Lipoarabinomannan (LAM) is a component of the cell wall shed by Mycobacterium tuberculosis, the bacteria responsible for tuberculosis, an infection mainly affecting the lungs. LAM can be detected in urine samples from patients coinfected with HIV, but current LAM detection methods have failed for HIV-negative patients. Using hydrogel “nanocage” nanoparticles and a chemical bait with high affinity for LAM, Paris et al. showed that patients negative for HIV with active tuberculosis infections had detectably higher concentrations of LAM in their urine than patients without active tuberculosis infections. Nanocages could also be used to detect cytokines and other antigens present in low concentrations in urine, demonstrating the versatility of the technology as a method to detect and monitor infections.

An accurate urine test for pulmonary tuberculosis (TB), affecting 9.6 million patients worldwide, is critically needed for surveillance and treatment management. Past attempts failed to reliably detect the mycobacterial glycan antigen lipoarabinomannan (LAM), a marker of active TB, in HIV-negative, pulmonary TB–infected patients’ urine (85% of 9.6 million patients). We apply a copper complex dye within a hydrogel nanocage that captures LAM with very high affinity, displacing interfering urine proteins. The technology was applied to study pretreatment urine from 48 Peruvian patients, all negative for HIV, with microbiologically confirmed active pulmonary TB. LAM was quantitatively measured in the urine with a sensitivity of >95% and a specificity of >80% (n = 101) in a concentration range of 14 to 2000 picograms per milliliter, as compared to non-TB, healthy and diseased, age-matched controls (evaluated by receiver operating characteristic analysis; area under the curve, 0.95; 95% confidence interval, 0.9005 to 0.9957). Urinary LAM was elevated in patients with a higher mycobacterial burden (n = 42), a higher proportion of weight loss (n = 37), or cough (n = 50). The technology can be configured in a variety of formats to detect a panel of previously undetectable very-low-abundance TB urinary analytes. Eight of nine patients who were smear-negative and culture-positive for TB tested positive for urinary LAM. This technology has broad implications for pulmonary TB screening, transmission control, and treatment management for HIV-negative patients.

The ideal urine test would measure a variety of pathogen and host analytes ( 2 ) to achieve the highest specificity and accuracy at all phases of TB. Therefore, we explored whether the nanocage technology could be extended to other characterized analytes associated with TB and the host response and to other immunoassay formats useful in low-resource settings. We introduce here new high-affinity chemical dye baits that bind the following well-characterized TB antigens and inflammatory markers: (i) LAM, (ii) early secretory antigenic target 6 (ESAT6) ( 11 ), (iii) culture filtrate protein 10 (CFP10) ( 11 ), and (iv) inflammatory cytokines nonspecifically associated with an active infection including interleukin-2 (IL-2), interferon-γ (IFN-γ), and tumor necrosis factor–α (TNFα) ( 12 ). For ESAT6, we examined the versatility of the cage nanotechnology as a new class of sandwich immunoassay using the chemical bait as one side of the sandwich.

The poor sensitivity of existing LAM assays in HIV-negative/TB-positive patients has been explained by three main hypotheses. The first hypothesis is that LAM is shed into the urine of active pulmonary TB patients only in the context of glomerular dysfunction caused by HIV infection including HIV-related nephropathy (HIVAN) ( 8 , 9 ). However, in HIVAN, urinary LAM is not associated with heavy proteinuria, suggesting that this is not an important mechanism ( 9 ). The second hypothesis is that LAM is shed into the urine of patients with active TB only when there is extrapulmonary renal tract involvement, such that the antigen can enter the urine directly from infected tissue ( 10 ). The third hypothesis is that the concentration of LAM in patients with active pulmonary TB is below the concentration detection limits of existing assays and may be masked by formation of immune complexes, excess non-LAM proteins, or other inhibitors present in the urine ( 7 ). Here, we apply a new class of analytical nanocage technology to definitively address these hypotheses and solve this dilemma.

