Crystal violet stains proteins in SDS-PAGE gels and zymograms.

Coomassie brilliant blue R250, an anionic dye is the most popular stain to detect proteins resolved in SDS-PAGE gels. Crystal violet, a cationic dye was found to be versatile and stained proteins in SDS-PAGE gels and in zymograms. Stained proteins can be transferred to nitrocellulose and the stained proteins on the western detected with enzyme coupled antibodies. Staining can be reversed. Staining takes 3 h at RT or 30 min at 60 °C. Crystal violet stained some E. coli high and low molecular weight proteins not stained by Coomassie blue R250. Crystal violet stained down to 16 ng of protein, some five-fold lower than Coomassie blue, though the two stains had a similar linear dynamic range. The staining sensitivity could be increased to 2 ng when crystal violet and Coomassie blue were combined in a double staining/counterion dye formulation. The low concentrations of the dye without a destaining step reduces the costs of the technique and results in a more environmentally friendly stain compared to traditional staining methods.

Towards development of aptamers that specifically bind to lactate dehydrogenase of Plasmodium falciparum through epitopic targeting.

BACKGROUND: Early detection is crucial for the effective treatment of malaria, particularly in those cases infected with Plasmodium falciparum. There is a need for diagnostic devices with the capacity to distinguish P. falciparum from other strains of malaria. Here, aptamers generated against targeted species-specific epitopes of P. falciparum lactate dehydrogenase (rPfLDH) are described. RESULTS: Two classes of aptamers bearing high binding affinity and specificity for recombinant P. falciparum lactate dehydrogenase (rPfLDH) and P. falciparum-specific lactate dehydrogenase epitopic oligopeptide (LDHp) were separately generated. Structurally-relevant moieties with particular consensus sequences (GGTAG and GGCG) were found in aptamers reported here and previously published, confirming their importance in recognition of the target, while novel moieties particular to this work (ATTAT and poly-A stretches) were identified. Aptamers with diagnostically-supportive functions were synthesized, prime examples of which are the aptamers designated as LDHp 1, LDHp 11 and rLDH 4 and rLDH 15 in work presented herein. Of the sampled aptamers raised against the recombinant protein, rLDH 4 showed the highest binding to the target rPfLDH in the ELONA assay, with both rLDH 4 and rLDH 15 indicating an ability to discriminate between rPfLDH and rPvLDH. LDHp 11 was generated against a peptide selected as a unique P. falciparum LDH peptide. The aptamer, LDHp 11, like antibodies against the same peptide, only detected rPfLDH and discriminated between rPfLDH and rPvLDH. This was supported by affinity binding experiments where only aptamers generated against a unique species-specific epitope showed an ability to preferentially bind to rPfLDH relative to rPvLDH rather than those generated against the whole recombinant protein. In addition, rLDH 4 and LDHp 11 demonstrated in situ binding to P. falciparum cells during confocal microscopy. CONCLUSIONS: The utilization and application of LDHp 11, an aptamer generated against a unique species-specific epitope of P. falciparum LDH indicated the ability to discriminate between recombinant P. falciparum and Plasmodium vivax LDH. This aptamer holds promise as a biorecognition element in malaria diagnostic devices for the detection, and differentiation, of P. falciparum and P. vivax malaria infections. This study paves the way to explore aptamer generation against targeted species-specific epitopes of other Plasmodium species.

Plasmodium glyceraldehyde-3-phosphate dehydrogenase: A potential malaria diagnostic target.

Malaria rapid diagnostic tests (RDTs) are immunochromatographic tests detecting Plasmodial histidine-rich protein 2 (HRP2), lactate dehydrogenase (LDH) and aldolase. HRP2 is only expressed by Plasmodium falciparum parasites and the protein is not expressed in several geographic isolates. LDH-based tests lack sensitivity compared to HRP2 tests. This study explored the potential of the Plasmodial glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as a new malaria diagnostic biomarker. The P. falciparum and P. yoelii proteins were recombinantly expressed in BL21(DE3) Escherischia coli host cells and affinity purified. Two epitopes (CADGFLLIGEKKVSVFA and CAEKDPSQIPWGKCQV) specific to P. falciparum GAPDH and one common to all mammalian malaria species (CKDDTPIYVMGINH) were identified. Antibodies were raised in chickens against the two recombinant proteins and the three epitopes and affinity purified. The antibodies detected the native protein in parasite lysates as a 38 kDa protein and immunofluorescence verified a parasite cytosolic localization for the native protein. The antibodies suggested a 4-6 fold higher concentration of native PfGAPDH compared to PfLDH in immunoprecipitation and ELISA formats, consistent with published proteomic data. PfGAPDH shows interesting potential as a malaria diagnostic biomarker.

