K-ASSAY 

KAMIYA BIOMEDICAL COMPANY

KAMIYA BIOMEDICAL COMPANY

Rat Liver Fatty Acid BindingProtein (L-FABP) ELISA
For the quantitative determination ofL-FABP in rat biological samples

Cat. No. KT-627
For research use only.

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KAMIYA BIOMEDICAL COMPANYPRODUCT INFORMATION

Rat Liver Fatty Acid Binding Protein (L-FABP) ELISA
Cat. No. KT-627
INTENDED USE
The Rat L-FABP ELISA is a highly sensitive two-site enzyme-linked immunoassay (ELISA) for the quantitative
determination of L-FABP in rat biological samples. For research use only.

PRINCIPLE
The principle of the double antibody sandwich ELISA is represented in Figure 1. In this assay the L-FABP present in thesample reacts with the anti-L-FABP antibodies which have been adsorbed to the surface of polystyrene microtiter wells.
After the removal of unbound proteins by washing, anti-L-FABP antibodies conjugated with horseradish peroxidase (HRP)
are added. This HRP-conjugated antibody forms a complex with the previously bound L-FABP. Following anotherwashing step, the enzyme bound to the immunosorbent is assayed by the addition of a chromogenic substrate, 3,3’,5,5’tetramethylbenzidine (TMB). The quantity of bound enzyme is proportional to the concentration of L-FABP in the sample
tested; thus, the absorbance, at 450 nm, is a measure of the concentration of L-FABP in the test sample. The quantity ofL-FABP in the test sample can be interpolated from the calibration curve constructed from the calibrators, and corrected
for sample dilution.

Figure 1.

Anti-L-FABP Antibodies Bound To Solid Phase│
Calibrators and Samples Added│L-FABP * Anti-L-FABP Complexes Formed│
Unbound Sample Proteins Removed│
Anti-L-FABP-HRP Conjugate Added│
Anti-L-FABP-HRP * L-FABP * Anti-L-FABP
Complexes Formed│
Unbound Anti-L-FABP-HRP Removed│
TMB Substrate Added│
Determine Bound Enzyme Activity

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KAMIYA BIOMEDICAL COMPANYCOMPONENTS
1. Diluent Concentrate
One bottle containing 60 mL of a 1X concentrated diluent running buffer.
2. Wash Solution Concentrate
One bottle containing 50 mL of a 20X concentrated wash solution.
3. Enzyme-Antibody Conjugate Concentrate
One vial containing 150 µL of a 100X concentrated affinity-purified anti-rat L-FABP antibody conjugated with HRP in astabilizing buffer.
4. TMB Substrate Solution
One vial containing 12 mL of TMB and hydrogen peroxide in citric acid buffer at pH 3.3.
5. Stop Solution
One vial containing 12 mL of 0.3 M sulfuric acid. WARNING: Avoid contact with skin.
6. Microtiter Plate
Twelve removable eight-well micro strips in well holder frame.
FABP.

Each well is coated with affinity-purified anti-rat L-

7. Rat L-FABP Calibrator
One vial containing Rat L-FABP Calibrator.

MATERIALS REQUIRED BUT NOT PROVIDED••••••••

Precision pipettes (2 µL to 200 µL) for making and dispensing dilutions
Test tubes
Microplate washer/aspirator
Distilled or de-ionized H2O
Microplate reader
Assorted glassware for the preparation of reagents and buffer solutions
Timer
Vortex mixer

PRECAUTIONS
1.
2.
3.
4.
5.
6.

Read the instructions carefully before beginning the assay.
This kit is for research use only.
Great care has been taken to ensure the quality and reliability of this product. However, it is possible that in certain
cases, unusual results may be obtained due to high levels of interfering factors.
No additives or preservatives are necessary to maintain the integrity of the specimen. Avoid azide contamination.
Azide and thimerosal at concentrations higher than 0.1% inhibit the enzyme reaction.
Other precautions: Do not interchange kit components from different lots.
 Do not use kit components beyond the expiration date.
 Protect reagents from direct sunlight.
 Do not pipette by mouth.
 Do not eat, drink, smoke or apply cosmetics where reagents are used.
 Avoid all contact with the reagents by using gloves.
 Stop solution contains diluted sulfuric acid. Irritation to eyes and skin is possible. Flush with water aftercontact.