An accurate screening test for active pulmonary tuberculosis (TB) is urgently needed for patients who are not coinfected with HIV ( 1 , 2 ). Worldwide, TB is one of the most prevalent bacterial infections (9.6 million cases and 1.5 million deaths in 2014), with the highest mortality in developing countries ( 1 ). Ideally, such a test would use a noninvasive body fluid such as urine to facilitate utilization in a low-resource setting ( 1 , 2 ). This objective, at first, appears straightforward because the outer surface glycan lipoarabinomannan (LAM), a TB antigen shed into the urine during active TB, has been identified and well characterized ( 3 , 4 ). Although enzyme-linked immunosorbent assay and lateral flow tests have been developed to measure LAM, their sensitivity is limited ( 4 ). These tests can detect urinary LAM in patients with pulmonary TB who are coinfected with HIV ( 5 , 6 ) but not in those who are HIV-negative ( 5 , 6 ). Quantitative gas chromatography–mass spectrometry has been used to identify d-arabinose as a proxy for LAM in TB patients irrespective of HIV status, but the sensitivity is limited to 10 to 40 ng/ml ( 7 ). Unfortunately, the failure to detect LAM in the urine of HIV-negative patients limits the applicability of these assays, because most of the TB patients are HIV-negative (85% of the 9.6 million patients worldwide) ( 1 ).

RESULTS

A recognized barrier to glycosciences and to the use of glycans as diagnostic biomarkers is the scarcity and suboptimal quality of available monoclonal antibodies (mAbs) directed against complex carbohydrates such as LAM (13). To address this problem, we introduce here a new class of chemical affinity bait, a copper complex reactive dye, Reactive Blue 221 {RB221; cuprate(4-),[2-[[[[3-[[4-chloro-6-[ethyl[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-2-hydroxy-5-sulfophenyl]azo]phenylmethyl]azo]-4-sulfobenzoato(6-)]-,tetrahydrogen} (Fig. 1). RB221 binds and sequesters carbohydrate glycan LAM antigen with extremely high affinity (fig. S1) that is at least 100 times greater than any known lectin (fig. S2 and table S1). RB221 is immobilized in open-mesh hydrogel nanoparticle cages. When introduced into urine, the nanocages harvest LAM with high efficiency within minutes while simultaneously dissociating interfering substances in solution (fig. S3). Our affinity capture nanotechnology increases the sensitivity of LAM detection in urine by 100- to 1000-fold depending on the available volume of urine (Fig. 1, A and B) (14–18).

Fig. 1 Nanocages that were covalently functionalized with copper complex dye Reactive Blue 221 sequestered and concentrated lipoarabinomannan from urine. (A) Schematic depicting high internal/external surface area ratio and binding capacity of nanocages. Affinity ligands covalently immobilized in the inner volume establish high-affinity noncovalent interaction with tuberculosis (TB) antigens. (B) Schematic showing the concentration factor given by the volumetric ratio between the initial urine volume and the final testing volume. Structures within the urine sample are nanocages. (C) Molecular structure of lipoarabinomannan (LAM) (right) and affinity probe Reactive Blue 221 (RB221) {cuprate(4-),[2-[[[[3-[[4-chloro-6-[ethyl[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-2-hydroxy-5-sulfophenyl]azo]phenylmethyl]azo]-4-sulfobenzoato(6-)]-,tetrahydrogen} (left). (D) Western blot, glycan staining, and image analysis of protein macroarray assay of LAM. C, LAM control (50 ng); IS, initial solution (50 ng of LAM spiked in 50 μl of human urine); S, supernatant; E, eluate from the nanocages; P, nanocages; AU, arbitrary units. Mean and SD, n = 3 replicates.

We applied this technology to study the concentration of LAM in the pretreatment urine of 48 HIV-negative patients with microbiologically confirmed active pulmonary TB from a Peruvian hospital (Tables 1 and 2). All patients were confirmed to have normal kidney function by in-hospital assessment including creatinine measurement and urinalysis. To determine whether urinary LAM concentrations reflect body disease burden, we compared the urine LAM concentrations with sputum TB organism counts, cough frequency, appetite, and change in body weight (19) in this cohort (Table 2) of well-characterized hospitalized patients using the widely accepted simplified nutritional appetite questionnaire (SNAQ) scoring system (20). Controls included age-matched healthy volunteers and diseased non-TB control patients who were hospitalized and ill with a variety of severe systemic, pulmonary, neurologic, and genitourinary tract diseases (table S2).

