Optimal Refolding Research Articles

Investigation of lipid/protein interactions in trifluoroethanol-water mixtures proposes the strategy for the refolding of helical transmembrane domains.

Membrane proteins are one of the keystone objects in molecular biology, but their structural studies often require an extensive search for an appropriate membrane-like environment and an efficient refolding protocol for a recombinant protein. Isotropic bicelles are a convenient membrane mimetic used in structural studies of membrane proteins. Helical membrane domains are often transferred into bicelles from trifluoroethanol-water mixtures. However, the protocols for such a refolding are empirical and the process itself is still not understood in detail. In search of the optimal refolding approaches for helical membrane proteins, we studied here how membrane proteins, lipids, and detergents interact with each other at various trifluoroethanol-water ratios. Using high-resolution NMR spectroscopy and dynamic light scattering, we determined the key states of the listed compounds in the trifluoroethanol/water mixture, found the factors that could be critical for the efficiency of refolding, and proposed several most optimal protocols. These protocols were developed on the transmembrane domain of neurotrophin receptor TrkA and tested on two model helical membrane domains-transmembrane of Toll-like receptor TLR9 and voltage-sensing domain of a potassium channel KvAP.

Application of biophysical methods for improved protein production and characterization: A case study on an high-temperature requirement A-family bacterial protease.

The high-temperature requirement A (HtrA) serine protease family presents an attractive target class for antibacterial therapeutics development. These proteins possess dual protease and chaperone functions and contain numerous binding sites and regulatory loops, displaying diverse oligomerization patterns dependent on substrate type and occupancy. HtrA proteins that are natively purifiedcoelute with contaminating peptides and activating species, shifting oligomerization and protein structure to differently activated populations. Here, a redesigned HtrA production results in cleaner preparations with high yields by overexpressing and purifying target protein from inclusion bodies under denaturing conditions, followed by a high-throughput screen for optimal refolding buffer composition using function-agnostic biophysical techniques that do not rely on target-specific measurements. We use Borrelia burgdorferi HtrA to demonstrate the effectiveness of our function-agnostic approach, while characterization with both new and established biophysical methods shows the retention of proteolytic and chaperone activity of the refolded protein. This systematic workflow and toolset will translate to the production of HtrA-family proteins in higher quantities of pure and monodisperse composition than the current literature standard, with applicability to a broad array of protein purification strategies.

Overproduction, Purification and Refolding of codon-optimized Hepatitis B Virus X Protein Subgenotype B3 in Escherichia coli BL21(DE3)

Hepatitis B virus (HBV) infects human and causes chronic liver infection, leading to liver cirrhosis and hepatocellular carcinoma. HBV X (Hbx) protein is known to interact with tumor suppressor protein p53 and block its translocation into the nucleus. This study outlines the overproduction of Hbx protein from HBV subgenotype B3 in Escherichia coli BL21(DE3), including its purification and refolding. The gene encoding Hbx was first codon-optimized and inserted into pET16b. The recombinant plasmid was then transformed into E. coli BL21(DE3) as an expression host. Optimization of Hbx expression was performed with variation of IPTG concentration and overproduction temperature. The results showed that Hbx protein was optimally induced by 0.075 mM IPTG and overproduction of Hbx at 17, 25, and 37°C exhibited no difference in protein level and location. The optimal refolding of Hbx was obtained using 0.1 M arginine prior to elution from Nickel column using 100 mM imidazole and 0.25 M arginine. Hbx migrates differently in SDS-PAGE reducing and non-reducing, while the melting curve pattern in TSA analysis changed after the refolding step. Essentially, this purified Hbx protein could potentially be used for interaction study with p53 and the inhibitor candidate of the protein.

A Simplified Process for Purification and Refolding of Recombinant Human Interferon-α2b

Background: Interferon α-2b is a vital biotherapeutic produced through the recombinant DNA technology in E. coli. The recombinant IFN-α2b normally appears as intercellular IBs, which requires intensive refolding and purification steps. Method:Purification of IFN-α2b from solubilized IB was performed using two-phase extraction. To optimize refolding conditions, the effects of pH and different additives, including cysteine, cystine, urea, glycerol, Triton X-100, NaCl, and arginine, were investigated. Optimal refolding buffer (0.64 mM of urea, 5.57 mM of cysteine ​​, and 1.8 mM of cystine) was obtained using RSM. The refolding process was performed by an optimized refolding buffer in the dilution and fed-batch refolding method at different protein concentrations (25-1000 µg/mL). Result: At a final protein concentration of 500 µg/mL, the fed-batch refolding method yielded in a biological activity of 2.24 × 108 IU/mg, which was nearly twice that of dilution method. Conclusion:Fed-batch refolding method resulted in the biologically active IFN-α2b with high purity, which can be used for research and industrial purposes.

