Whole blood GBP5 protein levels in patients with and without active tuberculosis

From February 2019 to May 2020, suspected aTB patients admitted to the Xinglin Branch of the First Affiliated Hospital of Xiamen University were recruited. The heparinized whole blood was collected from each recruited patient at enrolment for GBP5 protein detection and TB-IGRA. The diagnosis of aTB was based on clinical, radiological, microbiological, and histopathological information and the patient’s response to anti-TB therapy for at least 3 months, and the aTB patients included had PTB and EPTB individuals. For non-TB patients mainly include patients infected with non-tuberculosis mycobacterium (NTM) and other lung disease (OLD). In addition, the study counted the Immunosuppressive condition of participants, including HIV infection, chronic renal disease, diabetes, and underlying disease relevant for immunosuppressive treatment.

The study was approved by the Ethical Committee of the School of Public Health, Xiamen University. Written informed consent was obtained from each participant.

The coding sequence of GBP5 was synthesized by Sangon Biotech (Sangon Biotech Co., Ltd., Shanghai, China) and cloned into the expression vector pET-30a. Then, the recombinant plasmid was transformed into Escherichia coli BL21 (DE3). After induction by isopropyl-β-d-thiogalactoside, GBP5 was expressed in the form of inclusion bodies and further purified by Ni²⁺-NTA affinity chromatography under denaturing conditions. As analyzed by SDS-PAGE and HPLC, the purity of GBP5 was above 80% and the GBP5 protein was detected by western blot analysis using an anti-GBP5 rabbit pAb(Abcam, Cambridge, UK) (Additional file 1: Fig. S1). Balb/c mice were immunized with purified GBP5 (50 µg) emulsified in Freund’s adjuvant, and mouse monoclonal antibodies (mAbs) against GBP5 were prepared by hybridoma technology. A total of 70 mAbs against GBP5 were obtained and purified.

The synthesized cDNA of GBP5 was cloned into pTT5, a eukaryotic protein expression plasmid, and HEK 293 T cells were transfected for transient expression. At 48 h post transfection, cells were collected and lysed with lysis buffer (1% NP40 and 0.25% DOC in deionized water). After centrifugation, the supernatant was collected and stored at − 80 ℃ for mAb pair screening and ELISA establishment. The supernatants of HEK 293 T cells with transient expression of GBP5 and mock-transfected HEK 293 T cells were used as positive and negative controls, respectively.

One mAb pair (7G9 and 9A9) with the highest positive to negative (P/N) ratio was selected to develop a double antibody sandwich ELISA to detect GBP5 protein in whole blood. mAbs 7G9 and 9A9 were employed for microplate coating and biotin conjugation, respectively, and the P/N ratio of these pairs exceeded 40 (Additional file 3: Fig. S2). The assay was developed and performed as below. In brief, 96-well polystyrene microplates (Xiamen Labware Co., Ltd., Xiamen, China) were coated with mAb 7G9 diluted with 20 mM phosphate buffer, pH 7.4, at a concentration of 4 µg/ml overnight at 37℃ followed by blocking with 200 µL of blocking buffer (2% BSA in 20 mM PBS) for 2 h. To detect GBP5 protein in whole blood, freshly collected heparinized whole peripheral blood was lysed with a fourfold volume of lysis buffer and centrifuged at 12,000 ×g for 10 min, and then the supernatant was added to the microplate. After incubation at 37 ℃ for 1 h, the microplate was washed 5 times with washing buffer (0.02% Tween-20 in 20 mM phosphate buffer saline, pH 7.4). Next, 100 µL of biotin-labelled mAb 9A9 (1 µg/ml in blocking buffer) was added to the washed microplate and incubated at 37 ℃ for 30 min, followed by another 5 washes. Then, 100 µL of horseradish peroxidase-conjugated streptavidin (Thermo Fisher Scientific, Rockford, IL) was added to the microplates and incubated at 37 ℃ for 30 min. After washing 5 times again, 100 µL of TMB substrate (Beijing Wantai, Beijing, China) was added and incubated at 37 ℃ for 15 min. Then, the colour development was stopped with 50 µL of 0.5 M sulfate, and the optical density (OD) at 450 nm with 630 nm as a reference was determined by an Autobio PHOmo microplate reader (Autobio, Zhengzhou, China).

According to the manufacturer’s instructions, the status of MTB infection was determined by WANTAI TB-IGRA (Beijing Wantai, Beijing, China) [19]. In brief, 1 ml of fresh heparinized venous whole blood was added into Eppendorf tubes containing nil for the negative control (N), mitogen for the positive control (P) and TB antigens (a recombinant protein of ESAT-6 and CFP-10) for the INF-γ test (T). After incubation at 37 °C for 22 ± 2 h, each tube was centrifuged, and the concentration of INF-γ in the plasma was measured. As recommended by the manufacturer, the cut-off of T-N was 14 pg/ml, and the results were valid if N ≤ 400 pg/ml and P-N ≥ 20 pg/ml simultaneously.

Analyses were carried out with SPSS 25.0 (IBM Corp., Armonk, NY, USA). The Mann-Whitney U test or Fisher’s exact test was performed to compare the differences between two groups; the analyses were two-tailed, and statistical significance was considered when p <0.05. Receiver operating characteristic (ROC) analyses were performed by GraphPad Prism 8.0 (GraphPad Software, CA, USA) to evaluate the assay’s performance in distinguishing aTB patients from non-TB patients. The area under the curve (AUC) was calculated, and the optimum cut-off values were selected based on Youden’s index.