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NGAL (Stool) ELISA Assay Kit

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$440.00
SKU:
NGL35-K01

 Product Description

Package insert view pdf

 

 

NGAL (Stool) ELISA Assay Kit:
For Research Use Only
Size:  1x96 wells
Sensitivity:  0.04 µg/g
Dynamic Range:  1.2 - 97.2 µg/g
Incubation Time:  2 hours
Sample Type:  Stool
Sample Size: 50 mg

Controls Included

NOTE:
NGAL ng/mL X 3.6 = NGAL µg/g stool
NGAL µg/g stool X 0.278 = NGAL ng/mL

Intended Use
The Eagle Biosciences Human NGAL ELISA Assay Kit is intended for use in the quantitative determination of human neutrophil gelatinase-associated lipocalin (Lipocalin-2 or NGAL) in feces. NGAL is extremely stable in feces.  The Eagle Biosciences Human human neutrophil gelatinase-associated lipocalin (Lipocalin-2 or NGAL) ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.
 
Assay Background
NGAL or neutrophil gelatinase-associated lipocalin also known as Lipocalin-2 (LCN2) or oncogene 24p3 is a protein, which in humans is encoded by the LCN2 gene.[1][2][3] NGAL is involved in innate immunity by sequestrating iron that in turn limits bacterial growth.[4] It is expressed in neutrophils and in low levels in the kidney, prostate, and epithelia of the respiratory and alimentary tracts.[3][5]Studies have shown that NGAL is an early biomarker for ischaemic renal injury after cardiopulmonary bypass.

Assay Principle
This NGAL ELISA Assay Kit is designed, developed and produced for the quantitative measurement of human NGAL in stool samples. The assay utilizes the “sandwich” technique with selected antibodies that bind to various epitopes of NGAL.  Assay standards, controls and patient samples are added directly to wells of a microtiter plate that is coated with antibody to human NGAL and incubated at room temperature for one hour. The plate is then washed and horseradish peroxidase (HRP) conjugated anti NGAL is added to each well. After an additional incubation period, a “sandwich” of solid-phase polyclonal antibody - human NGAL – HRP conjugated antibody” is formed. The unbound antibodies and buffer matrix are removed in the subsequent washing step.

For the detection of this immunocomplex, the well is then incubated with a substrate solution in a timed reaction, which is terminated with an acidic reagent (i.e. ELISA stop solution). The absorbance is then measured in a spectrophotometric microplate reader. The enzymatic activity of the immunocomplex bound to the wall of each microtiter well is directly proportional to the amount of human NGAL in the test sample. A standard curve is generated by plotting the absorbance versus the respective human NGAL concentration for each standard on a point-to-point or 4-parameter curve fitting. The concentration of human NGAL in test samples is determined directly from this standard curve.

The validation data of this test were generated by using Fecal Sample Collection Tube (Eagle Biosciences Cat.#  CAL35-C50).  Each kit contains 50 tubes. A different fecal NGAL test result may be obtained by using a different type of fecal sample collection tube.

  1. Add 100 µl of Standards, Controls and diluted patient samples (diluted beforehand with NGAL Sample Dilution Buffer) into the designated microwells.
  2. Seal the plate wells securely, cover with foil or other material to protect from light. Incubate the plate static, at room temperature for 1 hr. ± 5 minutes.
  3. Just prior to the end of the incubation time, dilute the proper amount of Tracer Antibody for the assay.
  4. Wash each well 5 times by dispensing 350 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
  5. Add 100 µL of the above Tracer Antibody to each well.
  6. Seal the plate wells securely, cover with foil or other material to protect from light. Incubate the plate static, at room temperature for 30 minutes ± 5 minutes.
  7. Wash each well 5 times by dispensing 350 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
  8. Add 100 µL of ELISA HRP Substrate into each of the wells.
  9. Cover the plate with aluminum foil or other material to avoid exposure to light. Incubate the plate static, at room temperature for 20 minutes.
  10. Immediately add 100 µL of ELISA Stop Solution into each of the wells. Mix gently.
  11. Read the absorbance at 450 nm with reference filter at 620 nm or 650 nm.

Typical Standard Curve

fecal-ngal.png


REFERENCES

  • Kjeldsen L, Johnsen AH, Sengeløv H, Borregaard N (May 1993). "Isolation and primary structure of NGAL, a novel protein associated with human neutrophil gelatinase". J. Biol. Chem. 268 (14): 10425–32.
  • Chan P, Simon-Chazottes D, Mattei MG, Guenet JL, Salier JP (September 1994). "Comparative mapping of lipocalin genes in human and mouse: the four genes for complement C8 gamma chain, prostaglandin-D-synthase, oncogene-24p3, and progestagen-associated endometrial protein map to HSA9 and MMU2". Genomics 23(1): 145–50.
  • Cowland JB, Borregaard N (October 1997). "Molecular characterization and pattern of tissue expression of the gene for neutrophil gelatinase-associated lipocalin from humans". Genomics 45 (1): 17–23.
  • Yang J, Goetz D, Li JY, Wang W, Mori K, Setlik D, Du T, Erdjument-Bromage H, Tempst P, Strong R, Barasch J (November 2002). "An iron delivery pathway mediated by a lipocalin". Mol. Cell 10 (5): 1045–56.
  • Friedl A, Stoesz SP, Buckley P, Gould MN (July 1999). "Neutrophil gelatinase-associated lipocalin in normal and neoplastic human tissues. Cell type-specific pattern of expression". Histochem. J. 31 (7): 433–41.

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Nashua, NH 03063
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