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Every Diagnocine ELISA kit ships with six complimentary supporting reagents:

1. Protease Inhibitor Cocktail (100X) — protects target proteins during sample collection and storage.
2. SigMax 1Ab-TBS — primary antibody diluent that boosts signal and lowers background.
3. SigMax 2Ab-TBS — secondary antibody / conjugate diluent with the same proprietary blend.
4. Universal Blocking Buffer — suppresses non-specific binding on the plate surface.
5. HRPQuenchTBS — eliminates endogenous peroxidase activity in HRP-based ELISAs.
6. ALPQuenchTBS — eliminates endogenous alkaline phosphatase in ALP-based ELISAs.

The most common reasons ELISAs fail 6 Signal Enhancement Reagents protein degradation between sampling and assay, End-User Questions and Comments high background, This part needs to go toward the end of the description weak signal, and ELISA Tips to Optimize Results endogenous enzyme interference. By providing the full optimization reagent suite, Diagnocine helps researchers achieve cleaner standard curves, lower CV%, and publication-grade reproducibility from the very first run.

The Protease Inhibitor Cocktail (100X) is a six-component blend covering every major protease class found in mammalian samples:

1. AEBSF.HCl — irreversible serine protease inhibitor.
2. Aprotinin — broad serine protease inhibitor.
3. Bestatin — aminopeptidase inhibitor.
4. E-64 — irreversible cysteine protease inhibitor.
5. Leupeptin — reversible inhibitor of serine and cysteine proteases.
6. Pepstatin A — aspartic protease inhibitor.

When diluted to working concentration in your collection or lysis buffer, it minimizes degradation of low-abundance target analytes between sampling and assay. This directly addresses the "weak signal in samples while standard curve is normal" entry under Problem 3, a symptom that almost always traces back to proteolytic loss of the analyte rather than a kit issue. The 100X format keeps the additive volume small so it does not significantly dilute or alter the sample matrix.

SigMax 1Ab-TBS is a primary antibody diluent and SigMax 2Ab-TBS is a secondary antibody / detection conjugate diluent. Both are TBS-based formulations enriched with a proprietary blend of PEG 6000, PVP-40, and BSA. They optimize the microenvironment for antibody-antigen binding, increase signal intensity, lower background, and have been shown to reduce antibody consumption by up to 50%.

1. Use SigMax 1Ab-TBS when diluting capture or primary detection antibodies.
2. Use SigMax 2Ab-TBS for the labeled detection antibody, HRP-conjugate, or streptavidin-conjugate step.

For phospho-specific ELISAs and other low-abundance targets, the PEG/PVP polymer environment also helps preserve native epitope conformation, a problem that BSA-only or milk-only diluents do not solve.

Universal Blocking Buffer is formulated to suppress non-specific protein adsorption on the polystyrene plate surface — the root cause of the high background described in Problem 1. Use it as your default blocker in these scenarios:

1. Phospho-specific ELISAs — non-fat milk contains casein with phosphate groups that cross-react with phospho-antibodies and inflate background.
2. Biotin-streptavidin detection systems — milk and casein both contain endogenous biotin and will compete with the detection conjugate.
3. Multiplex panels — a single buffer must be compatible across every well; switching blockers between targets introduces variability.
4. Unknown sensitivity — when you do not yet know whether your antibody pair is sensitive to milk-derived proteins, the universal formulation is the safe choice.

HRPQuenchTBS is an endogenous-peroxidase quenching solution in a TBS matrix. It is needed because many sample types carry endogenous peroxidase activity that mimics HRP signal and inflates background, producing false positives in HRP-based ELISAs:

1. Whole blood and serum — erythrocyte and leukocyte peroxidases.
2. Tissue lysates — particularly liver, spleen, and kidney.
3. Organoid and tumor lysates — macrophage-derived peroxidases.
4. Microbiome and gut samples — bacterial and host peroxidase activity.

Treating wells or samples with HRPQuenchTBS before the detection step inactivates this endogenous activity. Hydrogen peroxide based quenching is the established, peer-reviewed method for inactivating endogenous peroxidases in immunoassays and IHC.

