Product Overview: Understanding α-Ketoglutaric Acid in Insect Research
α-Ketoglutaric Acid (α-KA) is a key intermediate in the tricarboxylic acid (TCA) cycle, playing a critical role in cellular energy metabolism, amino acid synthesis, and nitrogen transport. In insect research, α-KA serves as an essential metabolite linking carbohydrate metabolism to nitrogen excretion pathways, including the synthesis of uric acid—the primary nitrogenous waste in many insect species. Accurate quantification of α-KA in insect samples is fundamental for studies on insect physiology, metabolic adaptation, diapause, pesticide toxicology, and developmental biology.
Yanda Bio’s Customized Insect α-Ketoglutaric Acid (α-KA) ELISA Kit is specifically optimized for insect sample matrices, providing researchers with a sensitive and reliable tool for measuring this critical TCA cycle metabolite. As a professional ELISA kit manufacturer with a portfolio exceeding 6,000 detectable targets and established collaborations with over 60 universities across China, we combine deep expertise with rapid customization capabilities.
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Why Choose Yanda Bio for Your Custom Immunoassay Needs?
EEAT-Compliant Quality Standards
Our commitment to Experience, Expertise, Authoritativeness, and Trustworthiness ensures that every custom kit meets the highest research standards.
| Principle | Our Commitment |
|---|---|
| Experience | Over 6,000 detectable factors developed; 60+ university collaborations; years of specialized expertise in immunoassay manufacturing across diverse species including insects. |
| Expertise | Specialized R&D team with proprietary ELISA optimization protocols; proven expertise in adapting assays for unique sample matrices such as insect hemolymph and whole-body extracts. |
| Authoritativeness | Validated by academic research partners; publications citing our kits; ISO-aligned manufacturing standards; rigorous quality control at every development stage. |
| Trustworthiness | Rapid custom service with 3-7 day turnaround; transparent specifications; unconditional quality guarantee; reliable technical support from development through application. |
Rapid Custom Development for Insect Research
Need an assay for a unique insect species or specific sample type? Yanda Bio offers custom ELISA kit development services with a rapid 3-7 day turnaround—ideal for specialized entomological research applications. Our team will work with you to:
- Optimize antibody pairs for your target insect species
- Validate performance with your specific sample matrix
- Establish appropriate detection ranges and sensitivity
- Provide complete documentation and technical support
Two Optimized Kit Formats for Your Research Needs
To accommodate diverse experimental requirements and budget considerations, we offer this customized α-KA ELISA kit in two specialized formats.
Format 1: Standard High-Efficiency Kit
Designed for routine, reliable quantification of α-KA in standard insect research settings. This format employs a streamlined one-step incubation where antigen binds simultaneously with the solid-phase antibody and enzyme-labeled antibody. A proprietary sample diluent is included to effectively minimize interference from common matrix components such as hemolymph proteins, lipids, and pigments (e.g., melanin), ensuring stable baseline signals and optimal binding. With extensive validation demonstrating a success rate above 99%, this format represents exceptional value for most applications.
- Typical Pricing: Approximately $120 (retail). Bulk pricing available under $100 per kit.
- Special Offer: Free shipping on orders of three or more ELISA kits.
Format 2: Advanced High-Sensitivity Kit
Engineered for demanding applications where sample material is precious, limited, or where detection of very low α-KA concentrations is required. This advanced format utilizes a biotinylated recombinant monoclonal antibody (purity >95%) paired with a high-activity Streptavidin-HRP conjugate (≥500 U/mg). This signal amplification system delivers superior sensitivity, directly circumvents matrix effects from complex insect samples, and is designed to ensure success in critical, one-attempt experiments.
Selection Guide:
- Choose Format 1 for cost-effective, reliable analysis in routine metabolic studies with sufficient sample volume
- Choose Format 2 when working with irreplaceable samples (e.g., individual larvae, micro-dissected tissues), detecting subtle changes in low-level metabolites, or requiring maximum assay robustness
Assay Principle: Competitive ELISA Technology for Small Molecules
This kit employs a competitive inhibition enzyme immunoassay format, ideal for small molecule metabolites like α-KA where sandwich formats are not feasible. The microplate is pre-coated with an antibody specific for α-Ketoglutaric Acid. During incubation, α-KA in standards or samples competes with HRP-labeled α-KA (the tracer) for binding to the immobilized antibody. After washing to remove unbound tracer, TMB substrate is added. The HRP enzyme catalyzes a colorimetric reaction, producing a blue product that turns yellow upon acidification. The intensity of the color is inversely proportional to the concentration of α-KA in the sample—higher α-KA concentrations result in lower color intensity.
