Category: Tech Guide

BUNSEN Mouse ELISA kit Precautions and Plate Washing Method

BUNSEN Mouse ELISA kit Precautions

specimen and reagent preparation‌: serum / Ensure the plasma is thoroughly centrifugation, avoiding haemolysis, lipaemia and repeated freeze-thaw; All reagents should be removed from storage at 2–8 °C in advance and allowed to equilibrate at room temperature for 30 minutes; if crystals have formed in the concentrated wash solution, dissolve them in a 37 °C water bath before diluting for use.

Operating procedures: Change the tip when using different reagents, specimen to prevent cross-contamination; strictly adhere to the incubation temperature and duration specified in the instructions; sealing film ensure the lid is tightly sealed to prevent liquid evaporation; place a humidification tray inside the incubator to avoid edge effects.

Colour development and reading: Keep the substrate solution away from light at all times; do not use if a pale blue colour appears. Complete the test using the enzyme analyser within 30 minutes of adding the stopping solution to prevent result drift.

Storage requirements: Unopened kit Store refrigerated at 2–8 °C away from light; do not freeze. Return any remaining, unused strips to the sealed bag with the desiccant and use within one month.

Standard Procedure for Washing Printers

Preparation of washing solution: Dilute the concentrated washing solution with fresh deionised water in accordance with the proportions specified in the instructions; prepare and use immediately. For BUNSEN BUNSEN kit, the standard dilution for the washing solution is 20-fold.

Welling and soaking: Add 300 μL of washing solution to each well, filling the microplates to the brim without spilling, and leave to soak for 30 seconds to 1 minute to allow the washing solution to come into full contact with the well walls.

Drain and pat dry: Empty the liquid from the hole by tilting it vertically, then turn the microplate upside down on a thick sheet of absorbent paper and pat it dry vigorously. Do not wipe it with toilet paper to avoid paper fibres residue contaminating the bottom of the hole.

Repeat the cycle: Repeat the above steps of adding liquid, soaking, discarding the liquid and patting dry five times. When patting dry for the final time, ensure that there is no residue liquid remaining in the wells to prevent residue enzyme complexes from causing an increase in background levels.

Next step: Once the microplate has been dried, add the reagent for the next step immediately; do not leave the plate to dry for an extended period.

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Activity protein Class aliquot Component retention

standard following aliquot, enzyme-labelled antibody, biotinylated antibody and other active protein . After sealing, store in a light-protected environment at -20°C; repeated freeze-thaw is strictly prohibited. Each time, remove only a single single-use portion of aliquot reagent, and use it in its entirety immediately after thawing.

Before freezing, clearly label the aliquot tubes with the component name, aliquot date and concentration details to avoid confusion between different batches of reagents.

Other aliquot Component preservation

Any unused portion after aliquot microplate items should be returned to their original aluminium foil pouches, with desiccant added. After sealing, store at -20°C. In high-humidity environments, place the pouches in an additional sealed food-storage container to prevent moisture from causing the antibody coating to become inactive.

The concentrated wash solution and specimen diluent prepared after step aliquot should be sealed and stored in a refrigerator at 2–8 °C. Should crystals form, these may be dissolved in a warm water bath; this will not affect subsequent use.

Storage restrictions

Under no circumstances should any reagents following aliquot be frozen directly to –80 °C, as this may cause irreversible damage to the protein structure.

Photosensitive reagents such as the TMB colouring solution following aliquot must be stored in sealed, brown containers away from light and refrigerated at 2–8 °C; exposure to light must be avoided to prevent oxidation and discolouration.

All aliquot reagents must be used within one month; after this period, their activity must be verified using a positive control, and they may only be used in experiments if they pass this test.

