Introduction to Monkeypox Antigens A29L, A35R, B6R, M1R

Monkeypox Virus Surface Membrane Proteins

Monkeypox virus (MPXV) belongs to the Orthopoxvirus genus. Generally, viruses within the same genus share similar biological characteristics. The transmission mechanism of the monkeypox virus may be similar to that of the "poxvirus genus model virus"—vaccinia virus. The stages of virus maturation include four forms: intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV), and extracellular enveloped virus (EEV). Among these, IMV, CEV, and EEV are infectious. IMV is considered the virus particle responsible for transmission between hosts; EEV has a fragile membrane that cannot survive in the physical environment outside the host, and once it ruptures, it releases IMV particles that are fully infectious and relatively stable. CEV mainly facilitates intercellular transmission of the virus, while EEV is used for long-distance transmission. Some studies suggest that monkeypox CEV may remain on the cell surface and not be released as EEV.

IMV Surface Membrane Proteins

A29L

IMV Surface Membrane Proteins

(Note: The image is sourced from the Uniprot official website)

Protein name 14 kDa protein
Full length 110 amino acids
Gene names A29L
Accession Q9YN60
Species Monkeypox virus

A29L is a surface membrane protein of intracellular mature virus (IMV) and is homologous to A27 of the vaccinia virus (VACV). It is widely conserved in the poxvirus family. Research on vaccinia virus has found that the A27 protein plays multiple roles in the virus life cycle, such as binding to cell surface heparan sulfate, regulating membrane fusion, and mediating the transport of mature virus (IMV) to form enveloped virus (IEV).

A27 can bind to cell surface heparan sulfate and mediate cell fusion under low pH conditions in the nucleus. Additionally, A27 is involved in assembling the fusion inhibitor protein A26 in mature viruses (IMV). After MV is endocytosed by vesicles, the acidic environment within the endosome induces the dissociation of the A26 protein from MV, leading to the fusion of the viral membrane with the vesicle membrane. A27 interacts with the integral membrane protein A17, which then forms a protein complex with A26 through disulfide bonds, anchoring A26 to the mature virus (IMV) particles.

In addition to these functions, the A27 protein also promotes the release of enveloped viruses. A portion of the MV progeny in infected cells is transported from the viral factory to the Golgi network through an A27-dependent mechanism, acquiring a second membrane layer to become intracellular enveloped viruses (IEV). IEV is released through exocytosis.

M1R

M1R is homologous to the L1 protein of vaccinia virus and is a transmembrane protein found on the surface of mature IMV particles. It is encoded by the L1R ORF, is highly conserved, and plays an important role in virus particle assembly, virus entry, and maturation. L1 is essential for inducing cell-cell fusion triggered by low pH and is crucial for vaccinia virus replication. Co-immunoprecipitation experiments show that L1 interacts with A28 and some other components of the entry/fusion complex (EFC) and indirectly with F9, suggesting that L1 is an additional component of the viral entry machinery. L1-MV can attach to cells but cannot enter or induce membrane fusion. L1 physically interacts with the EFC and works in concert with other known entry proteins.

IEV and EEV Surface Membrane Proteins

A35R

IEV and EEV Surface Membrane Proteins

(Note: The image is sourced from the Uniprot official website)

Protein name M1R
Full length 250 amino acids
Gene names M1R
Accession Q80KX3
Species Monkeypox virus

A35R is a membrane component of intracellular enveloped virus particles (IEV) and extracellular enveloped virus particles (EEV), and it is homologous to the A33R protein of chordopoxviruses. Vaccinia virus spreads from one cell to another through both antibody-sensitive and antibody-resistant pathways, and the A33R protein plays a role in antibody-resistant transmission. Research has shown that the cytoplasmic domain of the A33R protein interacts with amino acids 91-111 of A36R, which directly participates in the formation of actin tails. The A33R protein is essential for the association of A36R protein with the IEV membrane. Additionally, the A33R binding site of A36R (amino acids 105-116) overlaps with the Nck binding site, a protein necessary for actin tail nucleation, suggesting that A33R may regulate viral particle movement and actin tail nucleation to prevent premature events.

B6R

IEV and EEV Surface Membrane Proteins

(Note: The image is sourced from the Uniprot official website)

Protein name B6R
Full length 317 amino acids
Gene names B6R
Accession Q773E2
Species Monkeypox virus

B6R is a host range protein located on the membrane of extracellular enveloped virus particles (EEV) and is homologous to the complement control protein B5 of vaccinia virus. It is involved in the negative regulation of complement activation. B5 binds to complement components C3 and C1q, blocking complement activation at multiple sites and downregulating pro-inflammatory chemotactic factors (C3a, C4a, and C5a), thereby reducing cellular influx and inflammation. B5 is conserved in various Orthopoxviruses and B5 antibodies and can protect the host from vaccinia virus attack. B5 antibodies can neutralize vaccinia EV, although the mechanism is unclear. Studies have shown that monoclonal human anti-B5 IgG is heavily dependent on IgG binding to complement C3 and C1q. Similarly, complement-binding IgG controls the complement-dependent cytotoxicity of infected cells. Human polyclonal antibodies induced by smallpox vaccines also rely on complement to neutralize EV and depend on complement to destroy infected cells.

References

1.Gong SC, Lai CF, Esteban M. Vaccinia virus induces cell fusion at acid pH and this activity is mediated by the N-terminus of the 14-kDa virus envelope protein[J]. Virology, 1990, 178: 81-91.
2.Chang TH, Chang SJ, Hsieh FL, et al. Crystal structure of vaccinia viral A27 protein reveals a novel structure critical for its function and complex formation with A26 protein[J]. Plos Pathog., 2013.
3.Ward BM, Weisberg AS, Moss B. J. Mapping and functional analysis of interaction sites within the cytoplasmic domains of the vaccinia virus A33R and A36R envelope proteins[J]. Virol., 2003, 77: 4113-26.
4.Law M, Hollinshead R, Smith GL. J. Gen. Antibody-sensitive and antibody-resistant cell-to-cell spread by vaccinia virus: role of the A33R protein in antibody-resistant spread[J]. Virol., 2002, 83: 209-222.
5.Smith SA, Sreenivasan R, Krishnasamy G, et al. Mapping of regions within the vaccinia virus complement control protein involved in dose-dependent binding to key complement components and heparin using surface plasmon resonance[J]. Biochim. Biophys. Acta, 2003, 1650: 30-39.
6.Paran N, Lustig S. Complement-bound human antibodies to vaccinia virus B5 antigen protect mice from virus challenge[J]. Expert Rev Vaccines, 2010, 9: 255-259.
7.Iyer LM, Aravind L, Koonin EV. J. Common origin of four diverse families of large eukaryotic DNA viruses[J]. Virol., 2001, 75: 11720-34.
8.Bisht H, Weisberg AS, Moss B. Vaccinia virus l1 protein is required for cell entry and membrane fusion[J]. J Virol., 2008, 82(17): 8687-94.