Sample Dilution Note: Samples are pre-diluted 2-fold during the procedure; multiply results by 2 to obtain actual concentrations.
Sample Collection & Preparation for Insect Samples
Proper sample handling is critical for accurate results. These protocols are optimized for insect research applications.
Liquid Samples
| Sample Type | Collection & Processing |
|---|---|
| Hemolymph | Collect insect hemolymph via microcapillary tube or puncture method. Centrifuge at 3000 rpm for 10 minutes at 2-8°C to remove hemocytes. Carefully collect plasma/serum fraction. Store at -80°C for long-term storage. |
| Cell Culture Supernatant | Collect using sterile tubes. Centrifuge at 3000 rpm for 20 minutes at 2-8°C. Carefully collect supernatant. Store at -20°C or -80°C. |
| Other Biological Fluids | For insect saliva, excretions, or other fluids: collect, centrifuge at 3000 rpm for 20 minutes at 2-8°C, collect supernatant. |
Solid Insect Samples
| Sample Type | Processing Protocol |
|---|---|
| Whole Insect Larvae/Adults | Flash freeze in liquid nitrogen. Grind to fine powder using mortar and pestle or tissue grinder. Add PBS (0.01M, pH 7.4) at 1:9 weight-to-volume ratio (1g tissue to 9mL PBS). Homogenize further if needed. Centrifuge at 5000×g for 10 minutes at 2-8°C. Collect supernatant. |
| Insect Tissues | Dissect tissue of interest (fat body, gut, flight muscle). Rinse with cold PBS. Follow same homogenization protocol as whole insects. |
| Insect Cells | For adherent cells: wash with cold PBS, detach with trypsin, collect by centrifugation. For suspension cells: collect directly. Wash cells 3 times with cold PBS. Resuspend at 1×10⁷ cells/mL in cold PBS. Lyse by sonication. Centrifuge at 3000 rpm for 20 minutes at 2-8°C. Collect supernatant. |
Important Sample Notes
- Extract samples as soon as possible after collection. Perform assays promptly after extraction.
- For short-term storage (up to 6 days), samples can be stored at 2-8°C.
- For long-term storage, freeze at -20°C (≤1 month) or -80°C (≤2 months). Avoid repeated freeze-thaw cycles.
- If precipitate forms during storage, centrifuge again before testing.
- Do not use samples containing sodium azide (NaN₃), as it inhibits horseradish peroxidase (HRP) activity.
Materials Required But Not Provided
- Microplate reader capable of measuring absorbance at 450 nm
- Precision pipettes and tips (0.5-10 μL, 2-20 μL, 20-200 μL, 200-1000 μL)
- 37°C incubator or water bath
Important Notes for Assay Procedure
- Store the kit at 2-8°C. Allow all reagents to equilibrate to room temperature for 20 minutes before use. Concentrated wash buffer may crystallize; this is normal. Dissolve crystals by warming in a water bath.
- Return unused strips to the resealable bag with desiccant and store properly at 2-8°C. Use as soon as possible.
- The S0 standard (zero concentration) serves as a negative control/blank. Note that samples are pre-diluted 2-fold during the procedure; multiply results by 2 to obtain actual concentrations.
- Strictly adhere to the incubation times, volumes, and sequence specified in the protocol.
- Mix all liquid components thoroughly before use.
Reagent Preparation
20× Wash Buffer Dilution: Dilute 1 part 20× wash buffer with 19 parts distilled water to prepare 1× working wash buffer.
Washing Method
Manual Washing: Aspirate liquid from wells, fill each well with wash buffer, allow to stand for 1 minute, then aspirate. Invert plate and tap dry on absorbent paper. Repeat 5 times.
Automated Plate Washer: Inject 350 μL of wash buffer per well, allow to soak for 1 minute, and wash 5 times.
Assay Procedure
- Remove the required number of strips from the aluminum foil bag after 20 minutes of room temperature equilibration. Reseal unused strips and return to 4°C.
