Serum Proteomic Analysis by Nanoflow LC-MS/MS-Based Proteomics in IgA Chronic Kidney Disease.

BACKGROUND: IgA nephropathy (IgAN) is the most frequently occurring primary glomerulonephritis. A lack of specific biomarkers hinders the early diagnosis and treatment of this disease. This study analyzes and validates potential serum biomarkers using mass spectrometry proteomics. METHODS: Global proteomics profiles of serum from 60 patients with IgAN and 43 healthy control subjects were compared to identify significantly changed proteins. These proteins were validated with targeted proteomics using parallel reaction monitoring (PRM) in an independent validation set consisting of samples from 67 different stage IgAN patients and 60 healthy controls. RESULTS: A total of 37 significantly changed proteins were found in the discovery set, among which 18 proteins were identified as potential biomarkers for IgAN through PRM assays in the validation set. Of these 18 proteins, IgGFc-binding protein, MS-A1 light chain variable region, transthyretin, ficolin-3, and myosin-reactive immunoglobulin light chain variable region were up-regulated in different IgAN stages, B cell receptor heavy chain variable region, rheumatoid factor RF-ET6, heavy chain Fab, cryocrystalglobulin CC1 heavy chain variable region, FLJ94213, lumican, and Q68CN4 (uncharacterized protein) were down-regulated in different IgAN stages. These proteins support previous findings that CKD is accompanied by altered immune response. CONCLUSIONS: This study lays the groundwork for additional research using biomarkers to clinically diagnose IgAN. These proteins are potential molecular markers that could help us understand the potential molecular mechanism of IgAN.

Inhibition of human sperm function by an antibody against apolipoprotein A1: A protein located in human spermatozoa.

Apolipoproteins A1 (ApoA1), an important protein in initiating sperm capacitation, has been found in the oviduct liquid, whilst whether it expresses in human spermatozoa has not yet been defined. ApoA1 expression was detected by reverse transcription-polymerase chain reaction, Western blot and immunofluorescence in sperm cells. Sperms were incubated with different concentration of ApoA1 antibody to observe the changes of sperm motility and apoptosis including annexin V binding to the cell surface, mitochondrial membrane potential and ultrastructural changes. In addition, cholesterol levels of human spermatozoa and fertilization in vitro were completed to explore the role of ApoA1 in fertilization process. We identify ApoA1 mRNA is presented in spermatozoa, and the ApoA1 protein, the molecular weights of 31 kD, is located at the postacrosomal region and neck of human spermatozoa. ApoA1 antibody affects cholesterol efflux of human spermatozoa and inhibits fertilization in vitro. Moreover, ApoA1 antibody decreases sperm motility and increases sperm apoptosis. This work shows ApoA1 plays an important role in maintaining cholesterol stability and sperm motility, survival rate and fertilization process. Furthermore, ApoA1 implications in the clinical work and other reproductive areas are needed to be studied.

Clinical Characteristics and Influence Factor Analysis of ABO Typing Discrepancy Among Patients in a Tertiary Hospital.

