 ng/mL. To avoid a strong influence of breast milk of a very diverse and variable composition, a sample pretreatment is proposed. The six macrolides mentioned can be visually detected in breast milk after 20 min pretreatment at concentrations of 10-1000 ng/mL or instrumentally with cLOD of 4.0, 2.5, 30, 42, 42 and 180 ng/mL. The recovery rate from the spiked samples carried out using a strip scanner device ranged from 71 % to 110 %, and precision expressed as relative standard deviation was between 3-14 %. The described rapid on-site diagnostic assay format can be useful for monitoring the content of antibiotics in breast milk during macrolide treatment to ensure safe breastfeeding of infants.

Specific and Generic Immunorecognition of Glycopeptide Antibiotics Promoted by Unique and Multiple Orientations of Hapten.

Conjugation chemistry does not always provide adequate spatial orientation of hapten in immunogens for the best presentation of generic or individual epitopes. In the present study, the influence of unique and multiple orientations of immunizing hapten on the immune response repertoire was compared to select generic recognition system. The glycopeptides, teicoplanin (TPL) and ristomycin (RSM), were conjugated to BSA to produce immunogens with unique and multiple orientations of haptens. Polyclonal antibodies generated against TPL conjugated through a single site were of uniform specificity and demonstrated selective TPL recognition, regardless of the coating conjugates design. The sensitivity (IC50) of 4 enzyme-linked immunosorbent assays (ELISAs) for TPL varied little within the 3.5-7.4 ng/mL, with a dynamic range of 0.2-100 ng/mL. RSM was coupled to BSA through several glycoside sites that evoked a wider repertoire of response. This first described anti-RSM antibody was selective for RSM in homologous hapten-coated ELISAs with IC50 values in the range 4.2-35 ng/mL. Among the heterologous antigens, periodate-oxidized TPL conjugated to gelatine was selected as the best binder of generic anti-RSM fraction. The developed ELISA showed group recognition of glycopeptides RSM, TPL, eremomycin, and vancomycin with cross-reactivity of 37-100% and a 10-10,000 ng/mL dynamic range. Thus, multiple presentations of immunizing hapten help expand the repertoire of immune responses and opportunities for the selection of the required fine-specificity agent.

Broadening the Detection Spectrum of Small Analytes Using a Two-Antibody-Designed Hybrid Immunoassay.

The recognition spectrum of immunoassays developed on the basis of class-specific antibodies can include the several nearest analytes but rarely all of the desired representatives of the group. The situation may be sufficiently improved using a hybrid assay combining two antibodies with specificities that complement each other. Two monoclonal antibodies (mAb) with broad but different specificities toward sulfonamides were examined for their binding to a panel of hapten conjugates. mAb-hapten pairs without mutual cross-reactions were identified, and classical direct antigen-coated and mAb-coated ELISAs were developed as formats with referent specificities. Both interactions were combined in a single hybrid assay, which was designed as a one-step double-competitive sandwich-ELISA. For this assay, the intermediate bifunctional reagent mAb(1)-hapten(2) conjugate was synthesized and able to simultaneously bind to hapten(1) and be bound by mAb(2). Formation of a two-mAbs sandwich complex was inhibited by competitors of interaction(1) as well as by competitors of interaction(2). Thus, due to the summation effect, simultaneous determination of analytes recognized by both mAbs was achieved. The hybrid assay can be performed in two reversed arrangements using a coating antigen or coating antibody, the characteristics of which were compared and found to be similar in sensitivity and extended specificity. The suitability of the developed test for the determination of 14 sulfonamides at their maximum residue limit (MRL) concentration was demonstrated using the examples of turkey muscle and milk samples.

Hapten modification approach for switching immunoassay specificity from selective to generic.

The cross-reactivity profile of polyclonal antibodies against a low molecular weight analyte is strongly influenced by design of the coating or enzyme-linked hapten. The hapten modification effect on immunoassay specificity was studied. Heterology in hapten type and linking method were applied. The influence of these factors on analyses of two groups of antibiotics, 16-membered macrolides and glycopeptides was studied. This approach was used to convert the selective ELISAs to tylosin and eremomycin for group determination of tylosin\tilmicosin, tylosin\spiramycin and eremomycin\vancomycin. It was shown that the analytical spectrum of the developed polyclonal antibody-based immunoassays could be expanded and depended mainly on the type of coating hapten but not on the linking method. Modification of the hapten type in coating conjugates applied in present study served as a mechanism for switching specificity of the ELISA between selective and group.

Development of a competitive indirect ELISA for the determination of lincomycin in milk, eggs, and honey.

Polyclonal antibodies to lincomycin (LIN) were developed in rabbit as a result of immunization with BSA-LIN conjugate. Periodate oxidizing of hapten was the common step of both immunogen synthesis and preparation of conjugated antigens for coating plates (homologous and heterologous). Several ELISA variants on a base of the different antigens immobilized on polystyrene were compared. Heterology of solid-phase antigens was provided with relative hapten clindamycin (CLIN) and ethylene- or hexanediamine as spacer arm between hapten and carrier. The spacer insertion yielded no desirable effect, whereas gelatin-CLIN assay variant showed better test characteristics in comparison with the homologous one, although insignificant (IC(50) was 9.15 vs 18.3 ng mL(-1)). The detection limits of the developed test, being estimated as 0.43 ng mL(-1) (milk) and 0.65 ng mL(-1) (eggs), were sufficient to measure maximum residue levels for LIN in examined matrices. This value for honey was 1.9 ng mL(-1) (1.3 µg kg(-1)). The assay sensitivity was enough to dilute milk, egg, and honey samples by 10-100 times to minimize matrix effect. The examination of matrix effect and simple ways of its overcoming are detailed in the paper. The developed assay showed 111% cross-reactivity with CLIN; therefore, it is suitable for the determination of both lincosamides.