Galactomannan ELISA in bronchoalveolar lavage fluid with surface-enhanced raman scattering readout

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Abstract

BACKGROUND: Invasive aspergillosis is a threatening fungal infection with high mortality rates. One of the key factors for timely diagnostics of the disease is an approach based on detection of galactomannan, a polysaccharide from the pathogen’s cell wall. The existing galactomannan enzyme-linked immunosorbent assays (ELISA) employ the colorimetric detection. The emergence of the new highly sensitive techniques for the measurement of peroxidase label in ELISA, e.g., based on the effect of surface-enhanced Raman scattering (SERS), facilitates the development of a next generation assays with improved sensitivity and with potentially broader range of biological fluids applicable for analysis. An important step of developing the new test-system is the demonstration of its applicability to real clinical samples and comparability of the results with reference method. AIM: To evaluate the applicability of the previously developed highly sensitive and selective SERS-based ELISA protocol for the measurement of Aspergillus galactomannan in bronchoalveolar lavage fluid samples. METHODS: Ten bronchoalveolar lavage fluid samples were analyzed using both the GalMAg-EIA ELISA kit (XEMA LLC, Russia) and the previously developed SERS-based ELISA, which employs o-phenylenediamine substrate and silver nanoparticles colloid. RESULTS: Using standard samples prepared with in vitro cultivated galactomannan, the limit of detection for the SERS-based ELISA was estimated as 53 pg/mL with the working range spanning up to 10.8 ng/mL and the mean coefficient of variation within this range being 4%. Using the GalMAg-EIA kit, 6 out of 10 bronchoalveolar lavage fluid samples were identified as positive and 4 — as negative. The comparison of the results for these two methods demonstrates the non-linear dependence with the Spearman coefficient of correlation being 0.93–0.95 (p=2.3×10-5–1.1×10-4). CONCLUSION: For the bronchoalveolar lavage fluid samples, the comparability of the results was demonstrated when detecting the galactomannan using the commercially available GalMAg-EIA kit and the previously developed SERS-based ELISA. The results confirm the high sensitivity of the SERS-based Aspergillus galactomannan ELISA and its prospectiveness for practical laboratory applications. Compared to chromogenic detection, the advantages of surface-enhanced Raman scattering readout include a lower limit of detection for galactomannan and increased sensitivity at the lower end of the galactomannan-positivity index (<2). These findings could enable more accurate determination of the threshold for discriminating the positive and the negative samples. Thus, the research provides the basis for future larger scale trials of the SERS-based galactomannan ELISA in order to evaluate its applicability for clinical-laboratory diagnostics.

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BACKGROUND

Invasive aspergillosis is a threatening fungal infection [1], affecting, according to the modern estimates, more than 2 million people each year [2]. Most frequently invasive aspergillosis develops as a severe complication of the chronic pulmonary obstructive disease [3], lung cancer, hematological malignancies and organ transplantations, also as a result of intensive care [4, 5]. During the last decade, invasive aspergillosis is more often observed in a context of acute respiratory diseases caused by the influenza and SARS-CoV-2 viruses [6–8]. In case of the presence of invasive aspergillosis, the mortality for mentioned groups of patients ranges from 40% to 90% [2].

The specific feature of invasive aspergillosis is the complexity of timely diagnostics. The identification of the pathogen by culture tests is time-consuming, therefore, modern recommendations on the diagnostics of invasive aspergillosis are based on a combination of less specific, but rapid histopathological and computed-tomography-based examinations and especially the results of detecting the galactomannan — the polysaccharide component of the cell wall of Aspergillus [9–11]. The galactomannan test, in particular, the enzyme-linked immunosorbent assay (ELISA), detects the polysaccharide component in the biological fluids, such as the blood serum and the bronchoalveolar lavage fluid, providing an important information [12] both for the diagnostics and for the monitoring of treatment efficiency. Despite the undoubtful advances in decreasing the mortality caused by the invasive aspergillosis and the advances related to the implementation of such diagnostic kits into clinical practice, the issue of developing new generation methods for detecting the galactomannan with improved characteristics is still topical.

