NGAL Human ELISA kit

NGAL Human is an enzyme-linked immunosorbent assay (ELISA) kidney injury biomarker for distal tubular damage.

Human Neutrophil Gelatinase-Associated Lipocalin (NGAL) is a low molecular weight 25kDa glycoprotein and a member of the lipocalin superfamily, a family of small extracellular proteins that are characterized by the ability to bind small hydrophobic molecules. NGAL can also bind to specific cell surface receptors.

NGAL has been identified as an iron-transporting protein during nephrogenesis, demonstrating a role for NGAL in renal organogenesis. NGAL is widely expressed in a variety of human tissues, including, kidney trachea, lungs, stomach and colon. In the kidney NGAL is thought to be expressed in the collecting duct and distal tubule.

The NGAL EIA provides a method for the quantitative determination of NGAL in human urine, plasma and cell culture supernatant. NGAL is rapidly upregulated and released in response to injury and has demonstrated value as an early biomarker for the detection of acute kidney injury (AKI) onset in various settings.

NGAL has also been investigated as a biomarker of renal injury.

This test kit is intended for use in the quantitative determination of human neutrophil gelatinase-associated lipocalin (Lipocalin-2 or NGAL) in EDTA-plasma.

Range	0.4-100ng/ml
Sensitivity	0.4ng/ml
Incubation time	3.5 hours
Sample volume	100μl
Sample type	Urine (Suggested initial dilution of 1:20) Plasma (Suggested initial dilution of 1:10) Cell culture

Reagents, Preparation and Storage

This test kit must be stored at 2-8°C upon receipt. For the expiration date of the kit refer to the label on the kit box. All components are stable until this expiration date.

Prior to use, allow all reagents to come to room temperature. Reagents from different kit lot numbers should not be combined or interchanged.

1. Anti-NGAL Antibody Coated Microplate (catalog# 30641)
   One microplate with twelve by eight strips (96 wells total) coated with polyclonal anti-human NGAL antibody. The plate is framed and sealed in a foil zipper bag with a desiccant. This reagent should be stored at 2-8°C and is stable until the expiration date on the kit box.
2. HRP Conjugated Anti-NGAL Antibody (catalog# 30650)
   One vial containing 0.6ml HRP-labeled anti-human NGAL antibody in a stabilized protein matrix. This reagent should be stored at 2-8°C and is stable until the expiration date on the kit box.
3. ELISA Wash Concentrate (catalog# 10010)
   One bottle containing 30ml of 30-fold concentrate. Before use the contents must be diluted with 870ml of demineralized water and mixed well. Upon dilution, this yields a working wash solution containing a surfactant in phosphate-buffered saline with a non-azide, non-mercury preservative. The diluted wash solution may be stored at room temperature and is stable until the expiration date on the kit box.
4. ELISA HRP Substrate (catalog# 10020)
   One bottle containing 12ml of tetramethylbenzidine (TMB) with hydrogen peroxide. This reagent should be stored at 2-8°C and is stable until the expiration date on the kit box.
5. ELISA Stop Solution (catalog# 10030)
   One bottle containing 12ml of stop solution. This reagent may be stored at 2-8°C or room temperature and is stable until the expiration date on the kit box.
6. Human NGAL Standards (catalog# 30642 – 30647)
   Six vials containing recombinant human NGAL in a lyophilized bovine serum-based matrix with a non-azide preservative. Refer to the vials for exact concentration of the standard. These standards should be stored at 2-8°C and are stable until the expiration date on the kit box. Refer to assay procedure section for dilution direction.
7. Human NGAL Controls (catalog# 30648 – 30649)
   Two vials containing human NGAL in a lyophilized bovine serum based matrix with a non-azide preservative. Refer to vials for exact concentration range for each control. Both controls should be stored at 2-8°C and are stable until the expiration date on the kit box. Refer to assay procedure section for reconstitution instructions.
8. Tracer Antibody Diluent (catalog# 30651)
   One bottle containing 12ml ready-to-use buffer. It should be used only for tracer antibody dilution according to the assay kit insert procedures. This reagent should be stored at 2-8°C and is stable until the expiration date on the kit box.
9. NGAL Sample Dilution Buffer (catalog# 30654)
   One bottle contains 30ml of 2-fold concentrated buffer matrix with protein stabilizers and preservative. This reagent should be stored at 2-8°C and is stable until the expiration date on the kit box. Before use the concentrated buffer must be diluted with 30ml of demineralized water and mixed well.

Assay Principle

This ELISA kit is designed, developed and produced for the quantitative measurement of human NGAL in EDTA-plasma 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.