Comparing Antibody Responses in Chickens Against Plasmodium falciparum Lactate Dehydrogenase and Glyceraldehyde-3-phosphate Dehydrogenase with Freund's and Pheroid® Adjuvants.

Pheroid® technology was assessed as an alternative to Freund's adjuvant to raise antibodies in experimental animals. Chickens were immunized with two recombinantly expressed Plasmodium falciparum proteins, lactate dehydrogenase (PfLDH) and glyceraldehyde-3-phosphate dehydrogenase (PfGAPDH), alone or in combination with Freund's adjuvant or Pheroid®. Chicken egg yolk antibodies (IgY) were isolated and compared for specificity, sensitivity and yield. Freund's adjuvant and Pheroid® stimulated prolonged antibody responses in chickens against both antigens. Affinity purified antibodies had specificity for the recombinant and the native proteins on Western blots. Antibodies generated in the presence of Freund's adjuvant had high sensitivity for both antigens. Pheroid® generated antibodies that detected the lowest concentration of recombinant PfLDH. Freund's adjuvant and Pheroid® both improved chicken IgY yields, with Pheroid® showing a 2-fold increase relative to controls. Pheroid® was well-tolerated in chickens and has potential for development as a safe adjuvant for testing alternative stimulatory factors to improve adjuvant formulations.

Identification and initial characterisation of a Plasmodium falciparum Cox17 copper metallochaperone.

Copper is an essential micronutrient for all living organisms as an important catalytic co-factor for key enzymes. In higher eukaryotes intracellular copper is distributed by copper metallochaperones. Copper chelators such as neocuproine and tetrathiomolybdate inhibit Plasmodium falciparum erythrocytic development, indicating a requirement for copper by the parasite. A screen of the P. falciparum genome database identified eight potential copper-requiring protein orthologs, including four candidate copper metallochaperones implicated in the delivery of copper to cytochrome-c oxidase. A P. falciparum Cox17 ortholog (PfCox17) was recombinantly expressed and the purified protein bound reduced copper in vitro. PfCox17 was localised to the parasite cytoplasm. Characterisation of plasmodial proteins involved in copper metabolism will help us understand the role of this essential microelement in plasmodial homeostasis.

Infection pre-Ad26.COV2.S-vaccination primes greater class switching and reduced CXCR5 expression by SARS-CoV-2-specific memory B cells.

Neutralizing antibodies strongly correlate with protection for COVID-19 vaccines, but the corresponding memory B cells that form to protect against future infection are relatively understudied. Here we examine the effect of prior SARS-CoV-2 infection on the magnitude and phenotype of the memory B cell response to single dose Johnson and Johnson (Ad26.COV2.S) vaccination in South African health care workers. Participants were either naïve to SARS-CoV-2 or had been infected before vaccination. SARS-CoV-2-specific memory B-cells expand in response to Ad26.COV2.S and are maintained for the study duration (84 days) in all individuals. However, prior infection is associated with a greater frequency of these cells, a significant reduction in expression of the germinal center chemokine receptor CXCR5, and increased class switching. These B cell features correlated with neutralization and antibody-dependent cytotoxicity (ADCC) activity, and with the frequency of SARS-CoV-2 specific circulating T follicular helper cells (cTfh). Vaccination-induced effective neutralization of the D614G variant in both infected and naïve participants but boosted neutralizing antibodies against the Beta and Omicron variants only in participants with prior infection. In addition, the SARS-CoV-2 specific CD8+ T cell response correlated with increased memory B cell expression of the lung-homing receptor CXCR3, which was sustained in the previously infected group. Finally, although vaccination achieved equivalent B cell activation regardless of infection history, it was negatively impacted by age. These data show that phenotyping the response to vaccination can provide insight into the impact of prior infection on memory B cell homing, CSM, cTfh, and neutralization activity. These data can provide early signals to inform studies of vaccine boosting, durability, and co-morbidities.