Efficient refolding of recombinant reteplase expressed in Escherichia coli strains using response surface methodology

Reteplase is a deleted variant of human tissue plasminogen activator with a complex structure containing nine disulfide bonds. Reteplase is expressed as inclusion bodies in Escherichia coli and needs the additional step of refolding for activation. In this study an experimental design was performed to find the optimal refolding condition for reteplase. The influence of 14 chemical additives was assessed by one factor at a time method and then Taguchi design followed by response surface methodology was employed to find compounds with most significant effects on reteplase refolding and their optimum concentration. We found that 0.13 M histidine, 1.64 M methionine, 0.33 M cysteine, and 0.34 M arginine in addition to the GSH/GSSG is the optimal condition for refolding of reteplase. We also investigated the refolding yield for inclusion bodies obtained from different E. coli strains and found that BL21 (DE3) has the best recovery yield in comparison to Rosetta-gami and Shuffle T7.

Selection conditions of the recombinant bovine α-interferon from E. coli inclusion bodies

Like other proteins of the cytokine family, bovine α-interferon activates and modulates antiviral state of the target cells and inhibits division and growth of the infected cells which makes it an excellent candidate as a new antiviral therapeutic agent.This study is concerned with the determination of the optimal isolation, purification and refolding conditions of the recombinant bovine interferon-α (rbIFN-α) from inclusion bodies (IBs). Main methods used were UV/Visible spectroscopy, electrophoresis, liquid chromatography and refolding by dilution.It was found that two step IBs washing with solutions containing 50 mmol/l Tris, 50 mmol/l NaCl and 3.5 mol/l urea and their subsequent solubilization in 50 mmol/l Tris-HCl, pH 9.8 mol/l Urea and 20 mmol/l β-mercaptoethanol allow us to receive the target protein in monomeric form and 53.18 ± 9.3 % purity. Further application of the anion-exchange tandem chromatography on DE 52 cellulose and toyopearl DEAE-650 M gives a possibility to remove the major impurities and obtain rbIFN-α with 80.7 ± 8.6 % purity. Refolding by dilution in the buffer containing 20 mmol/l NaPB, рН 7.4, 0.4 mol/l sucrose, 1 mmol/l L-Cys, 0.1 mmol/l L-Cystine, 1 mmol/l EDTA, 0.05 % Kolliphor EL at 10 °C followed by the protein collection allows to get the recombinant rbIFN-α in homogeneous state, with 98.43 % purity and antiviral activity about (5 ± 3.6)•106 U/mg.

Protein refolding based on high hydrostatic pressure and alkaline pH: Application on a recombinant dengue virus NS1 protein.

In this study we evaluated the association of high hydrostatic pressure (HHP) and alkaline pH as a minimally denaturing condition for the solubilization of inclusion bodies (IBs) generated by recombinant proteins expressed by Escherichia coli strains. The method was successfully applied to a recombinant form of the dengue virus (DENV) non-structural protein 1 (NS1). The minimal pH for IBs solubilization at 1 bar was 12 while a pH of 10 was sufficient for solubilization at HHP: 2.4 kbar for 90 min and 0.4 kbar for 14 h 30 min. An optimal refolding condition was achieved by compression of IBs at HHP and pH 10.5 in the presence of arginine, oxidized and reduced glutathiones, providing much higher yields (up to 8-fold) than association of HHP and GdnHCl via an established protocol. The refolded NS1, 109 ± 9.5 mg/L bacterial culture was recovered mainly as monomer and dimer, corresponding up to 90% of the total protein and remaining immunologically active. The proposed conditions represent an alternative for the refolding of immunologically active recombinant proteins expressed as IBs.