BACKGROUND: An accurate interpretation of the ABO blood group of an individual is of utmost importance to ensure patient safety and good transfusion practices. The aim of the study was to determine the incidence and causes of ABO typing discrepancies among patients and analysis of the clinical characteristics as well. METHODS: A retrospective observational study was carried out in the Department of Laboratory Medicine in the General Hospital of Chinese PLA from March 2018 to December 2020. Records of patients were collected and analyzed for frequency, clinical characteristics, and influencing factors of ABO typing discrepancies. RESULTS: There were 132 ABO typing discrepancies patients (57 females, 75 males), aged from 3 to 84 (50.3 ± 19.3) years, with history of operation 89 cases (67.42%) and blood transfusion 23 cases (17.42%). In the control group, there were 142 cases (63 females and 79 males), aged 10 - 78 years (50.5 ± 15.55), with operation history 68 cases (47.88%) and blood transfusion history 2 cases (1.40%). Among the inconsistent blood types group, there were 59 cases (44.7%) type B, 38 cases (28.8%) type A, 28 cases (21.2%) type AB, and 7 cases (5.3%) type O. There were 21 cases (15.9%) in the department of hematology, 15 cases (11.36%) in the department of orthopedics, 14 cases (10.6%) in the department of hepatobiliary surgery, 11 cases (8.33%) in the department of general surgery, 11 cases (8.33%) in the department of vascular surgery, and less than 5% in other departments. The common cause of ABO typing discrepancies was due to low/weak affinity antibody (3.79%), low/weak affinity antibody B (18.18%), weak A antigen (3.03%), weak B antigen (12.12%), hematopoietic stem cell transplantation (10.60%), irregular antibody (46.70%), subtype (3.79%), and cold agglutination (1.51%). The diseases in the ABO discrepancy group mainly included hematological disorders, malignant tumors, osteoarthritis and so on. Binary logistic regression showed that hematological disorders, malignant tumors, history of operation (p = 0.01), and history of blood transfusion (p = 0.017) were the influencing factors of ABO typing discrepancies. CONCLUSIONS: It is possible that the antibody of ABO blood group system could not be detected. The independent influence factors were hematological disorders, malignant tumor, blood transfusion history and operation history for ABO typing discrepancies. It was necessary to analyze the causes correctly and judge the correct blood group by serological methods.

Study on amniotic fluid metabolism in the second trimester of Trisomy 21.

BACKGROUND: Trisomy 21 is a common aneuploid condition in humans and accounts for approximately one quarter of all aneuploid live births. To date, early diagnosis of Trisomy 21 remains a challenging task. Metabolomics may prove an innovative tool to study the early pathophysiology of Trisomy 21 at a functional level. METHODS: Ultra-performance liquid chromatography coupled with mass spectrometer (UPLC-MS) was used for untargeted metabolomic analysis of amniotic fluid samples from women having normal and trisomy 21 fetuses. RESULTS: Many significantly changed metabolites were identified between amniotic fluid samples from Trisomy 21 pregnancies and normal euploid pregnancies, such as generally lower levels of several steroid hormones and their derivatives, higher levels of glutathione catabolites coupled with lower levels of gamma-glutamyl amino acids, and increased levels of phospholipid catabolites, sugars, and dicarboxylic acids. The identification of a human milk oligosaccharide in amniotic fluid may worth further investigation, since confirmation of this observation may have significant implications for regulation of fetal development. CONCLUSIONS: The metabolisms in amniotic fluid from Trisomy 21 and normal pregnancies are quite different, and some of the significantly changed metabolites may be considered as candidates of early diagnostic biomarkers for Trisomy 21.

Infected RBC flag/parameter provided by Mindray BC-6800 haematology analyzer aid the diagnosis of malaria.

BACKGROUND: The Mindray BC-6800 haematology analyzer (BC-6800) provides a dedicated flag 'Infected RBC' (InR) and the number of InR (InR#)/the permillage of InR (InR‰) in routine blood testing as a screening tool for malaria in endemic areas. This study sought to evaluate the effectiveness of the BC-6800 flag parameter for aiding the diagnosis of malaria. METHODS: A total of 181 samples were tested using the Mindray BC-6800 haematology analyzer, including 117 malaria-infected samples collected from Yunnan, China, and 64 samples from healthy controls. Microscopy examination was conducted as reference when stained thick blood film revealed the presence of malaria parasites identified as Plasmodium vivax and Plasmodium falciparum. The receiver operating characteristic (ROC) curve analysis was developed using Analyse-it v4.92.3. The Kappa value was determined to evaluate the agreement between BC-6800 and light microscopy. RESULTS: The sensitivity of InR‰ generated by BC-6800 for P. vivax and P. falciparum was 88.3 and 24.1%, respectively; specificity of InR‰ for malaria parasites was 84.3 and 84.3%, respectively; positive predictive value and negative predictive value was 89.4 and 82.7% for P. vivax, and 52.8 and 60.3% for P. falciparum. There was a strong correlation between ΔWBC and InR‰ (R2 = 0.9731 for P. vivax and R2 = 0.9757 for P. falciparum). There was also a significant correlation between parasitaemia and InR# in P. vivax-infected samples (R2 = 0.734). InR# was evaluated using ROC curve analysis, the area under the ROC curve is 0.95 with a 95% confidence interval of 0.926 to 0.974, and the cut-off value is 0.01 × 109/L for P. vivax. However, the ring stage and the early trophozoite stage of Plasmodium cannot be detected easily on BC-6800, possibly because of the small size and low nucleic acid content of these stages. CONCLUSIONS: The findings suggest that the flag 'InR' and the parameters 'InR#/InR‰' provided by the BC-6800 haematology analyzer could be used to screen for malaria in a clinical setting.