The first direction to improve the diagnostics is the application of more specific antibodies. By using of synthetic oligosaccharide molecular probes related to the determinant fragments of galactomannan, it was shown [13] that the existing commercially available galactomannan tests specifically recognize the smaller carbohydrate epitope than it was considered previously [14] and demonstrate cross-reactivity to some non-Aspergillus fungi [15–17] and bacteria, in particular, Bifidobacterium spp. [18, 19]. In order to solve this problem, the 7В8 monoclonal antibodies were proposed, being highly specific for Aspergillus fumigatus and Aspergillus flavus galactomannans [20]. The second promising direction is changing the method for detecting the product generated by the peroxidase label in ELISA, for instance, the highly sensitive measurements of о-phenylenediamine enzymatic oxidation product based on surface-enhanced Raman scattering (SERS) and silver nanoparticle colloids [21]. Using the calibration samples of cultural galactomannan added to the denatured human plasma and highly specific 7B8 antibodies, the compatibility of this SERS-based readout with the standard microplate ELISA procedures was demonstrated, achieving the galactomannan limit of detection of 43 pg/mL. This result is one and a half orders better compared to the colorimetric method using о-phenylenediamine as the substrate and 5 times more sensitive compared to ELISA with chromogenic detection using the 3,3’,5,5’-tetramethylbenzidine [22].

Research Aim — to evaluate the applicability of the previously developed highly sensitive and selective SERS-based ELISA protocol [22] for the purpose of Aspergillus galactomannan detection in the bronchoalveolar lavage fluid samples.

METHODS

Research Description

Preparation of calibration samples, containing the Aspergillus galactomannan

The stock solution of cultural A. fumigatus galactomannan was derived by cultivation of the fungus on the liquid medium, followed by isolation, inactivation and determining the galactomannan concentration, as described previously [20, 22]. The optimal matrix for calibration samples is the supernatant of denatured human blood plasma additionally depleted for native galactomannan [22]. It was prepared according to previously described protocol [22], using the pooled donor blood plasma collected at the Ministry of Healthcare of the Russian Federation Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. All the donors were healthy and had no inflammatory diseases or infections within the last two weeks.

The stock Aspergillus galactomannan solution was diluted with 100 mM Tris-HCl, 150 mM NaCl, pH 8, containing 0.05% Tween-20 (Tris Buffered Saline with Tween 20), supplemented with 1% bovine serum albumin (BSA) to the required concentration and mixed at a ratio of 1:9 with the matrix. Final concentration of galactomannan ranging from 16 to 10 800 pg/mL was used for calibration curve.

ELISA using the GalMAg-EIA kit

The positivity index (PI) for the bronchoalveolar lavage fluid samples was determined using the GalMAg-EIA (XEMA LLC, Russia) for the qualitative detection of Aspergillus galactomannan antigen in human serum or bronchoalveolar lavage fluid according to the manufacturer protocol. The absorbance (A) at 450 nm was measured using the xMark plate spectrophotometer (Bio-Rad, USA). For each tested sample, the positivity index was calculated: PI =Atest/Acut-off, where Atest is the absorbance of the tested sample; Acut-off is the absorbance of the cut-off sample, provided in the kit (absorbance within a range from 0.4 to 0.8). The obtained positivity indices were interpreted in the following way: PI >1.1 — positive sample (the tested sample contains galactomannan); 0.9≤ PI < 1.1 — indeterminate result; PI < 0.9 — negative result.

ELISA with SERS-based readout

The preparation of the bronchoalveolar lavage fluid samples was performed directly prior to ELISA by adding 200 µL of 0.1 М ethylenediaminetetraacetic acid (EDTA) to 500 µL of each sample, followed by boiling for 3 minutes and centrifuging for 20 minutes at 14 000 g. The supernatant after the centrifugation was used for further analysis. The sample preparation for each independent repeat was done separately.

