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1. Western blotting (immunoblotting): history, theory, uses, protocol and problems 2023

   https://ncsu.summon.serialssolutions.com/2.0.0/link/0/eLvHCXMwpV1bixMxFA66ovgiuorWGxFEFDp00rkkkWVhle36sF5Yu-iLhGSS2MLSkV4e_Peek2Rmt6Kw4ssQMu0UzpeenG9yzncIeS5qC7uEqDLGLRCUStSZMExnzAPE3oqGh6r3iy9zqq4W5o9Z7KP_hhbmAFwslf0HePuHwgSMAWS4AsxwvRTQX6LWAaagxxxmiBnnWPXRdjPA9pH4R2nhYNRQvhhGm1X0E6jX0MKiSMIBocvMauvEd95OO73XPg7_vInZyCd6NXOpTCooYoMVQ1R6pA1w8bPe9x-1qQDi4Mwu54uUYpveOoyLPq0KNo3oKWWZZ1Ik55lcaWyCkpZMccEvxiZIaYdlsWz0d-eN4m1gbLNeAL5AkVE653yX6k7mP3xUk9PjYzU9_Dq9Sq6NuZTYuGJy8qnn2TnnMsqp4iNHWw_cCj9Cl4a_U4sQYkxvk1uJG9CDiPEdcsUtdsn12C305y658T7lQdwl3_Zm-wn3vdFsn3ZI05dwYxt8vP_qNU3gD2mEfkgR-CHtYKcAO-1gv0dOJ4fTt--y1Cgja4pKrmFHK_1YcqZ9XjUQ8Lo6t8bJCoM7I7StdC20bLQvdWOY9xwlfaz0jnvd8DIvHhDKjIGRBQLtDXB-Kepcc2-1d7rQjfAD8qIznPoRhVAUEEi0sAILK7QwCs2WA_IGzdp_CPXLw0S7_K7Sv0TJurJ-7LzGQhIIpERTa1eYRgKhZzl3A_KsA0WBv8NDLL1w7WalAHDGg8TpgNyPaPU_VdRgBMaKh5f49iNy83xdPyY76-XGPSE76HKehuX0C_EpfVU

   Sule, Rasheed; Rivera, Gabriela; Gomes, Aldrin V

   Bio Techniques, Vol. 75, Issue 3, pp. 99 - 114.

   Western blotting (immunoblotting) is a powerful and commonly used technique that is capable of detecting or semiquantifying an individual protein from complex mixtures of proteins extracted from ce... Read more

   Western blotting (immunoblotting) is a powerful and commonly used technique that is capable of detecting or semiquantifying an individual protein from complex mixtures of proteins extracted from cells or tissues. The history surrounding the origin of western blotting, the theory behind the western blotting technique, a comprehensive protocol and the uses of western blotting are presented. Lesser known and significant problems in the western blotting field and troubleshooting of common problems are highlighted and discussed. This work is a comprehensive primer and guide for new western blotting researchers and those interested in a better understanding of the technique or getting better results. Read less

   Journal Article  |  Full Text Online

2. Western immunoblotting as a new tool for investigating direct antiglobulin test-negative... 2015

   https://ncsu.summon.serialssolutions.com/2.0.0/link/0/eLvHCXMwnV1RT9swED4hJiFeYBtoZGyThxDiJWqT1IkjIaQNrfACEqhQ7cmyE5tWKw1qU4m9TPsP_EN-ye6cpC3SJk285cGxbN-d_Z3v_B3AvohzPCUE94MkRweFi9gXOlB-YFHENhdZ4l69L13mLEp9VfwQ8ws3sgy3X5OBKz1tjLxVTiwVMha0-yKIoaINX78veMIpOljFlgOfaqnXnELP_nx2Er2iRX34G8xcQq1hfex0N8E0A3bZJlTjYDntvuWoDh2_RUPr-KLJvYaNGpeyL5UivYEVM34La-d15H0Lfh0NjvsVqwIb0puSQo8KlzN91BocMzVliiFCZ2VRjBgCYTZcEHiMb1l1cjIU45AoSCj9nSHILZ9-P47NrSMfZ2pWFq5rwwbmrhj9xKHgH-ZuqKbbcN391js58-vaDX4WxVHoo-sbZakNdZwqkWoR5gIdSZ2ngTEWUZ7OEZhlaYe3dWjSIAs7JsoSbiLFEUVENtoBFmiddNo5-nRWoxuairitEpsra1SkMmE92GsEKO8rbg7Z-DS4hNItoQcHTrTzFmryg5LZEi77F6fytHdzws8v-7LrwedG9hJNjOImOMViNpXEJ0TQstP24F2lFPPeQi4c6Z8Hh06e_x6I7F113cf7_2-6C-sI0HiVmvYBVsvJzHyEVVKzT07h_wDFFQZa

   Bloch, Evgenia M.; Sakac, Darinka; Branch, Haley A....

   Transfusion (Philadelphia, Pa.), Vol. 55, Issue 6pt2, pp. 1529 - 1537.

