Production of high-quality SARS-CoV-2 antigens: impact of bioprocess and storage on glycosylation, biophysical attributes, and ELISA serologic tests performance

June 23, 2021 Taylor 0 Comments

Serological assays are helpful instruments to check SARS-CoV-2 unfold and, importantly, to determine people that have been already contaminated and could be probably resistant to a virus re-infection. SARS-CoV-2 Spike protein and its Receptor Binding Area (RBD) are the antigens with larger potential to develop SARS-CoV-2 serological assays. Furthermore, structural research of those antigens are key to know the molecular foundation for Spike interplay with angiotensin changing enzyme 2 receptor, hopefully enabling the event of COVID-19 therapeutics.

Thus, it’s pressing that vital quantities of this protein grew to become accessible on the highest high quality. On this work, we produced Spike and RBD in two human derived cell hosts: HEK293-E6 and Expi293F™. We evaluated the affect of various and scalable bioprocessing approaches on Spike and RBD manufacturing yields and, extra importantly, on these antigens’ high quality attributes. Utilizing destructive and optimistic sera collected from human donors, we present a wonderful efficiency of the produced antigens, assessed in serologic ELISA exams, as denoted by the excessive specificity and sensitivity of the check.

We present sturdy Spike productions with last yields of approx. 2 mg per liter of tradition that have been maintained independently of the manufacturing scale or cell tradition technique. To the most effective of our data, the ultimate yield of 90 mg per liter of tradition obtained for RBD manufacturing, was the best reported so far. An in-depth characterization of SARS-CoV-2 Spike and RBD proteins was carried out, specifically the antigen’s oligomeric state, glycosylation profiles and thermal stability throughout storage.

The correlation of those high quality attributes with ELISA efficiency present equal reactivity to SARS-CoV-2 optimistic serum, for all Spike and RBD produced, and for all storage circumstances examined. Total, we offer simple protocols to provide high-quality SARS-CoV-2 Spike and RBD antigens, that may be simply tailored to each tutorial and industrial settings; and combine, for the primary time, research on the affect of bioprocess with an in-depth characterization of those proteins, correlating antigen’s glycosylation and biophysical attributes to efficiency of COVID-19 serologic exams.

Analysis of IgM LAT and IgM ELISA as in comparison with microscopic agglutination check (MAT) for early analysis of Leptospira sp

Leptospirosis is a worldwide zoonotic illness attributable to spirochetes of the genus Leptospira. The scientific manifestation of leptospirosis is non-specific and continuously misdiagnosed as different sicknesses. The purpose of this research was to match the diagnostic accuracies of two industrial exams for early analysis of Leptospira species: the IgM latex agglutination check (IgM LAT) and the IgM enzyme-linked immunosorbent assay (IgM ELISA).

A complete of 140 serum samples have been obtained from sufferers suspected of leptospirosis on the Universiti Kebangsaan Malaysia Medical Centre (UKMMC). These serum samples have been examined for the presence of Leptospira sp. utilizing IgM LAT, IgM ELISA and MAT. From Desk 1, IgM LAT confirmed 21% (n = 29) optimistic, 18% (n = 25) inconclusive and 61% (n = 86) destructive, whereas IgM ELISA confirmed 6% (n = 8) optimistic, 6% (n = 8) inconclusive, 88% (n = 124) destructive and MAT confirmed 11% (n = 16) optimistic, 47% (n = 65) inconclusive, 42% (n = 59) destructive.

The sensitivity, specificity, optimistic predictive worth (PPV) and destructive predictive worth (NPV) of IgM LAT have been 68.8%, 57.6%, 30.6% and 87.2% respectively, whereas for IgM ELISA they have been 37.5%, 89.8%, 50% and 84.1%, respectively as in comparison with MAT (Desk 2). The outcomes confirmed that IgM LAT had larger sensitivity however decrease specificity in comparison with IgM ELISA.

In conclusion, IgM LAT could be helpful as an early screening check for early analysis of Leptospira sp., whereas IgM ELISA is an acceptable methodology for decreasing false destructive detection of Leptospira sp. As each exams present reasonable percentages (~65%) in accuracy, a further check is required for higher detection of Leptospira sp.

