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

OBJECTIVETo investigate the effect of laparoscopic Roux-en-Y gastric bypass on the short-term immune function of type 2 diabetic patients and to explore the correlation between the immune regulatory effect and blood glucose control.

METHODSClinical data of 28 patients with type 2 diabetes mellitus who underwent laparoscopic Roux-en-Y gastric bypass surgery in our hospital during January 2014 to January 2015 were retrospectively collected. Fasting blood glucose (FBG), glycosylated hemoglobin (HbA1c), immunoglobulin (IgG, IgA, IgM), T lymphocytes CD3(+), killer T cells CD3(+)CD8(+), helper T cells CD3(+)CD4(+), natural killer(NK) cell subsets CD16(+),CD56(+) and B cell subsets CD19(+),CD45(+) levels were detected and compared between before and after surgery. Correlation of immunoglobulin and immune cell subsets with the level of FBG and HbA1c were examined.

RESULTSAfter operation, FBG and HbA1c decreased significantly from (5.78±1.15) mmol/L to (14.21±1.89) mmol/L, and (9.96±0.97)% to (6.87±0.69)%, respectively (all P<0.05), and immunoglobulin (IgG, IgA, IgM) increased significantly [IgG: from(9.41±1.23) g/L to (12.74±1.61) g/L, IgM: from (1.71±0.22) g/L to (2.43±0.39) g/L, IgA: from (1.25±0.26) g/L to (1.97±0.23) g/L, all P<0.05]. Besides, T lymphocytes CD3(+), killer T cells CD3(+)CD8(+) and B cell subsets CD19(+)CD45(+) elevated significantly as well [T lymphocytes CD3(+): from (55.3±3.8)% to (67.6±4.6)%, killer T cells CD3(+)CD8(+): from (15.6±5.3)% to (28.7±4.2)%, B cell subsets CD19(+)CD45(+): from (8.4±3.7)% to (18.1±4.1)%, respectively, all P <0.05]. There was no significant difference in the expression of helper T cells CD3(+)CD4(+) and natural killer cell subsets CD16(+)CD56(+) between pre-operation and post-operation(all P>0.05). Levels of immunoglobulin (IgG, IgA, IgM), CD4(+)/CD8(+) ratio and B cell subsets were negatively correlated with HbA1c and FBG levels (IgG with FBG: r=-0.865, IgA with FBG: r=-0.887, IgM with FBG: r=-0.902, CD4(+)/CD8(+) with FBG: r=-0.956, CD19(+)CD45(+) with FBG: r=-0.834; IgG with HbA1C: r=-0.859, IgA with HbA1C: r=-0.957, IgM with HbA1C: r=-0.843, CD4(+)/CD8(+) with HbA1C: r=-0.912, CD19(+)CD45(+) and HbA1C: r=-0.885, all P<0.05), but the proportion of NK cells was not significantly correlated with HbA1c and FBG (P>0.05).

CONCLUSIONSLaparoscopic Roux-en-Y gastric bypass has good effect on the immune function of patients with type 2 diabetes mellitus. Improvement of immune function is correlated with the control of FBG and HbA1c.

Blood Glucose ; analysis ; Diabetes Mellitus, Type 2 ; immunology ; surgery ; Gastric Bypass ; Glycated Hemoglobin A ; analysis ; Humans ; Immunoglobulins ; blood ; Laparoscopy ; Lymphocyte Subsets ; immunology ; Postoperative Period ; Retrospective Studies

BACKGROUNDGlutamic acid decarboxylase antibody (GADA) and protein tyrosine phosphatase antibody (IA-2A) are two major autoantibodies, which exert important roles in the process of type 1 diabetes mellitus (T1D). Our study aimed to investigate the changes in positivity and titers of GADA and IA-2A during the course of Chinese acute-onset T1D patients and their relationships with clinical features.

METHODSTwo hundreds and forty-seven Chinese newly diagnosed acute-onset T1D patients were consecutively recruited. GADA and IA-2A were detected at the time of diagnosis, one year later, 3-5 years later after diagnosis during the follow-up; all the clinical data were recorded and analyzed as well.

RESULTSDuring the course of acute-onset T1D, the majority of patients remained stable for GADA or IA-2A, however, a few patients changed from positivity to negativity and fewer patients converted from negativity to positivity. The prevalence of GADA was 56.3% at diagnosis, decreasing to 50.5% one year later, and 43.3% 3-5 years later while the corresponding prevalence of IA-2A were 32.8%, 31.0% and 23.3%, respectively. The median GADA titers were 0.0825 at diagnosis, declining to 0.0585 one year later and 0.0383 3-5 years later (P < 0.001), while the corresponding median titers were 0.0016, 0.0010, 0.0014 for IA-2A, respectively. Fasting C-peptide (FCP) and postprandial C-peptide 2 hours (PCP2h) levels of GADA or IA-2A negativity persistence patients were higher than those of positivity persistence and negativity conversion patients (P < 0.05) which indicated GADA or IA-2A negativity persistence T1D patients had a less loss of β cell function.

CONCLUSIONOur data suggest that repeated detection of GADA and IA-2A are necessary for differential diagnosis of autoimmune diabetes and the indirect prediction of the β cell function in Chinese patients.

Adolescent ; Adult ; Aged ; Antibodies ; therapeutic use ; Asian Continental Ancestry Group ; Child ; Child, Preschool ; Diabetes Mellitus, Type 1 ; drug therapy ; immunology ; Female ; Glutamate Decarboxylase ; immunology ; Glycated Hemoglobin A ; metabolism ; Humans ; Infant ; Male ; Middle Aged ; Protein Tyrosine Phosphatases ; immunology ; Young Adult