The silver nanoparticle colloids were synthesized using the method by Leopold and Lendl [23], modified as described previously [21].

The wells of the 96-well clear polystyrene high binding microplate (No. 2592, Corning, USA) were filled with 100 µL of the solution of monoclonal antibodies against Aspergillus galactomannan (clone 7B8) with a concentration of 10 µg/mL in 100 mM Tris-HCl, 150 mM NaCl, pH 7.4. The plate was incubated for 16 hours at 4°C. After the triple wash with TBS-Т, the plate wells were blocked with 1% BSA solution in TBS-Т (16 hours at 4°C). Sample aliquots (50 µL) were added to the wells (calibration sample or prepared bronchoalveolar lavage fluid sample) along with 100 µL of 7B8 monoclonal antibodies conjugated with horseradish peroxidase (50 ng/mL). The plate was incubated for 60 minutes at 37°C with mixing at a rate of 600 rpm. Upon the completion of incubation, the plate was washed five times with TBS-Т solution and once — with 50 mM of pH 6 citrate buffer. The enzymatic reaction was initiated by adding 100 µL of the substrate solution containing 1 mM of о-phenylenediamine and 80 µM of H2O2 in 50 mM citrate buffer (pH 6) with 5 µg/mL of BSA. The enzymatic reaction was performed for 5 minutes at 37°C with mixing at a rate of 600 rpm, after which 50 µL aliquots were transferred to the test tubes containing 100 µL of 1.5 М citrate buffer (pH 3) for stopping the reaction. The obtained mixture was added to the silver nanoparticles colloid at a ratio of 1:1, and in 2 minutes after this, the surface-enhanced Raman spectra were acquired from the suspension of aggregated nanoparticles using the i-Raman Pro BWS475-785 H portable spectrometer (BWTek, Plainsboro, NJ, USA) with the excitation wavelength of 785 nm, 20-fold magnification objective, 135 mW power on the sample, and accumulation time of 0.5 seconds in 30 automatically averaged repeats. The surface-enhanced Raman spectra recorded for each galactomannan concentration were processed as described previously [21] for obtaining the band intensity of 733 cm-1, corresponding to the protonated form of 2,3-diaminophenazine (DAP) [24], the enzymatic reaction product.

Statistical analysis

The calculations of all statistical parameters were performed using the built-in functions of the Mathematica 10.2 software (Wolfram Research, USA): the arithmetic mean (Mean), the standard deviation (StandardDeviation), the Spearman correlation coefficient (SpearmanRho), the p-value (SpearmanRankTest), the median and percentiles of sample with automatic graph plotting (BoxWhiskerChart). The limit of detection was calculated as the concentration point on the calibration curve corresponding to the signal equaling to the sum of mean signal for the solution with zero galactomannan content and the tripled value of standard deviation. When calculating the mean coefficient of variation, the ratio of the standard deviation to the arithmetic mean was calculated for each sample with averaging the obtained values; for the evaluation of the measurement repeatability for calibration samples — using all the concentrations of the working range, while for the evaluation of intra-laboratory coefficient of variation — using all the measured samples in the group.

RESULTS

Research sample

Using the GalMAg-EIA kit (XEMA LLC, Russia) and the previously developed SERS-based ELISA protocol that employs the о-phenylenediamine substrate and the silver nanoparticles colloid, a total of 10 bronchoalveolar lavage fluid samples were tested.

The samples of bronchoalveolar lavage fluid were provided by XEMA LLC from the pool of biological material collected during the process of testing and registering the GalMAg-EIA kit. All the samples were collected after the written consent from the patients.

