Strongyloidiasis Serological Analysis with Three Different Biological Probes and Their Electrochemical Responses in a Screen-Printed Gold Electrode.

(1) Background: The validation of biological antigens is the study's utmost goal in biomedical applications. We evaluated three different probes with single and multiple epitopes through electrochemical detection of specific IgG in serum for human strongyloidiasis diagnosis. (2) Methods: Screen-printed gold electrodes were used and probes consisting of two single-epitope synthetic peptides (D3 and C10) with different sequences, and a multi-epitope antigen [detergent phase (DP)-hydrophobic membrane proteins]. Human serum samples from three populations were used: Strongyloides stercoralis positive, positive for other parasitic infections and negative controls. To test the immobilization of probes onto a screen-printed gold electrode and the serum IgG detection, electrochemical analyses were carried out through differential pulse voltammetry (DPV) and the electrode surface analyses were recorded using atomic force microscopy. (3) Results: The electrochemical response in screen-printed gold electrodes of peptides D3 and C10 when using positive serum was significantly higher than that when using the DP. Our sensor improved sensitivity to detect strongyloidiasis. (4) Conclusions: Probes' sequences are critical factors for differential electrochemical responses, and the D3 peptide presented the best electrochemical performance for strongyloidiasis detection, and may efficiently substitute whole antigen extracts from parasites for strongyloidiasis diagnosis in electrochemical immunosensors.

Performance of phospho-L-tyrosine immobilized onto alginate/polyacrylamide-based cryogels: Effect of ligand coupling on human IgG adsorption and Fab fragments separation.

The ortho-phospho-tyrosine (P-Tyr) pseudoaffinity ligand was immobilized via ether linkage onto polyacrylamide-alginate (PAAm-Alg)-epoxy cryogels prepared according to two different approaches in order to explore their performance in the immunoglobulin G (IgG) purification from human serum. In the first approach, the P-Tyr was attached to cryogel prepared by cryocopolymerization of acrylamide and alginate with allyl glycidyl ether (AGE) as functional comonomer, and methylenebisacrylamide and Ca(II) as crosslinkers, obtaining the PAAm-Alg-AGE-P-Tyr. In the second approach, the PAAm-Alg was synthesized under the same conditions, but without AGE, and the P-Tyr was coupled to epichlorohydrin (ECH)-activated cryogel, obtaining the PAAm-Alg-ECH-P-Tyr. Both pseudoaffinity cryogels were characterized by scanning electron microscopy, swelling tests, porosity, ligand density, and flow dynamics. The human IgG differently interacted with the PAAm-Alg-ECH-P-Tyr and PAAm-Alg-AGE-P-Tyr cryogels, depending on the pH and adsorption buffer system used. The selectivity analyzed by electrophoretic profiles was similar for both cryogels, but PAAm-Alg-ECH-P-Tyr achieved higher IgG adsorption capacity (dynamic capacity of 12.62 mg of IgG/mL of cryogel). The IgG purity assayed by ELISA was 95%. The maximum IgG adsorption capacity and dissociation constant of the PAAm-Alg-ECH-P-Tyr, determined by Langmuir isotherm, were found to be 91.75 mg IgG/g of dry cryogel and 4.60 × 10-6 mol/L at pH 6.0 from aqueous solutions. The PAAm-Alg-AGE-P-Tyr showed potential to purify the Fab fragments from papain-digested human IgG solution at pH 7.0. Fab fragments were separated from Fc fragments (but with uncleaved IgG) in eluted fractions (analyzed by the Western blot technique), with yield of 82% and purity of 95% (determined by radial immunodiffusion).

Polyacrylamide-alginate (PAAm-Alg) and phospho-L-tyrosine-linked PAAm-Alg monolithic cryogels: Purification of IgG from human serum.