ALPQuenchTBS targets endogenous alkaline phosphatase activity, which is the parallel problem to endogenous peroxidase but for ALP-based ELISA detection systems. Endogenous ALP is abundant in intestinal, placental, bone, and liver-derived samples and competes directly with the assay's labeled ALP, distorting both background and dynamic range.

1. Use HRPQuenchTBS when your kit uses an HRP-conjugate (commonly with TMB substrate).
2. Use ALPQuenchTBS when your kit uses an ALP-conjugate (commonly with pNPP, BCIP/NBT, or AMPPD substrate).

* ELISA kit include either B5 or B6 depending on HRP or ALP format of detection.

All Diagnocine Precision sterile buffers including the bonus reagents above, are filtered 0.1 micron once and 0.04 micron once. This is materially more rigorous than the industry-standard single 0.22 micron filtration.

For ELISA, this directly impacts:

1. Background — contaminating cellular debris and proteins inflate non-specific signal (Problem 1).
2. Standard curve linearity — variable bioburden between buffer batches scrambles linearity (Problem 4).
3. Inter-assay reproducibility — the documented effect of mycoplasma on host cell metabolism alters analyte expression in any cell-derived sample.

Linearity range, linearity, lower limit of detection, precision, recovery, stability, specificity and natural samples detection are carefully evaluated under strict QC standards to ensure good performance.

We offer positive and negative controls in qualitative ELISA kits, while we offer standards which can work as quality control in quantitative ELISA kits. Kit standards and the entire reagent system are matched. The customer can draw a good standard curve through the experiment, which proves that the whole system including the standards works well, so the standards can play a role of quality control. We suggest you run duplicates for the standards.

Different ELISA kits are designed for different species varying from human, rat, horse, rabbit, mouse, sheep, pig, bovine, dog, chicken, fish, monkey, plant, etc. Please refer to each protocol for species instruction. For different species, the antibody's specificity is different, and there is matrix interference. We can recommend the right kit if you have difficulty finding the kit of species you need.

It is recommended to run duplicated wells both for the standards and samples.

No, the reagents in the ELISA kits with different lot numbers cannot be mixed and we do not sell the components in the kit separately. Hope you could understand.

Some components in the ELISA kits contain BSA. We use commercial Proclin 300 as our preservative and 2N sulfuric acid as stop solution in most of ELISA kits. Please, Check the manual.

Incubation is performed at 37°C for many ELISA kits except for food safety & drug residues ELISA kits, as it is the best temperature for combination of antigen and antibody. We do not recommend room temperature since it is not stable.

TMB (3,3',5,5'-Tetramethylbenzidine) is a chromogenic substrate used in ELISA. TMB can act as a hydrogen donor for the reduction of hydrogen peroxide to water by peroxidase enzymes such as horseradish peroxidase. The reaction will be terminated and the color developed in the wells will turn from blue to yellow upon addition of the stop solution. The absorbance value should be read at 450 nm. We recommend you use dual-spectrum to test your sample to avoid the error caused by interference and scratch in the bottom. Please, check and verify your plate reader and filters.

ELISA is the abbreviation of the enzyme-linked immunosorbent assay used to identify the presence of specific proteins and to determine their concentrations. There are three kinds of ELISA including "sandwich", competitive and indirect ELISA. The common procedures are Storage, Reagents preparation, ELISA plate, Samples or reagents adding, Incubation, Washing and Reading. Please refer to our manual for details.

1. Incorrect reagents added. You should check the components and Lot No. of reagents to ensure correct reagents of the kit.
2. Insufficient plate washing. You should be sure all wells are filled with buffer with the same volume during every wash step. After final wash, blot plate forcefully on paper towel to remove residual buffer.
3. Contamination of the pipette tips and chromogenic agent container with the enzyme conjugate or contamination of blank wells with positive control. You should change pipette tips between reagents and use separate reservoirs for each reagent. Use pipette during operation.
4. Substrate exposed to light or contaminated prior to use. You should keep substrate in the dark until ready to dispense into wells.
5. Concentration of detection antibody or avidin-HRP is too high. You should check calculations or try again after further dilution.
6. Incubation time or color developing time is too long. You should follow the kit protocol strictly.
7. Wrong wavelength used when taking readings. Wavelength should be 450 nm with a 650 nm wavelength correction for TMB.