Primary research findings

The application of the previously developed protocol of SERS-based [22] to the samples of bronchoalveolar lavage fluid required two minor modifications: decreasing the volume of the prepared sample in the well to 50 µL, and decreasing the concentration of the detection antibody conjugate with the horseradish peroxidase. For the confirmation of the analytical characteristics for the modified protocol, the calibration curve was obtained using the samples with known concentrations of the cultural galactomannan (16–10 800 pg/mL) in the supernatant of denatured human blood plasma depleted for native galactomannan. The analytical signal was the intensity of 733 cm-1 band, corresponding to DAP, the product of enzymatic reaction (Fig. 1). The limit of detection for galactomannan was 53 pg/mL with a working range spanning up to 10.8 ng/mL and the mean coefficient of variation within this range of 4%.

 

Fig. 1. The calibration curve for SERS band intensity at 733 cm-1 (n=2) versus the concentration of galactomannan (16–10 800 pg/mL): the insert provides the lower part of the calibration curve with the galactomannan concentration below 450 pg/mL; the limit of detection and its corresponding signal level are plotted with red diamond.

 

Ten bronchoalveolar lavage fluid samples were tested using the GalMAg-EIA kit. In accordance with kit manufacturer’s protocol, 4 samples were identified as negative (positivity indexes — 0.64, 0.69, 0.74, 0.78) and another 6 — as positive (positivity indexes — 1.38, 1.97, 2.46, 5.64, 11.8, 15.9). The same 10 samples were tested using the SERS-based ELISA, in series of two independent repeats in 2 days for the evaluation of the intra-laboratory coefficient of variation, which was 15% for negative samples and 9% for the positive ones. Due to the fact that the dependence of SERS-based ELISA results on the measured positivity index (Fig. 2) is non-linear, the Spearman correlation coefficient was calculated for each of 4 repeats (0.93, 0.95, 0.95, 0.93 with the p values of 1.1×10-4, 2.3×10-5, 2.3×10-5, 1.1×10-4, respectively).

 

Fig. 2. SERS band intensities at 733 cm-1 for 10 bronchoalveolar lavage fluid samples (single repeat for each) versus their positivity indices measured with commercial GalMAg-EIA kit: dashed lines depict the negative (< 0.9, blue) and the positive (>1.1, red) thresholds values of the positivity index; greyed area represents undetermined samples (positivity index from 0.9 to 1.1).

 

For the evaluation of the possibilities of interpreting the assay result based on the single SERS-based measurement, intensity of the 733 cm-1 band for the pools of negative and positive samples was tested (Fig. 3). For the negative samples, the median value was 167 counts, minimum and maximum — 118 and 271 counts, respectively. The median for the positive samples was 1861 counts with the minimal and the maximal values being 829 and 3137 counts.

 

Fig. 3. SERS band intensities at 733 cm-1 for the negative and the positive bronchoalveolar lavage fluid samples: vertical columns of black dots indicate the raw data in four repeats; shaded boxes denote 25th and 75th percentiles; white lines indicate the medians; the whiskers and the numerical values indicate the minimal and maximal intensity values within the group.

 

DISCUSSION

The continuous development of the improved analogues of existing commercial products is the basis for the progress, including the field of laboratory clinical diagnostics. Surface-enhanced Raman scattering, being the optic method potentially capable of detecting the compounds at the extremely low concentrations, in the last decades is being widely used for the development of various analytical procedures. In a recent research [21], the conditions for specific and highly sensitive measurements of 2,3-diaminophenazine, a product of the peroxidase oxidation of о-phenylenediamine, were found by the detailed analysis of the physico-chemical principles of the SERS signal formation on the surface of silver nanoparticles. This finding enabled the method of SERS-based measurement of the horseradish peroxidase with a detection limit of 67 fM. Later on, using the calibration samples containing the cultural Aspergillus galactomannan and highly specific 7B8 antibodies, the compatibility of this SERS-based readout with the standard microplate ELISA procedures was demonstrated, which lead to the development of the ELISA protocol for the highly sensitive and selective detection of galactomannan [22]. During the present research work, this protocol with minor changes, needed for its adaptation to the new type of samples, was applied to test the real clinical samples of bronchoalveolar lavage fluid.