Condensed Assay Protocol

1	100μl Standards, controls, and 1:100 diluted patient samples
	↓	Incubate @ RT for 60 min static Wash 5 x
2	100μl Tracer Antibody
	↓	Incubate @ RT for 30 min static Wash 5 x
3	100μl TMB Substrate
	↓	Incubate @ RT for 20 min static
4	100μl Stop Solution
	↓	Immediately
5	Read absorbance at 450/650 or 450/620nm	within 10 minutes

Assay Procedure

Reagent Preparation

1. Prior to use allow all reagents to come to room temperature. Reagents from different kit lot numbers should not be combined or interchanged.
2. ELISA Wash Concentrate (catalog# 10010) must be diluted to working solution prior to use. Please see REAGENTS section for details.
3. Reconstitute assay standards and controls by adding 1.0ml of deminerialized water to each standard and control bottle. Allow the standard and controls to sit undisturbed for 5 minutes, and then mix well by inversions or gentle vortexing. Make sure that all solid is dissolved completely prior to use. These reconstituted standards and controls may be stored at 2-8°C for up to 3 days or below -20°C for long-term storage. Do not exceed 3 freeze-thaw cycles.
4. Concentrated Patient Sample Diluent (catalog# 30654) must be diluted to working solution prior to use. Please see REAGENTS section for details.
5. Each unknown sample needs to be diluted 1:100 using 1x NGAL Sample Dilution Buffer as a sample diluent.
6. Prepare Tracer Antibody working solution by 1:21 fold dilution of the NGAL Tracer Antibody (catalog# 30650) by adding the tracer antibody into the Tracer Antibody Diluent (catalog# 30651). Following is a table that outlines the relationship of strips used and antibody mixture prepared.
   NOTE: the tracer antibody should be prepared just prior to the end of the first incubation cycle.

Dilution Scheme	Tracer Antibody Diluent	Tracer Antibody
1	1ml	50μl
2	2ml	100μl
3	3ml	150μl
4	4ml	200μl
5	5ml	250μl
6	6ml	300μl
7	7ml	350μl
8	8ml	400μl
9	9ml	450μl
10	10ml	500μl
12	11ml	550μl
13	12ml	600μl

7. Test Configuration

ROW	STRIP 1	STRIP 2	STRIP 3	STRIP 4
A	STD 1	STD 5	SAMPLE 1	SAMPLE 5
B	STD 1	STD 5	SAMPLE 1	SAMPLE 5
C	STD 2	STD 6	SAMPLE 2	SAMPLE 6
D	STD 2	STD 6	SAMPLE 2	SAMPLE 6
E	STD 3	C 1	SAMPLE 3
F	STD 3	C 1	SAMPLE 3
G	STD 4	C 2	SAMPLE 4
H	STD 4	C 2	SAMPLE 4

8. Place a sufficient number of Anti-NGAL antibody-coated microwell strips (catalog# 30641) in a holder to run human NGAL standards, controls and unknown samples in duplicates.

Assay Procedure

1. Add 100μl of Standards, Controls and diluted patient samples (diluted beforehand 1:100 with NGAL Sample Dilution Buffer, catalog# 30654) 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 1hr ± 5 minutes.
3. Wash each well 5 times by dispensing 350μl of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
4. Dilute the proper amount of Tracer Antibody for the assay.
5. Add 100μl 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 350μl of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
8. Add 100μl of ELISA HRP Substrate (catalog# 10020) 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 100μl of ELISA Stop Solution (catalog# 10030) into each of the wells. Mix gently.
11. Read the absorbance at 450nm with reference filter at 620nm or 650nm.

Example Data and Standard Curve

A typical absorbance data and the resulting standard curve from this EDTA plasma NGAL ELISA are represented. This curve should not be used in lieu of standard curve generated with each assay.

Well I.D.	OD 450/650nm Absorbance			Results
	Readings	Average	Corrected
Std-1: 0ng/ml	0.017 0.017	0.017	0.000
Std-2: 0.3ng/ml	0.106 0.102	0.104	0.087
Std-3: 1ng/ml	0.256 0.252	0.254	0.237
Std-4: 3ng/ml	0.679 0.694	0.687	0.670
Std-5: 9ng/ml	1.597 1.614	1.605	1.588
Std-6: 27ng/ml	2.810 2.881	2.846	2.829
Control 1	0.491 0.446	0.468	0.451	1.991 ng/ml
Control 2	1.245 1.257	1.251	1.234	6.688 ng/ml

Expected Values
EDTA plasma samples from normal healthy adults ages 20–60 were collected and measured with this ELISA. The recommended normal high cut-off for NGAL concentration by using this ELISA is 500ng/ml with an average level > 106ng/ml (range 48–390ng/ml, SD > 56ng/ml). We strongly recommend for each clinical laboratory to establish its own normal range by measuring EDTA plasma with this ELISA.

Sample Preparation

Urine
Collect urine using normal aseptic techniques. Centrifuge the urine to remove debris (1500xg at 4°C for 15min). Transfer urine to a fresh polypropylene tube.

Plasma
Separate plasma from whole blood within 20 min of sampling (1500xg at 4°C for 15min). Remove plasma and transfer to fresh polypropylene tube. Recentrifuge the transferred plasma in order to avoid every contamination with white blood cells (1500xg at 4°C for 15min). Most reliable results are obtained if EDTA plasma is used.

Storage
Store samples below -20°C, preferably at -70°C in polypropylene tubes. Use samples within 24 hours after thawing. Avoid multiple freeze-thaw cycles. Do not use hemolyzed, hyperlipemic, heat-treated or contaminated samples.

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. NGAL is involved in innate immunity by sequestrating iron that in turn limits bacterial growth. It is expressed in neutrophils and in low levels in the kidney, prostate, and epithelia of the respiratory and alimentary tracts.