FluoBolt™ α-KLOTHO immunoassay is a 96-well format, complete kit with a standard and control samples in a pooled serum matrix.    This assay is intended for measurement of α-KLOTHO in just 10 uL of undiluted serum or plasma samples.

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.

Although there are assays for measuring Klotho protein levels available in the market, they are reported to have inconsistent quality records, or are semi-quantitative in nature (eg. immunoprecipitation-immunblot).  FluoBolt™-Technology provides a high sensitivity Klotho protein assay for basic and clinical research, which increases detection and may improve data consistency in literature.  The kit offers individual lyophilized standard/calibrator vials, so no dilution series or calculations are needed.  The calibrator is a recombinant protein with a c-terminal His-tag and an MW of approx. 120kD representing the whole extracellular domain (isoform 1) containing KL1 and KL2. Additionally, the kit also includes 2 lyophilized controls in a serum matrix.

MATERIAL SUPPLIED WITH THE KIT

  • 1 x 96 well- Anti-human Klotho antibody, pre-coated MEF-microtiter plate
  • 1 x 25 ml- Wash buffer concentrate 20x, natural cap
  • 1 x 5 ml- Anti-human Klotho antibody, labeled with either: FITC, Cy3, Cy5 or AlexaFluor®680
  • 6 x 0.25 ml- Standards 1-6, (400, 200, 100, 50, 25, 0 pmol/l),white caps, lyophilized
  • 2 x 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 for use manual
  • 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 diapharma.com/fluobolt/

Method Metal Enhanced Direct Sandwich Fluorescence Immunoassay in 96-well format
Sample Type Serum and Plasma
Standard Range 0 to 400 pmol/L (0-25 ng/mL)

6 standards and 2 controls

Conversion Factor 1 ng/mL = 16 pmol/L
Sample Volume 10 uL (undiluted sample)/well
Incubation/time/temperature Single-step assay / Overnight / Room Temperature
Sensitivity LOD (0 pmol/L+3 SD); 2.5 pmol/L (0.16 ng/mL)

LLOQ 25 pmol/L (1.6 ng/mL)

Specificity Tested for human KLOTHO, human shares high sequence identity with mice, rats, and non-human primates (see chart below).  Cross-reactivity has not been tested.

This assay detects only α-KLOTHO and does not cross-react with β-KLOTHO (shown in Figure 2 under performance tab).  No interference of recombinant FGF-23 with the assay’s signal up to 100-fold molar excess was monitored (shown in Figure 3 under performance tab).

 

Figure 1- Example of typical calibration curve

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).

Figure 2- α-KLOTHO and does not cross-react with β-KLOTHO

Klotho Cross Reactivity

Figure 3-  FGF-23 does not interfere with α-KLOTHO detection

Klotho Signal Change

Intra assay:
4 samples of known concentrations were tested 3 times within 1 assay run
CVs ranged from 1 – 11.0%

Inter assay:
4 samples of known concentrations were tested in duplicates within 3 different assay runs
CVs ranged from 6 – 10%

Spike/Recovery:
The recovery of KLOTHO in serum was evaluated by adding known amounts of human recombinant KLOTHO to 4 different human serum samples. Mean recovery was 82% (69 101%).
Mean recovery in Citrate and Heparin Plasma was each 75% (n=4) and 88% in EDTA Plasma (n=4).

Cross-Reactivity:
Human α-KLOTHO shares between 84-100% aa sequence identity with primates, mice, rats, hamster, and pig (shown in the chart below). Cross-reactivity of this assay with species other than human has not been tested with FluoBolt™ α-KLOTHO.

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%

Basic Structure and Biology

The name for the klotho (KL) family of proteins was derived from Greek mythology.  Clotho was the daughter of Zeus and Themis and one of the three fates.  Clotho was the fate that spun the thread of Life, while the other Fates would each draw out or cut this thread.  There are three subfamilies of Klotho: α-Klotho, β-Klotho, and γ-Klotho.  Α-Klotho and β-Klotho are essential components of the endocrine fibroblast growth factor (FGF) receptor complexes.  They are required for the high-affinity binding of FGF-19, FGF-21, and FGF-23 to their cognate FGF receptors (FGFRs).  When the subfamily is not specified, the word “Klotho” generally means the α-klotho subfamily.

Membrane alpha-Klotho Soluble alpha-Klotho

Figure 1     Klotho can exist in a membrane-bound or soluble state. The membrane bound Klotho is mainly expressed on cells of the kidney and acts locally on kidney biology via signaling interactions with FGF-23. Soluble Klotho can be produced by protease cleavage sites within the membrane bound isoform, or a proposed directly secreted isoform. As a soluble form, Klotho can enter the blood stream and act distally, similar to a hormone or cytokine.