Seroprevalence of Toxoplasma gondii in ruminants through the use of latex agglutination check (LAT) and enzyme-linked immunosorbent assay (ELISA) in Assiut governorate

Toxoplasmosis is a crucial zoonotic parasitic illness world-wide. Nonetheless, knowledge on Toxoplasma gondii an infection in ruminants are scarce in Assiut governorate, Egypt. Thus we carried out a survey on the seroprevalence of T. gondii an infection through the use of latex agglutination check (LAT) and enzyme-linked immunosorbent assay (ELISA). A complete of 274 sera have been examined (56 imported camels, 56 cattle, 55 buffaloes, 50 sheep and 57 goats) within the interval from November 2014 – February 2016.

The general seroprevalence in ruminants was (35.8%) by LAT and (83.6%) by ELISA. The very best charge of T. gondii an infection by LAT was present in goats (47.4%) adopted by sheep (44%), camels (35.7%), cattle (32.1%) and buffaloes (20%). The very best charge of T. gondii an infection by ELISA was recorded in camels (96.4%) adopted by goats (87.7%), sheep (86%), buffaloes (74.5%) and cattle (73.2%). Antibody titres to T. gondii in optimistic sera by LAT ranged from 1:2 to 1:64 in imported camels, 1:2 to 1:32 in cattle, 1:2 to 1:Eight in buffaloes, from 1:Four to 1:64 in sheep and from 1:2 to 1:16 in goats. The variations of seroprevalence between completely different species have been vital by LAT and extremely vital by ELISA. Our outcomes counsel that the T. gondii parasite is extensively unfold on account of excessive publicity to infective cat faeces.

Serological Check to Decide Publicity to SARS-CoV-2: ELISA Based mostly on the Receptor-Binding Area of the Spike Protein (S-RBD _N318-V510) Expressed in Escherichia coli

Huge worldwide serological testing for SARS-CoV-2 is required to find out the extent of virus publicity in a selected area, the ratio of symptomatic to asymptomatic contaminated individuals, and the period and extent of immunity after an infection. To realize this, the event and manufacturing of dependable and cost-effective SARS-CoV-2 antigens is vital. We report the bacterial manufacturing of the peptide S-RBD_N318-V510, which incorporates the receptor-binding area of the SARS-CoV-2 spike protein (area of 193 amino acid residues from asparagine-318 to valine-510) of the SARS-CoV-2 spike protein.

We purified this peptide utilizing an easy method involving bacterial lysis, his-tag-mediated affinity chromatography, and imidazole-assisted refolding. The antigen performances of S-RBD_N318-V510 and a industrial full-length spike protein have been in contrast in ELISAs. In direct ELISAs, the place the antigen was instantly sure to the ELISA floor, each antigens discriminated sera from non-exposed and uncovered people. Nonetheless, the discriminating decision was higher in ELISAs that used the full-spike antigen than the S-RBD_N318-V510.

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800-181-5P	Alpha Diagnostics	1 pk	EUR 343.2

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TCP20-96	Bio Basic	1 UNIT	EUR 64.18

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C107-1EA	Arbor Assays	1EA	EUR 162

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PFS096	ProFoldin	kit	EUR 347.84
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96-Well Plate Plasmid DNA Miniprep Kit
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STA-416	Cell Biolabs	96 assays	EUR 1027.2
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D8235152-1	Biochain	1 plate	EUR 328

Radius 96-Well Cell Migration Assay
CBA-126	Cell Biolabs	96 assays	EUR 686.4
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CBA-126-5	Cell Biolabs	5 x 96 assays	EUR 2697.6
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D8235090-1	Biochain	1 plate	EUR 328

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D8235086-1	Biochain	1 plate	EUR 328

96 Well Impact MicroBatch Plate, 80/CSE
M-673101	MiTeGen	80 PLATES	EUR 808
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D8235248-1	Biochain	1 plate	EUR 328

