1

Human Kidney Injury Molecule 1 ELISA
Cat. No.: BA1001
Enzyme Immunoassay for the quantitative determination Kidney Injury
Molecule 1 (KIM-1) in human urine and serum.
The urinary kidney injury molecule 1 (KIM-1) participates in renal tissue
damage and repair and is proposed as a biomarker of early and subclinical
AKI. KIM-1 is also elevated in the urine of a significant fraction of patients
apparently recovered from an AKI. Besides its potential utility in the earlyand subclinical diagnosis of renal damage, this study suggests a newapplication of urinary KIM-1 in the non-invasive follow-up of renal repair
after AKI.1
PRINCIPLE OF KIDNEY INJURY MOLECULE 1 ELISAMATERIAL REQUIRED BUT NOT SUPPLIED
1.
2.
3.
4.
5.

WARNINGS AND PRECAUTIONS
1.
2.
3.
4.
5.

The microtiter plate is coated with the antibody specifically binding theKidney Injury Molecule 1. The human serum or plasma is incubated in theplate with the capture antibody.
The specimen is washed out and the specifically bound protein is incubatedwith HRP-labelled detection antibody. Unbound reagent is then washedout. Horseradish peroxidase (HRP) bound in the complex reacts with thechromogenic substrate (TMB) creating the blue colour. The reaction isstopped by addition of STOP solution (H2SO4).
The absorbance values are measured at 450 nm (optionally 450/630 nm)
and are proportional to the concentration of KIM-1 in the specimen. The
concentration of KIM-1 in unknown samples is determined from thecalibration curve which is created by plotting the absorbance values against
the standard concentration values.

Glassware and test tubes
Microtiter plate washer
Precision pipettes (various volumes) with tips
Orbital shaker
Microtiter plate reader capable of measuring absorbanceat 450 nm or 450/630 nm with software for data generation

6.
7.

For research use only
For professional laboratory use
The reagents with different lot numbers should not be mixed
To prevent cross sample contamination, use disposable labware
and pipette tips
To protect laboratory stuff, wear protective gloves andprotective clothing
The substrate solution should remain colourless, keep it
protected from light
The test should be performed at standard laboratory conditions(temperature 25oC ±2oC).

STORAGE CONDITIONS
1.
2.

The kit must be stored at 2 – 8oC.
The opened components can be stored for one week at 2 – 8oC.

PREPARATION OF REAGENTS
Use new pipette tip for pipetting different reagents and samples
to prevent cross-contamination.
All reagents and samples should be allowed to reach thetemperature 25oC ±2oC.

Preparation of Standards
Reconstitute lyophilized Human KIM-1 Standard in Dilution Buffer, for thevolume information see the Certificate of Analysis. Let it rehydrate for15 min. The concentration of human KIM-1 in Master Standard is1000 pg/mL,Prepare set of Standard solution as follows:
Use the Master Standard to produce a dilution series (as below). Mix eachtube thoroughly before the next transfer. The Dilution Buffer serves asBlank.
Volume of Standard
Std1

Kit Contents
Item
Antibody Coated Microtiter Plate
Antibody-HRP Conjugate
Master Standard (lyophilized)
Quality Control A (human serum, lyophilized)
Quality Control B (human serum, lyophilized)
Dilution Buffer
Wash Buffer 15x conc.
Substrate Solution
STOP Solution
v_01

Qty.
96 wells
13 mL
1 vial1 vial1 vial
13 mL
50 mL
13 mL
13 mL

Dilution
BufferSee CofA

Concentration

Std2

Standard 1000 pg/mL(lyophilised)
300 µL of Std1

1000 pg/mL

300 µL500 pg/mL

Std3300 µL of Std2300 µL

250 pg/mL

Std4300 µL of Std3300 µL

125 pg/mL

Std5300 µL of Std4300 µL

62.5 pg/mL

Std6300 µL of Std5300 µL

31.25 pg/mL

Std7300 µL of Std6300 µL

15.625 pg/mLBlank-250 µL

0 ng/mL

Preparation of Quality Control A and B
Reconstitute the lyophilized human serum Quality Controls indeionized/distilled water, for the volume information see the Certificate ofAnalysis. Let the QCs rehydrate for 15 min and dilute them 1:2 in Dilution
Buffer, prior to use, see Preparation of samples.

2

Preparation of Wash Buffer 1x3.

Prepare a working solution of Wash Buffer by adding 50 mL of Wash Buffer
15x conc. to 700 mL of deionized/ distilled water (dH2O). Mix well. Store at4oC for two weeks or at -20oC for long term storage.