Table 1 Demographic characteristics of study participants. IQR, interquartile range. View this table:

Table 2 Clinical characteristics of hospitalized patients (n = 48 microbiologically confirmed TB patients and n = 2 TB-negative patients). Urine was collected from patients before therapy. SNAQ, simplified nutritional appetite questionnaire; MODS, microscopic observation broth-drug culture and susceptibility. View this table:

Copper complex dyes: High-yield LAM sequestration from urine The carbohydrate structure of LAM (Fig. 1) (3) poses unsolved challenges in terms of identifying adequate probes for affinity isolation in urine in the presence of a vast excess of interfering urinary proteins and other biomolecules (7, 21, 22). For this study, 37 different dye chemistries (table S1) were screened to identify a molecular bait that would sequester LAM from urine with high affinity, deplete the supernatant, dissociate LAM-binding proteins, and permit a high-yield quantitative recovery (Fig. 1, C and D, and fig. S1). The dye chemistry panel was selected by inference from dyes known to be useful for tissue histology mucin staining or fluorescent staining of microorganisms. Western blot analysis was conducted to screen dyes for their affinity to LAM. Two mAbs were tested and yielded highly specific bands for LAM with no detectable background in urine matrix (fig. S4). The specificity of the mAb clone CS-35 was verified by antigen competition (fig. S5) (23). Dyes containing copper moieties for histologic staining are known to preferentially interact with glycans. Here, nanocages that were covalently functionalized with copper-containing dyes (Alcian blue pyridine variant and RB221) proved to be superior to other affinity probes, such as fluorescent brightener 28 (FB28), fast blue B, and safranin O (Fig. 1C and figs. S1, S2, and S6). Figure 1D documents the full depletion of LAM (100 ng/ml) spiked in human urine using the RB221 nanocages (Fig. 1D, lane S). Binding was independent of the pH of solution in the range 5 to 7 (fig. S7). The molecular weight of the band by Western blot analysis and carbohydrate staining is the expected full size of LAM (~38,000) with no lower–molecular weight bands. After nanocage capture and elution, no differences were detected in the quantity, shape, or intensity of the LAM band captured in urine matrix as compared to LAM captured in phosphate-buffered saline (PBS) (Fig. 1D). Because PBS did not contain interfering substances, this verifies that the LAM was sequestered away from potential interfering urinary molecules including proteins, lipids, glycans, and cellular debris that could interfere with sequestration. On the basis of the intensity of the band compared to standards, the complete depletion of the supernatant at equilibrium, the yield and efficiency of capture and elution is greater than 95%. In human urine, on the basis of the bound versus free LAM at equilibrium, the capture affinity considerably exceeds K d (dissociation constant) = 10−9 M (fig. S1). Competition with 10% copper acetate in water or chelation by EDTA displaced LAM bound to RB221 (Fig. 1D, bar graph, and fig. S8), documenting the involvement of the copper moiety in the binding function. To further characterize the copper complex dye RB221 binding to the glycan, we used sodium m-periodate (NaIO 4 ) oxidative degradation. As demonstrated by solid-phase immunoassay (fig. S9), NaIO 4 at low concentrations and low pH extracted LAM from the RB221 cages, verifying that intact LAM diol bonds are required for RB221 binding (fig. S9).

Nanocage-based measurement of LAM in the pretreatment urine of patients with active pulmonary TB Quantitation of LAM in human urine was performed after nanocage capture and elution using an immunomacroarray assay (24). The concentration of LAM in the reference calibrator was qualified by the anthrone colorimetric method in the linear portion of the assay (0.16 mg/ml; fig. S10). The concentration factor was 100-fold (Fig. 1). The immunomacroarray assay limits of detection and quantifications for 1 ml of input urine were 14 and 15 pg/ml, respectively [background estimate, 547.32 arbitrary units (AU); SD, 22.6 AU; lower limit of detection (LLD) = background + 2 * SD; and lower limit of quantification (LLQ) = background + 10 × SD; the polynomial equation y = 8 × 10−9x2 − 3 × 10−6x + 0.0126 (R2 = 0.9985) was used to estimate LAM concentration (Fig. 2B)]. Unknowns were tested in an array with built-in negative controls and standards (Fig. 2). All samples and controls were identically processed through the nanocages. Fig. 2 LAM antigen was detected in the urine of HIV-negative/TB-positive patients using RB221 nanocages for diseased and control patients listed in Table 1 (A) Image of a quantitative immunomacroarray for LAM detection, incorporating (B) a dilution curve in every membrane. Neg, negative; BKG, background. (C) Example immunomacroarray comparing urine samples from a set of true-positive and known TB-negative samples using nanocage preprocessing. (D) Bar plot of the intensities of LAM determined via immunomacroarray and ImageJ analysis from urine samples from healthy TB-negative, TB-negative diseased, and TB-positive patients shown in Table 1 (mean ± SD, n = 4 patient replicates).