- Set up standard wells and sample wells. Add 50 μL of each standard concentration to the standard wells.
- For sample wells: Add 25 μL of test sample followed by 25 μL of sample diluent.
- Add 50 μL of HRP-labeled competitive antigen (tracer) to both standard and sample wells. Seal with a plate cover and incubate at 37°C for 60 minutes.
- Aspirate liquid, tap dry on absorbent paper, fill each well with wash buffer, let stand for 1 minute, aspirate, and tap dry. Repeat washing 5 times (automated washer acceptable).
- Add 50 μL of Substrate A and 50 μL of Substrate B to each well. Incubate at 37°C in the dark for 15 minutes.
- Add 50 μL of stop solution to each well. Measure the OD at 450 nm within 15 minutes.
Data Analysis
This competitive ELISA requires calculation of percent binding for accurate quantification.
Calculation Steps:
- Read OD Values: Record absorbance at 450 nm within 15 minutes of adding stop solution.
- Calculate Percent Binding:
- Let B0 = OD of S0 standard (zero concentration)
- Let B = OD of each standard or sample
- Let NSB = Non-specific binding (if applicable)
- Percent binding = (B / B0) × 100%
- Construct Standard Curve:
- Plot standard concentrations (x-axis, log scale) against percent binding (y-axis, linear scale) using logit-log or 4-parameter logistic (4PL) curve fitting.
- Calculate logit values: logit = ln[B/B0 / (1 – B/B0)]
- Determine Sample Concentrations:
- Calculate percent binding for each sample
- Interpolate concentration from the standard curve
- Multiply by the dilution factor (2×) to obtain actual sample concentration
Automated Analysis: Use ELISA data analysis software with logit-log or 4PL models for automatic curve fitting and concentration calculation.
Kit Performance Specifications
| Parameter | Performance |
|---|---|
| Accuracy | Standard curve linear regression coefficient (R) ≥ 0.9900 |
| Sensitivity | Minimum detectable concentration < 5 ng/mL |
| Detection Range | 50 – 2000 ng/mL (customizable based on insect species) |
| Specificity | No significant cross-reactivity with other TCA cycle intermediates or soluble structural analogs |
| Intra-assay CV | < 15% |
| Inter-assay CV | < 15% |
| Storage | 2-8°C, protected from light and moisture |
| Shelf Life | 6 months |
Customization Options
As a Customized Insect ELISA Kit, this product can be tailored to your specific research requirements:
| Custom Parameter | Available Options |
|---|---|
| Target Species | Any insect species (Lepidoptera, Diptera, Coleoptera, Hymenoptera, etc.) |
| Sample Type | Hemolymph, whole-body homogenate, tissue-specific extracts, cell lysates |
| Detection Range | Adjustable based on expected physiological levels |
| Sensitivity | Optimized for your specific application |
| Kit Format | Standard or high-sensitivity (biotin-streptavidin amplification) |
| Custom Controls | Species-specific matrix-matched controls available |
Custom Development Timeline: 3-7 days from specification confirmation to prototype delivery.
Special Offer
Purchase any three ELISA kits from Yanda Bio and enjoy free shipping. Combine our Customized Insect α-KA kit with our Pyruvate and Citrate kits to build a comprehensive TCA cycle metabolism research panel for your insect studies at exceptional value.
Comparison with Premium International Brands
Yanda Bio’s custom ELISA kits deliver performance comparable to leading international brands at a fraction of the cost—often 40-60% less than major commercial alternatives. Our efficient manufacturing process and direct-to-researcher business model eliminate unnecessary markups, allowing us to offer professional-grade immunoassays at highly competitive prices. With our 3-7 day custom development capability, we also deliver tailored solutions far faster than traditional custom service providers.
Disclaimer
- This product is for research use only. It is not intended for clinical diagnosis or human use. The user assumes all responsibility for any consequences arising from misuse.
- Strict adherence to the protocol is required. Yanda Bio is not responsible for results obtained from deviations from the specified procedure.
Ordering & Contact Information
To order this Customized Insect α-Ketoglutaric Acid (α-KA) ELISA Kit, or to inquire about custom development for other insect metabolites, volume discounts, and format selection guidance, please contact our sales team.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