Membrane-bound Klotho is a single transmembrane protein with a short (10 amino acid) intracellular domain that is not yet credited for any signal transduction (shown in Figure 1). The extracellular part of Klotho contains two homologous domains, termed KL1 and KL2.  Between the membrane and KL2 and in between KL1 and KL2 are cleavage sites for ADAM10 and ADAM17.  Cleavage produces soluble Klotho proteins that either contains independent KL1 domain, KL2 domain, or both.  The form containing both KL1 and KL2 is 130 kDa in size and appears that the predominant form of soluble Klotho protein.  In fact, neither secondary cleavage product has been detected in human serum so far, only in in vitro systems. In addition to cleaved membrane Klotho, an alternatively spliced Klotho mRNA transcript is hypothesized to code for a secreted Klotho protein, comprised of the KL1 domain with a 10 amino acid tail.  The soluble forms of klotho are detected blood, urine and cerebrospinal fluid.

Klotho is often described as the fountain of youth protein and seems to translate across species in studies of function and biomarker usefulness.  When Klotho was first discovered, Klotho deficiencies in a mouse model produced a phenotype that was similar to human ageing.  These mice died prematurely, but also showed decreased muscle strength, endurance, cognitive ability, and osteoporosis.  Although the vast majority of research has been based on lack of Klotho in mouse models, overexpression of Klotho extends lifespan by 20–30% compared to wild type mice and is protective for kidney disease, cardiac and vascular disease, lung damage, neurodegenerative disease, and diabetes.  Exogenous Klotho in Klotho-deficient mice or even aged animals improves the aging phenotype.  In humans, variations in the Klotho gene are associated with both life extension and increased cognition in human populations.  In addition, soluble Klotho protein has been shown to be a circulating factor detectable in serum that declines with age.

The functions of Klotho are mainly linked to FGF-23 induced signaling and related to their role in mineral homeostasis.  Klotho is under the transcriptional control of PPARy and is most predominantly expressed in the epithelial cells of the renal tubules in the kidney, but is also detected in the brain and to a lesser extent a variety of other tissues. Klotho exists in at least two forms including a membrane form and a soluble form. The two forms of Klotho have distinct functions in the regulation of various physiological and pathophysiological processes.  Membrane Klotho forms a complex with the fibroblast growth factor 23 (FGF-23) and FGF receptors to regulate renal phosphate excretion and calcium reabsorption. As mentioned above, the soluble form can be produced as a result of a protease cleavage of the extracellular portion of the membrane bound Klotho or a proposed alternative spliced explicitly secreted form of Klotho.  In either case, soluble Klotho can be released into circulation and act as a hormone on distal tissues and organs.

Klotho as a Biomarker in Research

Klotho is linked to a variety of diseased states associated with aging, so it is a likely target to be examined when searching for sensitive and specific biomarkers that can be measured in the blood or urine.

  • Kidney
    Throughout the past 20 years, evidence from clinical and basic science studies has showed that Klotho is significantly correlated with the development and progression of Chronic Kidney Disease (CKD) and its complications. The expression level of Klotho decreases in human and animal subjects with CKD as the severity of kidney disease increases.

In a rat model of Acute Kidney Injury (AKI) a rapid decrease in plasma and urine Klotho is detectable as early as at 3 h after injury. Klotho changes were detected earlier than serum creatinine (24 h) and NGAL (5 h). This report also suggests that Klotho levels in the urine of AKI subjects are lower than healthy subjects, but more work focused on timing and specificity need to be established.

  • Brain
    Klotho is also highly expressed in the brain and animal studies suggest it has a role with neuro dysfunction or disease.  Among these studies, mice deficient in Klotho have a reduced number of synapses and cognitive deficits, while overexpressing klotho enhanced learning, memory and was neuroprotective.  Overexpression of Klotho in a mouse model of Alzheimer’s disease can improve cognitive function and prevent cell loss in the brain.  In a microarray analysis comparing young and old monkeys, Klotho is shown to be downregulated in the aged brain.  These animal studies suggest that Klotho expression level correlates with brain health and increase interest in Klotho as a human biomarker.

Consistent with animal reports, humans with higher plasma Klotho showed both higher cognitive assessment scores and less decline at follow up exams.  Clinical research studies show that Klotho levels have potential as a biomarker of multiple sclerosis severity, psychological dysfunction from chronic stress to bipolar disorder, epilepsy, and Alzheimer’s disease. Together, lower brain Klotho correlates with neurological disease while higher brain Klotho with neurological health.