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Monkeypox Virus (MPXV) Detection Methods

Monkeypox virus (MPXV) belongs to the Orthopoxvirus genus along with smallpox and cowpox. The first human case of MPXV was reported on September 1, 1970, at a hospital in Basankusu, Democratic Republic of the Congo. MPXV is primarily transmitted from wild animals like rodents and primates to humans, causing a rare zoonotic disease known as monkeypox. Human-to-human transmission is limited. Monkeypox symptoms are similar to smallpox but less severe. Initially confined to remote areas in Central and West Africa, MPXV has evolved into two clades: the more virulent Central African (Congo Basin) clade and the less virulent West African clade.

Monkeypox Virus Overview

Monkeypox virus is an enveloped double-stranded DNA virus with low dependence on the host cell's DNA and RNA replication machinery, allowing it to replicate in the host cell cytoplasm. Similar to other viruses in the Orthopoxvirus genus, MPXV likely replicates into morphologically distinct intracellular mature virions (IMVs) and extracellular enveloped virions (EEVs). IMVs have a robust physical structure, facilitating transmission between hosts, while the more fragile EEVs, encased in an envelope, evade host immune clearance, making them suitable for intercellular spread. MPXV efficiently infects human primary monocytes, suppressing CD4+ and CD8+ T cell activation, eliminating local T cell responses, and avoiding systemic immunosuppression and immune surveillance.

Clinical Diagnosis of Monkeypox Virus

The best diagnostic samples for monkeypox come from skin lesions, specifically vesicles and pustules, as well as dried scabs. Lesion samples must be stored in a dry, sterile tube (without viral transport media) and kept cold. Clinical diagnosis of monkeypox must consider other rash diseases such as chickenpox, measles, bacterial skin infections, scabies, syphilis, and drug-related allergies. Lymphadenopathy in the early stages may be the only clinical feature distinguishing monkeypox from chickenpox or smallpox.

1.1 Electron Microscopy Biopsy

Under electron microscopy, MPXV appears as a brick-shaped cytoplasmic particle with lateral bodies and a central core measuring approximately 200-300 nm. Since Orthopoxvirus species cannot be morphologically distinguished, this method does not confirm the diagnosis but suggests the virus belongs to the Poxviridae family.

1.2 Genetic Detection

1.2.1 Real-Time Fluorescent Quantitative PCR

Routine detection of MPXV DNA in clinical samples or MPXV-infected cell cultures can be performed using PCR or real-time PCR, preferably in a biosafety level 3 facility. Real-time PCR targeting the conserved regions of the extracellular enveloped protein gene (B6R), DNA polymerase gene, E9L, DNA-dependent RNA polymerase subunit 18, rpo18, and F3L genes is recommended.

1.2.2 Restriction Fragment Length Polymorphism (RFLP)

PCR-amplified gene or gene fragment restriction length fragment polymorphism (RFLP) can also be used to detect MPXV DNA, but RFLP is time-consuming and requires virus culture. RFLP of PCR products also requires enzyme digestion followed by gel electrophoresis, making it less suitable in clinical settings where speed, sensitivity, and specificity are critical.

1.2.3 High-Throughput Sequencing (NGS)

Whole-genome sequencing using NGS remains the gold standard for distinguishing MPXV from other Orthopoxviruses (OPV), but this technique is costly and requires substantial computational resources for downstream data processing. Therefore, NGS may not be suitable for resource-limited countries in sub-Saharan Africa.

Real-time PCR remains the preferred method for routine diagnosis of MPXV, but on-site genome sequencing techniques, such as Oxford Nanopore MinION, are essential for providing real-time viral genomic data, which is crucial for evidence-based epidemiological interventions.

1.3 Immunodetection Methods

Monkeypox virus immunodetection methods mainly include enzyme-linked immunosorbent assay (ELISA) for detecting IgG and IgM antibodies and immunohistochemistry for detecting viral antigens. Immunochemical analysis using MPXV antibodies can distinguish between Poxvirus infections and herpesvirus.

It has been shown that antiviral antibodies and T cell responses increase before and after the onset of disease, with MPXV IgM and IgG detected in serum approximately 5 days and more than 8 days after the rash onset, respectively. The presence of IgM and IgG antibodies in unvaccinated individuals with a history of rash and severe disease may suggest MPXV infection. IgM-capture ELISA positivity indicates recent exposure to MPXV, while IgG-capture ELISA positivity suggests prior exposure through vaccination or natural infection. Simultaneous presence of IgM and IgG in a sample suggests previous exposure in individuals vaccinated or naturally infected with MPXV. However, Orthopoxviruses have serological cross-reactivity, and antigen and antibody detection methods cannot provide monkeypox-specific confirmation but may be feasible for serological surveillance in MPXV-endemic areas.

References

1. Sarah Keasey, Christine Pugh, Alexander Tikhonov, et al. Proteomic Basis of the Antibody Response to Monkeypox Virus Infection Examined in Cynomolgus Macaques and a Comparison to Human Smallpox Vaccination[J]. Plos one, 2010, 5(12): e15547.
2. Emmanuel Alakunle, Ugo Moens, Godwin Nchind, et al. Monkeypox Virus in Nigeria: Infection Biology, Epidemiology, and Evolution[J]. Viruses, 2020, 12(11):1257.
3. Erika Hammarlund, Anindya Dasgupta, Clemencia Pinilla, et al. Monkeypox virus evades antiviral CD4+ and CD8+ T cell responses by suppressing cognate T cell activation[J]. Proc Natl Acad Sci USA, 2008, 105(38): 14567-72.

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Genomic Comparison Between Monkeypox Virus (MPXV) and Smallpox Virus

Smallpox is a highly fatal human epidemic caused by the Variola virus (VAR), a member of the Orthopoxvirus genus of the Poxviridae family. The global eradication of smallpox in 1977 was made possible by the introduction of a highly safe and affordable vaccine derived from the Vaccinia virus (VAC), mass vaccination, and thorough epidemiological surveillance. After the eradication of smallpox, vaccination was discontinued worldwide. This provided an opportunity for zoonotic Orthopoxviruses, such as the Monkeypox virus (MPV), Cowpox virus (CPV), and Buffalopox virus, to spread and undergo adaptive mutations. MPV has garnered the most attention among zoonotic Orthopoxviruses. The clinical similarity between monkeypox and smallpox has sparked speculation about the genetic relationship between these viruses and whether MPV might evolve into a VAR-like virus with high-frequency human-to-human transmission.