Due to the modification of the protocol, its analytical characteristics were independently tested using the calibration samples with known concentrations of cultural galactomannan. The limit of detection for galactomannan (53 pg/mL) was comparable to the previous result (43 pg/mL) [22], the working range remained unchanged (up to 10.8 ng/mL), the mean coefficient of variation within the working range has decreased from 11% to 4%. Thus, the modification of the protocol did not significantly affect its analytical characteristics.

Ten bronchoalveolar lavage fluid samples were tested using both the commercially available GalMAg-EIA kit and the SERS-based ELISA in 4 repeats. The relationship between the results of two methods was non-linear (see Fig. 2) due to the non-linearity in the dependencies of both signals on the galactomannan concentration. For the colorimetric method, this occurs primarily due to the deviation from the Beer–Bouguer–Lambert law at high absorbance. For SERS-based method, the concentrations dependence for the measured compound (DAP) is S-shaped [21]. The non-linearity of the relationship between the results from two methods determined the choice for evaluation of their correlation in favor of the Spearman rank correlation coefficient, which was 0.93–0.95 with the significance level < 0.0005.

The evaluation of the results of all SERS-based measurements of the bronchoalveolar lavage fluid samples (see Fig. 3) allows for drawing the preliminary (due to only 10 analyzed samples) conclusion that the classification of a sample as positive/negative is possible based on a single measurement when using the threshold within a range from 271 counts (maximal signal for negative samples) to 829 counts (minimal signal for positive samples). The benefits of SERS-based detection comparing to the chromogenic one are the decrease in the detection limit for galactomannan and the increase in the sensitivity within the positivity index range of up to 2, which in future may allow for more precisely determining the cut-off value for the positive and the negative results.

Research limitations

The developed SERS-based ELISA for the measurement of Aspergillus galactomannan was tested on a small number of samples (10 bronchoalveolar lavage fluid samples) and, hence, requires a wider verification for making a conclusion on the possibilities of using it in the clinical-laboratory diagnostics. Expanding the sample size is planned during further research.

CONCLUSION

The comparability of galactomannan measurements in the bronchoalveolar lavage fluid samples using the commercially available GalMAg-EIA kit and the previously developed enzyme-linked immunosorbent assay with the surface-enhanced Raman scattering readout was demonstrated. The obtained results indicate that the proposed protocol of SERS-based Aspergillus galactomannan enzyme-linked immunosorbent assay has a high sensitivity and its results correlate with the reference method. In a pilot group of 10 samples, the possibility of unambiguous interpretation of the testing result as the positive/negative was clearly demonstrated.

The research provides the basis for future larger scale trials of the proposed protocol for the purpose of making a conclusion on the possibilities of using it in the clinical-laboratory diagnostics.

ADDITIONAL INFORMATION

Author contributions: L.V. Yurina, writing a draft of the manuscript, conducting research; A.D. Vasilyeva, writing a draft of the manuscript, developing a methodology, conducting research; E.G. Evtushenko, developing a methodology, data analysis, revision and editing of the manuscript, visualization; E.S. Gavrilina, conducting research; V.B. Krylov, writing a draft of the manuscript, revision and editing of the manuscript, research support; D.V. Basmanov, research support, revision and editing of the manuscript; N.E. Nifantiev, attracting funding, revision and editing of the manuscript, providing research; I.N. Kurochkin, defining the concept, providing research, revision and editing of the manuscript, attracting funding. Thereby, all authors provided approval of the version to be published and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Acknowledgments: The authors are grateful to Y.S. Lebedyn and E.S. Kostrykina for productive discussions of the results, and for providing the the experimental materials. The study was performed using equipment purchased under the M.V. Lomonosov Moscow State University development program.

Ethics approval: All procedures performed in studies involving human subjects comply with the ethical standards of the Russian Committee on Bioethics, as well as the 1964 Declaration of Helsinki and its subsequent amendments.