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Frequently Asked Questions on the Entire ELISA Assay Process

BUNSEN BUNSEN Drawing on extensive practical laboratory experience, this guide covers the most common issues encountered at every stage of an ELISA experiment—from reagent preparation to result interpretation—enabling you to quickly pinpoint the cause of experimental failure:

1. Signal colouring-related anomalies

Standard curve shows no colour/very faint colour‌: standard Insufficient vortex mixing after dissolution; dissolution cannot be guaranteed by pipette pipetting alone; or failure to add key components such as antibody and enzyme, resulting in antigen and antibody failing to bind and produce a colour.

The standard curve shows normal colour development; specimen no signal: the concentration of the target in specimen is far below kit the limit of detection, or specimen the presence of interfering substances such as strong surfactants has disrupted the specific binding of antigen antibody.

Abnormally high background, specimen signal masked: OD values in blank wells exceeding 0.1 are usually due to excessive colour development of the TMB substrate; the reaction was not terminated in time before a distinct colour appeared in the positive control, resulting in a significant accumulation of non-specific binding.

The colour appears normal to the naked eye, but the enzyme spectrophotometer reading is low: The wavelength settings for the enzyme spectrophotometer are incorrect. To detect the TMB substrate, the measurement wavelength must be set to 450 nm and the reference wavelength to 650 nm; using the wrong wavelengths will directly result in distorted readings.

2. reproducibility and stability-related exceptions

Excessively high CV value for multiple wells (exceeding 15%)‌: pipette Failure to calibrate regularly, inconsistent sample dispensing, and significant variation in the volume transferred between wells; or temperature fluctuations during incubation, resulting in inconsistent reaction progress across different wells.

The colour development at the peripheral wells is significantly greater than at the central wells: the seal is not airtight; 96-well plate as the liquid in the peripheral wells evaporates and concentrates, the concentration of the reaction mixture increases locally, resulting in a typical edge effect.

For the same specimen but with different dilution gradients, the calculated concentrations vary considerably: at high concentrations of specimen, a hook-shaped effect occurs, whilst an excess of antigen / antibody hinders the effective binding of the enzyme labelled secondary antibody, resulting in lower OD values and causing false negatives.

Abnormally high background values after washing: If the concentration of Tween-20 in the washing solution is less than 0.05 per cent, or if the number of washes is fewer than three, unbound non-specific protein residue remains in the wells, causing the background to rise.

Key points to note throughout the entire process

Pre-experiment preparations

All reagents should be removed from a 2–8 °C environment in advance and allowed to equilibrate to room temperature (20–25 °C) for 20–30 minutes before use, to avoid antigen antibody uneven binding efficiency; during winter when room temperature is low, place the reagents in a 37°C incubator to assist with equilibration.

standard Must be aliquot cryopreserved; avoid at all costs repeated freeze-thaw which leads to protein; if crystals form in the washing solution, it must be fully dissolved and thoroughly mixed in a 37°C water bath before use to prevent variations in concentration.

Perform calibration in advance pipette; for multi-channel pipette systems, calibrate the pipette volume for each well to avoid overall deviations in results caused by pipetting errors.

Coating and Sealing Stages

Coat antigen / antibody with pH 9.6 carbonate buffer buffer; the concentration should be determined by preliminary experiments, as too high a concentration will increase non-specific background, whilst too low a concentration will result in an insufficient signal.

After coating, it is recommended to leave the plates to stand overnight (12–16 hours) at 4 °C; this results in more stable binding and lower background levels than incubation at 37 °C. Immediately after coating, remove any excess liquid and add the blocking solution straight away to prevent the well bottoms from drying out, which could lead to uneven blocking.

Sample addition and incubation stages

When dispensing, hold the pipette tip vertically in the air; do not touch the walls or bottom of the well to avoid dislodging the seal; change the pipette tip after each specimen to prevent cross-contamination.

Incubation must be carried out in a humid environment; place damp filter paper at the bottom of the incubation chamber. Keep the chamber sealed throughout the incubation at 37 °C to prevent condensation from dripping into the wells and diluting the sample specimen, whilst also preventing evaporation of the liquid, which could cause a edge effect.