Genomic Differences Between Monkeypox Virus and Variola Virus

Scientists from the State Research Center of Virology and Biotechnology "Vector" in Russia, including Sergei N. Shchelkunov, sequenced the 197 kb genome of MPV isolated from a patient during a large-scale monkeypox outbreak in Zaire in 1996. By comparing the genome sequence of MPV with those of the Indian 1967 strain of VAR (VAR-IND), which causes severe disease, and the relatively mild smallpox strain Garcia-1966 (VAR-GAR), they found that the nucleotide sequence in the central region of the MPV genome, which encodes essential enzymes and structural proteins, shares 96.3% homology with that of VAR. In contrast, the terminal regions encoding virulence and host range factors exhibit significant differences. The mutations in two interferon (IFN) resistance genes in MPV, as well as the presence of interleukin-1β (IL-1β) inhibitors, may account for the differences in characteristics between these two viruses and could also limit the use of MPV as a smallpox model. Although the extensive genetic differences confirm that MPV is not a direct "relative" of VAR, the possibility of future adaptation of MPV to humans cannot be ruled out.

1. Full-Length Genome

The full-length genome of MPV-ZAI is 196,858 bp, containing 190 largely non-overlapping ORFs with ≥60 amino acid residues. Its structural features and GC content (31.1%) are similar to those of other Orthopoxviruses. The coding sequence of the MPV-ZAI gene is longer than that of VAR, at 195,118 bp. The longer length of MPV DNA is mainly due to the duplication of four terminal ORFs on the left side, which are part of the terminal inverted repeats (TIRs), while the VAR genome has very short non-genic TIRs lacking ORF duplication (Figure 1).

Schematic diagram of the terminal species-specific variable genome regions of MPV-ZAI and VAR-IND

Figure 1. Schematic diagram of the terminal species-specific variable genome regions of MPV-ZAI and VAR-IND

Note: TIRs are indicated by arrows, and short tandem terminal repeat regions are shown as rectangles. Overlapping sequences are indicated by wide black bars; deletions in one genome relative to the other are shown as lines. The boundaries of variable genome regions are marked by nucleotide numbers corresponding to their positions in the genome.

2. Central Genome

The central genome region of Orthopoxviruses mainly contains highly conserved essential genes. The central genome region of MPV-ZAI DNA is 101,466 bp, defined by the C10L and A25R ORFs, with 96.3% homology to VAR-IND. The amino acid sequences of the virion proteins encoded in this region of MPV-ZAI show 91.7-99.2% similarity to those of VAR-IND.

3. Terminal Regions

1) Virulence Factors

Notably, mutations in the two IFN resistance genes of MPV-ZAI result in their translation into intracellular proteins, which are intact in VAR and other Orthopoxviruses. One of these proteins (C3L in VAR-IND, Table 1) is a homolog of eukaryotic translation initiation factor 2α (eIF-2α) and acts as a decoy to inhibit the antiviral activity of double-stranded RNA-dependent protein kinase (PKR). The MPV genomes (ZAI and CNG strains) do not encode this protein. Studies have shown that mutated VAC strains lacking this gene exhibit IFN sensitivity, with virus yields reduced by approximately 100-fold compared to the parent virus. Another IFN resistance gene (E3L in VAR-IND, Table 1) is present in VAR and other Orthopoxviruses, expressed as long or short forms depending on the first or second methionine (Figure 2A). In VAC, the long form's N-terminal domain mediates binding to Z-DNA, nuclear localization, and interaction with PKR, essential for VAC virulence. The C-terminal domain of both forms binds to double-stranded RNA, inhibiting IFN-induced PKR and 2-5A synthetase activation, which is necessary for IFN resistance and VAC host range. In MPV, the first translation start codon and downstream nonsense mutations result in only the short form being translated (Figure 2A). These two IFN mutations may contribute to the lower human-to-human transmission of MPV compared to VAR.

MPV encodes a complement-binding protein with only three short repeats, while other Orthopoxviruses have four (Figure 2B). Additionally, MPV encodes a secreted IL-1β-binding protein and 3-β-hydroxy-Δ5-steroid dehydrogenase, which have incomplete ORFs in VAR strains (Table 1). Notably, the VAC gene encoding the IL-1β-binding protein is associated with fever and pathogenicity. The presence of the IL-1β-binding protein in MPV may be one reason for its lower pathogenicity compared to VAR.

Alignment of amino acid sequences of IFN resistance factors and complement-binding proteins between MPV and VAR

Figure 2. Alignment of amino acid sequences of IFN resistance factors and complement-binding proteins between MPV and VAR

*Notes: Figure 2A shows the alignment of amino acid sequences of the E3L IFN resistance factors encoded by corresponding genes in VAR-IND, VAR-GAR, and MPV-ZAI. Regions containing the N-terminal adenosine deaminase Z-α domain (gray) and C-terminal double-stranded RNA-binding motifs are indicated. Identical amino acid residues are marked with dots; deletions are marked with dashes. The first and second methionine residues starting the long or short forms of the protein are indicated with asterisks above the sequence. Figure 2B shows the alignment of complement-binding protein amino acid sequences in VAR-IND and MPV-ZAI ORFs. Conserved cysteine residues are indicated by black vertical bars, and other conserved residues are indicated by gray vertical bars. The numbers above the boxes represent the four typical repeats of the complement control protein.

Table 1. Comparison of virulence factors between MPV and VAR

 Comparison of virulence factors between MPV and VAR

2) Ankyrin Repeat Proteins

ORFs encoding ankyrin repeat-containing proteins, some of which have host range functions (MPV-ZAI D7L and C1L, homologous to Orthopoxvirus), form the largest gene family in Orthopoxviruses. Among the 10 genes in this family, the gene corresponding to VAR-IND B19R is deleted in the MPV-ZAI genome, and the gene corresponding to D1L in MPV-ZAI is missing in both VAR strains. Additionally, four VAR genes in this family (D6L, D7L, C1L, and O3L in VAR-IND) are truncated compared to their MPV homologs. Currently, we can only speculate on how these differences might affect host range or virulence.

Table 2. Comparison of ankyrin repeat proteins between MPV and VAR

Comparison of ankyrin repeat proteins between MPV and VAR

4. Phylogenetic Tree Analysis

To better understand the genetic relationships, a phylogenetic analysis of the terminal variable genome regions of four human pathogenic Orthopoxviruses was performed using 117,600 bp of DNA (Figure 3). The major and minor subspecies of VAR are closely related, and MPV is slightly more distant from VAR than VAC.