Preparation of recombinant human thymic stromal lymphopoietin protein

ABSTRACTHuman thymic stromal lymphopoietin (hTSLP) protein plays a central role in inflammation. Characterizing properties of hTSLP requires a recombinant overexpression system that produces correctly folded, active hTSLP. In this report, an efficient overexpression system for the production of hTSLP was developed. We constructed expression plasmids of the full-length hTslp gene with or without the signal peptide and transformed the plasmids into Escherichia coli. The design of the recombinant proteins included an N-terminal His-tag, which facilitated purification. An affinity gradient elution method was used to improve recovery and concentration levels of denatured hTSLP, with 90% purity observed following affinity chromatography. Refolding of the denatured hTSLP was tested using four different protein refolding approaches. The optimal refolding conditions involved stepwise buffer exchanges to reduce the urea concentration from 4 to 0 M in 50 mM Tris (pH 8.0), 1 mM EDTA, 50 mM NaCl, 10% glycerol, 400 mM L-Arg, 0.2 mM oxidized glutathione, and 2 mM reduced glutathione. The activity of the refolded recombinant hTSLP protein was measured by an ELISA assay. Interestingly, the presence of N-terminal signal peptide inhibited the overexpression of hTSLP in E. coli. The amount of recombinant hTSLP protein purified reached a level of 2.52 × 10−3 mg/L.

Large-Scale Refolding and Enzyme Reaction of Human Preproinsulin for Production of Human Insulin.

Human insulin is composed of 21 amino acids of an A-chain and 30 amino acids of a B-chain. This is the protein hormone that has the role of blood sugar control. When the recombinant human proinsulin is expressed in Escherichia coli, a serious problem is the formation of an inclusion body. Therefore, the inclusion body must be denatured and refolded under chaotropic agents and suitable reductants. In this study, H27R-proinsulin was refolded from the denatured form with β-mercaptoethanol and urea. The refolding reaction was completed after 15 h at 15°C, whereas the reaction at 25°C was faster than that at 15°C. The refolding yield at 15°C was 17% higher than that at 25°C. The refolding reaction could be carried out at a high protein concentration (2 g/l) using direct refolding without sulfonation. The most economical and optimal refolding condition for human preproinsulin was 1.5 g/l protein, 10 mM glycine buffer containing 0.6 M urea, pH 10.6, and 0.3 mM β-mercaptoethanol at 15°C for 16 h. The maximum refolding yield was 74.8% at 15°C with 1.5 g/l protein. Moreover, the refolded preproinsulin could be converted into normal mature insulin with two enzymes. The average amount of human insulin was 138.2 g from 200 L of fermentation broth after enzyme reaction with H27R-proinsulin. The direct refolding process for H27R-proinsulin was successfully set up without sulfonation. The step yields for refolding and enzyme reaction were comparatively high. Therefore, our refolding process for production of recombinant insulin may be beneficial to the large-scale production of other biologically active proteins.

Important amino acid residues for the crab toxicity of PaTX, a type 3 sodium channel peptide toxin from the sea anemone Entacmaea actinostoloides

Sodium channel toxins from various sea anemones can generally be classified into three types. Type 3 toxins (27–32 residues) are distinct from type 1 and 2 toxins (46–49 residues) in chain length. Of the known type 3 toxins (ATX III, Da I, Da II, PaTX, and Er I), ATX III has been clarified for structure–activity relationships. However, the data obtained with ATX III do not seem to be universal for type 3 toxins, since ATX III is significantly different in conformation as well as primary structure from the other type 3 toxins. Thus the aim of this study was to identify the amino acid residues that are important for the toxicity of PaTX. First, an optimal refolding condition was established using chemically synthesized Er I. Then, PaTX (a peptide one residue shorter in length than Er I) and its analogs, each of which has an Ala substitution, were synthesized, refolded, and purified using reverse-phase high-performance liquid chromatography (HPLC). The crab toxicities determined for the purified preparations showed two cationic residues (Lys-4 and His-27) and three hydrophobic residues (Tyr-15, Pro-20, and Trp-21) that were strongly implicated in the toxicity of PaTX. The results with PaTX are substantially applicable to Da I, Da II, and Er I.