Funding source: This work was supported by the Ministry of Science and Higher Education of the Russian Federation (FFZZ-2024-0004, agreement No 075-03-2024-401/3 from 30 May 2024—synthesis of antigenic oligosaccharides and glycoconjugates thereof; FFZR-2024-0005, agreement No 075-00422-24-02 from 28 May 2024—preparation of silver nanoparticles, performing SERS-based ELISA).

Disclosure of interests: The authors declare that they have no competing interests.

Statement of originality: The authors did not use previously published information (text, illustrations, data) while conducting this work.

Data availability statement: The authors report that all data is presented in the article and/or its appendices.

Generative AI: Generative AI technologies were not used for this article creation.

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About the authors

Lyubov V. Yurina

Institute of Biochemical Physics named after N.M. Emanuel

Author for correspondence.
Email: lyu.yurina@gmail.com
ORCID iD: 0000-0003-1836-9722
SPIN-code: 2199-7648

PhD

Russian Federation, Moscow

Alexandra D. Vasilyeva

Institute of Biochemical Physics named after N.M. Emanuel

Email: alexandra.d.vasilyeva@gmail.com
ORCID iD: 0000-0002-2286-0592
SPIN-code: 7122-9629

PhD

Russian Federation, Moscow

Evgeniy G. Evtushenko

Institute of Biochemical Physics named after N.M. Emanuel; Lomonosov Moscow State University

Email: evtushenko@enzyme.chem.msu.ru
ORCID iD: 0000-0003-1264-4770
SPIN-code: 8722-2388
Russian Federation, Moscow; Moscow

Elizaveta S. Gavrilina

Institute of Biochemical Physics named after N.M. Emanuel

Email: e.gavrilina98@gmail.com
ORCID iD: 0000-0002-2058-8660
SPIN-code: 6835-4460
Russian Federation, Moscow

Vadim B. Krylov

Institute of Organic Chemistry

Email: v_krylov@ioc.ac.ru
ORCID iD: 0000-0001-9477-4610
SPIN-code: 9750-0292

PhD

Russian Federation, Moscow

Dmitry V. Basmanov

Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine

Email: dmitry.basmanov@rcpcm.org
ORCID iD: 0000-0001-6620-7360
SPIN-code: 1801-6408
Russian Federation, Moscow

Nikolay E. Nifantiev

Institute of Organic Chemistry

Email: nen@ioc.ac.ru
ORCID iD: 0000-0002-0727-4050
SPIN-code: 5160-0379

PhD, Professor, Corresponding member of the Russian Academy of Sciences

Russian Federation, Moscow

Ilya N. Kurochkin

Institute of Biochemical Physics named after N.M. Emanuel; Lomonosov Moscow State University

Email: inkurochkin@gmail.com
ORCID iD: 0000-0002-2631-5866
SPIN-code: 4977-5273

PhD, Professor

Russian Federation, Moscow; Moscow

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2. Fig. 1. The calibration curve for SERS band intensity at 733 cm-1 (n=2) versus the concentration of galactomannan (16–10 800 pg/mL): the insert provides the lower part of the calibration curve with the galactomannan concentration below 450 pg/mL; the limit of detection and its corresponding signal level are plotted with red diamond.

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3. Fig. 2. SERS band intensities at 733 cm-1 for 10 bronchoalveolar lavage fluid samples (single repeat for each) versus their positivity indices measured with commercial GalMAg-EIA kit: dashed lines depict the negative (< 0.9, blue) and the positive (>1.1, red) thresholds values of the positivity index; greyed area represents undetermined samples (positivity index from 0.9 to 1.1).

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4. Fig. 3. SERS band intensities at 733 cm-1 for the negative and the positive bronchoalveolar lavage fluid samples: vertical columns of black dots indicate the raw data in four repeats; shaded boxes denote 25th and 75th percentiles; white lines indicate the medians; the whiskers and the numerical values indicate the minimal and maximal intensity values within the group.

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