Differentiation of the Intradural Extramedullary Spinal Tumors, Schwannomas, and Meningiomas Utilizing the Contrast Ratio as a Quantitative Magnetic Resonance Imaging Method.

BACKGROUND: Schwannomas and meningiomas are the most common intradural extramedullary spinal tumors; however, differentiating between them using magnetic resonance imaging (MRI) is a frequent challenge. In this study, we aimed to investigate the use of the contrast ratio (CR) as a quantitative MRI method in the differentiation of schwannomas and meningiomas. METHODS: We analyzed the data of patients with intradural extramedullary spinal tumors who underwent surgery and were diagnosed with either schwannomas or meningiomas by histopathological analysis. Regions of interest were set for the entire spinal tumor on T2-weighted sagittal MRI. To obtain the CR values of spinal tumors (CRtumor), we used the signal intensity (SI) values of the tumor (SItumor) and spinal cord (SIcord) according to the following formula: [CRtumor = (SItumor-SIcord)/(SItumor+SIcord)]. RESULTS: The study included 50 patients (23 males and 27 females) with a mean age of 61.5 years old (11-85 years old). Histopathological analysis revealed that 33 and 17 patients were diagnosed with schwannomas and meningiomas, respectively. The mean CR values of the schwannomas and meningiomas were 0.3040 ± 0.1386 and 0.0173 ± 0.1929, respectively. The CR value of the schwannomas was statistically significantly higher than that of meningiomas (P < 0.01). The cutoff CR value obtained from the receiver operating characteristic curve was 0.143, with a specificity and sensitivity of 90.9% and 88.2%, respectively. Furthermore, the value for the area under the receiver operating characteristic curve was 0.925 (95% confidence interval: 0.852-0.998). CONCLUSIONS: The evaluation of CRs by using MRI to distinguish between schwannomas and meningiomas is a beneficial quantitative tool.

Development of a novel animal model of lumbar vertebral endplate lesion by intervertebral disk injection of monosodium iodoacetate in rats.

PURPOSE: Vertebral endplate lesions (EPLs) caused by severe disk degeneration are associated with low back pain. However, its pathophysiology remains unclear. In this study, we aimed to develop a vertebral EPL rat model mimicking severe intervertebral disk (IVD) degeneration by injecting monosodium iodoacetate (MIA) into the IVDs and evaluating it by assessing pain-related behavior, micro-computed tomography (CT) findings, and histological changes. METHODS: MIA was injected into the L4-5 and L5-6 IVDs of Sprague-Dawley rats. Their behavior was examined by measuring the total distance traveled and the total number of rearing in an open square arena. Bone alterations and volume around the vertebral endplate were assessed using micro-CT. Safranin-O staining, immunohistochemistry, and tartrate-resistant acid phosphatase (TRAP) staining were performed for histological assessment. RESULTS: The total distance and number of rearing times in the open field were significantly reduced in a time-dependent manner. Micro-CT revealed intervertebral osteophytes and irregularities in the endplates at 12 weeks. The bone volume/tissue volume (BV/TV) around the endplates significantly increased from 6 weeks onward. Safranin-O staining revealed severe degeneration of IVDs and endplate disorders in a dose- and time-dependent manner. Calcitonin gene-related peptide-positive nerve fibers significantly increased from 6 weeks onward. However, the number of osteoclasts decreased over time. CONCLUSION: Our rat EPL model showed progressive morphological vertebral endplate changes in a time- and concentration-dependent manner, similar to the degenerative changes in human IVDs. This model can be used as an animal model of severe IVD degeneration to better understand the pathophysiology of EPL.

Evaluation of Epiconus and Conus Medullaris Disorders due to Thoracolumbar Vertebral Fracture using Motor Evoked Potentials.