The incubation time must be strictly adhered to as specified in the instructions; do not extend it arbitrarily, as doing so will significantly increase non-specific binding and directly raise the background level.

Washing and Colour Development Stages

Washing is a key step in reducing the background; for each wash, fill each well to the brim, allow to soak for 30 seconds, then blot dry; repeat this washing procedure at least 3–5 times; After washing, invert the microplate on absorbent paper and tap it firmly to remove any unbound residue.

The TMB substrate solution must be prepared immediately before use and kept away from light throughout the process to prevent premature oxidation and loss of efficacy; when a distinct blue gradient appears in wells with a high concentration standard, stop the reaction immediately to prevent over-colouration.

Termination and Reading Procedures

The order and rate at which the stopping solution is added should be kept as consistent as possible with the substrate, to ensure that the reaction is terminated simultaneously in every well; readings must be taken within 15 minutes of termination, as the coloured product will fade if this time is exceeded, resulting in lower OD values.

Wipe away any fingerprints or water marks from the base of the microplate before taking a reading. The use of dual-wavelength reading (450 nm measurement wavelength + 650 nm reference wavelength) effectively eliminates background noise and significantly improves reading accuracy.

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Tag: Cryovial Tube Three in One Cryovial Tube Cryovial Tube

BUNSEN For thawing procedures involving cells cryovial, the principle of ‘slow freezing and rapid thawing’ must be followed, ensuring that cell viability is maintained and operational safety is prioritised throughout the process. The specific standard procedure is as follows:

thawing Pre-safety preparations

Wear a laboratory coat and cold-resistant cotton gloves at all times; it is recommended that you wear safety goggles or a face shield to avoid biological hazards caused by frostbite from liquid nitrogen or by cryovial bursting.

Preheat culture medium to 37°C in advance, and have 70 per cent alcohol, sterile, centrifuge tube, pipette and other sterile consumables, and complete the disinfection preparations in advance within the laminar flow cabinet.

After removing cryovial from the liquid nitrogen tank, allow it to stand in the air for 3–5 seconds to ensure that any trace amounts of liquid nitrogen that may have seeped into the tube have fully evaporated; this prevents the tube from bursting due to the rapid sublimation of liquid nitrogen upon direct contact with water.

Quick thawing Operations

Place cryovial rapidly into a water bath maintained at 37°C, shaking gently and continuously to ensure it melts within 1–2 minutes; remove it as soon as only a very small number of ice crystals remain. Take great care to avoid slow thawing, which can cause secondary ice crystal formation within the cells and damage their viability.

Strictly control the water level in the water bath to ensure that the water does not rise above cryovial the rim of the lid, in order to prevent water from seeping into the tubes and causing specimen cell contamination.

sterile Transfer and cell recovery

thawing Once completed, immediately wipe the outer surface of cryovial with 70 per cent alcohol and transfer it to a cleanbench to carry out the subsequent steps.

Gently unscrew tube cap and transfer the cell suspension to a tube pre-filled with a small amount of sterile centrifuge tube that has been preheated to culture medium:

If the DMSO concentration in the cryopreservation solution is 5 per cent, there is no need for centrifugation; the cells may be inoculated directly after diluting with 15 ml of culture medium, as DMSO at this concentration exhibits no significant cytotoxicity;

For DMSO-sensitive cell lines, use the low-speed centrifugation method: 125–500 g centrifugation for 5–10 minutes; discard the supernatant containing the cryoprotectant and gently resuspend the cell pellet in 1–2 ml of fresh, pre-warmed culture medium solution.

Post-resuscitation care and follow-up observation

Transfer the resuspended cell suspension to a pre-prepared culture vessel, mix thoroughly, and place it in an incubator at 37 °C and 5 % CO₂ for incubation. Avoid moving the culture vessel as much as possible within 12 hours of inoculation to minimise disruption to cell attachment.