   BACKGROUND Direct antiglobulin test–negative (DAT(–)) autoimmune hemolytic anemia, characterized by hemolysis without detectable immunoglobulin or complement on patient red blood cells (RBCs), pose... Read more

   BACKGROUND Direct antiglobulin test–negative (DAT(–)) autoimmune hemolytic anemia, characterized by hemolysis without detectable immunoglobulin or complement on patient red blood cells (RBCs), poses a diagnostic challenge. To select therapy, classification of the hemolysis as immune‐ or non–immune‐mediated is important. We developed a method using Western immunoblot (WB) to classify DAT(–) patients by measuring and comparing levels of RBC immunoglobulin (Ig)G to normal donors. STUDY DESIGN AND METHODS RBC samples from 42 normal donors were made into ghosts and analyzed by WB and densitometry to establish a normal mean relative quantity of IgG (RQIgG) on the RBCs. RQIgG on eight DAT(–) and eluate‐negative patients with hemolytic anemia was determined and compared to RQIgG on normal RBCs. RESULTS RQIgG of 42 normal donors indicated a calculated mean ± SD of 0.0016 ± 0.0015 and we used a cutoff of 0.0047, the mean + 2SD. This was compared with a receiver operating curve cutoff of 0.0041 with 100% sensitivity and 93% specificity. Of the eight patients tested, three were classified as non–immune‐mediated (one had pyruvate kinase deficiency) and five as immune‐mediated. Two of the patients in the latter group underwent splenectomy, followed by remission. CONCLUSION WB analysis is more sensitive than conventional test tube DAT or elution analysis. Our assay confirms: 1) previous studies showing normal RBCs are sensitized with IgG, perhaps due to natural autoantibody to senescence; 2) that some normal RBCs have increased levels of IgG without signs of disease; and 3) that WB distinguishes between non–immune‐ and immune‐mediated hemolytic anemia in DAT(–) patients and may be useful for clinical diagnosis. Read less

   Journal Article  |  Full Text Online

3. Automated Western immunoblotting detection of anti-SARS-CoV-2 serum antibodies 2021

   https://ncsu.summon.serialssolutions.com/2.0.0/link/0/eLvHCXMwlV3di9QwEB_cOxRf_DjlrJ5LFd80XJukTQrHwbq4rKIgnqviS0mahF3Q7XHuCv73TtKP63L6cC99SELbZGaSmcxvZgBeyNzgKSEzkgqDBkomcyJ1qkjqkMTOyEqEqPfBZU4fxx3A7p1HMmzUg1i3JOBl0frlNGFEjmA_S9EK9-Hhi3f99oscGGKkC8aJL6nVRsr8-x07p9Fo6bGQVxXNq3jJodOUtWfR7C6UXURPgKD4wgdDLP5xyH8Ykl4Mcj1eZ8b34E6rpsaThq_uww27PoCbTeHKPwdw60Prkn8Ai8l2U6Paa018sjz92iReiFc-7KTWP-oAqz45Xp7Gxm4C7Gsd1y5Geq7I2eTTGZnWXwiNURC2P0Orrj2u8SEsZm8-T-ekrdVAKp7zDaEZ06YyhhlaCFUZlWkruRUiVTzRLOGOUiUKLRNDDbPa4cGJjMEUS3TumGGPIE61Fhz7pXAazc5C5okSzihnFVOVdBG87IhVnje5OMrLrMt-sUpcrDIsVikjeI707Af6NNrzyfvStyWMes0x_Z1GcNSRu2yF91dJfSWlXKBqFMGzvhvFzvtS1NrWWxzDC49izbmI4LDhjv5TjOVo5jIegdjhm51_2e1Zr5YhtbcovGOziOBVxxKXv_X_qT6-3vAncJsGrvJ3SUewt7nY2qew5_l0DCPxTYTn63GQqzHsz6dvZ9_HHhX78S8qNByo

   Edouard, Sophie; Jaafar, Rita; Orain, Nicolas; Parola, Philippe...

   European Journal Of Clinical Microbiology & Infectious Diseases, Vol. 40, Issue 6, pp. 1309 - 1317.

   ELISA and chemiluminescence serological assays for COVID-19 are currently incorporating only one or two SARS-CoV-2 antigens. We developed an automated Western immunoblotting as a complementary sero... Read more

   ELISA and chemiluminescence serological assays for COVID-19 are currently incorporating only one or two SARS-CoV-2 antigens. We developed an automated Western immunoblotting as a complementary serologic assay for COVID-19. The Jess TM Simple Western system, an automated capillary-based assay, was used, incorporating an inactivated SARS-CoV-2 lineage 20a strain as the source of antigen, and total immunoglobulins (IgG, IgM, IgA) detection. In total, 602 sera were tested including 223 from RT-PCR-confirmed COVID-19 patients, 76 from patients diagnosed with seasonal HCoVs and 303 from coronavirus-negative control sera. We also compared this assay with the EUROIMMUN® SARS-CoV-2 IgG ELISA kit. Among 223 sera obtained from RT-PCR-confirmed COVID-19 patients, 180/223 (81%) exhibited reactivity against the nucleocapsid and 70/223 (31%) against the spike protein. Nucleocapsid reactivity was further detected in 9/76 (14%) samples collected from patients diagnosed with seasonal HCoVs and in 15/303 (5%) coronavirus-negative control samples. In the subset of sera collected more than 2 weeks after the onset of symptoms, the sensitivity was 94% and the specificity 93%, the latter value probably reflecting cross-reactivity of SARS-CoV-2 with other coronaviruses. The automated Western immunoblotting presented a substantial agreement (90%) with the compared ELISA (Cohen’s Kappa=0.64). Automated Western immunoblotting may be used as a second line test to monitor exposure of people to HCoVs including SARS-CoV-2. Read less

   Journal Article  |  Full Text Online

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