Phylogenetic tree analysis of the terminal variable genome sequences of MPV, VAR, CPV, and VAC

Figure 3. Phylogenetic tree analysis of the terminal variable genome sequences of MPV, VAR, CPV, and VAC

In conclusion, the comparison of the genomes of MPV and VAR reveals multiple differences in virulence genes between the major and minor strains of VAR. MPV and VAR likely evolved independently from a common Orthopoxvirus ancestor. The genetic differences between VAR and MPV raise significant questions about the validity of using MPV as a smallpox model. However, given the severe diseases caused by MPV, close monitoring of MPV infection rates is essential to ensure that it does not undergo human adaptation, either spontaneously or through recombination, especially in unvaccinated populations with high HIV prevalence.

References

1.Shchelkunov S.N. et al. (2001). Analysis of the Monkeypox Virus Genome. Virology Journal.

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Genomic Analysis of Monkeypox Virus (MPXV)

Monkeypox virus (MPV) belongs to the Orthopoxvirus genus of the Poxviridae family and causes a human disease similar to smallpox. The 196,858 bp genome of MPV was analyzed for structural features and open reading frames (ORFs). Each end of the genome contains a terminal inverted repeat (ITR) sequence that is 6379 bp long, completely identical but oriented in the opposite direction, a putative telomere resolution sequence, and short tandem repeat sequences. MPV contains the core genes of known Orthopoxviruses but only a subset of the immune modulation and host range genes. Sequence comparison confirms that MPV is a distinct Orthopoxvirus, neither a direct ancestor nor a direct descendant of the Variola virus.

1. Introduction

Poxviridae comprises complex double-stranded DNA viruses that replicate in the cytoplasm of vertebrate or invertebrate cells. Orthopoxviruses include Variola virus (VAR), Monkeypox virus (MPV), Cowpox virus (CPV), and Vaccinia virus (VAC). VAR is the causative agent of human smallpox, a disease with a mortality rate of 10%-40%, eradicated through a strategic live VAC vaccination campaign. After smallpox eradication, vaccination ceased, leading to decreased immunity against other Orthopoxviruses. Consequently, susceptibility to zoonotic viruses such as MPV, CPV, and VAC has increased. These viruses' potential adaptation to increased pathogenicity or transmissibility in human populations could occur.

The similar clinical presentation of monkeypox and smallpox led to the hypothesis that MPV is an evolutionary ancestor of VAR. MPV and VAR evolution was analyzed based on genome restriction endonuclease maps or nucleotide sequence comparisons of single viral genes. The complete genome of human MPV isolate ZAI-96-I-16 (MPV-ZAI) was sequenced. Whole-genome comparison concluded that MPV is neither a direct ancestor nor a descendant of VAR. Detailed analysis of the MPV-ZAI DNA sequence, compared with corresponding whole-genome sequences of VAC, VAR, and partial CPV sequences, showed that the sequence includes the entire genome except for part of the covalent terminal hairpin loop.

2. Genome Map

identical but oriented in the opposite direction, known as the terminal inverted repeat (ITR), which includes a set of short tandem repeat sequences and terminal hairpins. The MPV-ZAI genome contains a 6379 bp ITR. One of the two complementary, incompletely base-paired hairpin loop strands in MPV-ZAI DNA was successfully cloned through S1 nuclease digestion and DNA polymerase I polymerization. This sequence, aligned with other Orthopoxvirus sequences, showed considerable conservation, with only four nucleotides missing from the loop-forming region (Figure 1).

Comparison of the terminal regions of MPV and other Orthopoxviruses

Figure 1. Comparison of the terminal regions of MPV and other Orthopoxviruses

Note: The upper line shows the ordered portion of the terminal loop of MPV-ZAI. Nucleotides differ from the VAC-WR sequence. ZAI, BSH, and WR represent Zaire-96-I-16, Bangladesh, and West Reserve virus strains, respectively.

The telomere resolution sequence of MPV is identical to those of VAC and VAR. The tandem repeat sequence region near the terminal hairpin of the MPV-ZAI genome is shorter, consisting of NR I (85 bp) and NR II (322 bp). The organization of this region in MPV-ZAI DNA is most similar to VAR-GAR (Figure 2). However, the terminal regions of all VAR strains have very short ITRs, lacking ORFs, and differing sets of tandem repeat sequences at the left and right ends.

Tandem repeat pattern within the ITR region of MPV and other Orthopoxviruses

Figure 2. Tandem repeat pattern within the ITR region of MPV and other Orthopoxviruses

Note: The white rectangles represent unique ITR sequences: NR I, NR II, and coding regions. Black diamonds correspond to 70 bp tandem repeat sequences, with the numbers indicated above. Gray triangles correspond to 54 bp repeats, with the numbers shown below. Pentagons in VAC-COP and VAC-WR ITR sequences represent 125 bp repeats. Open squares and triangles indicate variations from the consensus sequence through deletions, replacements, or insertions of these repeats. Black circles represent terminal hairpins. Dashed lines indicate the absence of corresponding DNA.

3. Coding Regions

Four ORFs on the left side of the MPV-ZAI genome (Figure 3A) are located within the ITR, so corresponding ORFs also exist on the right side of the genome (Figure 3B). All genes known to be crucial in other Orthopoxviruses are present in MPV and occupy the central region of the genome (ORFs C10L to A25R). These ORFs share more than 90% sequence homology with those of other Orthopoxviruses. Most species and strain-specific differences between MPV-ZAI and other Orthopoxviruses are found in the terminal regions at the left and right ends (Figures 3A and 3B). MPV-ZAI lacks 25 ORFs found in CPV-GRI and 19 potential ORFs in VAR-IND. Although the functions of some of these genes remain unknown, they may include many involved in immune evasion, host range, and cell proliferation.

Comparison of ORFs in the left (A) and right (B) terminal variable regions of MPV with corresponding genomic fragments of other Orthopoxviruses
Comparison of ORFs in the left (A) and right (B) terminal variable regions of MPV with corresponding genomic fragments of other Orthopoxviruses
Comparison of ORFs in the left (A) and right (B) terminal variable regions of MPV with corresponding genomic fragments of other Orthopoxviruses
Comparison of ORFs in the left (A) and right (B) terminal variable regions of MPV with corresponding genomic fragments of other Orthopoxviruses

Figure 3. Comparison of ORFs in the left (A) and right (B) terminal variable regions of MPV with corresponding genomic fragments of other Orthopoxviruses.

Note: ORF sizes and directions are indicated by arrows. Deletions of DNA and protein relative to another virus, exceeding 150 bp and 50 amino acids, respectively, are marked with dots. Black squares mark the ITR region. Nucleotide numbering is shown on the right.