Verification of the assay format A test set of 23 TB-positive patients and a verification set were analyzed (total n = 48 independent infected patient samples; Tables 1 and 2). The mean and SD of urinary LAM concentration in the test and verification sets were 700 ± 500 pg/ml and 410 ± 400 pg/ml, respectively. The two sets were statistically indistinguishable (Wilcoxon signed-rank test P = 0.07, n = 72). The patients were called positive if the LAM signal was 2 SD higher than the full process negative controls run simultaneously. LAM could not be measured in any of the TB-infected patient’s pretreatment urine without the nanocage concentration step.

Urinary LAM: HIV-negative active TB-positive pretreatment patients discriminated from healthy and diseased controls A total of 101 subjects qualified for the study (n = 48 microbiologically confirmed TB-positive patients, n = 14 diseased TB-negative patients, and n = 39 healthy volunteers). Informed consent was collected at the time of urine donation. The median age of the microbiologically confirmed TB patient was 29 years (interquartile range, 24 to 36), and 72% were males. The most commonly reported symptoms were cough (76%) and fever (64%). Demographic, clinical, and microbiological data are presented in Tables 1 and 2. Completed urine dipstick analysis was recorded (table S3). The full data results obtained with the immunomacroarray analysis described above are shown in Fig. 2D. For the true-positive patients (n = 48), only 2 had undetectable LAM concentrations according to the criteria stated above. The controls included age-matched healthy subjects and diseased non-TB patients who were ill with a variety of severe systemic, pulmonary, and urinary tract diseases. The diseases included pneumonia, lung cancer, pyelonephritis, genitourinary infection, sepsis, cryptosporidiosis, giardiasis, colon cancer with gastroenteritis, and liver failure (table S2). The difference in LAM concentration between the cases and controls was highly significant [P < 1 × 10−15, n = 101, Wilcoxon signed-rank test; difference in location estimate, −247.318; 95% confidence interval (CI), −351.3 to 200.8; Fig. 3]. As shown in Fig. 3B, a significantly higher concentration of LAM was measured in the urine of patients who had a higher score for sputum organism content (auramine score, P < 0.043, n = 42, Wilcoxon signed-rank test; difference in location estimate, −205.3; 95% CI, −452.0 to 5.1). Fig. 3 Urinary LAM concentration predicted pulmonary TB and correlated to mycobacterial burden and weight loss. (A) Box plot of the intensities of LAM in the urine of HIV-negative/TB-positive patients versus controls collected in endemic areas (Wilcoxon signed-rank test). (B) Box plot of the intensities of LAM in the urine of HIV-negative/TB-positive patients stratified on the basis of the auramine staining (low amount of microorganism, scores 0 and 1; high amount of microorganism, scores 2 and 3; Wilcoxon signed-rank test; n = 42). (C) Receiver operating characteristic analysis of the LAM intensity data. AUC, area under the curve. (D) Ordinal regression analysis shows statically significant correlation between the concentration of urinary LAM and the loss of body mass (P = 0.038, n = 37). Sensitivity and specificity were evaluated by receiver operating characteristic (ROC) analysis, and the area under the curve was calculated to be 0.95 (95% CI, 0.9005 to 0.9957; fig. S11) as an overall ROC performance [n = 48 cases, n = 53 controls; significance level, 0.05; power, 1 (25)]. At a threshold of 14 pg/ml, this ROC analysis yielded a sensitivity of 0.96 and a specificity of 0.81 for true-positive pulmonary TB patients in the present study set (positive predictive value, 0.82; negative predicted value, 0.95; power, 0.96) (Fig. 3C). By these criteria, the single false-positive urine in the diseased controls was patient #234, whose urinalysis had +++ leukocyte esterase, +++ protein, +++ blood, and + bilirubin (table S3). These urinalysis values would meet the exclusion criteria for clinical urine diagnostic testing. Notably, eight of the nine culture-positive but smear-negative patients were positive for urinary LAM.

