XhoI ELISA Kit

Cat. No.: X1522790

GRADE & PURITY BioReagent, for Enzyme immunoassay(ELISA)

Overview

This kit adopts the double-antibody sandwich ELISA method to detect XhoI content in samples. XhoI-specific antibodies are pre-coated on microplate wells. During detection, standards and samples are added separately into antibody-coated wells. XhoI in samples binds to the immobilized antibodies. After incubation, unbound substances are washed away. HRP-labeled XhoI detection antibody is added to form antibody-antigen-HRP antibody complexes. TMB substrate is added for color development. The color intensity is proportional to XhoI concentration. The residual XhoI in samples is determined by detecting color development of XhoI standards.

Detection Range: 0.125~4 ng/ml

Quantitation Limit: 0.125 ng/ml

Limit of Detection: 0.021 ng/ml

Required Instruments

Microplate reader, microplate constant temperature shaker, vortex mixer, pipettes, plate sealing film, etc.

Reagent Preparation

1. Equilibration: Allow all required reagents to equilibrate at room temperature (25±3℃) for at least 30 minutes. Determine the number of wells needed, take out required microplate strips, and return unused strips to aluminum foil bags with desiccant. Reseal and store at 2–8℃.

2. Prepare 1× Washing Buffer: Dilute 20× Washing Buffer with deionized water at a volume ratio of 1:20 according to required volume, mix well for standby use.

3. Standard Solution Dilution (Example):

Take 1 vial of XhoI standard, accurately add 1 mL sample diluent, and vortex to mix thoroughly. The final concentration is 10000 ng/mL, which is used as the standard stock solution.The prepared standard stock solution shall be temporarily stored at 2–8 ℃ and used within 24 hours. For long-term storage, aliquot the solution and keep it at -20 ℃, and use it within 3 months. 

No.	Standard Concentration (ng/ml)	Standard Volume (μl)	Diluent Volume (μl)	Total Volume (μl)	Final Concentration (ng/ml)	Remaining Volume (μl)
StA	10000	20	1800	2000	100	1980
St7	100	20	480	500	4	250
St6	4	250	250	500	2	250
St5	2	250	250	500	1	250
St4	1	250	250	500	0.5	250
St3	0.5	250	250	500	0.25	250
St2	0.25	250	250	500	0.125	500
St1 (Blank)	/	/	300	300	0	300

Assay Procedure

1. Mix all reagents equilibrated to room temperature gently before use.

2. Pipette 100 μL of standard solutions (St1~St7) or test samples into corresponding wells. Seal the plate with a plate sealer, then incubate at 25±3 ℃ and 400–500 rpm for 1 hour.

3. Washing: Discard the liquid in wells. Add at least 350 μL of 1× Washing Buffer to each well (using a multichannel pipette or wash bottle). Discard residual liquid and pat the plate dry on lint-free absorbent paper. Repeat washing for 3 cycles.

(Note: Do not allow wells to dry excessively for a long time before adding XhoI detection antibody. Improper washing may cause high CV between duplicate wells and abnormal absorbance signals.)

4. Add 100 μL of HRP-labeled XhoI detection antibody to each well. Seal the plate and incubate at 25±3 ℃, 400–500 rpm for 30 minutes.

5. Washing: Discard the liquid in wells. Add at least 350 μL of 1× Washing Buffer to each well. Discard liquid and pat the plate dry. Repeat washing 3 times.

(Note: Avoid prolonged excessive drying of wells prior to TMB substrate addition.)

6. Color development: Add 100 μL TMB substrate to each well, and incubate for 20 minutes at room temperature in the dark.

7. Termination: Add 100 μL Stop Solution to each well, tap the plate gently to mix thoroughly and terminate the reaction.

8. Detection: Measure the absorbance at 450 nm within 10 minutes after adding Stop Solution.

Data Calculation

It is recommended to perform logarithmic transformation of concentration and fit standard curve with 4-Parameter Logistic regression model.