Previous comparisons of MPV and VAR genes added VAC and CPV growth factors and immune evasion genes in Table 1, encompassing all such known genes present in Orthopoxviruses. Based on the number of intact and broadly truncated virulence genes (indicated in parentheses), the following order was derived: CPV-GRI (17) > VAR-IND (11) = VAR-GAR (11) > MPV-ZAI (10) > VAC-COP (9) > VAC-MVA (6). Therefore, MPV-ZAI also contains fewer ankyrin repeat genes than CPV.

Table 1. Orthopoxvirus Growth Factors and Immune Evasion Genes

Orthopoxvirus Growth Factors and Immune Evasion Genes

Note: * indicates ORFs duplicated in the inverted terminal repeat (ITR) regions on the left and right sides. † indicates ORFs with significant functional differences from corresponding ORFs in CPV-GRI.

CPV-GRI encodes the Kelch family 6-500 amino acids of unknown function, sharing 22-26% similarity with each other. While VAC-COP encodes three Kelch family proteins with homology to CPV-GRI proteins at 99.4%, 97.9%, and 98.6%, respectively, the MPV-ZAI genome encodes only one C9L, homologous to CPV-GRI protein G3L with 97.3% similarity. In the VAR genome, these ORFs appear disrupted, suggesting they are not essential for replication.

VAC contains two ORFs with phospholipase D sequences: F13L is necessary for the formation of extracellular virions and effective intercellular spread of infection, while K4L is essential for replication in tissue culture. MPV-ZAI contains intact versions of these three ORFs (C4L, C5L, and C19L), whereas VAR strains lack C4L and C5L homologs due to DNA fragment deletions (Figure 3A). The functions of C4L and C5L proteins are unclear, but they are encoded by both MPV and CPV, two Orthopoxviruses with relatively broad host ranges.

4.Phylogenetic Analysis

Based on the DNA sequence of the terminal variable region, it is believed that the organizational structure of CPV is most similar to the ancestral orthopoxvirus species, and the distance between VAR and MPV is slightly farther than that of VAC. However, by analyzing the left and right variable regions separately, the situation appears to be more complicated. VAC strains appeared to be closer to the MPV-ZAI terminal variable region than their left side and VAR strains relative to the right terminal variable region (Figure 4), this difference may be due to the complex recombination origin of VAC.

Phylogenetic analysis

Figure 4. Phylogenetic analysis

5. Summary

The monkeypox virus (MPV) genome shows a distinct structure compared to other Orthopoxviruses, such as variola (VAR), cowpox (CPV), and vaccinia (VAC) viruses. The MPV genome is neither an ancestor nor a direct descendant of the VAR genome, suggesting independent evolutionary paths. The analysis also reveals differences in host range and immune evasion genes among Orthopoxviruses, with MPV having fewer such genes than CPV but more than VAR. These findings contribute to understanding MPV's evolutionary history and its relationship with other Orthopoxviruses.

References

1. Shchelkunov SN, Totmenin AV, Safronov PF, et al. Analysis of the monkeypox virus genome[J]. Virology. 2002, 297(2): 172-194.

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Morphology, Transmission, and Replication Cycle of Poxviruses

Since the global eradication of smallpox in 1980, there has been little development of therapies for the variola virus. The global monkeypox outbreak in 2022 brought smallpox back into public focus, raising concerns that variola or monkeypox viruses could become potential bioweapons of terrorism. The variola virus and the monkeypox virus (MPXV) both belong to the Poxviridae family, Chordopoxvirinae subfamily, and Orthopoxvirus genus. Poxviruses are the largest known animal viruses, capable of infecting most vertebrates and invertebrates. They are double-stranded DNA viruses with about 200 distinct genes. A subset of these gene products can independently perform essential viral functions without the host cell nucleus, while others widely regulate the host cell and immune system.

Notable orthopoxviruses in human epidemiology include variola virus, vaccinia virus (VACV), cowpox virus (CPXV), and monkeypox virus (MPXV). Variola virus, the causative agent of smallpox, has two clinical epidemiological variants identified in the United States: Variola major virus and Variola minor virus (or alastrim virus), with fatality rates of 5-40% and 1%, respectively, indicating greater virulence of the Variola major virus. The cowpox virus was named for its association with pustular lesions on cow udders and milkmaids' hands. Interestingly, cows are not the natural host of CPXV, and cowpox is not a common disease. The vaccinia virus is widely used as a poxvirus model in laboratories and was used to produce smallpox vaccines, with strains like M-63 (Soviet Union), Lister (UK), Tiantan (China), and Wyeth, IHD (USA) being most commonly used.

Poxvirus genome

The poxvirus genome is a linear double-stranded DNA with variable-length inverted terminal repeats forming hairpin structures. The full sequencing of vaccinia virus Copenhagen and WR strains shows a genome length of 191kbp, with 12kbp inverted terminal repeats (ITR) ending in covalently connected single-stranded hairpin loops. The sequence is AT-rich and contains short direct repeats and multiple ORFs. Poxviruses can replicate entirely in the cytoplasm, with low dependence on the host cell's DNA and RNA. The virus synthesizes new genes by generating and breaking down large tandem molecules, providing favorable conditions for genome amplification and evolution by acquiring new functions.

Schematic Diagram of the Vaccinia Virus Genome

Figure 1. Schematic Diagram of the Vaccinia Virus Genome

Poxvirus structure

Poxviruses are the most structurally complex known viruses, with elliptical or brick-like particles about 200-400nm long and an aspect ratio of 1.2-1.7. The membrane is a typical 50-55nm lipid-protein bilayer, surrounding the core and covered with randomly arranged tubular elements (STE), averaging 7nm wide and 100nm long. The virus particle consists of a core and related lateral bodies surrounded by a membrane, making it sufficiently infectious; however, for some virus strains and specific cell infections, the virus particle may acquire an additional lipid bilayer with a unique chemical composition, called the Envelope. This Envelope contains about twice the phospholipids of the non-enveloped virus particle. Studies have shown that antigens on the viral envelope can induce immunity, protecting the host from poxvirus infection. The poxvirus envelope contains at least seven different glycoproteins and a major non-glycosylated acylated polypeptide.

Structural Diagram of Poxvirus in IMV Form. Note: The existence of an inner membrane is still under debate.

Figure 2. Structural Diagram of Poxvirus in IMV Form. Note: The existence of an inner membrane is still under debate.

Poxvirus transmission

The transmission process varies significantly depending on the poxvirus species and host, usually involving the following methods: 1) remaining free in the cytoplasm; 2) migrating to the cell surface and being expelled through microvilli; 3) being enveloped by a double membrane from the Golgi apparatus, transported to the cell surface, and released by an envelope (from the Golgi apparatus intraluminal pool membrane); 4) forming large vesicles through budding and acquiring an envelope; 5) combining with non-membrane vesicles; 6) combining with a protein substance in an acidophilic cytoplasmic structure called an a-type inclusion body (ATI).

