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A Nanobody/Monoclonal Antibody “hybrid” sandwich technology offers an improved immunoassay strategy for detection of African trypanosome infections [1]

['Steven Odongo', 'Laboratory For Biomedical Research', 'Department Of Molecular Biotechnology', 'Environment Technology', 'Food Technology', 'Ghent University Global Campus', 'Incheon', 'South Korea', 'Bo-Kyung Jin', 'Hang Thi Thu Nguyen']

Date: 2024-08

The Nb474H used here, originated from a past study. Briefly, the Nb was engineered starting from mRNA of peripheral blood lymphocytes of an alpaca immunized with soluble lysate of Trypanosoma congolense (TC13). T. congolense glycosomal fructose-1,6-bisphosphate aldolase (TcoALD) was discovered as the cognate Ag of Nb474H. In this study, splenocytes were harvested from a mouse immunized with recombinant TcoALD and fused with NS01 cells to generate a hybridoma library. Random screening of the library on TcoALD retrieved a lone binder, designated IgM8A2. Using Nb474H as Ag-capture reagent in combination with the IgM8A2 monoclonal antibody Ag-detection reagent resulted in a tool that effectively detects native TcoALD released during infection by T. congolense parasites.

The scarcity of reliable devices for diagnosis of Animal African trypanosomiasis (AAT) presents a limitation to control of the disease. Existing high-sensitivity technologies such as PCR are costly, laborious, time-consuming, complex, and require skilled personnel. Hence, utilisation of most diagnostics for AAT is impracticable in rural areas, where the disease occurs. A more accessible point-of-care test (POCT) capable of detecting cryptic active infection, without relying on expensive equipment, would facilitate AAT detection. In turn, early management, would reduce disease incidence and severity. Today, several ongoing research projects aim at modifying complex immunoassays into POCTs. In this context, we report the development of an antigen (Ag) detection sandwich ELISA prototype for diagnosis of T. congolense infections, which is comprised of nanobody (Nb) and monoclonal antibody (mAb) reagents.

Animal African trypanosomiasis is a parasitic wasting disease, which mainly affects livestock of poor households in remote rural areas of sub-Saharan Africa, Asia and Latin America. Affordable interventions are primary to eradication campaigns. The latter is mandated to the Pan African Tsetse and Trypanosomiasis Eradication Campaign (PATTEC). PATTEC conducts activities including mass screening of human population for Human African trypanosomiasis (HAT) and treating positive cases. This strategy has dramatically reduced cases of HAT over the last two decades. Highly sensitive and specific current tests for AAT are technically demanding, confining their use to well-equipped reference laboratories in urban centres. Inaccessibility of tests for AAT by the locals living in remote areas, hinders case finding and delays treatment intervention, eventually perpetuating disease transmission and severity. Hence, the aim of our ongoing work is to ultimately convert a complex laboratory-based test for AAT caused by T. congolense, developed earlier by our research team, into a simple hand-held device suitable for use in the field. The current test format employs a nanobody-monoclonal antibody sandwich combination to detect T. congolense infections, through detection of trypanosome Fructose-1,6-bisphosphate aldolase that is present in the blood of infected animals. The diagnostic capability of the assay was successfully demonstrated on experimental samples.

Funding: This work was funded by the H2020-EU.3.2.1.1 Program for Controlling and progressively minimizing the burden of animal trypanosomosis, a Horizon 2020 project (COMBAT). SM received the grant. COMBAT is a European Union grant and its URL is https://www.combat-project.eu/ . The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Whereas developments of several Nb-based diagnostic devices for trypanosomiasis were initiated [ 22 , 23 , 27 ], commercialization of these tests has not yet been achieved for practical reasons. Firstly, some of these Nb-based devices are detecting their respective target Ags in a homologous sandwich fashion [ 22 ] making translation into an Ag capture LFA without loss of sensitivity impossible. Indeed, in an Ag detection LFA, the target Ag is pre-complexed with high amounts of detection Nbs and the complex is driven by capillary action to a line of printed Ag-capture Nbs. In a homologous sandwich format, the Ag-capture Nbs would compete for the same binding sites with the already-couped detection Nb. In this case, most of the complexes would escape capture, causing a drastic reduction in signal intensity and consequently lowering the assay sensitivity. A second limitation affecting development of Nb-based test devices is the small size of Nbs, which often compromise conjugation to Gold (Au) nanoparticles [ 28 ]. To mitigate these limitations, but still exploit the highly specific Nb-capturing capacity, we explored here a Nb/mAb “hybrid” heterologous sandwich system. As our previous research has shown that trypanosome aldolase is a target that allows for a highly sensitive detection of active trypanosome infection [ 22 ], the target of the new test format was kept the same. Hence, a mouse mAb (IgM8A2) was generated against TcoALD and integrated in the ELISA to substitute the previously described Nb detection reagent, thereby achieving a Nb474H/IgM8A2 “hybrid” sandwich setup. The capability of the “hybrid” sandwich system to detect native TcoALD was demonstrated.