STUDY DESIGN: A retrospective case-control study. OBJECTIVE: To characterize the motor evoked potential (MEP) when the epiconus or conus medullaris is compressed by a fracture of the T12 or L1 vertebra. SUMMARY OF BACKGROUND DATA: Although the characteristics of compressive cervical and thoracic myelopathy with transcranial magnetic stimulation MEP have been reported, the MEP parameters in compressive disorders of the epiconus and conus medullaris have not yet been characterized. METHODS: Twenty patients with T12 or L1 vertebral fractures who had lower extremity symptoms due to compression of the epiconus or conus medullaris were included. These patients were compared with 28 healthy controls and 32 patients with cervical spondylotic radiculopathy (CSR) without spinal cord compression. MEPs of abductor hallucis muscles were recorded using transcranial magnetic stimulation and electrical stimulation of the tibial nerve. MEP latency, central motor conduction time (CMCT), and peripheral conduction time (PCT) were evaluated. RESULTS: MEP latency, CMCT, and PCT were significantly longer in patients with fractures than in healthy controls and patients with CSR. MEP latency was most accurate for differentiating patients with fracture from healthy controls (cutoff value, 40.0 ms, sensitivity, 95.0%; specificity, 100%), and CMCT was most accurate for comparing patients with fracture and CSR (cutoff value, 15.5 ms, sensitivity, 80.0%; specificity, 93.8%). In the distinction between patients with fracture and CSR, 16 of the 20 patients with fracture exceeded the cutoff values for any of the parameters, and 12 of them exceeded the cutoff values for all parameters. There was no significant correlation between the linear distance from the most inferior end of the spinal cord to the site of compression and any of the MEP parameters. CONCLUSION: Both CMCT and PCT are often prolonged in compressive lesions of the epiconus and conus medullaris, and MEP latency and CMCT are useful in the diagnosis.

Short-segment spinal fusion for chronic low back pain with bone marrow edema adjacent to the vertebral endplate in adult spinal deformity.

PURPOSE: Corrective long spinal fusion is a widely accepted surgical method for patients with adult spinal deformities. However, instrumented long fusion is associated with a significant risk of complications. Therefore, we aimed to assess the success of short-segment spinal fusion, particularly for bone marrow edema (BME) adjacent to the vertebral endplate, in patients with low back pain (LBP) and spinal deformity. METHODS: A prospective study was performed at multiple hospitals wherein we monitored patients with spinal deformities and accompanying LBP. Patients aged ≥ 50 years with a minimum LBP severity score of 40 mm on the visual analog scale (VAS) were included in the study. We also included patients with lumbar BME on magnetic resonance imaging. Short spinal fusion was performed on segments with BME. Clinical evaluations of LBP on VAS and Oswestry Disability Index (ODI), and radiological parameters for sagittal vertical axis (SVA), pelvic incidence (PI), lumbar lordosis (LL) and pelvic tilt (PT) were carried out. RESULTS: Overall, 35 patients (22 men and 13 women), with a mean age of 66.7 years and a mean follow-up period of 32 months, were included in the study. The mean VAS and ODI scores were 72.4 mm and 49.0% before surgery and 25.5 mm and 29.9% at the final follow-up, respectively; these parameters significantly improved after surgery. The SVA, PI-LL, and PT scores were 70.1 mm, 20.9°, and 22.8° before surgery and 85.4 mm, 13.8°, and 22.7° at the final follow-up, respectively. The spinal alignment parameters did not change significantly after surgery. CONCLUSIONS: Short-segment spinal fusion is effective for treating LBP and spinal deformity with BME adjacent to the vertebral endplate without spinal correction.

A high-sensitivity ELISA for detection of human FGF18 in culture supernatants from tumor cell lines.

Fibroblast growth factor 18 (FGF18) is elevated in several human cancers, such as gastrointestinal and ovarian cancers, and stimulates the proliferation of tumor cells. This suggests that FGF18 may be a promising candidate biomarker in cancer patients. However, the lack of a high-sensitivity enzyme-linked immunosorbent assay (ELISA) does not permit testing of this possibility. In this study, we generated monoclonal antibodies against human FGF18 and developed a high-sensitivity ELISA to measure human FGF18 at concentrations as low as 10 pg/mL. Of the eight tumor cell lines investigated, we detected human FGF18 in culture supernatants from four tumor cell lines, including HeLa, OVCAR-3, BxPC-3, and SW620 cells, albeit the production levels were relatively low in the latter two cell lines. Moreover, the in-house ELISA could detect murine FGF18 in sera from mice overexpressing murine Fgf18 in hepatocytes, although the sensitivity in detecting murine FGF18 was relatively low. This FGF18 ELISA could be a valuable tool to validate FGF18 as a potential biomarker for cancer patients and to test the contribution of FGF18 for various disease models invivo and in vitro.

Rapid engineering of SARS-CoV-2 therapeutic antibodies to increase breadth of neutralization including BQ.1.1, CA.3.1, CH.1.1, XBB.1.16, and XBB.1.5.