Key tips to avoid pitfalls

cryovial Daily storage Must be stored in the vapour phase of liquid nitrogen or in a -80°C freezer; direct immersion in the liquid phase of liquid nitrogen is strictly prohibited, in order to prevent, at source, the risk of liquid nitrogen seeping into the tubing and causing it to burst during thawing.

The volume of cryopreserved material specimen must not exceed cryovial of the rated working volume, to prevent ice plugs from forming inside the tubes during the freezing process, which could cause high pressure to damage the tubes and pose a safety hazard.

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Upon receipt of serum, the following measures must be taken: inspection and verification, handling of any anomalies, and proper storage, to ensure that the activity of serum remains unaffected:

Goods Receipt and Inspection Stage

Immediately check serum that the outer packaging is intact and ensure there are no leaks or damage. If you find any damage to the packaging or leaks, contact the supplier immediately for after-sales assistance.

With regard to the cold chain transport conditions, it was confirmed that there was a sufficient supply of dry ice throughout the journey to maintain the low temperature, and there were no instances of temperatures falling below thawing or of temperatures exceeding the permitted limits by a significant margin.

Exception handling for partial melting

If dry ice sublimates during transport, causing serum to melt partially, there is no need to immediately declare it a write-off: allow serum thawing, then gently shake to mix, and return it to a -20°C environment for storage; the quality of serum will remain largely unaffected.

Points to note regarding storage upon receipt

serum Must be transferred immediately to low-temperature freezer at a temperature of -5℃ to -20℃; the storage period at 4℃ must not exceed one month under any circumstances.

serum As the volume expands by approximately 10 per cent when frozen, sufficient headspace must be left in the vial prior to freezing to prevent the glass vial from cracking and to avoid contamination.

Do not leave serum in an environment at 37°C for prolonged periods, as this will cause serum to become cloudy and destroy the internal active protein, which will directly affect the results of subsequent experiments.

Long-term storage: It is not recommended to store the product in an environment at -80℃, as this will increase the risk of the bottle cracking; furthermore, when the temperature subsequently rises to thawing, the significant temperature difference will cause a large amount of protein to precipitate.

Preparations prior to subsequent use

Upon receipt, in an sterile environment, divide aliquot into individual portions of approximately 50 mL as required, clearly labelling each with the batch number, aliquot date. For subsequent use, take only one portion thawing at a time to avoid repeatedly freeze-thaw opening the entire bottle serum, thereby preserving serum its potency.

BS8002	newborn calf serum
BS800820	Premium fetal bovine serum
BS800821	Superior fetal bovine serum

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The three types of PET, PETG and HDPE reagent bottle from BUNSEN BUNSEN have distinctly different material properties and applications; they must be selected to meet the relevant requirements:

PET reagent bottle Use

Features: The square design offers excellent gas barrier properties, complies with the USP Class VI standard, is compatible with bioreactor aeration systems, is available in sizes ranging from 50 ml to 1,000 ml, and is stackable, saving 30 per cent of storage space.

Suitable applications: routine reagents aliquot, cell resuscitation, seeding, pilot-scale culture medium storage; compatible with robotic arm handling on automated production lines.

Contraindications: Avoid contact with strong organic solvents; do not use to contain liquids exceeding 70°C; do not reuse repeatedly over a long period to prevent the migration of harmful substances; ensure the container is properly shock-proofed during transport to prevent stress fractures at the corners of the bottle.

PETG reagent bottle Use

Properties: Light transmittance >90%, resistant to pH 2–11, short-term resistance to 121°C sterilized, gamma irradiation sterilized with no DNA/RNA enzyme, and the 45° beveled edge design improves liquid transfer efficiency by 20 per cent.

Suitable applications: cell culture, biological reagents such as pancreatic enzyme /EDTA storage, and industrial culture medium preparation; the transparent bottle body allows for direct microscopic observation.