Deployment of accessible low-cost diagnostics that readily reveal trypanosomes in the host, as well as reservoir species, would be valuable for guiding treatment decision, monitoring control program and surveillance. Unfortunately, current tests for trypanosomiasis do not meet the ASSURED criteria required by the World Health Organization [ 14 ]. Largely, the existing tests for trypanosomes are costly, less sensitive, less specific, inherently complex, laborious and time-consuming, require qualified personnel, and rely on electricity making them unsuitable for use in resource-deprived rural communities. Deployment of reliable point-of-care tests (POCT) [ 15 ] for AAT on farms, would enable case finding and allow prompt treatment intervention. The only field POCT for AAT are card agglutination test (CATT/T. evansi) for detection of T. evansi [ 16 ], and VerY Diag (CEVA), which is a Lateral Flow Assay (LFA) for multiplex detection of T. congolense and T. vivax species. While these tests have aided epidemiological investigations of the target trypanosome species, they are Ab detection tests incapable of differentiating active infections from past exposures, hence restricting their scope. Where the test result is needed to inform treatment decisions, deployment of a confirmatory test is crucial. Microscopy and polymerase chain reaction (PCR) are valuable tests employed by reference laboratories to confirm AAT. However, microscopy is ineffective when specimen examination is delayed, time-consuming, laborious, not always field adapted, and it requires technical expertise. PCR on the other hand, is a high throughput sensitivity test; however, the technique requires reliable access to electricity and it is technically demanding. Therefore, conventional PCRs are not ideally suited for field operations. In contrast, Ag detection tests are amiable tool of choice for development of POCT for trypanosomiases, because they confirm active infection as well as drug failure. The deployment of Ag detection POCT has facilitated the control of malaria [ 17 ] and COVID-19 [ 18 ] among other infectious diseases. However, past efforts to develop similar mAb-based Ag detection tests for trypanosomiasis have been futile [ 19 ]. The mAb-based Ag detection test prototype for trypanosomiasis suffers from low sensitivity, mostly attributed to low Ag loads in specimens resulting from sequestration of Ag in immune complex, and an inherently low pathogen loads [ 20 ]. The first description of Nb technology 30 years ago by Hamers-Casterman et al [ 21 ] reinvigorated discovery research, targeting the technology, for the development of Ag detection tests for AAT [ 22 , 23 ]. Typically, Nbs possess a relatively extended complementarity determining region three (CDR3), compared to conventional Abs [ 24 ], thus allowing their preferential bindings to cryptic epitopes [ 25 , 26 ]. The fact that Nbs can bind Ag-Ab immune complexes through a unique epitope recognition, motivated its exploration as a tool of choice for outwitting low sensitivity of Ag-detection tests, caused by parasite-induced host Abs interference.