384 Well Deep Well Plate V
PCR0316	Scientific Laboratory Supplies	PK100	EUR 512.4

96 Well Plate Blood Genomic DNA Miniprep Kit
BT92031	Bio Basic	2Plates, 192prep	EUR 210.85

96 Well Plate Blood Genomic DNA Miniprep Kit
BT92032	Bio Basic	5Plates, 480prep	EUR 389.9

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C271-1EA	Arbor Assays	1EA	EUR 150

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BS8357	Bio Basic	2XPlates, 192prep	EUR 234.6

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354730	CORNING	5/pk	EUR 102
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BS8361	Bio Basic	2XPlates, 192prep	EUR 234.6

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Wizard Cryo 1 and 2 96 Well Matrix Block Plate (96 conditions 1.7 mL volumes in a 96-Well block plate)
M-R-1008649	MiTeGen	98 X 1.7 ml	EUR 316
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96 Well Plate RNA Cleanup and Concentration Kit
BRC1251	Bio Basic	2xPlates, 192prep	EUR 300.37

96-Well Plate Animal Genomic DNA Miniprep Kit
BS437	Bio Basic	5XPlates, 480prep	EUR 435.58

96-Well Plate Animal Genomic DNA Miniprep Kit
BS4372	Bio Basic	2XPlates, 192prep	EUR 234.6

96-well Plasmid DNA Extraction Kit (1 plate)
FAPWE-96001	Favorgen	1 prep	EUR 175.2

MULTIWELL PLATE, POLYSTYRENE 96-WELL U-B
M2186-100EA	Scientific Laboratory Supplies	PK100	EUR 131.87

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354721	Scientific Laboratory Supplies	PK5	EUR 90

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X037-2EA	Arbor Assays	2EA	EUR 37

96-Well Plate Bacteria Genomic DNA Miniprep Kit
SK1292	Bio Basic	2XPlates, 192prep	EUR 210.85

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MOL2370	Scientific Laboratory Supplies	PK50	EUR 543.6

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X105-1EA	Arbor Assays	1EA	EUR 73

Wizard Classic 1 and 2 96 Well Matrix Block Plate (96 formulations 1.7 mL volumes in a 96-Well block plate)
M-R-1008647	MiTeGen	96 X 1.7 ml	EUR 316
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Wizard Classic 3 and 4 96 Well Matrix Block Plate (96 formulations 1.7 mL volumes in a 96-Well block plate)
M-R-1008648	MiTeGen	97 X 1.7 ml	EUR 316
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CytoSelect 96-well Cell Transformation Assay
CBA-130	Cell Biolabs	96 assays	EUR 866.4
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CBA-130-5	Cell Biolabs	5 x 96 assays	EUR 3463.2
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CytoSelect 96-well Phagocytosis Assay (Zymosan)
CBA-224	Cell Biolabs	96 assays	EUR 866.4
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CytoSelect 96-well Phagocytosis Assay (Zymosan)
CBA-224-5	Cell Biolabs	5 x 96 assays	EUR 3525.6
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PBGG-5	Alpha Diagnostics	1 pk	EUR 270

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PBSA-5	Alpha Diagnostics	1 pk	EUR 270

Benchmark Beadblaster 96 96 Well Plate Prefilled with 4mm Beads
HOM3228	Scientific Laboratory Supplies	PK10	EUR 2634

Adapter for 96-well PCR plate 2 pcs.
E5825711009	Scientific Laboratory Supplies	PK2	EUR 158.4

BRAND(TM) 96-WELL PCR PLATE SKIRTED WH
BR781378-50EA	Scientific Laboratory Supplies	PK50	EUR 457.2

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G657160	Scientific Laboratory Supplies	PK100	EUR 182.4

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356773	Scientific Laboratory Supplies	PK50	EUR 307.2

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GP615	GenDepot	25 plates	EUR 254.4

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265300	Scientific Laboratory Supplies	PK30	EUR 3613.2

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265301	Scientific Laboratory Supplies	PK30	EUR 482.4

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265302	Scientific Laboratory Supplies	PK30	EUR 663.6

Goat anti-Mouse, Coated White 96 Well Plate, 1EA
X010-1EA	Arbor Assays	1EA	EUR 48

