CMIC Human L-FABP (FABP1) ELISA

High Sensitivity Human L-FABP ELISA Kit is for the quantitative determination of human L-FABP in urine and can be used for clinical research applications with L-FABP as a kidney disease biomarker.

L-FABP is a low molecular weight soluble protein (14kDa) expressed in the proximal tubule of the kidney. L-FABP plays important physiological roles during energy and lipid metabolism in the proximal tubule, which functions to reabsorb free fatty acids bound to albumin. L-FABP is excreted into urine in response to initial stress caused by proteinuria and microcirculatory ischemic stress, making it a useful biomarker in kidney disease research of tubular dysfunction. For research use only.

This product is a sandwich type ELISA kit, manufactured by the CMIC Holdings Group in Japan. This kit uses monoclonal antibodies that recognize human L-FABP with high sensitivity. Moreover, the L-FABP Antibody Coated Microplate can be separated for the measurement of a small number of specimen materials.

The Human Urinary L-FABP ELISA Kit can be used in research applications for:

• Early detection of ischemic events on acute kidney injury (AKI)
• Disease research of chronic kidney diseases (CKD)
• Early detection of diabetic nephropathy
• Analysis of renal tubular damage during septic shock

Quick Facts

• Store Temperature: 2-8°C
• Method: Enzyme-Linked-Immuno-Sorbent Assay of 2-step sandwich method
• Sample: Human urine
• Assay time: 120min
• Shelf life: 24 months
• Measurable range: 0.3- 60 ng/ml

L-FABP Antibody Coated Microplate	96 well x 1
Pretreatment Microplate	96 well x 1
Pretreatment Solution	12ml x 1
Assay Buffer	12ml x 1
The 2nd Ab-POD Conjugate	12ml x 1
Substrate Solution	12ml x 1
Wash Agent (x 40 concentrate)	50ml x 1
Stop Solution	12ml x 1
Standard Diluent (0ng/ml)	2.5ml x 1
L-FABP Standard (400ng/ml)	0.5ml x 1

Assay Principle

ELISA (Enzyme-Linked-Immuno-Sorbent Assay) of 2-step sandwich method is used for this kit. L-FABP Standard or urine samples are pretreated with Pretreatment Solution, and poured into L-FABP Antibody Coated Microplate on which Assay Buffer is placed and incubated. During this incubation process, L-FABP in the reacting solution binds to the immobilized antibody. L-FABP Antibody Coated Microplate is washed following by the L-FABP binding reaction process. As the second antibody, The 2nd Ab-POD Conjugate is added after washing procedure to make L-FABP antigen be sandwiched between immobilized antibody and conjugate antibody. The plate with sandwiched L-FABP antigen is washed again and added with Substrate for enzyme reaction process. Changes of color of samples appear according to quantity of L-FABP antigen. Microplate reader records optical density to draw a calibration curve of L-FABP concentration.

Measurement Range

0.3- 60 ng/ml

Operation manual

1. Required Instruments and Equipment
   • Micropipette: Adjustable to 20µl, 50µl
   • Multichannel micropipette: Adjustable to 50µl, 100µl
   • Graduated cylinder: 2,000ml
   • Plate mixer
   • 96 well microplate reader: Wave length of 450nm (over 610nm)
   • Plate Seal (Attached to each kit)
2. Preparation of wash solution
   Add distilled water to Wash Agent (x 40 concentrate) and prepare 2,000ml of wash solution.
3. Measuring operation method
   Make sure that all reagents are at room temperature approximately 30 minutes prior to use and tilt and mix each regent itself gently few times to check no quality would change in all reagents. Measure diluted L-FABP Standard while measuring test samples to set standard curve.

(1) Preparation of L-FABP Standards

1. As shown in Fig.1, use the first column (A1~H1wells) of “2.Pretreatment Microplate” for the preparation.
2. Add by 50µl of “9.Standard Diluent (0ng/ml)”to each well individually from B1 to H1 in “2.Pretreatment Microplate”.
3. Add 50µlof “10.L-FABP Standard (400ng/ml)”and 50µl of “9.Standard Diluent (0ng/ml)” into A1well (Conc. 200 ng/ml), and mix A1well gently (ten times pipetting).
4. Take 50µl of the mixed solution from A1well and add to B1well and mix them gently.
5. Continue to perform this doubling dilution procedure from B1well to G1well in the same manner one by one and take out 50µl of the solution from G1well.

(2) Pretreatment

1. After the preparation of L-FABP Standards, add by 50µl of sample specimens into the other wells individually (A2, B2,…) in “2.Pretreatment Microplate”.
2. Add by 50µl of “3.Pretreatment Solution” individually to all wells containing L-FABP Standards and the samples specimens. Seal the plate and stir it for more than 5 minutes with a plate mixer

(3) Set the strips of “1. L-FABP Antibody Coated Microplate” (two strips for standard + strips for specimens) from left side (1,2…) in the plate holder, and add by 100µl of “4.Assay Buffer” in each well.

(4) Pipette the standard solution from each well in the first column in “2. Pretreatment Microplate” and add the standard solution (20µl/well) to respective two wells in the first two strips in“1. L-FABP Antibody Coated Microplate”.

(5) Pipette by 20µl of the pretreated sample specimen from “2. Pretreatment Microplate” and add the solution to respective wells after third strips of “1. L-FABP Antibody Coated Microplate”.

(6) Seal “1. L-FABP Antibody Coated Microplate” and stir it for 5 minutes with a plate mixer, and then incubate “1. L-FABP Antibody Coated Microplate” for 55 minutes at room temperature (20~28℃).

(7) After the incubation, throw away the liquid from “1.L-FABP Antibody Coated Microplate”.