Immunoturbidimetric Assay for Determination of Peripheral Blood C Reactive Protein on the Pentra MS CRP Hematology Analyzer.

BACKGROUND: The Pentra MS CRP hematology analyzer (hereinafter the Pentra analyzer) can simultaneously provide 5-part leukocyte differential and C-reactive protein (CRP). The aim of the study was to investigate the performance of CRP determination by the Pentra analyzer. METHODS: The precision, limit of quantitation (LoQ), carryover, linearity, stability, and comparability of the Pentra analyzer were determined. The Passing-Bablok regression analysis and the Bland-Altman graphs illustrated the correlation for CRP concentration analyzed by the Pentra analyzer and BN-II analyzer. RESULTS: The within-run precision of CRP determination by the Pentra analyzer had a CV < 2.0% in peripheral blood, which met the requirements of the instructions (CV ≤ 10%). The Pentra analyzer had a total CV of 5.35% and 5.52% at a CRP concentration of 4.1 and 80 mg/L, respectively. The LoQ value for the Pentra analyzer was 0.96 mg/L. The carryover was 0.57% for peripheral blood and 0.86% for plasma by the analyzer. The stability of CRP results was good, when the anticoagulation samples were stored at room temperature or 4°C within 48 hours (deviation < 5%). The linearity range for whole blood samples was 0 - 188.13 mg/L (r² = 0.9992). There was high correlation of the CRP results analyzed with the Pentra analyzer and BN II analyzer. The Passing-Bablok regression analysis and the Bland-Altman graphs showed the bias plot display excellent agreement between the two assays (the mean value for the Pentra 2.19 mg/L and the BN-II 2.35 mg/L, n = 101). CONCLUSIONS: The results of CRP determination by the Pentra analyzer have the advantages of accuracy and reliability, and it is suitable for routine use in emergency laboratory and small to medium-size laboratories.

Comparing PyroMark Q24 Pyrosequencing and MALDI-TOF MS for Identification of CYP2D6*10.

BACKGROUND: CYP2D6*10 is mainly responsible for the large pharmacokinetic variability of routinely administered metoprolol in middle-aged and elderly Asian patients. Utilizing an efficient method for identifying the CYP2D6*10 genotypes is clinically important for evaluating the pharmacokinetic effect of administration of metoprolol. This study attempted to evaluate the effectiveness of the two methods used to detect the rs1065852 and rs1135840 SNPs of the CYP2D6*10 gene. METHODS: Blood samples were processed for the collection of genomic DNA from 198 subjects across Chinese population, and detection of CYP2D6*10 (rs1065852 and rs1135840) was performed using the PyroMark Q24 pyrose-quencing and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF MS). The discordant results were further validated with Sanger sequencing. We eventually attempted to assess some features of these two methods including reliability, rapidness, being appropriate, and cost-effectiveness. RESULTS: Genotyping of rs1065852 and rs1135840 detected by MALDI-TOF MS were concordant with those identified by PyroMark Q24 pyrosequencing in all 198 (100%) individuals. The hands-on-time and the turnaround time were shorter in the PyroMark Q24 pyrosequencing method than in the MALDI-TOF MS method for SNP of CYP2D6*10. In terms of being cost-effective and high-throughput, the MALDI-TOF MS method outperformed the PyroMark Q24 pyrosequencing method. CONCLUSIONS: CYP2D6*10 genotypes detected by PyroMark Q24 pyrosequencing and MALDI-TOF-MS showed that both methods were reliable, rapid, appropriate, and cost-effective methods. These methods are valuable for clinical applications.