Continuous Elution SDS-PAGE with a Modified Standard Gel Apparatus to Separate and Isolate an Array of Proteins from Complex Mixtures.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis is a powerful tool to separate proteins according to their relative sizes. The technique provides information about both the size of a number of proteins and potentially the comparative amounts of each protein. To confirm the identity of proteins, proteins can be eluted from the gel and transferred electrophoretically to nitrocellulose for antibody-based detection. During electrophoresis, if the current is not stopped, proteins continue to pass down the gel and elute from the bottom of the gel. The standard electrophoresis gel apparatus can be employed with the addition of some tubing and alterations to the separating gel to collect proteins separated by size as they elute from the base of the gel as described in this chapter. Complex protein mixtures can be separated into multiple fractions containing single proteins in a few hours. Small amounts (<500 µg of protein) of protein sample can be fractionated.

Phosphoethanolamine-N-methyltransferase is a potential biomarker for the diagnosis of P. knowlesi and P. falciparum malaria.

BACKGROUND: Plasmodium knowlesi is recognised as the main cause of human malaria in Southeast Asia. The disease is often misdiagnosed as P. falciparum or P. malariae infections by microscopy, and the disease is difficult to eliminate due to its presence in both humans and monkeys. P. knowlesi infections can rapidly cause severe disease and require prompt diagnosis and treatment. No protein biomarker exists for the rapid diagnostic test (RDT) detection of P. knowlesi infections. Plasmodium knowlesi infections can be diagnosed by PCR. METHODS AND PRINCIPAL FINDINGS: Phosphoethanolamine-N-methyltransferase (PMT) is involved in malaria lipid biosynthesis and is not found in the human host. The P. falciparum, P. vivax and P. knowlesi PMT proteins were recombinantly expressed in BL21(DE3) Escherichia coli host cells, affinity purified and used to raise antibodies in chickens. Antibodies against each recombinant PMT protein all detected all three recombinant proteins and the native 29 kDa P. falciparum PMT protein on western blots and in ELISA. Antibodies against a PMT epitope (PLENNQYTDEGVKC) common to all three PMT orthologues detected all three proteins. Antibodies against unique peptides from each orthologue of PMT, PfCEVEHKYLHENKE, PvVYSIKEYNSLKDC, PkLYPTDEYNSLKDC detected only the parent protein in western blots and P. falciparum infected red blood cell lysates or blood lysates spiked with the respective proteins. Similar concentrations of PfPMT and the control, PfLDH, were detected in the same parasite lysate. The recombinant PfPMT protein was detected by a human anti-malaria antibody pool. CONCLUSION: PMT, like the pan-specific LDH biomarker used in RDT tests, is both soluble, present at comparable concentrations in the parasite and constitutes a promising antimalarial drug target. PMT is absent from the human proteome. PMT has the potential as a biomarker for human malaria and in particular as the first P. knowlesi specific protein with diagnostic potential for the identification of a P. knowlesi infection.

Ten Minute Stain to Detect Proteins in Polyacrylamide Electrophoresis Gels with Direct Red 81 and Amido Black.

Proteins separated by SDS-polyacrylamide gel electrophoresis need to be stained with organic dyes to be visualized. Once stained the intensity of each stained protein band can be used to compare the differences in protein concentration and to measure the relative concentration of any protein band. The most popular standard protein staining is with Coomassie Blue R-250 which takes an hour to stain proteins to saturation and several hours to remove background staining. Direct Red 81 and Amido Black stain proteins within 2.5 min and staining is complete by 10 min. Here the rapid staining of proteins with Direct Red 81 and Amido Black in comparison to staining with Coomassie Blue R-250 is described.

Rapid detection of proteins in polyacrylamide electrophoresis gels with Direct Red 81 and Amido Black.

Proteins separated by SDS-polyacrylamide gel electrophoresis need to be stained with organic dyes to be visualized and to enable comparisons to be made between the intensity of protein bands to observe and determine differences in protein concentration. The standard protein staining is with Coomassie Blue R-250. Coomassie staining takes 1 h to complete. Direct Red 81 and Amido Black stain proteins within 10 min. This chapter describes Direct Red 81 and Amido Black staining in comparison to staining with Coomassie Blue R-250.