SARS-CoV-2 Omicron variant XBB.1.5 has shown extraordinary immune escape even for fully vaccinated individuals. There are currently no approved antibodies that neutralize this variant, and continued emergence of new variants puts immunocompromised and elderly patients at high risk. Rapid and cost-effective development of neutralizing antibodies is urgently needed. Starting with a single parent clone that neutralized the Wuhan-Hu-1 strain, antibody engineering was performed in iterative stages in real time as variants emerged using a proprietary technology called STage-Enhanced Maturation. An antibody panel that broadly neutralizes currently circulating Omicron variants was obtained by in vitro affinity maturation using phage display. The engineered antibodies show potent neutralization of BQ.1.1, XBB.1.16, and XBB.1.5 by surrogate virus neutralization test and pM KD affinity for all variants. Our work not only details novel therapeutic candidates but also validates a unique general strategy to create broadly neutralizing antibodies to current and future SARS-CoV-2 variants.

Vertebral osteomyelitis due to Mycobacterium abscessus subsp. massiliense with paravertebral abscess: A case report and review.

The incidence of vertebral osteomyelitis (VO) caused by non-tuberculosis mycobacteria (NTM) without immunocompetence is extremely rare. Herein, we reported on a case of VO caused by NTM. A 38-year-old man was admitted to our hospital with persisting low back and leg pain which had lasted for a year. Before coming to our hospital, the patient was treated with antibiotics and iliopsoas muscle drainage. The biopsy confirmed the presence of a NTM, Mycobacterium abscessus subsp. massiliense. Several tests were conducted which showed the infection had progressively increased, such as vertebral endplate destruction on plain radiography, computed tomography scan, and epidural and paraspinal muscle abscesses on magnetic resonance imaging. The patient underwent radical debridement, anterior intervertebral fusion with bone graft, and posterior instrumentation with antibiotic administration. A year later, the patient's low back and leg pain was relieved without any analgetic. VO due to NTM is rare but can be treated with multimodal therapy.

Differentiating Neurodegenerative Disease from Compressive Cervical Myelopathy Using Motor Evoked Potentials.

STUDY DESIGN: A retrospective case-control study. OBJECTIVE: To differentiate neurodegenerative diseases from compressive cervical myelopathy (CCM) using motor evoked potentials (MEPs). SUMMARY OF BACKGROUND DATA: When considering surgery for CCM, it may be necessary to differentiate the condition from a neurodegenerative disease. METHODS: A total of 30 healthy volunteers, 52 typical CCM patients with single-level compression of the spinal cord at C4-5 or C5-6, seven patients with amyotrophic lateral sclerosis (ALS), and 12 patients with demyelinating disease of the central nervous system (DDC), including 11 patients with multiple sclerosis and one patient with neuromyelitis optica spectrum disorder, formed our study population. MEPs were recorded from the bilateral abductor digiti minimi (ADM) and abductor hallucis (AH) muscles using transcranial magnetic stimulation and electrical stimulation of the ulnar and tibial nerves. Central motor conduction time (CMCT), peripheral conduction time, amplitude of MEPs, and frequency of F-waves were evaluated. Receiver operating characteristic (ROC) curve analysis was used to determine the cut-off value for distinguishing between CCM and ALS. RESULTS: Significant differences were observed in the amplitude of MEPs and frequency of F-waves evoked by peripheral nerve stimulation between patients with CCM and ALS. The MEP amplitude of AH was more accurate in differentiating between the two diseases compared to ADM (cut-off value, 11.2mV, sensitivity, 87.5%; specificity, 85.7%). All seven patients with ALS showed reduced frequency of F waves from ADM or AH, but none of the healthy volunteers or patients with other diseases demonstrated this finding. Moreover, there were no significant differences between CCM and DDC in any of the assessments. CONCLUSION: The amplitude of MEPs and frequency of F waves evoked by peripheral nerve stimulation could be helpful in differentiating ALS from CCM.

Differentiation Between Compressive Cervical and Thoracic Myelopathy Using the Central Motor Conduction Time Ratio.