Performance Evaluation of the Mindray BC 6800 Hematology Analyzer and Flag Comparison with the XE-2100 and Manual Microscopy.

BACKGROUND: The Mindray BC-6800 automated hematology analyzer is an automated hematology analyzer and 5-part leukocyte differential counter for in vitro diagnostic use in clinical laboratories. It is necessary to undergo an evaluation before the instrument is used to test patient samples. METHODS: The performance was evaluated with regards to precision, linearity, carry-over, and method comparison. The flag performances were evaluated and compared with the Sysmex XE-2100 hematology analyzer and manual microscope in the hematology laboratory of a tertiary hospital in China. RESULTS: There was minimal carryover (< 0.05%) and excellent linearity for white blood cells and platelet (PLT) counts (r > 0.999). The BC-6800 displayed very good correlation (r > 0.97) with the XE-2100 for blood cell count and cell differential parameters. In a comparison of 295 leukocyte differential count results analyzed in parallel with manual microscopy, the main flags (immature granulocytes, blasts, abnormal lymphocytes) showed approxi-mately the same sensitivity and specificity on both analyzers (sensitivity > 90%, specificity > 78%). CONCLUSIONS: The BC-6800 showed excellent performance and supplied confidence in flag information for abnormal samples in the routine hematology laboratory.

Increased NGAL level associated with iron store in chronic kidney disease with anemia.

An iron scarcity often occurs in chronic kidney disease (CKD). Neutrophil gelatinase-associated lipocalin (NGAL), a biomarker of acute kidney injury, is associated with iron metabolism. The present study determined the association between serum NGAL and iron status in chronic kidney disease with anemia. A total of 154 adult CKD patients were divided into anemia and without anemia groups. The anemia groups were further subdivided into two groups based on the presence or absence of iron deficiency, defined as a transferrin saturation (TSAT) < 20%. The NGAL was measured for all the 154 patients, and the possible relationships with iron status were analyzed. 27.7% patients with TSAT < 20% presented lower hemoglobin, serum iron, serum ferritin, and higher NGAL values than those without iron deficiency. NGAL was inversely correlated with hemoglobin, hematocrit, MCV, MCH, serum iron, and TSAT. NGAL adequately diagnosed the status of iron deficiency among CKD patients by ROC analysis. The optimal NGAL cutoff value able to identify iron deficiency was found to be > 244.8 ng/mL, with 73.01% sensitivity and 68.29% specificity. CKD patients with anemia presented altered NGAL values as this protein is involved in the maintenance of iron balance. Thus, NGAL might be proposed as a new tool for assessing the iron deficiency and in the management of iron therapy for CKD patients.

Clinical application of neutrophil gelatinase-associated lipocalin in the revised chronic kidney disease classification.