Continuous protein refolding in a tubular reactor

Continuous protein refolding by direct dilution was established in a laboratory-scale tubular reactor. Advanced refolding strategies by pulsed refolding and temperature leap, favoring the native pathway of specific model proteins were also implemented. The reactor was tested with two autoprotease fusion proteins, EDDIE-pep6His and 6His-EDDIE-GFPmut3.1, and also on carbonic anhydrase II. Direct refolding kinetics approximated by a folding kinetic of first order and aggregation kinetic of second order was similar to batch processes, while pulse and temperature leap refolding yields were higher than direct batch refolding method. Productivity comparisons calculated as amount of refolded protein per reactor volume and process time showed that an optimal refolding concentration to achieve highest productivity value is a balance between a rational reactor volume and a reasonable refolding time. Productivity in a tubular reactor is always higher as emptying and refilling times required for batch reactor decreases productivity. This productivity improvement is higher for a fast refolding protein than a slower one.

Getting ready for PAT: Scale up and inline monitoring of protein refolding of Npro fusion proteins

Screening for optimal refolding conditions for recombinant protein overexpressed in Escherichia coli as inclusion bodies is often carried out on micro-scale in non-agitated reactors. Currently, scale up of refolding of Npro fusion proteins is based on geometric similarity and constant Re number. Refolding/cleavage kinetics is recorded offline by HPLC and via fluorescence intensity. We show that the results for refolding obtained on the micro-scale can be transferred to the laboratory scale stirred tank reactor, with increases in scale up to a factor of 5000, with high agreement of kinetic constants and yield. Progress of refolding kinetics on the laboratory scale is monitored inline by attenuated total reflectance – Fourier transform infrared spectroscopy (ATR-FTIR). Addressing the demands for better process understanding, we demonstrate that ATR-FTIR enables the inline monitoring of refolding processes on the laboratory scale, replacing offline analysis which delivers the results with a time delay. Implementing inline monitoring will allow the integration of process control, thereby resulting in a more efficient and knowledge based production process.

Refolding of recombinant human interferon gamma inclusion bodies in vitro assisted by colloidal thermo-sensitive poly(N-isopropylacrylamide) brushes grafted onto the surface of uniform polystyrene cores

Recombinant human interferon gamma (rhIFN-γ) is a protein with great potential for clinical therapy, but rhIFN-γ expressed in Escherichia coli is usually in the form of insoluble inclusion bodies which should be refolded in vitro. A novel type of hairy particles (PNIPAM-grafted-PS) consisted of submicron polystyrene cores and brushes of thermo-sensitive poly(N-isopropylacrylamide) grafted onto the cores was prepared and then applied to assist the refolding of rhIFN-γin vitro. Two kinds of PNIPAM-grafted-PS particles with different thickness of brush layer (55nm and 110nm) were synthesized, which were spherical shape with good dispersion properties and the LCST was about 33°C. The effect of thickness of brush layer, particle concentration and temperature on the refolding process was investigated, it was shown that particles with larger thickness of brush layer were more effective and the final rhIFN-γ activity could be up to more than 21 times of that in dilution refolding when initial rhIFN-γ concentration was 50μg/mL. The optimal refolding condition was the concentration ratio of particle to rhIFN-γ 1:1 and refolding temperature of 15°C. All results above demonstrated that PNIPAM-grafted-PS particles could assist rhIFN-γ refolding which presented an alternative way to facilitate recombinant protein refolding in vitro.

Statistical vs. Stochastic experimental design: An experimental comparison on the example of protein refolding

Optimization of experimental problems is a challenging task in both engineering and science. In principle, two different design of experiments (DOE) strategies exist: statistical and stochastic methods. Both aim to efficiently and precisely identify optimal solutions inside the problem-specific search space. Here, we evaluate and compare both strategies on the same experimental problem, the optimization of the refolding conditions of the lipase from Thermomyces lanuginosus with 26 variables under study. Protein refolding is one of the main bottlenecks in the process development for recombinant proteins. Despite intensive effort, the prediction of refolding from sequence information alone is still not applicable today. Instead, suitable refolding conditions are typically derived empirically in large screening experiments. Thus, protein refolding should constitute a good performance test for DOE strategies. We compared an iterative stochastic optimization applying a genetic algorithm and a standard statistical design consisting of a D-optimal screening step followed by an optimization via response surface methodology. Our results revealed that only the stochastic optimization was able to identify optimal refolding conditions (~1.400 U g(-1) refolded activity), which were 3.4-fold higher than the standard. Additionally, the stochastic optimization proved quite robust, as three independent optimizations performed similar. In contrast, the statistical DOE resulted in a suboptimal solution and failed to identify comparable activities. Interactions between process variables proved to be pivotal for this optimization. Hence, the linear screening model was not able to identify the most important process variables correctly. Thereby, this study highlighted the limits of the classic two-step statistical DOE.