African trypanosomiasis (AT) is a devastating disease of humans and animals. Elimination of Human African Trypanosomiasis (HAT) of sleeping sickness, as a public health threat, is being targeted by the year 2030 [ 1 ]. The realization of this elimination would be a milestone in the fulfilment of the United Nations’s Sustainable Development Goals of eradicating poverty, ending hunger, and promoting good-health and well-being of the people [ 2 ]. Persistence of AT has affected development in communities where the disease occurs. Whereas trypanosome species responsible for HAT are restricted to Africa, some of the species causing the Animal African Trypanosomiasis (AAT) are prevalent far beyond the borders of Africa [ 3 , 4 ]. Hence, AT cases have been reported in South America [ 5 ], the Mediterranean Europe [ 6 ], and Asia [ 7 , 8 ] qualifying it among the most widely spread animal diseases in the world. Generally, AT is responsible for direct loss of lives [ 9 , 10 ], and it has a negative impact on the economy [ 11 ]. Employing chemicals to control the tsetse fly vector for AT, has raised environmental concerns including indiscriminate killing of non-target animals [ 12 ]. While elimination of AT would relieve the affected communities from the disease burden, it is both complex and highly demanding. An integrated approach involving vector control, case detection and treatment, has seen a near-elimination of gambiense-HAT cases in the disease endemic regions [ 13 ] holding promise for a future elimination altogether.

Methods

Ethics statement Permission for the use of mice was granted by the Ghent University Global Campus Institutional Animal Use and Care committee (Project number: IACUC 2022–014).

Mice Eight-weeks old female mice BALB/c and C57BL/6N were procured from the Korean Animal Technology (KOATEC) Co. Ltd, Republic of Korea. The animals were acclimatized for a fortnight in a facility at the Biomedical Research Centre, the Ghent University Global Campus, Republic of Korea. While the BALB/c mice were used for generation of hybridoma, C57BL/N6 were used for culturing trypanosomes.

Trypanosomes, trypanosome lysate, and sera Trypanosomes used in the study were Trypanosoma congolense TC13, T. b. brucei An Tat 1.1E, T. vivax ILRAD 700, and T. evansi STIB 816. Trypanosoma congolense was propagated, purified, and homogenized into lysate according to the protocol described elsewhere [22]. Briefly, aliquots of frozen trypanosome stocks (50 μl) were each reconstituted in 1xPBS (500 μl). The viability of cells was checked by wet smear and live parasites were quantified by a haemocytometer (Improved Neubauer China, Cat. No. 1103). For infection, 200 μl solution containing 5000 trypanosomes (T. congolense, T. b. brucei, T. vivax or T. evansi) was inoculated per mouse via intraperitoneal route. Mice were euthanized by CO 2 gas at the first peak of parasitaemia (1x108 trypanosomes/ml) and bled by cardiac puncture using a one ml syringe (Kovax-syringe, Korea Vaccine Co. Ltd) prefilled with heparin solution (40 μl). Obtained blood was pooled in a 15 ml centrifuge tube followed by centrifugation (1224 x g, 10 mins, 22°C). The buffy coat was collected and passed through a PD-10 column (Cytiva, Cat. No. 17043501) packed with DEAE Sepharose Fast Flow matrix (Cytiva, Cat. No. 17070901). The DEAE Sepharose Fast Flow matrix was pre-equilibrated with Phosphate Saline (PS) solution (NaCl, 36.5 mM; NaH 2 PO 4 , 3.6 mM; Na 2 HPO 4 , 59.5 mM) at either pH 7.5 (for T. congolense and T. vivax), or pH 8.0 (for T. b. brucei and T. evansi). Trypanosomes were eluted from the column using PS solution containing D-Glucose (88.8 mM) at either pH 7.5 or pH 8.0 for the respective species of trypanosomes. Eluted cells were centrifuged (1736 x g, 15 mins, 22°C) and obtained pellet was dissolved in 1xPBS (1000 μl). The lysate was prepared by resuspending the pellet followed by three rounds of freeze and thaw cycles alternating between -80°C and thaw 37°C, respectively. While on ice, the partially lysed cells were homogenized by sonication (Ultrasonic Processor K-SuperSonic KSS-N900DT, Korea Process Technology Co., Ltd.), and centrifuged (27,237 x g, 30 mins, 4°C). The supernatant was collected and the concentration of protein in the soluble lysate was estimated by a NanoDrop spectrophotometer and stored at—20°C. Sera used in the experiment were harvested from uninfected (naïve) as well as trypanosome-infected mice. In brief, mice were bled into a 1.5 ml centrifuge tube and blood was stored at 4°C for 2 days followed by centrifugation (9425 x g, 10 mins, 4°C). Afterwards, sera were harvested by a micropipette and stored at -20°C.