2'3'-Cyclic GAMP ELISA Kit, (384-well Plate)
K067-H1D	Arbor Assays	1x384 well plate	EUR 1557

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E0030133390	Scientific Laboratory Supplies	EACH	EUR 97.2

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X014-1EA	Arbor Assays	1EA	EUR 48

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X016-1EA	Arbor Assays	1EA	EUR 48

Bovine IgM ELISA kit
55R-1928	Fitzgerald	96 tests	EUR 708
Description: ELISA Kit for detection of IgM in the research laboratory

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EBI0045	Abclonal	96Tests	EUR 625.2

Bovine IgM ELISA Kit
EBI0104	Abclonal	96Tests	EUR 625.2

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QY-E60067	Qayee Biotechnology	96T	EUR 511.2

96 WELL PLATE; SQUARE; V BASE; 2ML; PP; 5/PK
60180-P105	Scientific Laboratory Supplies	PK5	EUR 44.4

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60180-P135	Scientific Laboratory Supplies	PK50	EUR 360

Donkey anti-Sheep, Coated Clear 96 Well Plate, 1EA
X061-1EA	Arbor Assays	1EA	EUR 48

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X063-1EA	Arbor Assays	1EA	EUR 48

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AB0800B	Scientific Laboratory Supplies	PK25	EUR 277.2

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E0030133366	Scientific Laboratory Supplies	EACH	EUR 97.2

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ARG1476	Scientific Laboratory Supplies	PK100	EUR 385.2

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AB-0700	Scientific Laboratory Supplies	PK25	EUR 198

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M-780261	MiTeGen	50 PLATES	EUR 240
Description: 96 Deep Well Masterblock Storage Plate, 50/CSE

Ultra-Fast 96-Well Plate Plasmid DNA Miniprep Kit
BS92028	Bio Basic	2XPlates, 192prep	EUR 254.7

Ultra-Fast 96-Well Plate Plasmid DNA Miniprep Kit
BS92029	Bio Basic	5XPlates, 480prep	EUR 437.4

96 Well Plate Bacterial Total RNA Miniprep Super Kit
BS585	Bio Basic	5XPlates, 480prep	EUR 585.38

96 Well Plate Bacterial Total RNA Miniprep Super Kit
BS5852	Bio Basic	2XPlates, 192prep	EUR 300.37

Microbiol blue skirted PCR plate 96 well 10pcs
E0030129318	Scientific Laboratory Supplies	EACH	EUR 92.4

96-well Checkpoint Kinase Activity Assay Kit
STA-414	Cell Biolabs	96 assays	EUR 908.4
Description: Our 96-well Checkpoint Kinase Activity Assay Kit provides a non-isotopic, sensitive and specific method to monitor checkpoint kinase activity using its physiological substrate; it can also be used in screening checkpoint kinase inhibitors.

96-well Checkpoint Kinase Activity Assay Kit
STA-414-5	Cell Biolabs	5 x 96 assays	EUR 3672
Description: Our 96-well Checkpoint Kinase Activity Assay Kit provides a non-isotopic, sensitive and specific method to monitor checkpoint kinase activity using its physiological substrate; it can also be used in screening checkpoint kinase inhibitors.

CORNING® BIOCOAT™ T-CELL ACTIVATION ANTI-MOUSE CD3 96 WELL FLAT BOTTOM ASSAY PLATE, INDIVIDUALLY WRAPPED, 5/CASE
354720	CORNING	5/pk	EUR 576
Description: Advanced Cells - DL; BioCoat 96 Well Clear - DL

CORNING® BIOCOAT™ T-CELL ACTIVATION ANTI-HUMAN CD3 96 WELL FLAT BOTTOM ASSAY PLATE, INDIVIDUALLY WRAPPED, 5/CASE
354725	CORNING	5/pk	EUR 568.8
Description: Advanced Cells - DL; BioCoat 96 Well Clear - DL

Mouse anti-Prolactin Clear Coated 96 Well Plate, 1EA
C140-1EA	Arbor Assays	1EA	EUR 150

