FluoBolt™ WNT3A immunoassay is a 96-well format, complete kit with a standard and control samples in a pooled serum matrix.    This assay is similar to a standard ELISA in the fact that this assay is a sandwich immunoassay comprised of a monoclonal capture antibody and a polyclonal detection antibody, but different from a standard ELISA in the fact that it utilizes Metal Enhanced Fluorescence (MEF) to dramatically increase the sensitivity with the same antibodies.  MEF technology uses fluorescence detection that can enhance the signal more than 100 times over chromogenic or chemiluminescent signals.

Although there are assays for measuring WNT3A transcript levels available in the market, detection methods for protein levels have been limited and thus hamper WNT3A research progress.  Existing options for measuring WNT3A protein either require expensive closed-systems instruments, have inconsistent quality records, or are only semi-quantitative in nature (eg. Western blotting).  FluoBolt™-Technology to provides a high sensitivity WNT3A protein assay for basic and clinical research, which increases detection and may improve data consistency better suited for research needs.

The FluoBolt™ WNT3A assay detects human WNT3A. Human WNT3A shares between 100-95% aa sequence identity with primates, mice, rats, hamster, and pig. Cross-reactivity of this assay with other species than human has not been tested with FluoBolt™ WNT3A.

 

Amino Acid Sequence Identity with Human
Source uniprot.org
Mouse Rat NHP Pig Canine Zebra Fish Hamster
Periostin 90% 87% 92-93% 95% 96% 54% 89%
Asporin 89% 89% 96-99% 89% 86% 54% 89%
Noggin 99% 99% 99-100% 99% 96% 55% 99%
Wnt3a 96% 89% 99-100% 96% 71% 82% 96%
Klotho 86% 84% 93-99% 88% n.a. 50% 85%

  • 1 x 96 well- Anti-human WNT3A monoclonal antibody, pre-coated MEF-microtiter plate
  • 1 x 25 ml- Wash buffer concentrate 20x, natural cap
  • 1 x 2.5 ml- Anti-human WNT3A polyclonal antibody, labeled with either: FITC, Cy3, Cy5 or AlexaFluor680
  • 6 vials, 0.25 ml- Standards 1-6, (2800, 1400, 700, 350, 175, 0 pmol/l),white caps, lyophilized

Standards are highly purified recombinant protein in human pooled-serum matrix.

  • 2 vials, 0.25 ml- Control A and B, yellow cap, lyophilized (for concentrations see label).
  • 1 x 10 ml- Sample diluent, natural cap, ready to use

 

ADDITIONAL MATERIAL SUPPLIED WITH THE KIT

  • 2 self-adhesive plastic films
  • QC data sheet
  • Protocol sheet
  • Instruction manual for use
  • 2 desiccant bags for plate storage

 

MATERIAL AND EQUIPMENT REQUIRED BUT NOT SUPPLIED

  • Precision pipettes calibrated to deliver 10 μl, 20 μl, 50 μl, 200 μl, 500 μl, and disposable tips
  • Distilled or deionized water
  • Plate washer, multichannel pipette, or manifold dispenser for washing
  • Refrigerator with 4°C (2-8°C)
  • Fluorescence microplate reader

Read more about the MEF technology here.

WNT3A is a secreted glycoprotein and belongs to the WNT family. Members of this family can interact with cell membrane receptors, thus playing a role in autocrine regulations and paracrine signaling. WNT3A is expressed in placenta at moderate levels, as well as in lung, spleen, and prostate at low levels. The canonical sequence of WNT3A consists of 352 amino acids (aa) and has a mass of 39.365 kDa. It is rich in cysteine and forms many disulfide bonds. At aa 87 and aa 298 glycosylation appears, since N-Acetylglucosamine is covalently attached to asparagine. At position aa 209 WNT3A is covalently lipidated at a conserved serine residue resulting in strong hydrophobic properties of the molecule. Therefore, in its physiological form it constitutes a soluble complex with afamin, which functions as a carrier for hydrophobic molecules in body fluids and is essential for the activity and solubility of WNT3A. WNT3A plays important roles in cell growth and differentiation, embryonic development, neural development, immune regulation, bone formation and carcinogenesis.