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REAGENT PREPARATION
1. Diluent Concentrate
The Diluent solution supplied is a 1X concentrate. No dilution is required.
2. Wash Solution Concentrate
The Wash Solution supplied is a 20X concentrate and must be diluted 1:20 with distilled or de-ionized water. Crystal
formation in the concentrate is not uncommon when storage temperatures are low. Warming of the concentrate to30-35°C before dilution can dissolve crystals.
3. Enzyme-Antibody Conjugate Concentrate
Calculate the required amount of working conjugate solution for each microtiter plate test strip by adding 10 µL
Enzyme-Antibody Conjugate to 990 µL of 1X Diluent for each test strip to be used for testing. Mix uniformly, but gently.
Avoid foaming.
4. TMB Substrate Solution
Ready to use as supplied.
5. Stop Solution
Ready to use as supplied.
6. Microtiter Plate
Ready to use as supplied. Unseal Microtiter Pouch and remove plate from pouch. Remove all strips and wells that will
not be used in the assay and place back in pouch and re-seal along with desiccant.
7. Rat L-FABP Calibrator
The Rat L-FABP Calibrator should be aliquoted and stored frozen. It is at a concentration of 23.08 µg/mL and needs tobe diluted in 1X diluent according to the chart below for each run. Rat L-FABP Calibrators need to be prepared
immediately prior to use (see the following chart). Mix well between each step. Avoid foaming.
Calibrator
76543210Concentration(ng/mL)
2001005025
12.5
6.25
3.125
0Calibrator Volume added
to 1X Diluent
5 µL Rat L-FABP Calibrator
250 µL Calibrator 7250 µL Calibrator 6250 µL Calibrator 5250 µL Calibrator 4250 µL Calibrator 3250 µL Calibrator 2

Volume of 1X Diluent
572 µL250 µL250 µL250 µL250 µL250 µL250 µL500 µLSTORAGE AND STABILITY
1. Complete Kit
The expiration date for the kit is stated on the outer label. The recommended storage temperature is 4°C. Note: Seelong term storage recommendations below for the Rat L-FABP Calibrator.
2. Diluent
The 1X Diluent Concentrate is stable until the expiration date. The solution should be stored at 4°C.
3. Wash Solution
The 20X Wash Solution Concentrate is stable until the expiration date. The 1X working solution is stable for at least
one week from the date of preparation. Both solutions can be stored at room temperature (RT, 16-25°C) or at 4°C.
4. Enzyme-Antibody Conjugate
Undiluted anti-L-FABP-HRP conjugate should be stored at 4°C and diluted immediately prior to use. The workingconjugate solution is stable for up to 8 hours.
5. TMB Substrate Solution
The TMB Substrate Solution should be stored at 4°C and is stable until the expiration date.
6. Stop Solution
The Stop Solution should be stored at 4°C and is stable until the expiration date.
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7. Microtiter Plate
Anti-rat L-FABP coated wells are stable until the expiration date and should be stored at 4°C in the sealed foil pouchwith a desiccant pack.
8. Rat L-FABP Calibrator
Long term storage: Upon receipt, aliquot the calibrator and store frozen. They will be stable until expiration date. Shortterm Storage: The calibrator is stable for up to 14 days at 4°C. The working calibrator solutions should be prepared
immediately prior to use.

INDICATIONS OF INSTABILITY
If the test is performing correctly, the results observed with the calibrator solutions should be within 20% of the expectedvalues.