Refolding and two-step purification by hydrophobic interaction chromatography of recombinant human bone morphogenetic protein-2 from Escherichia coli

To renature the inactive rhBMP-2 which overexpressed in Escherichia coli, post-expression treatments including inclusion bodies solubilization and in vitro refolding were systematically investigated. An optimized refolding process was established from screening and successfully scaled up with yield greater than 70%. Then, hydrophobic interaction chromatography (HIC) was adopted as two consecutive stages to separate the active rhBMP-2 homodimer from refolding mixture. Aiding additive N,N-dimethylformamide (DMF) was found to enhance the resolution of rhBMP-2 homodimer most effectively. The rhBMP-2 homodimer was purified to homogeneity through two HIC separations at different salt contents, the purified rhBMP-2 homodimer was fully bioactive and had equivalent biological activity to rhBMP-2 produced from Chinese hamster ovary cell (CHO). Under the optimal refolding and purification conditions, 80mg rhBMP-2 homodimer with high purity could be obtained from 1g wet weight of inclusion bodies. Finally, this efficient refolding and purification procedure was successfully scaled up in the pilot pharmaceutical plant.

A generic hierarchical screening method for the analysis of microscale refolds using an automated robotic platform

The refolding of protein derived from inclusion bodies is often characterized by low yields of active protein. The optimization of the refolding step is achieved empirically and consequently is time-consuming slowing process development. An automated robotic platform has been used to develop a dilution refold process-screening platform upon which a hierarchical set of assays rapidly determine optimal refolding conditions at the microscale. This hierarchy allows the simplest, cheapest, and most generic high-throughput assays to first screen for a smaller subset of potentially high-yielding conditions to take forward for analysis by slower, more expensive, or protein specific assays, thus saving resources whilst maximizing information output. An absorbance assay was used to initially screen out aggregating conditions, followed by an intrinsic fluorescence assay of the soluble protein to identify the presence of native-like tertiary structure, which was then confirmed by an activity assay. Results show that fluorescence can be used in conjunction with absorbance to eliminate low-yielding conditions, leaving a significantly reduced set of conditions from which the highest yielding ones can then be identified with slower and often more costly activity or RP-HPLC assays, thus reducing bottlenecks in high-throughput analysis. The microwell-based automated process sequence with generic hierarchical assays was also used to study and minimize the effect on redox potential or misfolding, of oxygenation due to agitation, before demonstrating that the platform can be used to rapidly collect data and evaluate different refolding conditions to speed up the acquisition of process development data in a resource efficient manner.

A high-throughput protein refolding screen in 96-well format combined with design of experiments to optimize the refolding conditions

Production of correctly folded and biologically active proteins in Escherichia coli can be a challenging process. Frequently, proteins are recovered as insoluble inclusion bodies and need to be denatured and refolded into the correct structure. To address this, a refolding screening process based on a 96-well assay format supported by design of experiments (DOE) was developed for identification of optimal refolding conditions. After a first generic screen of 96 different refolding conditions the parameters that produced the best yield were further explored in a focused DOE-based screen. The refolding efficiency and the quality of the refolded protein were analyzed by RP-HPLC and SDS–PAGE. The results were analyzed by the DOE software to identify the optimal concentrations of the critical additives. The optimal refolding conditions suggested by DOE were verified in medium-scale refolding tests, which confirmed the reliability of the predictions. Finally, the refolded protein was purified and its biological activity was tested in vitro. The screen was applied for the refolding of Interleukin 17F (IL-17F), stromal-cell-derived factor-1 (SDF-1α/CXCL12), B cell-attracting chemokine 1 (BCA-1/CXCL13), granulocyte macrophage colony stimulating factor (GM-CSF) and the complement factor C5a. This procedure identified refolding conditions for all the tested proteins. For the proteins where refolding conditions were already available, the optimized conditions identified in the screening process increased the yields between 50% and 100%. Thus, the method described herein is a useful tool to determine the feasibility of refolding and to identify high-yield scalable refolding conditions optimized for each individual protein.