Determination of WNT3A has been used for studying the following topics:

Embryonic Stem Cells and Organoid cultures–  WNT proteins have been implicated in oncogenesis, adipogenesis and in several other developmental processes including regulation of cell fate and patterning during embryogenesis.  WNT3A in particular has been a key reagent for embryonic or human pluripotent stem cell cultures (PSC or iPSC) being cultured for research use.  Purified WNT3A is an especially key protein for three-dimensional organoid culture or techniques, including stomach, small intestine, colon, pancreas, and liver organoids.    Recombinant Human WNT3A is a difficult protein to manufacture, thus it is has limited availability and can be expensive.  Many research scientists use less pure protein preparations or cell culture supernatant.   The lack of consistency with WNT3A protein can lead to variability that can be costly.  Techniques to precisely quantify Human WNT3A protein have previously been limited; thus, FluoBolt™ WNT3A could serve as a tool to increase accuracy of WNT3A in each protocol and thus increase reproducibility of culture conditions.

Liver Disease and Repair Research Biomarker– WNT Signaling is one of several pathways proposed to be a driving force of initiating liver repair.  Liver repair is both the healing and regeneration of liver health, as well as a process that increases the severity of disease by inducing scarring and fibrosis.  WNT3A and WNT10B are two canonical WNT signaling proteins upregulated in response to liver injuries (including alcohol, pharmaceutical, viral, metabolitic, etc.).  WNT3A is a prospective biomarker for early liver damage.

Oncology Research Biomarker– Elevated expression of WNT3A has been found in prostate-, breast-, and hepatocellular cancer tissue. Accumulating evidence has suggested that WNT3A promotes or suppresses tumor progression via the canonical WNT signaling pathway depending on cancer type. A vast number of studies on the role of this molecule have been and are still performed. WNT3A remains a hot topic in cancer research.

Ankylosing Spondylitis Research– Ankylosing spondylitis (AS) is associated with both pathologic formation of new bone and enhanced bone resorption. Thus it is not surprising that molecules involved in either of those biological pathways may be valuable biomarkers to study this disease. It is well known, that the WNT signaling pathway plays an essential role in bone remodeling (and therefore in AS) and so might the WNT-protein family members and their antagonists, which has been proven by finding elevated levels of DKK-1 and WNT3A in AS subjects.
As WNT-signaling is the center piece of bone remodeling, WNT-family members play a major role in osteoporosis (OP). Interestingly, WNT3A seems to mediate the beneficial effects of mechanical loading on bone quality.

Method Metal Enhanced Direct Sandwich Fluorescence Immunoassay in 96-well format
Sample Type Serum and Plasma
Standard Range 0 to 2,800 pmol/L (0-112 ng/mL)

6 standards and 2 controls in a serum based matrix

Conversion Factor 1 ng/mL = 25 pmol/L
Sample Volume 20 uL (undiluted sample)/well
Incubation/time/temperature Single-step assay / Overnight / Room Temperature
Sensitivity LOD (0 pmol/L+3 SD); 50 pmol/L (2 ng/mL)

LLOQ 175 pmol/L (7 ng/mL)

Specificity Tested for human WNT3A, but human shares 100-96% aa sequence homology with primates and mice.

Example of typical calibration curve:

MEF-FIA for human WNT3A
MEF-FIA for human WNT3A

The quality control (QC) protocol supplied with the kit shows the results of the final release QC for each kit lot at production date.

Fluorescence intensity obtained by customers may differ due to various influences and/or due to the normal decrease of signal intensity during shelf life.

However, this does not affect validity of results as long as the supplied kit controls read according to specifications (target ranges, see labels).