384 Well Plate, 10/Bag
PCR-384-SN	Bio Basic	1PK, 10UNIT	EUR 112.2

Microbiol clear skirted PCR plate 96 well 10pcs
E0030129300	Scientific Laboratory Supplies	EACH	EUR 92.4

Falcon 96 Well 320ul Clear Plate Standard Surface Tissue Culture Plate No Lid
353910	Scientific Laboratory Supplies	PK50	EUR 140.4

Tissue Culture Plate 96 Well Treated Ind. Wrapped
ARG1492	Scientific Laboratory Supplies	PK100	EUR 430.8

96 WELL CLEAR PCR HALF SKIRT AMPLIFICATION PLATE
PCR-96-HS-C	CORNING	10/pk	EUR 190.8
Description: PCR Plates & Tubes; A354 PCR Plate-Axygen

Mouse anti-Osteopontin Clear Coated 96 Well Plate, 1EA
C075-1EA	Arbor Assays	1EA	EUR 150

WELL PLATE BLUE PLUG; 8mm FOR 1ML INSERTS; 96/PK
60180-C101	Scientific Laboratory Supplies	PK96	EUR 25.2

Goat anti-Mouse, 1 x Whole Well, Clear Coated 96 Well Plate, 1EA
X011-1EA	Arbor Assays	1EA	EUR 48

INTELLI-PLATE 96-2 Shallow well; 2-well sitting drop, 120 plates
MAR-102-0001-20	MiTeGen	120 PLATES	EUR 990
Description: INTELLI-PLATE 96-2 Shallow well; 2-well sitting drop, 120 plates

Black 384 Well Plate, 2EA
X115-2EA	Arbor Assays	2EA	EUR 183

96-well PCR Plate (Non-Skirted+black lettering)
AB-0600-L	Scientific Laboratory Supplies	PK25	EUR 177.6

Modular In Situ-1 96 Well Crystallization Plate
ML-MIS-FP3D	MiTeGen	1 PLATE	EUR 392
Description: Modular In Situ-1 96 Well Crystallization Plate

BD Biocat poly d lysine la minin 96 well plate
354596	Scientific Laboratory Supplies	PK5	EUR 297.6

CytoSelect 96-Well Hematopoietic Colony Forming Cell Assay (96 assays)
CBA-320	Cell Biolabs	96 assays	EUR 644.4
Description: Hematopoietic stem cells (HSCs) are well-characterized, tissue-specific stem cells that are responsible for the lifelong maintenance of the hematopoietic system. HSCs or hematopoietic progenitors known as colony-forming cells (CFCs) proliferate to form discrete colonies when cultured in a suitable 3D environment, such as methylcellulose supplemented with nutrients and cytokines. Our CytoSelect 96-Well Hematopoietic Colony Forming Cell Assay promotes the formation of HSC colonies in just 7-10 days. Cells can then be either quantified in a fluorescence plate reader or recovered from the semisolid medium for further downstream analysis.

Mouse anti-Myeloperoxidase Clear Coated 96 Well Plate, 1EA
C223-1EA	Arbor Assays	1EA	EUR 150

Nunc 384 Well Plate Natural
264573	Scientific Laboratory Supplies	PK120	EUR 1395.6

96 WELL PLATE; 8MM RND; U BASE; 1300UL; PP; 5/PK
60180-P103	Scientific Laboratory Supplies	PK5	EUR 37.2

96 WELL PLATE; 8MM RND; V BASE; 450UL; PP; 120/PK
60180-P130	Scientific Laboratory Supplies	PK120	EUR 397.2

96 WELL PLATE; 8MM RND; U BASE; 1300UL; PP; 50/PK
60180-P133	Scientific Laboratory Supplies	PK50	EUR 306

Attachment of the antigens to the ELISA floor utilizing a layer of anti-histidine antibodies gave equal decision for each S-RBD_N318-V510 and the full-length spike protein. Outcomes exhibit that ELISA-functional SARS-CoV-2 antigens could be produced in bacterial cultures, and that S-RBD_N318-V510 could symbolize a cheap different to using structurally extra advanced antigens in serological COVID-19 testing.