BACKGROUND: A revised classification of chronic kidney disease (CKD) was proposed by the Kidney Disease: Improving Global Outcomes (KDIGO) in 2012. Neutrophil gelatinase-associated lipocalin (NGAL) was considered as one of the most promising biomarkers in clinical nephrology. The aim of this study was to examine the level of NGAL in patients with different impairment of GFR based on the new classification, and to evaluate whether NGAL in serum or urine was associated with different risk categories in CKD patients. METHODS: A cross-sectional study was performed in 240 patients with CKD. NGAL, serum cystatin C, ß2-macroglobulin (ß2-MG), urine α1-macroglobulin (α1-MG) and albuminuria were tested in patients with various degrees of renal impairment. RESULTS: Good correlation was found between the NGAL and the cystatin C, ß2-MG and the α1-MG (r > 0.7). The level of sNGAL in CKD stage 3b was more than that in CKD stage 3a (P = 0.025). The concentration of the NGAL increased progressively with the increasing of risk categories (proposed by the revised CKD classification). The cutoff value of NGAL was calculated from stage 2 to stage 5. ROC analysis showed good AUC (sNGAL > 0.8, uNGAL > 0.7) and high specificity (sNGAL > 87%, uNGAL > 90%) on the cutoff value of NGAL. CONCLUSION: The results confirm NGAL as a useful biomarker in clinical nephrology which is helpful to diagnosis and evaluate the categories for CKD proposed by the KDIGO.

Puerarin attenuates ovalbumin-induced lung inflammation and hemostatic unbalance in rat asthma model.

Aim. We aimed to investigate and evaluate the preventive activity of puerarin on the ovalbumin-induced asthma rat model. Materials and Methods. Male Wistar rats were sensitized intraperitoneally on days 0, 7, and 14 and challenged to ovalbumin intratracheally on day 21. Groups of sensitized rats were treated randomly either with placebo, puerarin, dexamethasone, or puerarin combined with dexamethasone, from days 15 to 20. Inflammatory markers, including cell counts in bronchoalveolar lavage fluid (BALF), inflammatory cytokines, histopathology, and coagulation parameters, such as coagulation tests and the activity of coagulation factors, were analyzed. Results. Puerarin significantly inhibited the recruitment of inflammatory cells in BALF and lung tissue. At the same time, the release of IL-4, IL-10, and IFN- γ in serum and the expression of mRNAs in lung tissue homogenate were changed by puerarin. Administration of puerarin also effectively rectified the coagulation disorder in asthmatic rats, such as prothrombin time (PT) (P < 0.01), thrombin time (TT) (P < 0.05), fibrinogen (FIB) (P < 0.01),the activity of factor II (FII) (P < 0.01), the activity of factor V (FV) (P < 0.05), the activity of factor VII (FVII) (P < 0.05), the activity of factor X (FX) (P < 0.05), the activity of factor VIII (FVIII) (P < 0.01), the activity of factor IX (FIX) (P < 0.05), and the activity of factor XII (FXII) (P < 0.05). Conclusions. Our results provide a clue that puerarin was useful for the preventive of allergic airway disease in rodents.

Application and development of review criteria on Sysmex XT-2100 in Chinese at a large third-grade class-A hospital.

BACKGROUND: Hematology analysis is an essential component of patient assessment and used in screening, diagnosis, and the planning of care. The objective of the study was to find out the suitable hematology review criteria for large scale general hospitals in China via the analysis of experimental data from Sysmex XE-2100 hematology analyzer. METHODS: A total 1486 blood samples were detected with the Sysmes XE-2100. Based on hematology review criteria suggested by international consensus group and a positive smear finding new optimal review rules were determined. RESULTS: With the International Article 41 Review Rules, the true positive ratio (TP), the false positive ratio (FP), the true negative ratio (TN), and the false negative ratio (FN) was 14.0% (208/1486), 31.49% (468/1486) 52.42% (779/1486), and 2.09% (31/1486), respectively. With the help of Laboman 4.2 software (the Sysmex Corporation), 19 rules for review of automated CBC and WBC differential were set up. With our review rules, the TP, FP, TN, and FN was 13.86% (206/1486), 25.17% (374/1486), 58.75% (873/1486) and 2.22% (33/1486), respectively. The review rules were validated, the FN was 0.96%, blasts and immature cells were not omitted. CONCLUSIONS: The review criteria can be developed in light of the rules of the International Consensus Group for Hematology Review, but should be improved depending on different laboratory's requirements.