Recombinant proteins All recombinant proteins used in this study originated from past studies. Nb474 fused with his x6 peptide tag (Nb474H), Nb474 fused with both his x6 and haemagglutinin (HA) peptide tags (Nb474HA), TcoALD, T. vivax aldolase (TvALD), and Leishmania mexicana aldolase (LmALD) originated from [22]; T. congolense pyruvate kinase (TcoPYK) from [23]; and T. evansi enolase (TevENO) and Nb77 fused with his x6 peptide tag (Nb77H) from [27]. These proteins were produced, and purified by nickel affinity chromatography. The levels of production and purity of the recombinant proteins were analysed by SDS-PAGE. The concentration of the purified protein in the sample was measured by a NanoDrop and stored, in aliquots, at -20°C.

Mice immunization and analysis of immune response Prior to immunization, blood (2.5 μl) was collected by tail-snip from the BALB/c mice (n = 3), and it was diluted (1/200) in 1xPBS. The diluted blood samples were stored at -20°C until analysed by Ab-ELISA. On the day of immunization, TcoALD was diluted to a desired concentration in a sterile distilled water and then emulsified in Gerbu Adjuvant (Biotechnik GmbH, Cat. No. 3001-1mL) following the manufacturer’s guideline (Table 1). The Ag preparation was successively administered subcutaneously six times into scruff of the neck of the mice. Two days after the last booster shot, blood (2.5 μl) was collected from each of the immunized mice and it was diluted (1/200) in 1xPBS. The Ab levels in the blood sample preparations was analysed by ELISA. PPT PowerPoint slide

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TIFF original image Download: Table 1. The immunization schedules per mouse. https://doi.org/10.1371/journal.pntd.0012294.t001

Construction of hybridoma library A hybridoma library was generated using the ClonaCell-HY Hybridoma Kit (STEMCELL Technologies, Cat. No. 03800). A mouse with the highest Ab response was euthanized on day eight post last booster shot, and the spleen was harvested in 5 mL ClonaCell-HY Medium B (STEMCELL Technologies, Cat. No. 03802) followed by pulverisation in Medium B (5 ml) using a gentleMACS Dissociator (Miltenyi Biotec, Cat. No. 130-093-235). The macerated cell suspension was sieved through a 70 μm strainer (SPL cell strainer, Cat. No. 93070). The filtrate was diluted (1/6) in Medium B followed by centrifugation (316 x g, 10 mins, 22°C). This step was repeated twice. The washed cell pellet was resuspended in Medium B (25 ml) and cells were enumerated by a haemocytometer. Next, 1x108 splenocytes were obtained for fusion with NS01 parental myeloma. For preparation of NS01 cells, the Medium A passaged cells (1x107) were inoculated into a T-250 flask prefilled with Medium A (148 ml) a day before fusion. The cell suspension was later evenly distributed over 15 culture dishes (SPL Life Sciences Co., Ltd. Cat. No. 20100) followed by an overnight incubation in a humidified CO 2 incubator (5% CO 2 , 37°C). On the day of fusion, the cells were harvested by centrifugation (316 x g, 10 mins, 22°C) when they were in early-mid log phase (8.2x104 cells/ml). The pellet was resuspended in Medium B (30 ml) followed by centrifugation (316 x g, 10 mins, 22°C). The washing step was repeated twice and the pellet was resuspended in Medium B (25 ml) followed by enumeration using a haemocytometer. For fusion, the splenocytes (9.5x107cells in 19 ml) were mixed with the NS01 cells (1.6 x107 cells in 25 ml) in a 50 ml centrifuge tube followed by centrifugation (316 x g, 10 mins, 22°C). The pellet was disrupted by gentle tapping. ClonaCell-HY PEG (1 ml) was added dropwise to the pellet by a one ml sterile transfer pipette (VWR, Cat. No. VWRI612-1747) over a period of one minute without stirring. The cells were resuspended by the tip of a serological pipette for one minute by a continuous gentle stirring. Then Medium B (4 ml) was dispensed, dropwise over a period of four minutes, into the fusion mixture while stirring in-between the additions until all the solution was ejected. Additional Medium B (10 ml) was slowly added to the fusion mixture followed by incubation (15 mins, 37°C) in a water bath. Afterwards, Medium A was added twice to the cells (starting with 30 ml and then 40 ml), and each of these additions was followed by centrifugation (316 x g, 7 mins, 22°C). The supernatant was carefully drained after the last wash leaving behind the pellet, which was then slowly resuspended in ClonaCell-HY Medium C (10 ml) and transferred into a T-75 cm2 cell culture flask (CellStar cell culture flask Greiner bio one, Cat. No. 658170) prefilled with Medium C (20 ml). The cell mixture was incubated in a humidified incubator (5% CO 2 , 16 hrs, 37°C). The following day, fused cell suspension was transferred into a 50 ml centrifuge tube and centrifuged (316 x g,10 mins, 22°C). Obtained pellet was resuspended in Medium C (12 ml), and transferred into ClonaCell-HY Medium D (90 ml). The two solutions were mixed by gently inverting the bottle several times followed by incubation (5 mins, 37°C). The semi-solid medium was distributed (10 ml/plate) over ten 100 mm cell culture dishes. The seeded dishes were arranged in large square plates for a prolonged incubation. Each of the large square plates received at most three seeded dishes and a fourth dish filled with water only to provide local humidity. The assembled cultures were incubated for 10 days, without disturbance, in a humidified CO 2 incubator (5% CO 2 , and 37°C).