  • Bone
    The kidneys play an important role in maintaining healthy bone mass and structure by balancing phosphorus and calcium levels in the blood. Chronic kidney disease causes mineral and bone disorder (CKD-MBD) because the kidneys do not properly balance the mineral levels in the body. Klotho is mainly expressed in the kidney and is vital to the regulation of phosphate and calcium.  Decreased Klotho levels are associated with decreased bone mineral density of bones in mice.   More studies with human bone metabolism need to be done to investigate Klotho’s role as a biomarker for bone diseases.
  • Cardiovascular
    Vascular calcification (VC) appears early in the course of CKD but becomes much more prevalent as kidney function deteriorates, creating a strong risk of cardiovascular mortality and morbidity in subjects with CKD. Klotho deficient mice demonstrate arteriosclerosis. In humans, Klotho gene variants have been found to be protective or detrimental for the development of cardiovascular and cerebrovascular disease.  Also, higher Klotho levels are reportedly associated with lower likelihood of cardiovascular disease and higher Klotho protein protects against endothelial dysfunction.
  • Cancer
    Klotho seems to have tumor suppressor activity via by inhibiting insulin/IGF-1 signaling and induction of cancer cell apoptosis. Klotho has been linked as a tumor suppressor in studies of breast cancer, lung cancer, pancreatic cancer, gastric cancer, liver cancer, colon cancer, and ovarian cancer.  In xenograft studies of breast cancer, injections of Klotho protein decreased tumor size.    Structure-function studies pin-point this activity to the KL1 part of Klotho protein and is independent of FGF-23 signaling.  Additionally, exogenous Klotho reduced lung cancer metastasis and increased overall survival.  Klotho is a proposed biomarker for human hepatocellular carcinoma (HCC) and Klotho-positive tumors correlated with higher survival.
  • Diabetes
    In humans and mice, Klotho is significantly decreased in pancreatic islets of subjects with Type 2 diabetes versus those of healthy donors. Diabetic mice treated with Klotho decreased blood glucose levels increased insulin levels.  Klotho seems to protect β-cells by decreasing apoptosis and protect against diabetes.
  • Fibrosis
    Wnt signaling plays a role in stem cell proliferation and maintenance and is also a contributor to fibrosis in a variety of organs and disease states. Another substantial contributor to fibrosis is TGFβ-1 signaling. Studies support that circulating Klotho directly binds multiple soluble Wnt molecules, including at least Wnt1, Wnt3, Wnt4 and Wnt5a, and resulted in the suppression of Wnt biological activity. Klotho also blocks TGFβ-1 signaling by binding to the TGF-β type-II receptor, blocking TGF-β1 receptor interactions, thereby inhibiting TGF-β1 signaling.  Tissues and organs from klotho-deficient animals showed evidence of increased Wnt and TGFβ-1 signaling.   As an antagonist of fibrotic signaling, Klotho is predicted to be protective against fibrosis.

Klotho Protein Detection- Clinical Research Hurdles

Commercial products for detecting soluble Klotho in biological samples has been riddled with obstacles that have hampered Klotho studies.  While commercially available antibodies against Klotho are available, several reports claim them to be unspecific and cross-react with other proteins. Many of the commercially available antibodies are not well characterized and it is unclear whether commercial assays are detecting the 130 kDa and/or 65 kDa circulating isoforms.  It is reported that novel anti-Klotho antibodies exist, but are currently not commercially available. Some use an immunoprecipitation-immunblot (IP-IB) assay in fresh urine samples, but the technique is labor intensive and difficult to scale up.

In addition to the antibody issues, generation of recombinant Klotho is difficult and the unpredictable quality of commercially produced recombinant proteins may affect the reproducibility of ELISA products.  For example, when one study compared a time-resolved fluorescence immunoassay (TRF) to an ELISA, no correlation was found between the assays and the levels of serum Klotho differed with by a factor 1000.  Furthermore, a related technical challenge is that soluble Klotho appears to be unstable in biological samples.  Standardization of techniques to prevent degradation and optimization of stable sample storage (fresh vs frozen) would eliminate conflicting data for kidney disease researchers, but no consensus protocol has yet been determined.

While improvements in sample preparation still need to be addressed, Fianostics is adding to the toolbox for Klotho researchers with FluoBolt™ alpha-Klotho.  The hope is to increase the options available to researchers so that they can produce accurate results and reliable conclusions.