Vaccinia virus replication cycle

The poxvirus replication cycle varies by virus species. Using the model virus VACV as an example, replication generally occurs 2-5 hours post-infection. Viral maturation occurs in five stages: immature virion (IV), intracellular mature virion (IMV), intracellular enveloped virion (IEV), cell-associated enveloped virion (CEV), and extracellular enveloped virion (EEV). IMV and EEV are the main infectious forms of vaccinia virus, with EEV being more infectious and entering cells through a non-pH-dependent membrane fusion process. EEV is responsible for intercellular transmission, eventually released as IMV into the external environment. Compared to EEV, IMV is more stable and responsible for inter-host transmission.

Uncoating

After fusion with the cell membrane, primary uncoating occurs, releasing the viral core into the cytoplasm. The core contains the viral genome, along with virus-dependent RNA polymerase, "initiation" proteins necessary for specific recognition of viral early gene promoters, and several RNA processing enzymes that modify viral transcripts. After release, the core synthesizes viral early mRNA (Step 2 in Figure 3), which exhibits the typical characteristics of cellular mRNA, being recognized and translated by the cell (Step 3 in Figure 3). About half of the viral genes are expressed early in infection. Some early proteins, similar in sequence to cellular growth factors, are secreted from the cell (Step 4 in Figure 3), inducing the proliferation of neighboring host cells or the suppression of host immune defense mechanisms. The synthesis of early proteins induces secondary uncoating, the core wall opens, and the nucleoprotein complex containing the genome is released from the core (Step 5 in Figure 3), halting the expression of viral early genes.

Replication

Early proteins catalyze the replication of the viral DNA genome (Step 6 in Figure 3), and the newly synthesized viral DNA molecules serve as templates for the next replication cycle (Step 7 in Figure 3) and as templates for the transcription of viral intermediate genes (Step 8 in Figure 3). Intermediate gene transcription requires specific viral proteins (products of early genes) that confer specificity for intermediate promoters on the viral RNA polymerase and host cell proteins (Vitf2) relocated from the infected cell nucleus to the cytoplasm. Intermediate mRNA encodes proteins (Step 9 in Figure 3), including those needed for late gene transcription (Step 10 in Figure 3). Late genes encode proteins that construct the virus particle, enzymes, and other early initiation proteins needed for virus particle assembly. These viral proteins are synthesized with the help of the cellular translation mechanism (Step 11 in Figure 3).

Assembly

Viral membrane proteins are non-glycosylated, and the role of the cellular membrane in early assembly is controversial (Step 12 in Figure 3). Initial assembly forms immature virus particles (Step 13 in Figure 3), spherical particles separated by a membrane, possibly obtained from the early secretion pathway of the cell. The virus particle matures into brick-shaped IMV (Step 14 in Figure 3), released upon cell lysis (Step 15 in Figure 3). Additionally, the particles can acquire a second bilayer from the Golgi apparatus or early endosome, forming intracellular enveloped virus particles (IEV) (Step 16 in Figure 3).

Separation

IEV moves to the cell surface on microtubules, fusing with the plasma membrane to form cell-associated virus particles (CEV) (Step 17 in Figure 3). These CEV induce actin polymerization, promoting the direct transfer of actin to surrounding cells (Step 18 in Figure 3); they can also separate from the membrane to form EEV.

Single-cell reproduction cycle of vaccinia virus

Figure 3. Single-cell reproduction cycle of vaccinia virus

References

1.Buller RM, Palumbo GJ. Poxvirus pathogenesis. Microbiol Rev. 1991, 55(1): 80-122.
2.Harrison SC, Alberts B, Ehrenfeld E, et al. Discovery of antivirals against smallpox. Proc Natl Acad Sci U S A. 2004, 101(31): 11178-11192.

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At present, common thyroid hormone detection methods include Chemiluminescent Immunoassay (CLIA) and Isotope Dilution Liquid Chromatography Tandem Mass Spectrometry (ID-LC /MS /MS). The accuracy of the LC-MS/MS results is clinically recognized, but why is CLIA still the most preferred method for thyroid hormone detection?

The difficulty of the application of LC-MS/MS in clinical thyroid hormones detection mainly lies in the fT3/fT4 detection, in which, the separation of fT3/fT4 needs to ensure the endogenous balance between the bound state and the free state. Current LC-MS/MS separation system takes a processing time of 30 min-24h, which is very time-consuming. Moreover, it is a challenge to avoid non-specific binding of the sample to the surface of the membrane or device during the separation process.

In conclusion, the complex and time-consuming sample pre-processing for LC-MS/MS, the unavoidable non-specific binding on the membrane surface, and the demands for automation in clinics have made the popularization of LC-MS/MS in clinical testing particularly difficult.

T3/T4 Sandwich Antibody

High Sensitivity · One Step Assay · ICA/CLIA Verified

The binding sites of small molecule compounds such as T3 and T4 and the corresponding antibodies are mostly in a cave structure, and most of their structure is covered, resulting in insufficient space for antibody binding. Therefore, traditional Sandwich Immunoassay technology is difficult to detect small molecules, and most of the T3/T4 detection kits use Competitive Immunoassay. With years of experience in developing small molecule sandwich antibodies, OkayBio has successfully developed T3 and T4 sandwich antibodies.

OkayBio T3 sandwich antibody enables the CLIA detection to realize the upgrade from two-step Competitive Immunoassay to One-step Sandwich Immunoassay, which reduces 50% of the consuming time for thyroid function tests. It is a tremendous breakthrough for multi-projects combined thyroid function tests and POCT rapid tests.

Advantage of Sandwich Immunoassay

advantage
Difference in signal: Sandwich VS Competitive

Figure 1. Difference in signal: Sandwich VS Competitive

Work flow in CLIA Sandwich VS Competitive

Figure 2. Work flow in Chemiluminescent immunoassay (CLIA) Sandwich VS Competitive: The Sandwich Immunoassay facilitates a streamlined "One-step assay" approach, wherein the capture monoclonal antibody (mAb), detection mAb, and the sample can be mixed or incubated simultaneously. This innovative process results in a significant time-saving of 50% compared to the traditional Competitive Immunoassay, where separate incubation and washing steps are mandatory for the antibody-antigen interaction.