Hybridoma library screening and isotype characterization The hybridoma library was screened for anti-TcoALD mAb producing clones when the colonies were visible on the semi-solid medium eleven days after plating. During screening, a colony was drawn into a 10 μl micropipette tip and subsequently inoculated into a well of a 96-well cell culture (SPL Life technologies, Cat. No. 31096) prefilled with Medium E (100 μl). When all the wells were seeded, additional Medium E (50 μl) was dispensed into each of the seeded wells followed by incubation in a humidified CO 2 incubator (4 days, 37°C, 5% CO 2 ). On day four, supernatant (50 μl) was harvested per mini-culture and probed by ELISA for the presence of mAbs against TcoALD. For the ELISA, Half-Area ELISA plate (Corning, Cat. No. 3690) was coated with TcoALD (0.25 μg/well) diluted in 1xPBS for an overnight at 4°C. The wells were washed thrice with PBS-T. Blocking solution, 5% skimmed milk (Oxoid, Cat No: LP00338) in 1xPBS, was added (160 μl/well) to washed wells followed by incubation (2 hrs, 22°C). The blocking solution was discarded and the wells were washed thrice. Thereafter, the mini-culture supernatant (50 μl), previously harvested, was added into each of the wells followed by incubation (1 hr, 22°C). The supernatant was discarded and the wells were washed thrice. Goat anti-mouse immunoglobulin (Ig) horseradish peroxidase (HRP) (SouthernBiotech SBA Clonotyping System-HRP, Cat. No. 5300–05) diluted (1/1000) in 2.5% milk solution was added into the wells (50 μl/well) followed by incubation (1 hr, 22°C). The unbound antibodies were washed four times. Thereafter, 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Sigma, T0440-100 ml) was added (50 μl/well) followed by incubation (15 mins, 22°C). The reaction was stopped by adding 1M H 2 SO 4 (50 μl/well). The retrieved positive clones were subjected to a secondary screening to identify potential “false positive” clones producing mAbs against either his x6 -tag on recombinant TcoALD immunizing antigen, or E. coli expression host proteins, which co-purified with the recombinant TcoALD. For this ELISA, all the positive clones were probed for binding unpurified recombinant TcoPYK and Nb474H crude protein extracts (both proteins are fused with his x6 -tag and were not purified from the crude E. coli lysate), or purified Nb474H protein (a protein fused with his x6 -tag), while the purified recombinant TcoALD served as a positive control. The clones that bound TcoALD specifically were next (iso)typed (SouthernBiotech SBA Clonotyping System-HRP, Cat. No. 5300–05). For (iso)typing, TcoALD was coated (0.25 μg/well) followed by addition of supernatants (50 μl/well). Each of the supernatants was probed with assortment of goat anti-mouse HRP conjugates against mouse IgG1, IgG2a, IgG2b, IgG3 and IgM. Finally, a third screening was done to verify if the characterized mAbs would detect TcoALD, TcoPYK or 1xPBS in ELISA plate coated with Nb474H or Nb77H as capturing reagents.

