Regulatory status: For research use only, not for use in diagnostic procedures.

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EDTA plasma, amniotic fluid, cell culture supernatants
Species Reactivity
Intended Use
The sHLA-G ELISA is a sandwich enzyme immunoassay for the quantitative measurement of soluble forms of human leukocyte antigen-G (sHLA-G).
Store the complete kit at 2-8°C. Under these conditions, the kit is stable until the expiration date (see label on the box).
Performance Characteristics
1. The total assay time is about 20 hours.
2. The kit measures shedded HLA-G1 and HLA-G5 in EDTA plasma, amniotic fluid, or cell culture supernatant.
3. Calibrator is human native protein.
4. Assay format is 96 wells.
5. Components of the kit are provided ready to use, concentrated or lyophilized.

Typical analytical data of sHLA-G ELISA are presented in this chapter

Limit of Detection (LOD), defined as concentration of analyte giving absorbance higher than mean absorbance of blank*plus three standard deviations of the absorbance of blank: Ablank+ 3xSDblank, is calculated from the real sHLA-G values in wells and is 0.6 Units/ml.
*Dilution Buffer is pipetted into blank wells.

Limit of assay
Samples with absorbances exceeding the absorbance of the highest standard should be measured again with higher dilution. The final concentration of samples calculated from the standard curve must be multiplied by the respectivedilution factor.

Presented results are multiplied by respective dilution factor

Intra-assay (Within-Run) (n=8) for EDTA plasma samples diluted using the Dilution Buffer 2:

Inter-assay (Run-to-Run) (n=5) for EDTA plasma samples diluted using the Dilution Buffer 2:

Spiking Recovery
EDTA plasma samples were spiked with different amounts of sHLA-G and assayed (Dilution Buffer 2 was used as a diluent).

EDTA plasma samples were serially diluted with Dilution Buffer 2 and assayed.

Stability of samples stored at 2-8°C
A significant decline in concentration of sHLA-G was observed in EDTA plasma after 7 days when stored at 2-8°C. Therefore, we strongly recommend to store the samples at -20°C, or preferably at -70°C for long-term storage.

Effect of Freezing/Thawing
Significant changes in concentration of sHLA-G wereobserved in EDTA plasma samples after repeated freeze/thaw cycles. Therefore, it is strongly recommended to avoid unnecessary repeated freezing/thawing of the samples.

Reference range
It is recommended that each laboratory include its own panel of control samples in the assay. Each laboratory should establish its own normal andpathological reference ranges for sHLA-G levels with the assay.
General Description
Human leukocyte antigen-G (HLA-G) differs from the other MHC class I genes by its low polymorphism and alternative splicing that generates seven HLA-G proteins, whose tissuedistribution is restricted to normal fetal and adult tissues that display a tolerogeneic function toward both innate and acquired immune cells. Soluble HLA-G is an immunosuppressive molecule inducing apoptosis of activated CD8(+) T cells and down-modulating CD4(+) T cell proliferation.
Recently, using specific ELISA to analyse the presence of sHLA-G molecules in culture supernatants of early embryos obtained by in vitro fertilization (IVF) before transfer, several reports demonstrated that positive embryo implantations occurred with embryos secreting sHLA-G molecules. These breakthrough results indicate that sHLA-G ELISA can be a useful biochemical assay in addition to embryo morphology in embryo selection for transfer in IVF treatment if there are other embryos with the same morphology.
Furthermore, monitoring of sHLA-G in amniotic fluidand plasma of pregnant women may have an important prognostic value to recognize pathological situations.
Other interesting observations suggest that HLA-G molecules seem to be directly involved in transplant acceptation, and their analysis should be taken into consideration when monitoring transplant-patients status. In addition, soluble HLA-G plasma levels are increased in lymphoproliferative disorders or in patients suffering from malignant melanoma, glioma, breast and ovarian cancer.


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