Table 1 Typical Standard Curve Data

Standard Concentration (ng/ml)	OD Reading 1	OD Reading 2	Average OD Value
4	1.432	1.41	1.421
2	1.056	1.087	1.071
1	0.694	0.683	0.689
0.5	0.439	0.435	0.437
0.25	0.298	0.289	0.293
0.125	0.202	0.218	0.21
0	0.113	0.11	0.111

Typical Standard Curve (4PL Logistic Model)

Performance

1. Limit of Detection (LOD): 0.021 ng/ml. Calculated by substituting the average absorbance of 20 blank controls plus 2 standard deviations into the standard curve.

2. Limit of Quantitation (LOQ): 0.125 ng/ml. The lowest concentration with coefficient of variation (n=6) ≤ 20%.

3. Linearity: Standard curves are plotted with serially diluted standard solutions, with R² ≥ 0.98.

4. Repeatability: Evaluated by detecting three samples of known concentrations for 20 replicates.

5. Intermediate Precision: Evaluated by detecting three samples of known concentrations 3 times by different operators on different days. 

Parameter	Repeatability (n=20) Low  Concentration	Repeatability (n=20) Mid  Concentration	Repeatability (n=20) High  Concentration	Intermediate Precision (n=3) Low  Concentration	Intermediate Precision (n=3) Mid  Concentration	Intermediate Precision (n=) High  Concentration
Mean (ng/mL)	0.46	1.99	3.05	0.53	2.09	3.31
SD	0.017	0.079	0.259	0.025	0.049	0.255
CV (%)	3.7	4.0	8.5	4.7	2.3	7.7

6. Recovery: The recovery rates of spiked XhoI at different levels within the full detection range in biological matrix were verified.

Average Recovery (%) (n=3)	Recovery Range (%)
105.9	96.2~117.6

Precautions

1. For research use only, not for clinical diagnosis or treatment.

2. Store reagents as labeled. Equilibrate reagents and samples to room temperature before detection.

3. Equilibrate pre-coated microplates to room temperature before opening packages. Return unused strips to sealed bags and store at 2–8℃. Seal all leftover reagents properly.

4. Mix XhoI  standard and HRP detection antibody thoroughly before use.

5. Pat washed wells dry on lint-free paper. Do not over-dry wells, which will increase CV and blank OD deviation.

6. TMB substrate should be colorless and transparent. Pale blue TMB indicates contamination and cannot be used.

7. Samples with extreme pH (<5.0 or >8.5), high salt, polysaccharide, urea, organic solvent or detergent may result in low recovery.

8. Due to complex biological matrix, use positive controls and duplicate wells to ensure accurate results.

Safety Instructions

1. Stop Solution is acidic. Operate with caution.

2. Wear proper personal protective equipment: lab coat, gloves, mask, goggles, etc.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Stability And Storage

Each component has a shelf life of 12 months under corresponding storage conditions. X1522790B/C: Store at 2-8℃ long term (12 months). Store in the dark. X1522790A/D/E/F/G: Store at 2-8℃ long term (12 months). 

Contents & Storage

Item No.	Appearance	Components	96 T	Storage
X1522790A	Solid	XhoI Standard	10 μg, 2 Vial	2-8℃
X1522790B	Liquid	XhoI Detection Antibody (HRP labeled)	12 ml, 1 Vial	2-8℃. Store in the dark.
X1522790C	Liquid	TMB Substrate	12 ml, 1 Vial	2-8℃. Store in the dark.
X1522790D	Liquid	Stop Solution	12 ml, 1 Vial	2-8℃
X1522790E	Liquid	Sample Diluent	50 ml, 1 Vial	2-8℃
X1522790F	Liquid	Wash Buffer (20×)	50 ml, 1 Vial	2-8℃
X1522790G	—	Pre-coated Assay Plate	96 wells, 1 Plate	2-8℃