Production and purification of monoclonal antibody The production of the only mAb clone (IgM8A2) that showed specific binding to TcoALD was upscaled after adapting to Hybridoma-Serum Free Medium (SFM) (gibco, Cat. No. 12045–084) and assessing its binding to T. congolense lysate (TcoLys). During adaptation, the IgM8A2 expressing clone cultured in Medium E was progressively exposed to Medium A. For this, Medium E was gradually reduced, from the Medium E-A mixture, by 25% until attaining 0% while Medium A was increased by 25% until reaching 100%. At the final stage of adaptation to 100% Medium A, the culture (2 ml) was first pelleted by centrifugation (316 x g, 10 mins, 22°C) and then resuspended in 100% Medium A (3 ml). The cell suspension was dispensed into a well on cell culture plate (SPL Life technologies, Cat. No. 30006) and incubated in a humidified CO 2 incubator (37°C, 5% CO 2 ). Afterwards, cells in Medium A were gradually adapted to SFM. As such, for complete adaptation to SFM, two cultures of 3 ml each, in a mixture of Medium A (25%) and SFM (75%) were pooled in a 15 ml centrifuge tube followed by centrifugation (316 x g, 10 mins, 22°C). The pellet was resuspended in SFM (6 ml) and the cell suspension was dispensed into two culture dishes (3 mL/dish) prefilled with SFM (12 ml/plate) followed by incubation in a humidified CO 2 incubator (37°C, 5% CO 2 ). At 1.74x106/ml cell density, the culture (15 ml) was inoculated into a vented tissue flask-T 175 prefilled with SFM (30 ml) and incubated in a humidified incubator (37°C, 5% CO 2 ). On day 4 post-inoculation when the culture has attained a stationary growth phase, characterized by yellowish coloration of the medium, the supernatant was harvested and centrifuged (316 x g, 10 mins, 22°C) followed by storage at -20°C. The purification of mAb was done by AKTA Start (Cytiva) employing UNICORN start 1.1 (Build1.1.0.2) software. During purification, 50 mL of the supernatant was defrosted and clarified by filtration through a 0.2 μM sieve (Sartorius Minisart, S6534). The filtrate was dialysed in 1M (NH 4 ) 2 SO 4 , pH 7.5 (500 ml) at 4°C using a dialysis tubing (Spectra/Por Dialysis Membrane Standard RC tubing) of pore size 6-8kD. The dialysate (50 mL) was loaded (0.5ml/min) onto a HiTrap IgM purification column (Cytiva, Cat. No. 17-5110-01), which was pre-equilibrated with five column volume of the binding buffer [20 mM Sodium Phosphate, 1M (NH 4 ) 2 SO 4 , pH 7.5]. Thereafter, copious quantity of binding buffer was passed through the column to wash-off the unbound impurities until A 280 returned to baseline. The retained Ab was eluted out of the column by washing with elution buffer (20 mM Sodium Phosphate Buffer, pH 7.5) at 0.5 ml/min. The eluted Ab was dialysed in 1xPBS pH 7.4 at 4°C. The concentration of Ab in the sample was estimated by a NanoDrop spectrophotometer and the sample was stored at -20°C.