In recent decades cryogels as monolithic materials have gained interest as stationary phase in chromatography for purification of biomolecules. In this study, polyacrylamide-alginate (PAAm-Alg) monolithic cryogels were prepared by cryo-copolymerization of acrylamide and alginate monomers and methylene-bisacrylamide as crosslinker to be used as a matrix in affinity chromatography for purification of proteins. Ortho-phospho-L-tyrosine (P-Tyr) was covalently attached onto PAAm-Alg cryogels via bisoxirane-activation (PAAm-Alg-Bix-P-Tyr) and both derivatized and non-derivatized cryogels were utilized for the purification of immunoglobulin G (IgG) from human serum. Cryogels were characterized by scanning electron microscopy, swelling tests, elemental analysis, FTIR, and flow dynamics. The effects of buffer systems, conductivity, and pH on IgG adsorption were studied. Through breakthrough curve analysis a dynamic capacity of 9.2 mg IgG/mL with an IgG purity of 94% was obtained (based on ELISA analysis of IgG and albumin) for PAAm-Alg-Bix-P-Tyr cryogel when human serum was diluted in 10 mmol/L NaP buffer at pH 6.0. The adsorption isotherm data were well described by the Langmuir model with value of maximum adsorption capacity of 36.12 ±â€¯3.63 mg of IgG/g for PAAm-Alg-Bix-P-Tyr. The PAAm-Alg-Bix-P-Tyr cryogel provides an attractive alternative for adsorption of IgG from human serum.

Phospho-l-tyrosine-agarose chromatography: Adsorption of human IgG and its proteolytic fragments.

The behavior of human immunoglobulin G (IgG) and antigen-binding fragment (Fab fragment) adsorption onto phospho-l-tyrosine immobilized on agarose (P-Tyr-agarose) was evaluated by pseudoaffinity chromatography. The effects of buffer systems MES, MOPS, Bis-Tris, Tris-HCl and sodium phosphate (NaP) and pH on IgG adsorption were studied and high purity values were obtained (96%, based on ELISA analysis of albumin, transferrin and immunoglobulins A, G and M) when IgG was purified from human plasma diluted in 10 mmol L-1 NaP buffer at pH 6.0. The capture of IgG by the P-Tyr-agarose was also promising, since 91% of the IgG was adsorbed when plasma was diluted in 25 mmol L-1 MES buffer at pH 5.5, recommending its use for IgG depletion from human plasma under this condition. The experimental data on IgG adsorption kinetics were in agreement with the pseudo-second-order model. The adsorption isotherm data were well described by the Langmuir-Freundlich model with the value of parameter n being <1 (0.72), indicating negative cooperativity. Selectivity was achieved on P-Tyr-agarose from digested human IgG in HEPES 25 mmol L-1 buffer at pH 7.0 where Fab fragments were obtained in eluted fractions without Fc fragments (but with uncleaved IgG) with 86.2% recovery.

Behavior of human immunoglobulin G adsorption onto immobilized Cu(II) affinity hollow-fiber membranes.

Iminodiacetic acid (IDA) and tris(2-aminoethyl)amine (TREN) chelating ligands were immobilized on poly(ethylene vinyl alcohol) (PEVA) hollow-fiber membranes after activation with epichlorohydrin or butanediol diglycidyl ether (bisoxirane). The affinity membranes complexed with Cu(II) were evaluated for adsorption of human immunoglobulin G (IgG). The effects of matrix activation and buffer system on adsorption of IgG were studied. Isotherms of batch IgG adsorption onto finely cut membranes showed that neither of the chelates, IDA-Cu(II) or TREN-Cu(II), had a Langmuirean behavior with negative cooperativity for IgG binding. A comparison of equilibrium and dynamic maximum capacities showed that the dynamic capacity for a mini-cartridge in a cross-flow filtration mode (52.5 and 298.4 mg g(-1) dry weight for PEVA-TREN-Cu(II) and PEVA-IDA-Cu(II), respectively) was somewhat higher than the equilibrium capacity (9.2 and 73.3 mg g(-1) dry weight for PEVA-TREN-Cu(II) and PEVA-IDA-Cu(II), respectively). When mini-cartridges were used, the dynamic adsorption capacity of IDA-Cu(II) was the same for both mini-cartridge and agarose gel.