PURPOSE: Thoracic myelopathy is a rare condition whose diagnosis is often missed or delayed. This study aimed to differentiate between cervical and thoracic myelopathy using motor-evoked potential testing. METHODS: The authors included 835 patients with compressive cervical myelopathy and 94 patients with compressive thoracic myelopathy. Myelopathy using motor-evoked potentials were recorded from the bilateral abductor digiti minimi and abductor hallucis muscles through transcranial magnetic stimulation. The peripheral conduction time was measured through electrical stimulation of the ulnar and tibial nerves; moreover, the central motor conduction time (CMCT) was calculated by subtracting the peripheral conduction time from the myelopathy using motor-evoked potential latency. RESULTS: The most accurate differentiation between compressive cervical myelopathy and compressive thoracic myelopathy was achieved by the CMCT ratios (CMCT-ADM:CMCT-AH; cutoff value of 0.490, sensitivity of 83.0%, and specificity of 80.5%). After excluding patients with compressive cervical myelopathy who had spinal cord compression at C6-7, the cutoff value was 0.490, with a sensitivity of 83.0% and specificity of 87.3%. CONCLUSIONS: Determining the CMCT ratio (cutoff value of 0.490) through motor-evoked potential testing could facilitate differentiation between compressive cervical myelopathy and compressive thoracic myelopathy.

Bivalent intra-spike binding provides durability against emergent Omicron lineages: Results from a global consortium.

The SARS-CoV-2 Omicron variant of concern (VoC) and its sublineages contain 31-36 mutations in spike and escape neutralization by most therapeutic antibodies. In a pseudovirus neutralization assay, 66 of the nearly 400 candidate therapeutics in the Coronavirus Immunotherapeutic Consortium (CoVIC) panel neutralize Omicron and multiple Omicron sublineages. Among natural immunoglobulin Gs (IgGs), especially those in the receptor-binding domain (RBD)-2 epitope community, nearly all Omicron neutralizers recognize spike bivalently, with both antigen-binding fragments (Fabs) simultaneously engaging adjacent RBDs on the same spike. Most IgGs that do not neutralize Omicron bind either entirely monovalently or have some (22%-50%) monovalent occupancy. Cleavage of bivalent-binding IgGs to Fabs abolishes neutralization and binding affinity, with disproportionate loss of activity against Omicron pseudovirus and spike. These results suggest that VoC-resistant antibodies overcome mutagenic substitution via avidity. Hence, vaccine strategies targeting future SARS-CoV-2 variants should consider epitope display with spacing and organization identical to trimeric spike.

Foramen Magnum Decompression with Outer Dura Matter Layer Resection for Chiari Type I Malformation: Factors Correlated with Syrinx Reduction.

STUDY DESIGN: A retrospective cohort study. PURPOSE: We aimed to investigate the surgical results of foramen magnum decompression (FMD) to identify the potential factors associated with syrinx reduction in Chiari malformation type I (CMI). OVERVIEW OF LITERATURE: The predictive value of preoperative factors for syrinx reduction in patients with CMI remains debatable. METHODS: We enrolled patients who underwent microscopic FMD with outer dural layer resection for CMI. The distance from the tip of the cerebellar tonsil to the C2 vertebral endplate on sagittal magnetic resonance imaging (MRI) was defined as the tonsillar distance (TD). Patients who showed a >20% syrinx diameter reduction on the 1-year follow-up MRI were defined as the syrinx reduction group while the others were categorized in the syrinx nonreduction group. Patients with syringomyelia were categorized into the clinically improved and unimproved groups using the Chicago Chiari Outcome Scale. The imaging and clinical parameters were evaluated pre- and postoperatively. RESULTS: This study included 25 patients of whom 19 (76.0%) had syringomyelia. At the 1-year follow-up, the syrinx diameter had decreased in 11 patients (57.8%). The increased TD significantly differed between the syrinx reduction and nonreduction groups. At the 1-year follow-up, 12 and seven patients with syringomyelia were categorized into the clinically improved and unimproved groups, respectively. The clinically improved and unimproved groups showed significant differences in the mean age and increased TD. CONCLUSIONS: Postoperative syrinx reduction was significantly correlated with the upward shifting of the cerebellar tonsil in patients with CMI. Our quantitative evaluation of the alterations in hindbrain position after FMD was easily performed and reflects the clinical outcomes.

Differences between spinal cord injury and cervical compressive myelopathy in intramedullary high-intensity lesions on T2-weighted magnetic resonance imaging: A retrospective study.

