Standard curve
Example of Human LIF standard curve in Assay Diluent TA2.
Monkey & Human LIF OneStep ELISA Kit
Monkey & Human LIF OneStep ELISA Kit
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Product Details
Product Details
Product Specification
| Antigen | LIF |
| Immunogen | Recombinant Protein |
| Antibody Type | Recombinant mAb |
| Reactivity | Hu, Mk |
| Purification | Protein A |
| Stability & Storage | 12 months from date of receipt / reconstitution, 2 to 8°C as supplied. |
Kit
| Precision | Intra-assay: 2.3% Inter-assay: 2.9% |
| Sample type | Cell culture supernatant; Serum; Plasma |
| Assay type | Sandwich (quantitative) |
| Sensitivity | 3.87 pg/mL |
| Range | 31.3 pg/mL – 2000 pg/mL |
| Recovery | Cell culture supernatant (1640): 112% Cell culture supernatant (DMEM): 111% Human Serum: 105% Human EDTA Plasma: 88% Human Heparin Plasma: 102% Monkey Serum: 103% Monkey EDTA Plasma: 104% Monkey Citrate Plasma: 99% |
| Assay time | 60 minutes |
Background
Leukemia inhibitory factor (LIF) is a pleiotropic cytokine belonging to the IL-6 family that plays essential roles in cell differentiation, proliferation, and embryo implantation. Human LIF is synthesized as a 202-amino-acid precursor protein, and recombinant human LIF is typically produced in E. coli as a monomeric, alpha-helical four-helix bundle protein. It is widely used to maintain mouse embryonic stem cells and induced pluripotent stem cells in an undifferentiated, pluripotent state. For monkey LIF, the rhesus macaque (Macaca mulatta) and cynomolgus macaque (Macaca fascicularis) orthologs have been annotated, with the crab-eating macaque sequence encoding a 202-amino-acid protein of approximately 22 kDa. Functional studies in primate models have shown that recombinant human LIF is biologically active in cynomolgus monkeys, where administration induces weight loss and thrombocytosis. In the monkey endometrium, LIF expression is upregulated during the peri-implantation period, and its downregulation is associated with impaired endometrial receptivity. Recombinant human LIF also modulates monkey endometrial stromal cell behavior in vitro through the JAK-STAT signaling pathway. Given the high sequence conservation and cross-species bioactivity between human and monkey LIF, both proteins serve as valuable tools for studying reproductive biology, stem cell pluripotency, and hematopoietic regulation in translational primate models.
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ELISA
Spike Recovery
The recovery of LIF was evaluated in activated samples spiked with concentrations spanning the entire assay range.
Spike-and-dilution Linearity
To evaluate assay linearity, four samples were spiked with high levels of LIF in different matrices and serially diluted with the corresponding Calibrator Diluent to fall within the assay's dynamic range.
Cross-reactivity
The cross-reactivity was checked by testing Rat LIF, Human LIF, Mouse LIF, and Bovine LIF in the same assay system. Each protein was tested with serial dilutions, and the dose-response curves were compared. A noticeable cross-reactivity was seen when Bovine LIF was tested in the Human LIF assay. No significant cross-reactivity was observed when other LIF was tested in the Human LIF assay, as the signals remained within the background range of the assay.
Determination of Minimum Detectable Dose (MDD)
The MDD was determined using three independent lots of assay components. For each lot, 19 replicate measurements of the diluent (zero calibrator) were performed. The mean (AVERAGE) and standard deviation (STDEV) of the 19 replicates were calculated. The MDD for each lot was then calculated according to the following formula:
MDD = 2 × STDEV + AVERAGE
HOOK Effect Threshold
The upper limit of the HOOK effect was established at 100× the highest calibrator concentration (equivalent to a 2-log10 increase). No HOOK effect was observed below this threshold, confirming that samples within this concentration range yield reliable quantitative results without signal depression.
Intra-Assay Precision (Precision within an assay)
Three samples of known concentration were tested fifteen times on one plate to assess intra-assay precision.
Inter-Assay Precision (Precision between assays)
Three samples of known concentration were tested in separate assays to assess inter-assay precision. Assays were performed with at least three lots of components.
Dilution Linearity
The concentrations of Monkey LIF were measured and interpolated from the target standard curves and corrected for sample dilution.
The leftmost bar in the graph represents neat biological samples. "Undiluted" was defined as the neat biological sample (100%). The subsequent bars represent two-fold serial dilutions of this reference. The mean target concentration was determined to be 902.9 pg/mL in the stimulated Monkey PBMC supernatant.
Dilution Linearity
The concentrations of Human LIF were measured and interpolated from the target standard curves and corrected for sample dilution.
The leftmost bar in the graph represents neat biological samples. "Undiluted" was defined as the neat biological sample (100%). The subsequent bars represent two-fold serial dilutions of this reference. The mean target concentration was determined to be 647 pg/mL in the stimulated Human PBMC supernatant.
Serum levels in healthy individuals
Duplicate measurements were performed on serum samples obtained from eight individual healthy donors. Data are reported as dilution factor-corrected interpolated. The resultant mean LIF concentration was 3.3 pg/mL, with a distribution spanning 0 to 26.41 pg/mL across the donor cohort.
Protocol Diagram