Particle-enhanced turbidimetric immunoassay for determination of serum neutrophil gelatinase-associated lipocalin on the Roche Cobas c501 analyzer.

OBJECTIVE: Neutrophil gelatinase-associated lipocalin (NGAL) has been reported to be a good marker for tubular damage and acute kidney injury. The aim of this study was to develop a high throughput assay for the quantification of serum NGAL (sNGAL). METHODS: Imprecision, interference, linearity, recovery, and reference values were evaluated on Cobas c501. RESULTS: The assay was linear over the dynamic range of the study (R(2)=0.9988). The total assay imprecision was below 5%. The assay recovery was estimated at 98.89%-102.61%. The assay displayed a good linearity over the range from 35 µg/L to 4250 µg/L. A typical high-dose hook effect was observed for the assay at NGAL concentration>28,800 µg/L. No interference was observed with hemoglobin ≤ 5 g/L, bilirubin ≤ 0.3g/L, vitamin C ≤ 0.5 g/L, sodium heparin≤ 5 g/L and intralipid ≤ 1%. The 95th centile for serum NGAL was <122.57 µg/L from 454 healthy donors. There were no gender-related differences for serum NGAL. There were significant age-related differences between the 21-44 and 45-75 year categories for serum NGAL. The reference value for sNGAL was <116.52 µg/L in the 21-44 year group and <126.9 µg/L in the 45-75 year group. CONCLUSIONS: The NGAL assay verified to be a reliable assay with convenient performance characteristics. The assay improves and simplifies the laboratory workload.

Clinical laboratory urine analysis: comparison of the UriSed automated microscopic analyzer and the manual microscopy.

BACKGROUND: Several automated urine sediment analyzers have been introduced to clinical laboratories. Automated microscopic pattern recognition is a new technique for urine particle analysis. We evaluated the analytical and diagnostic performance of the UriSed automated microscopic analyzer and compared with manual microscopy for urine sediment analysis. METHODS: Precision, linearity, carry-over, and method comparison were carried out. A total of 600 urine samples sent for urinalysis were assessed using the UriSed automated microscopic analyzer and manual microscopy. RESULTS: Within-run and between-run precision of the UriSed for red blood cells (RBC) and white blood cells (WBC) were acceptable at all levels (CV < 20%). Within-run and between-run imprecision of the UriSed testing for cast, squamous epithelial cells (EPI), and bacteria (BAC) were good at middle level and high level (CV < 20%). The linearity analysis revealed substantial agreement between the measured value and the theoretical value of the UriSed for RBC, WBC, cast, EPI, and BAC (r > 0.95). There was no carry-over. RBC, WBC, and squamous epithelial cells with sensitivities and specificities were more than 80% in this study. CONCLUSIONS: There is substantial agreement between the UriSed automated microscopic analyzer and the manual microscopy methods. The UriSed provides for a rapid turnaround time.

Development of microscopic review criteria by comparison urine flow cytometer, strip and manual microscopic examination.

BACKGROUND: Microscopic examination is essential for urine analysis, but a time-consuming procedure. This study was undertaken to evaluate an automated urinalysis system - the Sysmex UF-1000i (URISYS 2400) for the analysis of urine constituents including chemistry components and particles. The objective was to screen urine samples and determine the screening criteria which would minimize the number of specimens reviewed with the microscope yet ensuring correct results. METHODS: A total of 1300 urine samples were sent for urinalysis using the automated system and compared with results obtained from manual microscopy using the Fuchs-Rosenthal counting chamber. RESULTS: Using Pearson statistics, we observed correlation between the UF-1000i and manual microscopy: for red blood cells (RBCs) r was 0.949, for white blood cells (WBCs) r was 0.882, for epithelial cells (EC) r was less than 0.76, for casts r was less than 0.7, while correlation between the URISYS 2400 and manual microscopy: for red blood cells r was 0.772 and for white blood cells r was 0.771. With the help of Uriaccess (an expert system provided by the Sysmex Corporation), 37 rules for microscopic review were set up. The review rules were validated, the review rate was less than 30% and the false-positive and false-negative results were acceptably low. CONCLUSIONS: UF-1000i is capable of reproducible measurement of urine particles within the clinically relevant range and shows its advantage over URISYS 2400. It is an optimal strategy for urine sample screening using the combination of the two methods.

