Precautions for the production of B-factor assay kit

During the production process of the B-factor assay kit, attention should be paid to the four core links of reagent preparation, stability control, operation standards, and safety protection. Specific precautions are as follows:

1. Reagent preparation and stability control

Buffer preparation

The pH value needs to be accurately controlled (such as CHES-TAPSO-MES buffer pH 6.86 or 7.25), and when adjusting with sodium hydroxide or hydrochloric acid, it should be added dropwise to avoid pH fluctuations affecting reagent activity. The components of the buffer solution (such as LiCl, CaCl ₂, NaCl) should be dissolved in proportion, stirred until completely transparent, and then brought to volume to prevent crystallization.

Antibody and latex particle treatment

When rabbit anti human factor B polyclonal antibody is coated with latex particles, the temperature (such as shaking at 25 ℃ for 4 hours) and antibody concentration (60-100mg) should be strictly controlled to avoid uneven coating leading to decreased sensitivity.

. The sensitizer (containing PEG6000/10000) needs to be dissolved step by step (25 ℃ → 45 ℃ → 37 ℃) to ensure complete dispersion of polyethylene glycol and prevent agglomeration from affecting reaction efficiency.

Anti corrosion and anti-interference design

Preservatives (such as sodium azide) should be added at a concentration of 0.02% -0.05% to inhibit microbial growth while avoiding enzyme activity inhibition.

. Anti interference agents (including BSA and blocking agents) need to be stirred and dissolved at 40 ℃ to remove endogenous interference such as cytokines and complement in the serum and improve detection specificity.

II. Operation Standards and Error Control

Sample Addition Accuracy Management

When using a multi-channel pipette for sample addition, the error range should be controlled within ± 1% to avoid distortion of the standard curve due to sample addition deviation.

. The sealing film is only for one-time use to prevent cross contamination; The washing solution needs to be diluted at a ratio of 1:50, and the number of plate washes should be strictly controlled within 3-5 times to avoid residual effects on color development.

Optimization of incubation conditions

Enzyme labeled plate incubation requires the use of a water bath or wet box, maintaining a constant temperature of 37 ℃ to reduce edge effects (temperature difference between the surrounding and central wells of a 96 well plate ≤ 0.5 ℃).

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The color reaction time should be precise to minutes (such as 15 minutes for TMB substrate light avoidance reaction), and exceeding the time limit will result in an excessively high OD value.

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Standard curve drawing

Standard samples should be used and prepared on site, and the dilution gradient (such as 12.5ng/mL → 400ng/mL) should cover the detection range. The coefficient of variation (CV) of the OD value of the wells should be ≤ 5%.

. The correlation coefficient (R ²) of the linear regression equation should be ≥ 0.99 to ensure quantitative accuracy.

III. Safety Protection and Waste Disposal

Personal protective equipment

Wear goggles, cut resistant gloves, and lab coats during operation to prevent reagents from splashing into the eyes or coming into contact with the skin.

. The termination solution (containing 2M H ₂ SO ₄) and substrate B (containing TMB) are corrosive and should be immediately rinsed with plenty of water upon contact.

Biosafety Requirements

All samples, detergents, and waste materials must be treated as infectious agents and disposed of after high-pressure sterilization (121 ℃, 30 minutes).

. Samples containing NaN3 are prohibited from detection as NaN3 can inhibit HRP enzyme activity, leading to false negative results.

Storage condition management

The kit should be stored in a dark place at 2-8 ℃. After opening, the strips should be sealed for storage and used up within 48 hours. Long term storage requires packaging to -70 ℃ to avoid repeated freeze-thaw cycles (≤ 3 times) and prevent protein denaturation.