Expression, purification, and refolding of active Nrf2 transcription factor fused to protein transduction TAT tag

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor responsible for activation of diverse genes that protect the cell against xenobiotics and oxidative stress. The Nrf2-dependent transcription is tightly controlled by cytoplasmic interaction of Nrf2 with its inhibitor, Kelch ECH-associating protein 1 (Keap1). The Keap1-mediated inhibition can be overcome by addition of xenobiotics or by overexpression of Nrf2 protein. The overexpressed Nrf2 overwhelms the Keap1 inhibition, translocates into the nucleus and activates antioxidant response element (ARE)-dependent gene transcription that protects the cell against oxidative stress. We expressed and purified recombinant mouse Nrf2 protein fused to a protein transduction domain (TAT), derived from transactivator of transcription protein from HIV-1. Full-length TAT-Nrf2 was expressed in Escherichia coli in insoluble inclusion bodies and purified to homogeneity using denaturing size exclusion chromatography. Optimal refolding conditions were determined through the use of a light scattering-based refolding assay and analytical size exclusion chromatography. The results demonstrate that the refolded TAT-Nrf2 could transduce into cultured human neuroblastoma cells. The transduced TAT-Nrf2 activated transcription of ARE-dependent genes and conferred protection against intracellular reactive oxygen species (ROS) generated by hydrogen peroxide exposure.

Purification of truncated and mutated Chemotaxis Inhibitory Protein of Staphylococcus aureus—an anti-inflammatory protein

The Chemotaxis Inhibitory Protein of Staphylococcus aureus (CHIPS) binds and blocks the C5a receptor (C5aR) and formyl-peptide receptor (FPR). This way, CHIPS is a potent inhibitor of the immune cell recruitment associated with inflammation. Truncation of the protein and the introduction of mutations, shifts the expression towards the insoluble fraction of Escherichia coli, whereas the wild-type protein can be solubly expressed. A protocol for expression and tag independent purification of biologically active CHIPS variants has been established to enable further characterization of an improved CHIPS variant, called ADC-1004. The CHIPS variants were purified by washing of E. coli inclusion bodies followed by refolding and gel filtration. New techniques were utilized to optimize the purification process. Expression in inclusion bodies was increased by the use of Ultra Yield ™ flasks and optimal refolding conditions were determined by the use of the iFOLD Refolding System 2 ™. The folding and biological activity of the purified proteins were analyzed by circular dichroism (CD) spectroscopy and flow cytometry, respectively, and compared to solubly produced CHIPS 31–113 and wild-type CHIPS 1–121. We show that the CHIPS variants produced in inclusion bodies can be refolded and purified to achieve equal biological activity as solubly produced CHIPS 31–113 and wild-type CHIPS 1–121. The truncation causes minor structural changes while purification from inclusion bodies or the soluble fraction does not further affect the structure.

Purification and characterization of recombinant extracellular domain of human HER2 from Escherichia coli

The human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor (EGFR) family, and it plays an important role in the development of many human adenocarcinomas. The extracellular domain (ECD) of HER2 is an ideal target for therapeutic approaches. In order to obtain large quantities of active HER2 ECD protein for biochemical and structural analysis and for detecting anti-HER2 ECD antibodies in serum, a systematic assessment of optimal parameters for the refolding of the glutathione S-transferase (GST) fusion protein was carried out. After the GST-HER2 ECD inclusion bodies were solubilized with denaturation buffer containing 8M urea, an approach was then used to optimize refolding parameters. This approach utilized dilution of denatured and reduced GST-HER2 ECD into different refolding buffers using orthogonal design method. Optimal refolding was obtained in an alkaline buffer containing reduced and oxidized glutathione, and subsequent incubation at 4°C for 24h. After purification with glutathione Sepharose 4B and PreScission protease cleavage of the fusion protein, 8.9mg of recombinant HER2 ECD was obtained from 1L of Escherichia coli. Rabbit polyclonal antibodies against HER2 ECD were obtained. The purified protein was found to be immunogenic and useful for immunodiagnostic studies of serum HER2 ECD and its antibodies by using enzyme-linked immunosorbent assay (ELISA).