Increases in aging populations have raised the number of patients with cervical spinal cord injury (SCI) without fractures due to compression of the cervical spinal cord. In such patients, it is necessary to clarify whether SCI or cervical compressive myelopathy (CCM) is the cause of disability after trauma. This study aimed to clarify the differences in magnetic resonance imaging (MRI) features between SCI and CCM. Overall, 60 SCI patients and 60 CCM patients with intramedullary high-intensity lesions on T2-weighted MRI were included in this study. The longitudinal lengths of the intramedullary T2 high-intensity lesions were measured using sagittal MRI sections. Snake-eye appearance on axial sections was assessed as a characteristic finding of CCM. The T2 values of the high-intensity lesions and normal spinal cords at the first thoracic vertebra level were measured, and the contrast ratio was calculated using these values. The longitudinal length of T2 high-intensity lesions was significantly longer in SCI patients than in CCM patients. Snake-eye appearance was found in 26 of the 60 CCM patients, but not in SCI patients. On both the sagittal and axial images, the contrast ratio was significantly higher in the SCI group than in the CCM group. Based on these results, a diagnostic scale was created. This scale made it possible to distinguish between SCI and CCM with approximately 90% accuracy.

Comparison of the electrophysiological characteristics of tight filum terminale and tethered cord syndrome.

PURPOSE: This study aims to characterize tight filum terminale (TFT) in motor evoked potential (MEP) testing by comparing TFT patients with both tethered cord syndrome (TCS) patients and healthy subjects. METHODS: Fifty TFT patients, 18 TCS patients, and 35 healthy volunteers participated in this study. We recorded MEPs following transcranial magnetic stimulation from the bilateral abductor hallucis muscles as well as compound muscle action potentials and F-waves evoked by electrical stimulation of the tibial nerve from the bilateral abductor pollicis brevis muscles. The peripheral conduction time (PCT) was calculated from the latency of the compound action potential and F-wave. Furthermore, the central motor conduction time (CMCT) was calculated by subtracting PCT from MEP latency. RESULTS: TFT and TCS patients had a significantly longer MEP latency than healthy subjects. PCT in TFT patients was significantly longer than those in TCS patients or healthy subjects. Using the cutoff values for PCT, we were able to diagnose patients with TFT patients with a sensitivity of 72.0% and a specificity of 91.4%. CONCLUSION: Prolonged PCT in the MEP test may be a useful indicator for TFT and suggests that MEP may be used as an adjunct diagnostic tool for TFT.

A New Method to Characterize Conformation-Specific Antibody by a Combination of Agarose Native Gel Electrophoresis and Contact Blotting.

In this study, we review the agarose native gel electrophoresis that separates proteins and macromolecular complexes in their native state and transfer of the separated proteins from the agarose gel to membranes by contact blotting which retains the native state of these structures. Green fluorescent protein showed functional state both on agarose gel and blotted membrane. Based on the combined procedures, we discovered conformation-specific monoclonal antibodies against PLXDC2 and SARS-CoV-2 spike protein.

Evaluation of intervertebral disc degeneration using T2 signal ratio on magnetic resonance imaging.

PURPOSE: Intervertebral disc degeneration is assessed clinically by magnetic resonance imaging (MRI). Although some quantitative evaluation methods for MRI under special imaging conditions have been reported, they are widely and generally difficult to use. The aim of this study is to determine if intervertebral disc degeneration can be assessed using the ratio of MRI T2 values of the disc to the spinal cord T2 values. METHODS: Signal ratio was calculated using the T2 signal intensity of the disc and the spinal cord on MRI under common conditions for a new assessment of disc degeneration. T2-weighted images of 100 patients undergoing MRI twice within a year under different imaging conditions, 1.5 T or less and 3.0 T, were used for the assessment. The T2 signal intensity was measured at the center of the discs at L2-3, L3-4, L4-5, L5-S1 and the spinal cord at T12 level. Signal ratio was calculated using these T2 signal intensity values. The ratio of the difference between the first and second values to the mean of the first and second values was calculated to confirm the equivalence of MRI assessments of disc degeneration in the same patient under different imaging conditions. RESULTS: The equivalence of values between the first MRI and the second MRI in the signal ratio was significantly higher than that in the T2 signal intensity. In addition, the signal ratio was negatively correlated with age and were significantly associated with Pfirrmann grade. CONCLUSIONS: By using the signal ratio, disc degeneration can be evaluated by MRI even under different imaging conditions.

Antibody recognition of complement factor H reveals a flexible loop involved in atypical hemolytic uremic syndrome pathogenesis.

