BsmBI ELISA Kit

Cat. No.: B1522703

GRADE & PURITY BioReagent, for Enzyme immunoassay(ELISA)

Overview

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

Detection Range: 0.156~10 ng/ml

Quantitation Limit: 0.156 ng/ml

Limit of Detection: 0.07 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 BsmBI standard, accurately add 1 mL sample diluent, and vortex to mix well. The final concentration is 10000 ng/mL, which serves as the standard stock solution.The prepared standard stock solution should be temporarily stored at 2–8 ℃ and used within 12 hours. Do not expose it to room temperature for a long time (no more than 4 hours). If not used for an extended period, aliquot and store at -20 ℃, and use 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	1980	2000	100	1950
St8	100	50	450	500	10	250
St7	10	250	250	500	5	250
St6	5	250	250	500	2.5	250
St5	2.5	250	250	500	1.25	250
St4	1.25	250	250	500	0.625	250
St3	0.625	250	250	500	0.313	250
St2	0.313	250	250	500	0.156	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~St8) 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 BsmBI detection antibody. Improper washing may cause high CV between duplicate wells and abnormal absorbance signals.)

4. Add 100 μL of HRP-labeled BsmBI 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
10	2.74	2.734	2.737
5	1.747	1.708	1.728
2.5	0.978	1.028	1.003
1.25	0.602	0.576	0.589
0.625	0.359	0.349	0.354
0.313	0.251	0.287	0.269
0.156	0.231	0.218	0.224
0	0.178	0.18	0.179

Typical Standard Curve (4PL Logistic Model)

Performance

1. Limit of Detection (LOD): 0.07 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): 0.156 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)	0.51	2.50	8.59	0.63	2.51	8.31
SD	0.030	0.118	0.538	0.039	0.149	0.503
CV (%)	5.9	4.7	6.3	6.1	5.9	6.1

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

Average Recovery (%) (n=3)	Recovery Range (%)
105.6	98.9 ~ 115.7

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 BsmBI 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.

9. Immediately take out the BsmBI Standard and store it at -20 ℃ after receiving the kit; store other reagents at 2–8 ℃.

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,Store at -20°C

Shipped In

Ice chest + Ice pads

Stability And Storage

Each component has a shelf life of 12 months under corresponding storage conditions. B1522703A: Store at -20℃ long term (12 months). B1522703B/C: Store at 2-8℃ long term (12 months). Store in the dark. B1522703D/E/F/G: Store at 2-8℃ long term (12 months).

Contents & Storage

Item No.	Appearance	Components	96 T	Storage
B1522703A	Solid	BsmBI Standard	10 μg, 2 Vial	-20℃
B1522703B	Liquid	BsmBI Detection Antibody (HRP labeled)	12 ml, 1 Vial	2-8℃. Store in the dark.
B1522703C	Liquid	TMB Substrate	12 ml, 1 Vial	2-8℃. Store in the dark.
B1522703D	Liquid	Stop Solution	12 ml, 1 Vial	2-8℃
B1522703E	Liquid	Sample Diluent	50 ml, 1 Vial	2-8℃
B1522703F	Liquid	Wash Buffer (20×)	50 ml, 1 Vial	2-8℃
B1522703G	—	Pre-coated Assay Plate	96 wells, 1 Plate	2-8℃