(8) Wash each well in “1. L-FABP Antibody Coated Microplate” with wash solution (350µl/well). Then, fill each well with wash solution and remove wash agent completely from “1. L-FABP Antibody Coated Microplate” by snapping it. This procedure should be repeated 3 times. Then, remove the remaining liquid from all wells completely by snapping “1. L-FABP Antibody Coated Microplate” onto paper towels. In case of using a plate washer, wash each well with 350µl of wash solution 3 times.

(9) Pipette by 100µl of “5.The 2nd Ab-POD Conjugate” into the wells of test samples, standards involving zero concentration.

(10) Seal the plate and stir it for 5 minutes with a plate mixer, and incubate the plate for 25 minutes at room temperature (20~28℃).

(11) After incubation of step (10), remove the liquid, and wash the plate 3 times in the same manner as step (8).

(12) Pipette by 100µl of “6.Substrate Solution” into the wells.

(13) Seal the plate and stir it for 5 minutes with a plate mixer, and incubate the plate for 25 minutes at room temperature (20~28℃) in the dark.

(14) Pipette 100µl of “8.Stop Solution” into the wells. Mix the liquid by tapping the side of the plate.

(15) Remove any dirt or drop of water on the bottom of the plate and check there is no bubble on the surface of the liquid. Set 96 well microplate reader and read absorbance which is confirmed with the wavelength (Dominant wavelength: 450nm, Secondary wavelength: over 610nm).

(16) Plot standard curve based on the absorbance of “10.L-FABP Standard” and calculate the amount of L-FABP in the specimen.

Fig.2 Operation Protocol

	Test Sample	Standard	Zero (0) concentration
Pretreatment	Test Sample 50μl	L-FABP Standard 50μl	Standard Diluent (0ng/mL) 50μl
Pretreatment reagent 50μl
Mix for more than 5 minutes by Plate Mixer after sealing plate
Assay Buffer	100μl	100μl	100μl
Pretreated samples	20μl	20μl	20μl
Mix for 5 minutes by Plate Mixer after sealing plate
Incubate for 55 minutes at room temperature
Wash 3 times
Labeled antibody	100μl	100μl	100μl
Mix for 5 minutes by Plate mixer and after sealing plate
Incubate for 25 minutes at room temperature
Wash 3 times
Substrate Solution	100μl	100μl	100μl
Mix for 5 minutes by Plate mixer and after sealing plate
Incubate for 25 minutes at room temperature with light shielding
Stop Solution	100μl	100μl	100μl
Tap the plate for mixing and measure absorbance at the wavelengths (Dominant wavelength: 450nm, Secondary wavelength: over 610nm) within 30 minutes after adding of Stop Solution.

Calculation & Standard Curve

Calculation of Test Result

1. Subtract the absorbance of zero concentration from all data, including standards and unknown samples before plotting to calculate specific Optical Density (Net O.D.) of respective wells.
2. Plot the Net O.D. of L-FABP standard in vertical axis and L-FABP concentration in horizontal axis on log-log graph paper. Draw the best smooth curve through these points to construct the standard curve. Read the concentration for unknown samples from the standard curve.

Example of Standard Curve

L-FABP Conc. (ng/ml)	O.D. (450nm)
60	2.508
30	1.278
15	0.618
7.5	0.316
3.75	0.170
1.88	0.101
0.94	0.066
0 (Blank)	0.031

See Graph
* The standard curve above is shown as an example. Set up standard curve for each assay.

Performance Characteristics

1. Sensitivity
   The minimal sensitivity of the assay is 0.3ng/ml.
2. Specificity

   Compound	Cross Reactivity
   L-FABP	100.0%
   I-FABP	≦0.1%

3. Repeatability
   The CV value is not more than 15%, in case of 8 times simultaneously measurement of the same specimen.

   Measurement value (ng/ml)	SD	CV (%)	n
   19.7	0.8	4.1	8
   10.7	0.4	3.7	8
   8.2	0.3	3.7	8

4. Dilution test

   Sample	Dilution ratio (×)		Measurement value (ng/ml)
   Urine A	1/2	0.5	55.9
   	1/4	0.25	28.7
   	1/8	0.125	14.4
   	1/16	0.0625	7.6
   	1/32	0.0313	4.1
   Urine B	1/2	0.5	43.7
   	1/4	0.25	22.3
   	1/8	0.125	11.0
   	1/16	0.0625	6.0
   	1/32	0.0313	3.0

Background

Protective functions of urinary excretion of L-FABP within renal proximal tubules

Free Fatty Acids (FFAs) are bound to serum albumin, filtered through glomeruli, and reabsorbed into the proximal tubule along with albumin. FFAs up-regulate L-FABP gene expression. L-FABP, a carrier protein of 14kDa, is expressed in the proximal tubule and plays a role in the intracellular transport of FFAs to mitochondria and/or to peroxisomes for metabolism.

Reactive oxygen generated due to peritubular ischemia/reperfusion injury change free fatty acids to fatty acid peroxides (lipid peroxides or lipoperoxides), which are highly toxic to cells. Lipoperoxides accumulate in proximal tubules during renal ischemia/reperfusion, causing damage to the tubules.

L-FABP binds lipoperoxides for relocation towards the lumen and excretion from the proximal tubules into urine. Thus, it is thought that L-FABP is “renoprotective”—it works to protect the kidneys.

This mechanism further supports urinary L-FABP as a biomarker of renal tubule damage. Elevated levels of urinary L-FABP are associated with and can indicate early stages of acute kidney injury (AKI), chronic kidney disease (CKD), diabetic nephropathy, and septic shock.