Correlation of urinary LAM with clinical measures of disease burden and severity Simple and multiple linear regression of covariates in Table 3 revealed that cough and appetite scoring compared to LAM urine concentrations were not individually significant by simple linear regression. However, when taken together, these two clinical measures were of significance and predictive of LAM urine concentrations (Table 3). Participants who reported a cough were likely to have an increased secretion of 269 pg/ml (10 to 528 pg/ml) of LAM (P = 0.042, n = 50). For appetite data, for each unit increase in SNAQ score (Table 4) (20), an increase of 54.1 pg/ml (4.76 to 103 pg/ml) of LAM was observed (P = 0.032, n = 37). Table 3 Simple and multiple linear regression analysis. Analysis shows that cough and SNAQ scores (20, 36), when combined, were significantly correlated to the concentration of urinary LAM. CI, confidence interval. View this table: Table 4 SNAQ scoring ( 20 36 ). View this table: Exploration of LAM as an ordinal variable revealed that the highest producers of LAM were those who had experienced the greatest change (loss) in body mass as a proportion of their baseline mass (Fig. 3D). When patients were grouped into low-level LAM producers (115 pg/ml), mid-level LAM producers (115 to 320 pg/ml), and high-level LAM producers (320 pg/ml), high-level LAM producers lost, on average, 17% of their body mass as compared to patients in the low- and mid-level LAM-producing group who lost 8 and 9% of their body masses, respectively. Ordinal regression revealed a significant correlation of percent weight loss and LAM categorization (P = 0.038, n = 37; Table 5). This indicates that loss of weight in patients with high urinary LAM was consistent with a cachexia-like state characteristic of patients with advanced TB infection (19). These data are in keeping with the conclusion that the concentration of urinary LAM is a reflection of total mycobacterial body burden [auramine score (26)] and disease severity [cough and weight loss (19, 20)] in patients with active pulmonary TB who are HIV-negative. Table 5 Ordinal regression analysis. A significant correlation between the urinary LAM concentration and body mass change was observed. View this table:

Extending the technology to other TB antigen markers and host-associated cytokines Beyond LAM, additional low-abundance mycobacterial antigens that promise to offer important future diagnostic utility if they can be detected with adequate sensitivity in the complex matrix of urine have been characterized. We searched for bait chemistries that exhibited a high affinity for additional TB antigens (Fig. 4) such as ESAT6 and CFP10, which are secreted by replicating bacteria in addition to LAM shedding (11) during infection. We also explored bait chemistries for host immune response factors that, although not specific for TB diagnosis, may be involved in the cytokine cascade of TB infection [IL-2, TNFα, and IFN-γ (27)]. Results shown in Fig. 4 indicate that nanocages completely captured the target analytes, depleted the supernatant, and increased the effective concentration in the Western blot analysis. There was no cross-reactivity of the antibodies with the negative control human urine in the absence of target analytes. The analysis of urine samples from four untreated TB patients from the Peruvian cohort revealed that ESAT6 is detectable by Western blot analysis only when nanocages are used as a preprocessing step (Fig. 4D and Supplementary Materials and Methods). The four patients analyzed were characterized by microscopic observation broth-drug culture and susceptibility (MODS) TB culture (four of four are positive) and sputum smear (three of four are positive) (28). These data document that the nanocage technology is not limited to the LAM antigen and can be extended to other TB-related antigens to expand the detection panel and increase the accuracy of TB diagnosis. Fig. 4 Nanocages captured multiple TB-related analytes. (A) SDS–polyacrylamide gel electrophoresis (PAGE) analysis; chemical bait incorporated in the nanocages (NP1, blue 3G-A; NP2, pigment red 177; NP3, disperse yellow 3). P, nanocage eluate. (B) Affinity probes (affinity probe 1, pigment red 177; affinity probe 2, blue 3G-A; affinity probe 3, trypan blue). (C) Nanocages effectively captured TB-related analytes from human urine (Western blot). U, negative control; C, recombinant protein (positive control, 75 ng). (D) Nanocage detection of TB antigen ESAT6 in the urine of untreated HIV-negative/TB-positive patients (Western blot).

Nanocages can be magnetized Magnetization permits the creation of a urine collection device that achieves rapid separation of the particles from the urine in a self-contained vessel. To meet this goal, we incorporated a magnetic label (Fe 3 O 4 functionalized with oleic acid; diameter, 100 nm) into the hydrogels (Fig. 5A and Supplementary Materials and Methods). As shown in Fig. 5B, magnetic separation is as efficient as centrifugation, if not superior, in separating the particles from urine and enabling the detection of ESAT6 and CFP10 at concentrations otherwise undetectable by Western blot analysis. Fig. 5 Magnetic hydrogel nanocages. (A) Schematic of magnetization. (B) Western blot analysis of ESAT6 and CFP10 expression in eluates of centrifugation-separated nanocages (top), in eluates of magnetic-separated nanocages (middle), and in supernatants after magnetic separation of nanocages from urine samples (bottom). Top and middle: Lane 1, positive control (recombinant protein; 10 ng); lanes 2 to 7, two to six eluates from nanocages incubated with 1 ml of urine containing ESAT6 (10, 5, 2.5, 1.2, 0.6, and 0.3 ng/ml) and CFP10 (10, 5, 2.5, 1.2, and 0.6 ng/ml). Bottom: Lane 1, positive control (recombinant protein; 10 ng); lanes 2 to 7, two to six supernatants after nanocage processing of 1 ml of urine containing ESAT6 (10, 5, 2.5, 1.2, 0.6, and 0.3 ng/ml) and CFP10 (10, 5, 2.5, 1.2, and 0.6 ng/ml).