1. Reagents expired or improperly stored. You should keep the kit well stored according to the kit protocol and use it before expiration date. Avoid contamination.
2. Reagents and samples were not brought to room temperature prior to use. You should put all the reagents and samples under room temperature for 30 minutes.
3. Pipette suction of fluids insufficient, pipette emissions too fast, fluids on the tip wall too much or tip wall unclean. You should use calibrated pipettes to ensure the pipette tips firmly matched and pipette slowly. Single use is recommended.
4. Insufficient incubation time. You should set the timer accurate.
5. Insufficient color development time. You should develop color in 15-30 minutes. 20 minutes is the best unless otherwise instructed.
6. Color development reagent added in incorrect order. You should follow the kit protocol strictly.
7. Too much washing or improper dilution of concentrated wash buffer. You should reduce washing impact force: dilute the concentrated wash buffer, control the washing time, and record the washing times and dosage according to the manual.
8. Unqualified distilled water. You should check and ensure the pH is neutral when preparation of wash buffer is finished.
9. Chromogenic agent inactive with the contamination of enzyme conjugate during wash or sample addition steps. You should ensure no enzyme inhibitor in the enzyme conjugate container. Check and ensure the washing buffer container clean. Ensure the purified water no contamination.

1. NaN₃ preservatives from the samples inhibit the reaction of enzyme. You should avoid using this preservative in samples.
2. No strong positive samples on the detected ones, the result is normal. You should repeat the assay with signal enhancement and optimization reagents.

Wrong filter is used when taking readings. Wavelength should be 450 nm with a 650 nm wavelength correction for TMB.

1. Improper storage of the kit or poor storage environment. You should store all components as recommended on data sheet rather than room temperature for excess time.
2. Incorrect preparation of standard. You should reconstitute standard strictly with the recommended diluent according to the kit protocol. You should prepare reagents in 10 minutes prior to use and add them to wells promptly.
3. Insufficient mixing after adding samples. You should fully mix reagents in the vortex mixer when adding several reagents at the same time. Be careful when holding reagents to avoid splashing.
4. Poor repeatability of the plate readings. You should calibrate the plate reader.
5. Inconsistent incubation time, washing condition, color development condition and operators. You should repeat the assay of standard. Ensure consistent reactivity condition and operators.
6. Improper washing. You should add 200µL of wash buffer or fully fill into every well with pipette but no overflows are allowed. Check that all ports of the plate washer are unobstructed to ensure sufficient washing.
7. Uneven temperature. You should keep constant temperature during incubation to avoid temperature fluctuations.
8. Too much residual on the wall of the wells when adding or the bottom of wells scratched with pipette tip. You should lower the pipette tips along the wall of wells when adding slowly and carefully. Do not touch or scratch the bottom of the wells while mixing samples.
9. Reused materials. You should change pipette tips between samples and reservoirs between reagents.
10. Occasional positive and negative values close to the cut off value. You should set 3 duplicates for the same sample and the same result over 2 samples.
11. Cross contamination when adding samples. You should avoid cross contamination when adding samples.

1. Cross contamination during manual washing. You should reduce the cross contamination by promptly removing the liquid in wells after 3 times of filling washing buffer during manual washing and then setting the soak time the next times.
2. Cross contamination when patting the plate. You should use proper paper towels when patting the plate. Do not bring unrelated materials into the plate. Do not pat at the same place to avoid cross contamination.
3. Contamination due to long storage of samples. You should keep samples fresh or store them under low temperate to avoid contamination.
4. Abnormal developed color due to insufficient filling or too much residual when the plate washer is obstructed. You should fully fill into every well with pipette but no overflows are allowed. Check that all ports of the plate washer are unobstructed to ensure sufficient washing.
5. Coagulation or interference of precipitates or residual cell caused by incomplete centrifugation of samples. You should complete centrifugation of serum and plasma.
6. Wrong preparation of wash buffer or misuse of concentrated wash buffer. You should prepare wash buffer as manual required.

Yes. The strips are movable and it's recommended to separate those wells needed for assay and store the rest at 2-8°C in the dark without germ contamination. Not all ELISA kits are formatted and designed with strips. Keep the sealers on and detach the portion of the plate that you plan to use.