Biotin-labelling of monoclonal antibody The IgM8A2 monoclonal antibody was labelled with biotin, using the EZ-Link Sulfo-NHS-Biotin kit (Thermo Scientific, Cat. No. 21217). Biotin powder (4 mg) in the kit was reconstituted in distilled water (500 μl) and the solution (40 μl) was added into a IgM8A2 solution (1000 μl), which was at a concentration of 1 mg/ml. The mixture was incubated on ice for an overnight. Thereafter, the biotin-labelled IgM8A2 (IgM8A2-B) was dialysed in 1xPBS pH 7.4 using Slide-A-Lyzer 3.5 K Dialysis Cassettes (Thermo Scientific, Cat. No. 66330). The concentration of IgM8A2-B in the solution was measured by a NanoDrop spectrophotometer and the sample was stored at -20°C. The success of IgM8A2 biotin-labelling was ascertained by ELISA employing the HRP Streptavidin (Strep-HRP) (Biolegend, Cat. No. 405210 / 1 ml) and TMB reporter system.

Assessing binding properties of retrieved monoclonal antibody Binding of IgM8A2 to a denatured aldolase was assessed by western blot and an indirect ELISA. For western blot, aldolase was resolved by SDS-PAGE under denaturing condition and electroblotted onto nitrocellulose membrane (Thermo scientific, Cat. No. 88018). The blotted protein was probed with a solution of an unlabelled IgM8A2 (4.47 μg/ml) in 2.5% milk followed by goat anti-mouse IgM HRP (SouthernBiotech, Cat. No. 5300–05) diluted (1/1000) in 2.5% milk. The retention of conjugated anti-mouse IgM by IgM8A2 was revealed by incubation in HRP substrate solution [10 ml 99% methanol (10 ml), 4-Chloro-1-napthol powder (45 mg), 1xPBS (45 ml), and 30% hydrogen peroxide (100 μl)]. An indirect ELISA was used to investigate the binding of IgM8A2 to a heat-denatured aldolase. Briefly, recombinant TcoALD or TvALD was diluted to 200 μg/ml, and aliquoted (120 μl/vial) followed by incubation at 55°C for different time lengths (0, 10, 20, 30, 40, 50, and 60 mins). The samples were coated (10 μg/well) on ELISA plate for an overnight at 4°C. The coating was discarded and wells were blocked with 5% milk solution (160 μl/well) for 2 hrs at 22°C. The wells were washed thrice and IgM8A2 diluted to 4.74 μg/ml in blocking buffer was added (50 μl/well) followed by incubation (1 hr, 22°C). Wells were washed 4 times and a dilution (1/1000) of goat anti-mouse IgM HRP was added (50 μl/ well) followed by incubation (1 hr, 22°C). Wells were washed-off excess unbound HRP conjugate five times. TMB substrate (Sigma, T0440-100 ml) was added (50 μl/well) and incubation was allowed for 15 min. The reaction was stopped with 1M H 2 SO 4 (50 μl/well) and OD 450nm was read. Next, the binding of IgM8A2 to the aldolases of other livestock infective trypanosome species, besides T. congolense, was assessed by an indirect immunofluorescent assay. Fixed-permeabilized trypanosomes were incubated in IgM8A2-B solution (2.5 μg/ml) or Biotin anti-mouse IgM Antibody (Biolegend, Cat. No. 406504/500 μg) solution (2.5 μg/ml) followed by Cy3 Streptavidin (Biolegend, Cat. No. 405215) solution (1 μg/ml) as described (S1 Materials and Methods). The specificity of Nb474H/IgM8A2-B “hybrid” sandwich system for detection of aldolase was assessed on recombinant TcoALD, TevENO or 1xPBS by ELISA employing the Strep-HRP and TMB reporter system. Finally, to asses the competition for binding TcoALD between IgM8A2 (Ag-detection reagent) and Nb474 (Ag-capture reagent), a competition ELISA was conducted as described (S2 Materials and Methods).