Detection of D-3-phosphoglycerate dehydrogenase autoantibodies in patients with autoimmune hepatitis: Clinical significance evaluation.

AIM: D-3-phosphoglycerate dehydrogenase (3-PHGDH) was identified as a putative target of autoantibodies in autoimmune hepatitis (AIH). The aims of the present study were to detect anti-3-PHGDH in patients with AIH and other chronic liver diseases and to analyze their clinical relevance. METHODS: Human 3-PHGDH gene was cloned and expressed in Escherichia coli and used in enzyme-linked immunosorbent assays and Western blots. Serum from patients with AIH (n = 101), primary biliary cirrhosis (PBC, n = 122), chronic hepatitis C (CHC, n = 117), chronic hepatitis B (CHB, n = 112), and from patients with other autoimmune disease (n = 125) were investigated. RESULTS: The highest incidence and activity of anti-PHGDH was observed in AIH patients. Thirty-two of 40 untreated (80%) and 37 of 61 AIH patients treated with corticosteroid (60.7%) were positive. Antibody titers decreased significantly during corticosteroid treatment. 15.8% of PBC patients, 9.8% of CHB and 12.8% of CHC patients, were anti-PHGDH-positive, with less than 12% of patients positive with other autoimmune diseases via reactions with recombinant 3-PHGDH protein. CONCLUSION: Anti-PHGDH were detected in chronic liver diseases. They occur predominantly in AIH, and corticosteroid treatment seems to decrease antibody titers. Whether the antibodies are primary or secondary phenomena and whether they are related to the etiology or pathogenesis, at least in a subgroup of patients with chronic liver diseases, has still to be evaluated.

[Cloning and expression of 3-phosphoglycerate dehydrogenase gene and its correlative antibodies in diagnosis of autoimmune hepatitis].

OBJECTIVE: To evaluate whether the D-3-phosphoglycerate dehydrogenase (Phgdh) correlative antibodies is crucial for AIH, we cloned Phgdh cDNA and constructed plasmid, then purified and identified the immunoreactivity of the recombinant protein, and established the enzyme linked immunosorbent assay (ELISA) to detect Phgdh autoantigen correlative antibodies in diagnosis of autoimmune hepatitis. METHODS: The constructed plasmid was transformed into E. coli. BL21(D3). This fusion protein was purified by Ni-NTA chromatography and its immunoreactivity was identified by SDS-PAGE and Western blot. The ELISA with the fusion protein was established first, then, the Phgdh autoantigen correlative antibodies in serum of patients with AIH (65) and patients with PBC (122) as well as chronic hepatitis B (CHB) (56), chronic hepatitis C (CHC) (117), and normal controls (60) were detected. RESULTS: The sequence of Phgdh autoantigen gene was the same as the sequence reported on the genebank. The fusion protein was found about 60kD strip on SDS-PAGE. Western blot analysis showed that the fusion protein had immunoreactivity. When analyzing the serum by ELISA, the immune reactivity to Phgdh was detected in 66.15% of patients with AIH, 21.42% of patients with PBC, 12.50% of patients with CHB, 6.83% of patients with CHC, and 3.30% of normal individuals. The differences of prevalence between AIH patients and healthy controls as well as other diseases were of statistical significance (P less than 0.01). CONCLUSION: The Phgdh cDNA is successfully cloned into E. coli BL21 (D3). The frequency of antibodies to Phgdh is much higher in patients with AIH than in patients with PBC, CHB, CHC and normal control. The antibodies to Phgdh may have utility in improved diagnosis of AIH.