Hemoglobin A1c (HbA1c) has a unique structure that makes monoclonal antibody (mAb) preparation challenging. This study aims to develop a method for preparing HbA1c mAbs and establish a fluorescent immunochromatographic assay (FICA) for rapid detection of HbA1c. Three glycosylated peptides were synthesized and used to prepare complete antigens, which were identified by dot enzyme-linked immunosorbent assay (Dot-ELISA) and ultraviolet absorption spectroscopy. The complete antigens and natural HbA1c were used for cross-immunization of mice, and the optimal complete antigen was selected. The mouse with the highest serum titer was chosen for mAb preparation. The purity and specificity of the mAbs were verified, and a FICA method was developed. The optimal complete antigen, with a titer of 1:512 000, was successfully prepared and selected. Fusion with splenocytes resulted in four specific HbA1c antibodies (purity > 90%). The best antibody exhibited a binding constant (Ka) of 1.67×10¹⁰ L/mol with the antigen. Based on this antibody, a FICA method was successfully established, capable of producing results within 15 min. The method demonstrated a good linear range (3%-13% HbA1c, y=0.071 3x+0.005 6, R²=0.993 7), recovery rates of 98%-102%, precision < 10.00%, and no nonspecific reactions. Clinical testing of 210 samples showed positive agreement of 96.36%, negative agreement of 97.00%, and overall agreement of 96.68%. The receiver operating characteristic (ROC) curve analysis yielded an area under curve (AUC) of 0.980 9 [95% confidence interval (CI): 0.961 0-1.000 0], with high consistency verified in multicenter studies. We successfully developed a key technique for preparing HbA1c monoclonal antibodies and established a FICA method for rapid detection of HbA1c. It will provide an efficient and convenient detection method for the early diagnosis and long-term management of diabetes and its complications.
Antibodies, Monoclonal/biosynthesis* ; Animals ; Mice ; Glycated Hemoglobin/immunology* ; Mice, Inbred BALB C ; Humans ; Antibody Specificity ; Chromatography, Affinity/methods* ; Enzyme-Linked Immunosorbent Assay/methods* ; Female

As the need for antibody production rises, there is an urgent need to lower the costs and enhance the efficiency of the separation process. Currently, the chromatographic media used for antibody separation and purification often focus on individual properties of antibodies, such as affinity, hydrophobicity, and charge, leading to issues like low purification efficiency or inadequate adsorption capacity. To address this, an electrostatically coupled polypeptide affinity medium (FD7-3, 5-diaminobenzoic acid n-sepharose, FD7-DA-Sepharose) was developed for rapid purification of antibodies from cell culture supernatant. This medium utilized 3, 5-diaminobenzoic acid as a spacer to attach the heptapeptide-affinity ligand (FYEILHD, FD7) to agarose microspheres. Antibodies could be adsorbed through charge interactions with the carboxyl functional group of the FD7-DA-Sepharose spacer, while FD7 enhanced electrostatic coupling and affinity adsorption through synergistic effects, significantly increasing the adsorption capacity while maintaining the affinity and specificity. The influences of pH and ionic strength on adsorption capacity were investigated with human immunoglobulin as a model protein. The static adsorption capacity (Q_m) of FD7-DA-Sepharose in the solution of pH 6.0 reached 67.73 mg/mL, representing a 52.68% increase compared with that (44.36 mg/mL) of the commercial Protein A affinity medium. Furthermore, the elution conditions for FD7-DA- Sepharose were mild (20 mmol/L PB, 0.5 mol/L NaCl, pH 6.0), in contrast to the harsh acidic elution (pH 2.7-3.6) typically associated with Protein A, which can damage antibody integrity. The FD7-DA-Sepharose medium was then employed to purify antibodies from cell culture supernatant, achieving the yield of 94.8% and the purity of 98.4%. The secondary structure of the purified antibody was determined by circular dichroism spectroscopy. The results demonstrated that FD7-DA-Sepharose enabled efficient purification of antibodies from cell culture supernatant, which provided a cost-effective solution (approximately one-third the price of commercial Protein A affinity medium) with gentle elution conditions that preserve the natural conformation of antibodies. This approach paves a novel, economical, and efficient way for the separation and purification of antibodies from cell culture supernatant.
Chromatography, Affinity/methods* ; Static Electricity ; Humans ; Sepharose/analogs & derivatives* ; Peptides/chemistry* ; Adsorption ; Antibodies/isolation & purification*