Contraindications: sterilized Check the seal on the bottle cap beforehand to prevent deformation under high pressure; do not overfill the bottle with liquid, leaving sufficient space for gas exchange.

HDPE reagent bottle Use

Features: Made from high-density polyethylene, resistant to most weak acids, weak alkalis and alcoholic reagents; withstands temperatures up to 110°C; available in a range of capacities from 30 ml to 1,000 ml; can be fitted with caps in different colours for reagent identification.

Suitable applications: Storage and transport of trace quantities of reagents; packaging for highly volatile reagents aliquot and kit, which effectively reduces the risk of evaporation and contamination.

Contraindications: It is not recommended to store food-grade liquids in this container for prolonged periods; avoid prolonged contact with highly polar organic solvents; and check the container regularly for cracks, signs of ageing or leaks.

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The range of domestic plastic pipettes compiled by BUNSEN BUNSEN pipette tip can be clearly categorised according to three main criteria: specification and range, functional characteristics and packaging format, covering the requirements of the vast majority of laboratory pipetting scenarios.

Classified by standard specification and measurement range

This is the most basic classification method; domestic pipette tip main flow ranges cover all scenarios:

10 μL: Available in two variants—0.5–10 μL white short tips and 0.5–10 μL white long tips—suitable for microvolume pipetting.

20 μL: 0.5–20 μL white pipettes with long tips, suitable for dispensing small volumes.

100 μL: Available in 10–100 μL sizes, suitable for routine experiments involving medium-sized samples.

200 μL: 20–200 μL yellow pipette tips are one of the standard sizes used in laboratories in China.

1000 μL: 100–1000 μL blue pipette tips; some brands offer an extended 1250 μL version, suitable for dispensing from deep vessels.

5,000 μL: A high-capacity model with a range of 1,000–5,000 μL, designed for dispensing large volumes of liquid.

Classified by specific functional characteristics

The functional pipette tip, designed to meet specific experimental requirements, represents the core focus of the upgrade programme for the domestically produced pipette tip:

Featuring filter pipette tip: An integrated hydrophobic polypropylene filter cartridge prevents aerosols from entering the interior of pipette, thereby avoiding cross-contamination of samples; suitable for high-precision experiments such as PCR and nucleic acid extraction.

low-binding pipette tip ‌: The inner walls have been treated using a special process, which significantly reduces the surface residue of viscous liquids such as nucleic acid and protein; the volume of precious samples residue can be as low as ≤0.5 per cent, thereby minimising reagent wastage.

sterile DNase/RNase-free pipette tip ‌: Treated with irradiation sterilized, free from RNase, DNase, DNA, ATP and pyrogen contamination; a full test certificate is provided, making it suitable for high-demand applications such as molecular diagnostics.

Special-purpose pipette tip ‌: Includes sub-categories such as gel loading for electrophoresis pipette tip, wide-mouth pipettes pipette tip for transferring macromolecular solutions, and specialised pipettes pipette tip compatible with external pistons pipette.

Classified by packaging type

The packaging of the domestically produced pipette tip is tailored to the usage habits of domestic users and is mainly divided into three categories:

Bagged pipette tip ‌: The most common format on the domestic market, packaged in plastic self-sealing bags containing 500–1,000 units per bag, with prices ranging from 20 to 60 yuan. These offer good value for money and are suitable for routine experiments not involving a high risk of contamination.

Boxed pipette tip ‌: pipette tip are pre-arranged neatly in pipette tip boxes, eliminating the need for manual packing, cleanliness offers greater assurance; the price is 1.5–2.5 times that of bagged products, and it is suitable for high-standard scenarios such as sterile and DNase/RNase-free.

Refill pack pipette tip: Multiple sheets of pipette tip stacked in a tower shape can be loaded directly into an empty pipette tip box, making them 10–20 per cent cheaper than boxed pipette tip and significantly reducing the use of plastic packaging materials.

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Note: The information on the scientific research products above is for reference only!