ELISA titration of antigen capture Nanobody against detection monoclonal antibody Nb474H (5 μg/ml) was serially diluted (two-fold) in 1x PBS until a final concentration of 0.005 μg/mL. Except for the wells on column 12, which were filled with 1xPBS (50 μl/well), the dilutions were coated (50 μl/well) on respective wells (S1 Table) followed by overnight incubation at 4°C. Next, wells were emptied, washed thrice, and SuperBlock blocking buffers (thermoscientific, Cat. No. 37516) was added in coated wells (160 μl/well) for 2 hrs at 22°C with an hourly buffer refreshment. Afterward, the blocking buffer was emptied and the wells were washed thrice. A constant amount of TcoALD antigen was added across the plate (0.25 μg/well) followed by incubation for an hour at 22°C. Wells were washed thrice. Next, two-fold serial dilutions of IgM8A2-B, starting from 5 μg/mL until 0.08 μg/ml, were added row-wise in a decreasing concentration except for wells in row H, which received 1xPBS only (S2 Table). The reaction was incubated for an hour at 22°C and the wells were washed four times. Strep-HRP diluted to 0.5 μg/ml in SuperBlock was added (0.025 μg/well) into the wells followed by incubation for an hour at 22°C. The enzyme conjugate was emptied and the wells were washed five times. TMB substrate (Sigma, T0440-100 ml) was added (50 μl/well) and color development was allowed for 15 mins. The reaction was stopped by adding 1M H 2 SO 4 (50 μl/well) and OD 450nm was read.

Analytical sensitivity of N474H/IgM8A-B “hybrid” sandwich ELISA A fixed amount of Nb474H was coated (0.1 μg/well) in wells on rows B-E, columns 2–11 and incubated for an overnight at 4°C. The following morning, coating solution was emptied and the wells were washed thrice. Washed wells were blocked by adding SuperBlock (160 μl/well) for 2 hrs at 22°C with an hourly refreshment. The blocking solution was discarded and wells were washed thrice. Serial dilutions (two-fold) of TcoALD (100–0.0004 μg/ml) were added into duplicate wells with control wells (D11E11) receiving 1xPBS only (S3 Table). The reaction was incubated for an hour at 22°C. The unbound antigens were discarded and the wells were washed thrice. The IgM8A2-B diluted to 2.5 μg/mL in SuperBlock was added into the wells (50 μl/well) followed by incubation for an hour at 22°C. Wells were washed four times and Strep-HRP diluted to 0.5 μg/ml in blocking buffer was added (50 μl/well) followed by an hour of incubation at 22°C. The HRP conjugate was discarded and wells were washed five times. TMB substrate (Sigma, T0440-100 ml) was added (50 μl/well) into the wells and incubated for 15 mins. The reaction was stopped by adding an equal volume of 1M H 2 SO 4 solution and the absorbance was read at 450nm.

Detection of T. congolense infections by Nb474H/IgM8A2-B “hybrid” sandwich ELISA Sera collected from the naïve or mice experimentally infected with a panel of trypanosome species including T. congolense TC13 at parasitemia 1x108 trypanosomes/ml, T. b. brucei AnTat1.1E at parasitemia 4.8x108 trypanosomes/ml, T. evansi STIB 816 at parasitemia 2.7x108 trypanosomes/ml, or T. vivax ILRAD 700 at parasitemia 4.1x108 trypanosomes/ml were examined with the Nb474H/IgM8A2-B “hybrid” sandwich ELISA. Nb474H was coated (0.1 μg/well) and incubated for an overnight at 4°C. Wells were emptied and washed thrice. Washed wells were blocked with 10% BSA (BOVOGEN, Cat. No. BSAS 0.1) at 160 μl/well and incubated for an hour at 22°C. The blocking buffer was refreshed and incubation was continued for an hour before discarding. Undiluted sera were added (50 μl/well) in duplicate into emptied wells. Both recombinant TcoALD (10 μg/well) and T. congolense lysate (5 μg/well) were used as positive controls for the ELISA. Incubation was performed for one hour at 22°C. The unbound antigens were discarded and the wells were washed thrice. Next, IgM8A2-B diluted to 2.5 μg/ml in 10%BSA was added into the wells (50 μl/well) followed by incubation for an hour at 22°C. The wells were washed four times. A strep-HRP solution diluted to 0.25 μg/ml in 10% BSA was added into the wells (50 μl/well) followed by incubation for 30 mins at 22°C. The conjugate was discarded and the wells were washed six times. Thereafter, color development was initiated by adding TMB substrate (Sigma, T0440-100 ml) at 50 μl/well. Incubation was allowed for 15 mins and the reaction was stopped by adding 1M H 2 SO 4 (50 μl/well) followed by OD reading at 450nm.

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[1] Url: https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0012294

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