Accuracy

Dilution linearity
SamplePreparation of samples

Dilution1

Human serum or plasma may be used with this assay. For long-term storage
the samples should be frozen at minimum -70oC. Lipemic or haemolytic
samples may cause false results.

2x4x8x2

Recommended dilution of samples is 1:2, i.e., 70 µL of sample + 70 µL ofDilution Buffer for singlets and 150 µL of sample + 150 µL of Dilution Buffer
for duplicates.

2x4x8x

Measuredconcentration(pg/mL)
509
230
106
52
448
196
99
54

Expected
concentration(pg/mL)
254
127
64
224
112
56

Yield(%)

Measuredconcentration(pg/mL)
236
663
446
341

Expected
concentration(pg/mL)
596
416
326

Yield(%)

908382878996

Do not store the diluted samples.
Spiking Recovery

ASSAY PROCEDURE
1.
2.

3.

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8.

Prepare the reagents as described in the previous chapter.
Pipette 100 µL of set of Standards, Quality Controls, diluted
Samples and Dilution Buffer = Blank into each well. Incubate
OVER NIGHT at 4oC ±2oC, NO shaking.
Wash the wells 3-times with 1x Wash Buffer (350 µL/well). When
finished, tap the plate against the paper towel to remove theliquid completely.
Pipette 100 µL of HRP-labelled Antibody Conjugate into eachwell. Incubate for 2 hours at 25oC ±2oC, shaking at 500 rpm.
Wash the wells as described in point 3.
Pipette 100 µL Substrate solution, incubate for 25 min,at 25oC ±2oC. Avoid exposure to the light during this step.
Pipette 100 µL of STOP solution.
Read the signal at 450 or 450/630 nm within 15 min.

SampleSpike(ng/mL)

1360
180
90111
107
105

Typical standard curve
The standard curve needs to be measured in every test. Most of themicroplate reader can automatically calculate the analyte concentration
using 4-parameter algorithm or alternative functions to fit the standard
points properly. The concentrations need to be multiplied by the dilution
factor, either automatically by reader or manually.

Plate layout
12
Std 1
Std 2
Std 3
Std 4
Std 5
Std 6
QCA
QCB

3Bckg

4
Sa 45
Sa 8

Sa 1

Sa 5

Sa 9

Sa 2

Sa 6

Sa3

Sa 7

Sa10
Sa
116
Sa
12
Sa
13
Sa
14
Sa
157
Sa
16
Sa
17
Sa
18
Sa
198
Sa
20
Sa
21
Sa
22
Sa
23

9
Sa
24
Sa
25
Sa
26
Sa2710Sa
28
Sa29
Sa30
Sa
31

11Sa32
Sa33
Sa34
Sa35

Human KIM1 Standard Curve

12Sa36Sa37Sa
38Sa
394
3.5
3Absorbance (450/630 nm)

A
B
CDEFG
H

PERFORMANCE CHARACTERISTICS
Samples used in the tests were diluted 1:2 as recommended and assayed.
The results are multiplied by the dilution factor.
1.

Sensitivity

The limit of detection, defined as a concentration of human KIM-1 giving
absorbance higher than absorbance of blank + 3 standard deviations, isbetter than 45 pg/mL of sample.
2.

2.5
2
1.5
1
0.5
015.625

Precision

Mean (pg/mL)
519
287

SD
8.8
15.6

CV (%)
1.7
5.4

SD
1615CV (%)
5.7
13.3

Inter-assay (Run – to – run)
Sample12v_01

62.5

125

250

500

1000

Concentration (pg/mL)

Intra-assay
Sample1231.25

Mean (pg/mL)
284
114

RESOURCES
1Cuesta C, Fuentes-Calvo I, Sancho-Martinez SM, Valentijn FA, Düwel A, Hidalgo-Thomas OA,
Agüeros-Blanco C, Benito-Hernández A, Ramos-Barron MA, Gómez-Alamillo C, Arias M, Nguyen TQ,
Goldschmeding R, Martínez-Salgado C, López-Hernández FJ. Urinary KIM-1 Correlates with the
Subclinical Sequelae of Tubular Damage Persisting after the Apparent Functional Recovery fromIntrinsic
Acute
KidneyInjury.
Biomedicines.
2022
May
10;10(5):1106.
doi:10.3390/biomedicines10051106. PMID: 35625842; PMCID: PMC9139078.