This study aims to establish a time-resolved fluorescence immunochromatography method for simultaneous determination of human papillomavirus (HPV) type 16 E6 and L1 protein concentrations. The amount of lanthanide microsphere-labeled antibodies, the concentration of coated antibodies, and the reaction time were optimized, and then a test strip for the simultaneous determination of the protein concentrations was prepared. The performance of the detection method was evaluated based on the concordance of the results from clinical practice. The optimal conditions were 8 μg and 10 μg of HPV16 L1 and E6-labeled antibodies, respectively, 1.5 mg/mL coated antibodies, and reaction for 10 min. The detection with the established method for L1 and E6 proteins showed the linear ranges of 5-320 ng/mL and 2-64 ng/mL and the lowest limits of detection of 1.78 ng/mL and 1.09 ng/mL, respectively. There was no cross reaction with human immunodeficiency virus (HIV), treponema pallidum (TP), or HPV18 E6 and L1 proteins. The average recovery rate of the established method was between 97% and 107%. The test strip prepared in this study showed the sensitivity, specificity, and diagnostic accuracy of 97.46%, 90.57%, and 95.32%, respectively, in distinguishing patients with cervical cancer and precancerous lesions from healthy subjects, with the area under the curve (AUC) of 0.980 1 and 95% Confidence Interval (CI) of 0.956 5 to 1.000 0. The time-resolved fluorescence immunochromatography combined with the test strips prepared in this study showed high sensitivity, high accuracy, simple operation, and rapid reaction in the quantitation of HPV16 E6 and L1 proteins. It thus can be used as an auxiliary method for the diagnosis and early screening of cervical cancer and precancerous lesions and the assessment of disease course.
Oncogene Proteins, Viral/immunology* ; Humans ; Chromatography, Affinity/methods* ; Female ; Human papillomavirus 16 ; Repressor Proteins/immunology* ; Capsid Proteins/immunology* ; Papillomavirus Infections/diagnosis* ; Fluorescence ; Uterine Cervical Neoplasms/virology*

The systematic and in-depth study of phosphoproteome rely on highly reproducible and specific phosphopeptide enrichment methods. At present, a variety of enrichment methods have been developed based on different principles, and these methods often display different selectivity and specificity. It is therefore very important to select the most suitable enrichment method according to different research purposes. This review summarized the phosphopeptide enrichment based on affinity chromatography, immunoprecipitation, chemical derivatization, chromatography and other newly developed methods. The advantages and disadvantages of these methods, as well as the related optimization and improvement strategies, were discussed in detail. In addition, we also briefly summarized the progress of the combination of phosphopeptide enrichment and fractionation methods developed in recent years.
Phosphopeptides/metabolism* ; Proteomics/methods* ; Titanium/chemistry* ; Chromatography, Affinity ; Proteome ; Phosphorylation

A rapid and simple method to detect tumor markers in liver cancer was established by combining immunochromatography technique with fluorescent microsphere labeling. According to the principle of double antibody sandwich, the cytoskeleton-associated protein 4 (CKAP4) paired antibody was used as the labeled and coated antibody, and the goat anti-rabbit polyclonal antibody was used as the quality control line coated antibody in the preparation of the CKAP4 fluorescent immunochromatographic test strips. After the preparation, the test strips were evaluated on various performance indicators, such as linearity, precision and stability. The CKAP4 immunochromatographic strip prepared by time-resolved fluorescent microspheres had high sensitivity, and good specificity. Its precision was within 15%, recovery between 85% and 115%, and linear range between 25 and 1 000 pg/mL. The test strip could be kept stable at 37 °C for 20 days, and it correlated well with commercial ELISA kits. The CKAP4 fluorescence immunochromatography method can quantitatively detect the content of CKAP4 in serum. Furthermore, it is rapid, sensitive, simple, economical and single-person operation. This method has the potential of becoming a new method for the diagnosis and treatment of liver cancer.
Animals ; Antibodies ; metabolism ; Chromatography, Affinity ; Fluorescence ; Humans ; Liver Neoplasms ; diagnosis ; Membrane Proteins ; isolation & purification ; Molecular Diagnostic Techniques ; instrumentation ; methods ; Sensitivity and Specificity

Rice stripe virus (RSV) causes dramatic losses in rice production worldwide. In this study, two monoclonal antibodies (MAbs) 16E6 and 11C1 against RSV and a colloidal gold-based immunochromatographic strip were developed for specific, sensitive, and rapid detection of RSV in rice plant and planthopper samples. The MAb 16E6 was conjugated with colloidal gold and the MAb 11C1 was coated on the test line of the nitrocellulose membrane of the test strip. The specificity of the test strip was confirmed by a positive reaction to RSV-infected rice plants and small brown planthopper (SBPH), and negative reactions to five other rice viruses, healthy rice plants, four other vectors of five rice viruses, and non-viruliferous SBPH. Sensitivity analyses showed that the test strip could detect the virus in RSV-infected rice plant tissue crude extracts diluted to 1:20 480 (w/v, g/mL), and in individual viruliferous SBPH homogenate diluted to 1:2560 (individual SPBH/μL). The validity of the developed strip was further confirmed by tests using field-collected rice and SBPH samples. This newly developed test strip is a low-cost, fast, and easy-to-use tool for on-site detection of RSV infection during field epidemiological studies and paddy field surveys, and thus can benefit decision-making for RSV management in the field.
Antibodies, Monoclonal/chemistry* ; China ; Chromatography, Affinity/methods* ; Collodion/chemistry* ; Colloids/chemistry* ; Gold Colloid/chemistry* ; Materials Testing ; Membranes, Artificial ; Oryza/virology* ; Plant Diseases/virology* ; Reproducibility of Results ; Sensitivity and Specificity ; Species Specificity ; Tenuivirus/isolation & purification*