Atypical hemolytic uremic syndrome (aHUS) is a disease associated with dysregulation of the immune complement system, especially of the alternative pathway (AP). Complement factor H (CFH), consisting of 20 domains called complement control protein (CCP1-20), downregulates the AP as a cofactor for mediating C3 inactivation by complement factor I. However, anomalies related to CFH are known to cause excessive complement activation and cytotoxicity. In aHUS, mutations and the presence of anti-CFH autoantibodies (AAbs) have been reported as plausible causes of CFH dysfunction, and it is known that CFH-related aHUS carries a high probability of end-stage renal disease. Elucidating the detailed functions of CFH at the molecular level will help to understand aHUS pathogenesis. Herein, we used biophysical data to reveal that a heavy-chain antibody fragment, termed VHH4, recognized CFH with high affinity. Hemolytic assays also indicated that VHH4 disrupted the protective function of CFH on sheep erythrocytes. Furthermore, X-ray crystallography revealed that VHH4 recognized the Leu1181-Leu1189CCP20 loop, a known anti-CFH AAbs epitope. We next analyzed the dynamics of the C-terminal region of CFH and showed that the epitopes recognized by anti-CFH AAbs and VHH4 were the most flexible regions in CCP18-20. Finally, we conducted mutation analyses to elucidate the mechanism of VHH4 recognition of CFH and revealed that VHH4 inserts the Trp1183CCP20 residue of CFH into the pocket formed by the complementary determining region 3 loop. These results suggested that anti-CFH AAbs may adopt a similar molecular mechanism to recognize the flexible loop of Leu1181-Leu1189CCP20, leading to aHUS pathogenesis.

Radiographic Factors for Adjacent Vertebral Fractures and Cement Loosening Following Balloon Kyphoplasty in Patients with Osteoporotic Vertebral Fractures.

Introduction: Balloon kyphoplasty (BKP) is a minimally invasive surgical approach for the treatment of osteoporotic vertebral fractures (OVF). Some risks have been reported after treatment with BKP; therefore, it is necessary to determine when BKP does not work. Thus, in this study, we aim to clarify the radiographic predictors of secondary vertebral fractures and cement loosening after BKP for OVF. Methods: This study enrolled patients with single-level OVF at the thoracolumbar junction (T11-L2) who underwent BKP for the first time between January 2011 and March 2014. The clinical outcomes were evaluated using the visual analog scale (VAS) and a modified Oswestry Disability Index (ODI) at 1 week and 1, 3, 6, and 12 months after surgery. Radiographic assessments were performed preoperatively and within 1 year after BKP using plain radiography and computed tomography. Results: The 85 patients who met the inclusion criteria underwent BKP. The average age of participants (21 men, 64 women) was 77.8 years (range, 57-92 years). Postoperative VAS and ODI scores were all significantly better than preoperative scores. Polymethyl methacrylate (PMMA)-cement leakage was observed in 18 patients (21.2%) but was asymptomatic in all cases. Secondary vertebral fractures were detected in 20 patients (23.5%), including adjacent levels in 15 patients (17.6%) and non-adjacent levels in 5 patients (5.9%). Rostral bridging osteophyte formation was found to be significantly associated with the occurrence of adjacent vertebral fractures (odds ratio 12.746; p=0.010). PMMA-cement loosening was observed in three patients (3.5%). A high prevalence (100%) of bridging osteophytes, vacuum clefts, and spinous process fractures was observed in patients with PMMA-cement loosening. PMMA-cement loosening was found in 3 out of 10 patients with all three of these factors. Conclusions: Rostral bridging osteophyte formation was determined to be a risk factor for both adjacent vertebral fractures and PMMA-cement loosening.Level of Evidence: 3.

Western blotting of native proteins from agarose gels.

We have developed a new Western blotting method of native proteins from agarose-based gel electrophoresis using a buffer at pH 6.1 containing basic histidine and acidic 2-(N-morpholino)ethanesulfonic acid. This gel electrophoresis successfully provided native structures for a variety of proteins and macromolecular complexes. This paper is focused on the Western blotting of native protein bands separated on agarose gels. Two blotting methods from agarose gel to PVDF membrane are introduced here, one by contact (diffusion) blotting and another by electroblotting after pre-treating the agarose gels with SDS. The contact blotting resulted in the transfer of native GFP, native human plexin domain containing protein 2 (PLXDC2) and native SARS-CoV-2 spike protein, which were detected by conformation-specific antibodies generated in-house.