T3 Monoclonal Antibody

T4 Monoclonal Antibody

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Product Name Catalog# Recommend Pair Platform
T3 Antibody R515n5 R515n5(Capture)-R514n5(Detection)
R515n5(Capture)-R513c8(Detection)
CLIA
R514n5
R513c8 R513c8(Capture)-R515n5(Detection) ICA
T4 Antibody R533c3 R533c3(Capture)-R555a5(Detection) CLIA
R534a7 R555a5(Capture)-R533c3(Detection)
R555a5(Capture)-R534a7(Detection)
ICA
R555a5

Clinical detection of 25-OH-VD

Currently, the detection methods of serum 25-OH-VD is mainly focused on Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) and Chemiluminescence Immunoassay (CLIA). The national health industry standard WS/T 677-2020 recommends LC-MS/MS as the first screening method for vitamin D deficiency, and Chemiluminescence Immunoassay as the second screening method. These two methods have their own advantages and disadvantages in clinical detection. LC-MS/MS is a direct analysis method based on the molecular weight, structure and other chemical properties of the tested sample, and has high sensitivity and high specificity. CLIA is an immunoassay technology based on antigen-antibody specific binding. Compared with LC-MS/MS, it has simple pretreatments for samples and higher level of automation for detection.

PART 1

The ‘Gold Standard’ of VD Detection—LC-MS/MS

Why LC-MS/MS is the ‘Gold Standard’
LC-MS/MS is the most preferred method for the detection of biochemical small molecules in clinical, which integrates the physical separation of liquid chromatography and the mass analysis of mass spectrometry. LC-MS/MS adopts two-stage tandem mass spectrometry analysis, which can utilize the information of precursor ions as well as fragment ions at the same time to selectively monitor the compounds to be quantified, and even trace components are not interfered by abundant substances. For the analysis and detection of small molecules like 25-OH-VD, LC-MS/MS has relatively high analytical specificity and detection sensitivity.

Limitations in clinical applications
LC-MS/MS is easily affected by substances other than the target small molecules in the samples to be tested. These interfering substances include endogenous substances from the sample itself and the external substances introduced from the environment during the method establishing process. In order to eliminate this matrix effect and improve the accuracy of LC-MS/MS analysis results, the samples have to be strictly pre-treated. Inspectors need to select appropriate sample pretreatment methods according to matrix purification effect, expected sample concentration, minimum detection limit and other factors to eliminate matrix effects caused by different substances.

PART 2

Mainstream Immunoassays for VD Detection

Limitations of CLIA Competition Method in Clinical Applications

Poor consistency with LC-MS/MS detection results
At present, for small molecules with low molecular weight and insufficient antigenic epitopes like 25-OH-VD, the CLIA competition method is most commonly used immunoassay in clinical detection. Plenty of studies have revealed that the CLIA competition method has poor correlation with LC-MS/MS in detection results.
Researchers from the Laboratory Department of Peking Union Medical College Hospital of Chinese Academy of Medical Sciences and the China-Japan Friendship Hospital compared the 25-OH-VD detection reagents of five major brands (Abbott, DiaSorin, IDS, Roche, Siemens) on the market and found that in these reagents, the coincidence rate between the test results and the clinical judgments based on LC-MS/MS test results was less than 71% (Table 1).

Table 1. Consistency between clinical judgments of LC-MS/MS and different CLIA competition methods

Supplier All Samples (245 cases)
<20ng/mL
N(%)
20~30ng/mL
N(%)
>30ng/mL
N(%)
Coincidence
Rate
LC-MS/MS 99(40.4) 68(27.8) 77(31.4)
A 115(46.9) 79(32.3) 51(20.8) 68.60%
B 127(51.8) 94(38.4) 24(9.8) 64.90%
C 95(38.8) 104(42.4) 46(18.8) 67.80%
D 121(49.4) 79(32.2) 45(18.4) 70.60%
E 39(15.9) 72(29.4) 134(54.7) 51.80%

Note: A, B, C, D, and E represent Abbott, DiaSorin, IDS, Roche, and Siemens in order. Among them, Abbott, IDS and Siemens use the AE Chemiluminescence competition method, DiaSorin uses the Isoluminol derivatives direct Chemiluminescence competition method, and Roche uses the Electrochemiluminescence competition method.

Poor recognition of 25-OH-VD2
The study above also found that no matter what kind of CLIA system, when the sample contains 25-OH-VD2, the correlation between the competition method and the LC-MS/MS decreases in detection results. It can be concluded that the major detection reagents circulating on the market for CLIA competition method do not recognize 25-OH-VD2 well, which directly affects the consistency of the reagents and LC-MS/MS detection results (Table 2).

Table 2. 25-OH-VD2 content vs. its Impacts to CLIA competition test results

Sample Slope 95% CI Intercept 95% CI r Value 95% CI Deviation 95% CI Deviation% 95% CI
Without 25-OH-VD2 (154 cases)
A 0.96 0.90~1.01 2.05 1.16~3.19 0.917 0.887~0.939 1.0 0.3~1.7 8.6 4.9~12.3
B 0.83 0.79~0.88 1.61 0.92~2.21 0.930 0.905~0.948 -2.5 -3.2~-1.8 -8.7 -11.9~-5.5
C 0.68 0.63~0.73 8.83 7.80~9.97 0.885 0.845~0.915 1.7 0.8~2.7 18.2 13.0~23.4
D 0.95 0.89~1.02 1.08 -0.07~2.02 0.909 0.877~0.933 -0.5 -1.3~0.3 -1.2 -5.0~2.6
E 1.29 1.17~1.42 5.34 2.94~7.47 0.848 0.796~0.887 10.8 9.5~12.1 44.7 39.8~49.7
Contain 25-OH-VD2 (91 cases)
A 0.66 0.56~0.75 4.41 1.80~6.92 0.804 0.717~0.867 -5.3 -6.5~-4.1 -19.9 -24.8~15.0
B 0.78 0.70~0.87 -0.4 -2.63~1.68 0.854 0.787~0.902 -6.8 -7.8~-5.7 -30.1 -34.2~26.0
C 0.77 0.68~0.86 2.51 0.05~4.61 0.845 0.773~0.895 -3.8 -4.8~-2.7 -14.0 -18.4~-9.7
D 1.03 0.90~1.17 -5.48 -9.10~-2.51 0.819 0.738~0.877 -5.0 -6.2~-3.8 -25.0 -30.2~-19.7
E 1.47 1.16~1.92 -1.70 -13.59~5.86 0.626 0.482~0.737 11.3 8.7~13.8 33.8 27.5~40.1
Total Samples(245 cases)
A 0.85 0.78~0.91 2. 84 1.68~3.80 0.848 0.808~0.880 -1.3 -2.1~-0.6 -2 -5.4~1.4
B 0.78 0.73~0.82 1.58 0.77~2.31 0.894 0.866~0.920 -4.1 -4.7~-3.5 -16.6 -19.4~-13.8
C 0.67 0.62~0.72 7.54 6.38 ~8.44 0.841 0.800~0.874 -0.3 -1.1~0.5 6.2 2.1~10.4
D 0.93 0.87~1.00 -0.31 -1.43~0.61 0.866 0.831~0.894 -2.2 -2.9~-1.5 -10.0 -13.4~-6.6
E 1.30 1.18~1.43 4.04 1.81~6.44 0.776 0.721~0.822 11.0 9.7~12.2 40.7 36.7~44.6

Note: The data in Table 2 are results comparing with LC-MS/MS.

