Technical Manual for Microbial Lignin ELISA Detection Kit

BS-5184 Microbial Lignin ELISA Kit

1. Product Overview

This kit uses a double antibody sandwich enzyme-linked immunosorbent assay (ELISA), specifically designed for quantitative detection of lignin content in microbial fermentation broth, cell lysate, and other samples. Lignin is an aromatic polymer produced during microbial metabolism or degradation of plant cell walls, and changes in its content can reflect the efficiency of microbial utilization of lignocellulose.

2. Detection principle

The solid-phase antibody package captures lignin in the sample by a microplate, and then adds enzyme labeled detection antibodies to form an 'antibody antigen enzyme labeled antibody' complex. After washing, TMB substrate was added for color development, and the absorbance was measured at 450nm wavelength. The absorbance value was positively correlated with lignin concentration.

3. Composition of the reagent kit

Component 96 hole configuration, 48 hole configuration, storage conditions

Microporous enzyme-linked immunosorbent assay (ELISA) plates with 12 wells x 8 strips, 12 wells x 4 strips, sealed and dried at 2-8 ℃

Standard product (lignin) 0.5mL x 6 tubes 0.5mL x 6 tubes -20 ℃, avoid light

Sample diluent 6mL 3mL 2-8 ℃

Enzyme linked antibody HRP 10mL 5mL 2-8 ℃, avoid light

20 x Wash buffer 25mL 15mL Room temperature

TMB color solution 9mL 4.5mL 2-8 ℃, avoid light

Termination solution 9mL 4.5mL at room temperature

2 sheets of sealing film, 2 sheets at room temperature

4. Sample processing

4.1 Microbial fermentation broth

Centrifuge at 8000 × g for 10 minutes to remove bacterial cells

Immediately detect the supernatant or freeze it at -80 ℃ (avoid repeated freezing and thawing)

4.2 Cell lysate

After ultrasonic fragmentation, centrifuge at 12000 × g for 15 minutes

Dilute the supernatant 10 times with PBS and test

4.3 Precautions

Sample turbidity requires 0.22 μ m filter membrane filtration

Avoid using samples containing sodium azide (to inhibit HRP activity)

5. Experimental steps

5.1 Reagent preparation

Wash buffer: Dilute distilled water 20 times to 1 x working solution

Standard gradient: Dilute to 0-60 μ g/mL according to the instructions

5.2 Sample addition

Add 50 μ L of standard/sample to the corresponding micropores

Incubate at 37 ℃ for 30 minutes

5.3 Washing

Add 300 μ L of washing solution to each well, let it stand for 1 minute, and pat dry

Repeat 5 times

5.4 Color rendering

Add 100 μ L of enzyme labeled antibody to each well and incubate at 37 ℃ for 30 minutes

After washing, add 90 μ L of TMB chromogenic solution and avoid light for 15 minutes

Add 50 μ L of termination solution to terminate the reaction

5.5 Determination

Measure OD value at 450nm wavelength (reference wavelength 630nm)

6. Result calculation

Draw a standard curve with standard concentration as the horizontal axis and OD value as the vertical axis. The sample concentration is calculated using the curve equation, and if it exceeds the range, it needs to be diluted and retested.

7. Performance indicators

Parameter indicators

Sensitivity ≤ 0.5 μ g/mL

Detection range 1-60 μ g/mL

Precision within batch CV<8%, inter batch CV<12%

Recovery rate 85% -115%

8. Precautions

The reagent kit needs to be equilibrated at room temperature for 30 minutes before use

Different batch numbers of reagents cannot be mixed

Termination liquid contains strong acid, wear protective equipment during operation

Color developing solution should avoid contact with metal ions

Micro pipettes need to be calibrated to avoid cross contamination

9. Validity period

6 months (stored at 2-8 ℃)

10. Declaration

This product is for scientific research use only.

Note: The above is for reference only. This reagent is for scientific research use only and is not considered as actual data. Experiments must strictly follow the instructions or consult technical teachers.

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