A one-step dual flow immunochromatographic assay (DICGA), based on a competitive format, was developed for simultaneous quantification of ochratoxin A (OTA) and zearalenone (ZEN) in corn, wheat, and feed samples. The limit of detection for OTA was 0.32 ng/ml with a detection range of 0.53‒12.16 ng/ml, while for ZEN it was 0.58 ng/ml with a detection range of 1.06‒39.72 ng/ml. The recovery rates in corn, wheat, and feed samples ranged from 77.3% to 106.3% with the coefficient of variation lower than 15%. Naturally contaminated corn, wheat, and feed samples were analyzed using both DICGA and liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the correlation between the two methods was evaluated using a regression analysis. The DICGA method shows great potential for simple, rapid, sensitive, and cost-effective quantitative detection of OTA and ZEN in food safety control.
Animal Feed ; Calibration ; Chromatography, Affinity ; Chromatography, Liquid ; Colloids ; Food Contamination/analysis* ; Food Safety ; Gold ; Immunoassay/methods* ; Inhibitory Concentration 50 ; Limit of Detection ; Metal Nanoparticles ; Ochratoxins/analysis* ; Regression Analysis ; Reproducibility of Results ; Sensitivity and Specificity ; Triticum ; Zea mays ; Zearalenone/analysis*

BACKGROUNDThe molecular mechanisms underlying the endometriosis are still not completely understood. In order to test the hypothesis that the approaches in phosphoproteomics might contribute to the identification of key biomarkers to assess disease pathogenesis and drug targets, we carried out a phosphoproteomics analysis of human endometrium.

METHODSA large-scale differential phosphoproteome analysis, using peptide enrichment of titanium dioxide purify and sequential elution from immobilized metal affinity chromatography with linear trap quadrupole-tandem mass spectrometry, was performed in endometrium tissues from 8 women with or without endometriosis.

RESULTSThe phosphorylation profiling of endometrium from endometriosis patients had been obtained, and found that identified 516 proteins were modified at phosphorylation level during endometriosis. Gene ontology annotation analysis showed that these proteins were enriched in cellular processes of binding and catalytic activity. Further pathway analysis showed that ribosome pathway and focal adhesion pathway were the top two pathways, which might be deregulated during the development of endometriosis.

CONCLUSIONSThat large-scale phosphoproteome quantification has been successfully identified in endometrium tissues of women with or without endometriosis will provide new insights to understand the molecular mechanisms of the development of endometriosis.

Adolescent ; Adult ; Chromatography, Affinity ; Endometriosis ; metabolism ; Endometrium ; metabolism ; Female ; Humans ; Phosphoproteins ; analysis ; Phosphorylation ; Proteomics ; methods ; Tandem Mass Spectrometry ; Young Adult

Affinity chromatography is a chromatographic method for separating molecules using the binding characteristics of the stationary phase with potential drug molecules. This method can be performed as a high throughput screening method and a chromatographic separation method to screen a variety of active drugs. This paper summarizes the history of affinity chromatography, screening technology of affinity chromatography, and application of affinity chromatography in screening bio-active compounds in herbal medicines, and then discusses its application prospects, in order to broaden applications of the affinity chromatography in drug screening.
Animals ; Chromatography, Affinity ; methods ; trends ; Drug Evaluation, Preclinical ; methods ; trends ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; pharmacology ; Humans ; Plants, Medicinal ; chemistry

Quantitative specific detection of Staphylococcus aureus is based on recombinant lysostaphin and ATP bioluminescence. To produce recombinant lysostaphin, the lysostaphin gene was chemically synthesized and inserted it into prokaryotic expression vector pQE30, and the resulting expression plasmid pQE30-Lys was transformed into E. coli M15 for expressing lysostaphin with IPTG induction. The recombinant protein was purified by Ni(2+)-NTA affinity chromatography. Staphylococcus aureus was detected by the recombinant lysostaphin with ATP bioluminescence, and plate count method. The results of the two methods were compared. The recombinant lysostaphin was successfully expressed, and a method of quantitative specific detection of S. aureus has been established, which showed a significant linear correlation with the colony counting. The detection method developed has good perspective to quantify S. aureus.
Adenosine Triphosphate ; chemistry ; Chromatography, Affinity ; Escherichia coli ; Luminescent Measurements ; methods ; Lysostaphin ; chemistry ; Recombinant Proteins ; chemistry ; Staphylococcus aureus ; isolation & purification