Obviating the need for elution: Nanocages partially dissolve to display the captured analyte The effective pore size of the particles is a function of hydrogel polymer cross-links. Rendering the cross-links degradable provides a means to induce the nanocages to open and display the captured sequestered analyte (TB antigen) cargo. Partially degradable nanocages were created by incorporating a cleavable cross-linker (N,N′-(1,2-dihydroxyethylene)bisacrylamide) under oxidizing conditions (Fig. 6, A to C, and Supplementary Materials and Methods) or N,N′-bis(acryloyl)cystamine under reducing conditions (fig. S12). In this workflow, nanocages were mixed with urine containing the antigen of interest, and the solution-phase antigen was captured within the particles. The degradable cross-links were then cleaved, causing an effective increase in pore size and exposing the captured antigens in the internal volume. Antibodies were used to probe the exposed captured antigen directly within the cages for LAM (fig. S12) and ESAT6 (Fig. 6D). Fig. 6 Partially dissolvable nanocages captured antigen for antibody binding in a high-sensitivity sandwich immunoassay. (A) Schematic demonstrating nanocage cross-link degradation in an oxidative environment. (B) Change in hydrodynamic diameter after nanocage oxidation [t test, n = 10; mean and SD of nanocage hydrodynamic diameter before (a) and after (b) oxidative degradation]. (C) SDS-PAGE analysis comparing N,N′-(1,2-dihydroxyethylene)bisacrylamide (DHEA) cross-linked nanocages mixed with a solution of monoclonal antibody (Ab) (0.05 mg/ml) with pores open (lanes 2 and 3) and closed (lanes 4 and 5). (D) Immunomacroarray demonstrating that antigen bound to the chemical bait retains its capability to bind to the antibody. a, nanocages deposited on polyvinylidene difluoride (PVDF) membrane after incubation with urine containing ESAT6 (1 ml, 10 ng/ml) and DHEA cross-link degradation; b, nanocages deposited on PVDF membrane after incubation with urine containing ESAT6 (1 ml, 10 ng/ml) in the absence of DHEA cross-link degradation; c, ESAT6 deposited on PVDF membrane (starting amount, 1 ng); d, DHEA nanocages deposited on PVDF membrane after incubation with urine in the absence of ESAT6. (E) Plot of immunoassay signal intensity as a function of bait capture affinity. High-affinity chemical baits achieve >2 log increased sensitivity for antigen capture compared to conventional antibody, as mathematically demonstrated in Supplementary Materials and Methods. (F) Schematic depicting direct, nonelution sandwich immunoassay using partially degradable nanocages. Inset shows an enzyme-linked antibody interacting with TB antigens captured inside the nanocage. (G) Calibration curve of a direct nanocage immunoassay for ESAT6 showing linearity in the 1- to 0.03-ng range. (H) Schematic of a lateral flow immunoassay using one antibody. Nanocages capture and preserve antigen in solution, migrate through the filter membrane, and provide colorimetric detection. (I) Lateral flow immunoassay for ESAT6 detection in urine. Positive signal for 10 ng of ESAT6 in 10 ml of human urine both visually (blue line, a) and with chemiluminescence (black line, b). Negative control urine in the absence of ESAT6 yields no signal (c and d).

Single-antibody sandwich This class of nanocages was then used as the basis for a single-antibody sandwich immunoassay for ESAT6 in a 96-well plate format. This format is completely distinct from conventional sandwich immunoassays because the capture antibody is replaced by the chemical bait and can yield improved analytical sensitivity (Fig. 6, E and F, and Supplementary Materials and Methods). The calibration curve for the assay is reported in Fig. 6G, indicating a high degree of linearity (R2 = 0.99) in the 1- to 0.03-ng range. This translates to a urine concentration sensitivity of 30 pg/ml for a 10-ml sample.