SPECIMEN COLLECTION AND HANDLING
Blood should be collected by venipuncture and the serum separated from the cells, after clot formation, by centrifugation.
For plasma samples, blood should be collected into a container with an anticoagulant and then centrifuged. Care should
be taken to minimize hemolysis, excessive hemolysis can impact your results. Assay immediately or aliquot and store
samples at -20°C. Avoid repeated freezing/thawing.
For any sample that might contain pathogens, care must be taken to prevent contact with open wounds. No additives orpreservatives are necessary to maintain the integrity of the specimen. Avoid azide contamination.

ASSAY PROTOCOL
Dilution of Samples
Due to the high-sensitive nature of the assay, each sample should be diluted before use for a normal assay. For a single
step determination a dilution of 1:5 is appropriate for most serum/plasma samples. For absolute quantification of samples
that yield results outside the range of the calibration curve, a lesser or greater dilution might be required. If unsure ofsample level, a serial dilution with one or two representative samples before running the entire plate is highly
recommended.
To prepare a 1:5 dilution of sample, transfer 50 µL of sample to 200 µL of 1X Diluent. This gives you a 1:5 dilution. Mix
thoroughly.

Procedure
1. Bring all reagents to RT before use.
2. Pipette 100 µL of
Calibrator 0 (0.0 ng/mL) in duplicate
Calibrator 1 (3.125 ng/mL) in duplicate
Calibrator 2 (6.25 ng/mL) in duplicate
Calibrator 3 (12.5 ng/mL) in duplicate
Calibrator 4 (25 ng/mL) in duplicate
Calibrator 5 (50 ng/mL) in duplicate
Calibrator 6 (100 ng/mL) in duplicate
Calibrator 7 (200 ng/mL) in duplicate
3. Pipette 100 µL of diluted sample (in duplicate) into pre designated wells.
4. Incubate the Microtiter Plate at 22°C (RT) for sixty (60 ± 2) minutes. Keep plate covered and level during incubation.
5. Following incubation, aspirate the contents of the wells.
6. Completely fill each well with appropriately diluted Wash Solution and aspirate. Repeat three times, for a total of fourwashes. If washing manually: completely fill wells with wash buffer, invert the plate then pour/shake out the contents
in a waste container. Follow this by sharply striking the wells on absorbent paper to remove residual buffer. Repeat
three times for a total of four washes.

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7. Pipette 100 µL of appropriately diluted Enzyme-Antibody Conjugate to each well. Incubate at 22°C (RT) for ten (10 ±2) minutes. Keep the plate covered in the dark and level during incubation.
8. Wash and blot the wells as described in Steps 5 and 6.
9. Pipette 100 µL of TMB Substrate Solution into each well.
10. Incubate in the dark at RT for precisely ten (10) minutes.
11. After ten minutes, add 100 µL of Stop Solution to each well.
12. Determine the absorbance at 450 nm of the contents of each well. Zero the plate reader to air.
The absorbance of the final reaction mixture can be measured up to 2 hours after the addition of the Stop Solution.
However, good laboratory practice dictates that the measurement be made as soon as possible.

RESULTS
1. Subtract the average background value from the test values for each sample.
2. Using the results observed for the calibrators construct a calibration curve. The appropriate curve fit is that of a fourparameter logistics curve. A second order polynomial (quadratic) or other curve fits may also be used.
3. Interpolate test sample values from calibration curve. Correct for sample dilution factor to arrive at L-FABP
concentration in original sample.

QUALITY CONTROLIn accord with good laboratory practice, the assays for specific L-FABP require meticulous quality control. Eachlaboratory should use routine quality control procedures to establish inter- and intra-assay precision and performance
characteristics.

LIMITATION OF THE PROCEDURE
1. Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete
understanding of the information contained in the package insert instructions and with adherence to good laboratory
practice.
2. Factors that might affect the performance of the assay include proper instrument function, cleanliness of glassware,
quality of distilled or de-ionized water, and accuracy of reagent and sample pipettings, washing technique, incubation
time or temperature.

FOR RESEARCH USE ONLY

KAMIYA BIOMEDICAL COMPANY
12779 Gateway Drive, Seattle, WA 98168
Tel: (206) 575-8068
Fax: (206) 575-8094
Email: LifeScience@k-assay.com
www.k-assay.com

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