PART 3

Double-antibody Sandwich Immunoassay / SMALL MOLECULE

  1. Benchmarking The ‘Gold Standard’ of LC-MS/MS

Most of the binding sites between small molecules and corresponding antibodies are cave-like structures, and majority of the structure of small molecules is covered, resulting in insufficient antibody binding space. Thus, traditional sandwich immunoassay techniques are difficult to detect small molecules. In order to break through the technical barriers of sandwich antibodies for small molecules, the R&D team of OKayBio continued to practice and kept challenging, and finally successfully developed antibodies and bulk packs of reagents suitable for 25-OH-VD sandwich immunoassays by adjusting the immune strategies and optimizing antibody designs.

PERFORMANCE VERIFICATION

It has been verified that the 25-OH-VD sandwich antibodies of OKayBio have strong abilities to recognize the total 25-OH-VD. The bulk packed reagents were inspected on the AE Chemiluminescence platform. The correlation (R2) between the self-test results and the mass spectrometry results was 0.9153, and the correlation (R2) between the customer-verified results and the mass spectrometry results was up to 0.945.

Self-tested sample coincidence rate

Clinical comparative analysis

Figure 1. Clinical comparative analysis
(Sandwich-MS assigned)

In order to improve the accuracy of 25-OH-VD immunoassay results, the production of OKayTM bulk packed reagents (Q80h1) for Chemiluminescent sandwich method benchmark the industrial ‘Gold Standard’ for detection - LC-MS/MS. Verified by the AE Chemiluminescence platform, the Q80h1 detection results were in good consistency with the mass spectrometry detection results, and the correlation (R2) can reach 0.9153 (Figure 1). In conclusion, the sandwich method improved the accuracy of the immunoassay results effectively.

Customer-verified sample coincidence rate

Clinical comparative analysis

Figure 2. Clinical comparative analysis
(Sandwich-MS assigned)

Our customer used OKayTM Q80h1 to test the mass spectrometry assigned samples, the preliminary test results were in good consistency with the mass spectrometry test results, and the correlation (R2) was up to 0.945 (Figure 2).

Total 25-OH-VD recognition capacity

Recognition capacity of sandwich antibody to total 25-OH-VD

Figure 3. Recognition capacity of sandwich antibody to total 25-OH-VD

Studies from the Laboratory Department of Peking Union Medical College Hospital of Chinese Academy of Medical Sciences and the China-Japan Friendship Hospital revealed that the cross-reactivity between 25-OH-VD antibody and 25-OH-VD2 directly affects the accuracy of total 25-OH-VD detection results. Verified by the Chemiluminescence platform, OKayTM 25-OH-VD sandwich antibodies have good cross-reactivity with 25-OH-VD2 and 25-OH-VD3, and can effectively identify the total 25-OH-VD in the sample.

Summary

The successful development of 25-OH-VD sandwich antibodies marked the first step of ‘technique innovation for small molecule sandwich antibody’ by Nanjing OKayBio. The 25-OH-VD sandwich antibodies not only have good consistency with the ‘Gold Standard’ test results of mass spectrometry, but also makes up for the limitations of the CLIA competition method in clinical detection to some extent. In the future, OKayBio will continue to launch more high-quality detection projects for small molecules.

Small molecule projects under research

Hypertension AⅠ, AⅡ, ALD
Thyroid T3, T4
Gestation Prog, E2
Anemia FA, VB12

PART 4

Related Products NANJING OKAYBIO

25-OH-VD Antibody

Method Catalog # Label Recommended use Recommended pair Platform
Sandwich L216u1 AE Detection L216u1-L217u1 Chemiluminescence
L217u1 Biotin Capture
R483k3 - Detection R483k3-R484j4
R484j4 - Capture
R485m5 - Detection R485m5-R486n6
R486n6 - Capture
Competition K32w6 - Detection - Chemiluminescence, Immunochromatography, Turbidimetric Immunoassay
L210u1 AE - -
L212u1 ALP - -
L219u1 Biotin - -

25-OH-VD Antigen

Name Catalog # Type Conjugate
Antigen C1580 Synthesized BSA

Miscellaneous

Name Catalog # Recommended Dosage Platform
Dissociation Agent D10a1 50μL /sample AE/ALP Chemiluminescence

ABOUT OKAYBIO

Nanjing OKay Biotechnology Co., Ltd. (briefly referred as Nanjing OKayBio) is a total solution provider of in vitro diagnostic raw materials, and has been committed to the rapid development and large-scale production of proteins and antibodies for in vitro diagnostics. Nanjing OKayBio insists on independent innovation and constantly breaking through key technologies. Nanjing OKayBio owns Protein structure analysis platforms, Flow-Cytometry platforms, Hybridoma platforms, and Single-cell sequencing platform to support continuous iterations in products. Nanjing OKayBio has passed the ISO 9001/ ISO 13485 quality system double certification and insist on continuous optimization. Combined with self-built Immunochromatography platform, Turbidimetric Immunoassay platform, Chemiluminescence platform and Fluorescence-activated cell sorting Flow-Cytometry platform, the quality stability of our products is effectively controlled no matter in the production process or at the final delivery stage. Nanjing OKayBio also owns SingleB® rapid monoclonal antibody discovery platform, BGE® high-throughput expression platform, DeepLight® functional pre-screening platform and macromolecule purification platform, which provide a strong guarantee for our product performance and product quality.

References

[1]  Makris K, Bhattoa HP, Cavalier E, Phinney K, Sempos CT, Ulmer CZ, Vasikaran SD, Vesper H, Heijboer AC. Recommendations on the measurement and the clinical use of vitamin D metabolites and vitamin D binding protein - A position paper from the IFCC Committee on bone metabolism. Clin Chim Acta. 2021 Jun;517:171-197.
[2]Beccaria M , Cabooter D . Current developments in LC-MS for pharmaceutical analysis. Analyst. 2020 Feb 17;145(4):1129-1157.
[3] Grebe SK, Singh RJ. LC-MS/MS in the Clinical Laboratory - Where to From Here? Clin Biochem Rev. 2011 Feb;32(1):5-31.
[4] Han S, Qiu W, Zhang J, Bai Z, Tong X. Development of a Chemiluminescence Immunoassay for Quantification of 25-Hydroxyvitamin D in Human Serum. J Anal Methods Chem. 2020 Aug 1;2020:9039270. 

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