Subjects and sera
Ninety-three patients with HIV, diagnosed in Beijing Youan Hospital from January to December in 2018, were enrolled in the present study. The diagnosis of patients was based on the relevant provisions of WTO and China's national industry standards[7]. According to the diagnostic criteria, all patients were diagnosed as the asymptomatic stage or AIDS since blood samples from patients with acute infection are difficult to collect. Patients with kidney injury caused by other causes, including recent severe trauma or surgery, and non-AIDS related acute and chronic infectious diseases, children, pregnant or lactating women and patients who did not meet the ethical requirements of informed consent were excluded. Sera of positive control were obtained from patients with classical anti-GBM disease diagnosed in Peking University First Hospital. Normal human sera were obtained from 20 healthy volunteers from Beijing Youan Hospital.
All serum samples were collected and stored at -80 °C until use. Clinical data of HIV patients were collected at the time of diagnosis and during follow-up. Siemens Advia2400 automatic biochemical analyzer was employed for renal function test, among which creatinine was measured by enzyme method, urea was measured by Urease method, and glomerular filtration rate was calculated by simplified MDRD formula. Sysmex XN series and relevant reagents were used for blood routine test (e.g. Hemoglobin), while Arkary AX4030 and necessary reagents were used for urine routine test (such as protein, red blood cell, etc.). BD FACS Canto II flow cytometer and the matching reagents were used for CD4 count, while Abbott real-time HIV-1 reagents were used for HIV viral load determination. The present study was in compliance of the Declaration of Helsinki and approved by the ethics committee of Beijing Youan Hospital and Peking University First Hospital. Informed consent was obtained from each individual.
Preparation of recombinant human a(IV) NC1 and chimeric proteins
The recombinant human α(IV)NC1 and chimeric protein were prepared as follows. Briefly, the cDNAs of the α1-α5(IV)NC1 were ligated into the X-type collagen triple helix guide sequence and cloned into the pcDNA plasmid. Then the plasmid was transfected into human embryonic kidney 293 cell line, and recombinant proteins were harvested from the culture solution after purification[8] .
The construct EA and EB consists entirely of the α1(IV)NC1 domain, EA contain 45 amino acids of α3(IV)NC1 EA region, and EB contain 37 amino acids of α3(IV)NC1 EB region. Construction of non-EAB was constructed in the context of α3(IV)NC1, in which the EA and EB regions were replaced by the corresponding amino acids of α1(IV)NC1[8, 9] .
Detection of anti-GBM antibodies in serum samples using enzyme-linked immunosorbent assay
A home-based enzyme-linked immunosorbent assay (ELISA) was performed to measure anti-GBM antibodies in the sera. In brief, a mixture of the recombinant human α1-α5(IV)NC1 proteins was diluted with carbonate buffer solution (CBS 0.05 M, pH 9.6) to the concentration of 1.0μg/mL for each antigen, and coated onto half of the wells of the polystyrene microtiter plate (Nunc, Roskilde, Denmark). The other half of the wells was coated with bovine serum albumin (BSA) as antigen-free wells, and they were diluted at the same concentration in 0.05M CBS to exclude non-specific binding. Incubation at 37°C for 60 min. After washing, the serum samples (1:100 diluted with a dilution buffer containing 0.64 M NaCl, 0.008 M Na2HPO4·12H2O, 0.003 M KCl, 0.002 M KH2PO4, 0.1% Tween-20, pH 7.4) were then added to the antigen coated and antigen-free wells respectively. Incubation again at 37°C for 60 min. After washing, alkaline phosphatase–conjugated goat anti-human IgG (Fc specific, Sigma, USA, 1:6000) was added, and incubation again at 37°C for 60 min. After washing, 50μL of substrate solution (containing 1 mg/mL p-nitrophenyl phosphate, 1.0 M diethanolamine, 0.5 M MgCl2, pH 9.8, Sigma, USA) was added into the wells for color development which was measured spectrophotometrically at 405 nm (Bio-Rad, Tokyo, Japan) 15min later. The net absorbance values were OD values of the antigen-coated wells minus the OD values of the antigen-free wells. Each serum sample was tested in duplication, while the samples were re-tested when the standard deviation between the wells >10%. Serum samples from healthy volunteers were used to build up the cutoff values using mean+2 SD for mixture α chains and the chimeric proteins.
Detection of antigen and epitope specificity in sera of HIV patients with anti-GBM antibodies
To detect antigen and epitope specificity, plate wells were respectively pre-coated with each of the five-recombinant human α(IV)NC1 or the chimeric proteins containing EA, EB and non-EAB (1μg/mL per protein). Serum samples of HIV patients with anti-GBM antibodies were diluted at 1:50 and added in duplication into the wells at 37°C for 60 min. After washing, alkaline phosphatase–conjugated goat anti-human IgG (Fc specific, Sigma, USA) was added, and incubation at 37°C for 60 min. Color was developed and measured as described above. Serum samples from healthy volunteers were used to build up the cutoff values using mean+2 SD for each α chain and the chimeric protein.
Detection of distribution of IgG subclass against α3(IV)NC1 in HIV patients with anti-GBM antibodies
The detection of IgG subclasses against α3(IV)NC1 was performed as previously reported[8]. In brief, the recombinant human α3(IV)NC1 proteins were diluted at 1μg/mL and coated onto half of the wells of a polystyrene microtiter plate. The other half of the wells was coated with BSA as antigen-free wells. Diluted serum samples at 1:100 were then added to the antigen and BSA coated wells respectively, and incubated at 37°C for 60 min. After washing, horseradish peroxidase labeled mouse monoclonal antibodies against human IgG1, IgG2, IgG3, and IgG4 (Fc specific, Southern Biotech, USA, 1:2000) were added at 37°C for 60 min. After washing, color was developed by adding 50μL of substrate (Tetramethyl benzidine, TMB, Invitrogen, USA) into the wells for 15 min. Reaction was terminated by adding stop solution and color measured at 450 nm. Again, serum samples from healthy volunteers were used to build up the cutoff values using mean+2 SD at the same steps.
Quality Control
All laboratory test indexes were monitored by quality control materials with at least two levels. In the routine urine analysis, in case of positive alarm of red blood cells, white blood cells, proteins and nitrites, artificial microscopy was employed to eliminate the interference of false positive (such as mucous filaments).
Statistics
Continuous variables were expressed as means ± SD or medians. Comparison between continuous variables was conducted by t test for normally distributing data or nonparametric test for non-normally distributing data. Differences between qualitative data were analyzed using χ2 or Fisher exact test. All statistical analyses were two-tailed and P <0.05 was considered significant different. Analysis was performed using the IBM SPSS statistics 24.0 software.