In the field of immunoassays, the Enzyme-Linked Immunosorbent Assay (ELISA) is a gold-standard technique. Among the various types of ELISA kits available, Uncoated ELISA Kits stand out for their unparalleled flexibility, making them the go-to choice for researchers developing custom assays or targeting novel biomarkers. This guide provides a deep dive into everything you need to know about uncoated ELISA kits.
Table of Contents
1. What are Uncoated ELISA Kits?
2. How Does an Uncoated ELISA Assay Work?
3. Benefits of using Uncoated ELISA Kits
4. Application Scenarios of Uncoated ELISA Kits
5. Coated vs Uncoated ELISA Kits vs Antibody Pairs
6. Common troubleshooting tips for uncoated ELISA kits
01 What are Uncoated ELISA Kits?
The Uncoated ELISA Kit is a detection kit that supplies only the core components required for ELISA, including validated capture antibodies, detection antibodies, standards, and other reagents, but it does not include a microplate pre-coated with the capture antibody. Users must perform the antibody coating step themselves, enabling highly customizable control over the detection system. These kits are widely used in scientific method development, high-throughput screening, and cost-sensitive testing scenarios.
The components of Elabscience® Uncoated ELISA kits include:
◆ Capture Antibody
◆ Reference Standard
◆ Biotinylated Detection Antibody
◆ HRP Conjugate
◆ Micro ELISA pre-Plate (uncoated ELISA plates)
Table 1. Ancillary reagents for Uncoated ELISA Kits
|
Cat. No. |
Product Name |
Size |
|
E-ELIR-K001 |
96T*5 |
Note: The kit contains a full set of ancillary reagents to complete the 96T*5 ELISA assay.
Fig. 1 Uncoated ELISA Kit Product
02 How Does an Uncoated ELISA Assay Work?
The uncoated ELISA assay (uncoated ELISA test) is typically used for sandwich ELISA, where the target analyte is detected using two specific antibodies.
The principle of double-antibody sandwich ELISA is to bind the specific antibody to the solid phase carrier to form the solid phase antibody, combine with the corresponding antigen in the sample to be tested to form the antigen-antibody immune complex, then add the enzyme-labeled antibody, combine with the antigen in the immune complex to form the solid phase antigen-antigen-enzyme labeled antibody complex, add the substrate to show color, and finally determine the antigen content. The final chromogenic result is proportional to the amount of antigen to be detected [1, 2].
The specific steps of the Uncoated ELISA kit include:
Fig. 2 Uncoated ELISA Protocol
03 Benefits of using Uncoated ELISA Kits
Pretreatment and optimized: Matched antibody pairs and Micro ELISA pre-Plate are carefully screened and validated, greatly reducing the waste of time and money in the establishment and optimization of ELISA system.
Flexible packaging: Flexible packaging of various specifications to meet the testing requirements of different sample quantities.
Multiple targets: Over 700 Uncoated ELISA Kits extensively cover different targets in a variety of biological processes and sample types.
Multiple detection methods: Kit provides the Capture & Detection Antibody and HRP Conjugate. In addition to complete colorimetric ELISA with Micro ELISA pre-Plate, chemiluminescence CLIA or other methods can be tried.
Cost-effective: Cost-effective, for those who want to flexibly adjust coating concentration and match their own buffer solution to save costs, and explore detection system of special sample types, is the best choice.
The Elabscience® Uncoated Human IL-6 ELISA Kit delivers exceptional precision, outstanding lot-to-lot consistency, and reliable stability for accurate and reproducible results.
Product Performance Display
Precision
The Elabscience® Uncoated Mouse IL-6 ELISA Kit (Cat. No. E-UNEL-M0070):
Table 2. Intra assay and inter assay precision verification of Uncoated Mouse IL-6 ELISA Kit
|
|
Intra Assay Precision |
Inter Assay Precision |
||||
|
Sample |
1 |
2 |
3 |
1 |
2 |
3 |
|
n |
20 |
20 |
20 |
20 |
20 |
20 |
|
Mean (pg/mL) |
93.02 |
175.23 |
762.56 |
95.55 |
165.32 |
690.87 |
|
Standard deviation |
5.96 |
8.44 |
40.61 |
7.25 |
12.65 |
48.32 |
|
CV (%) |
6.41 |
4.82 |
5.33 |
7.59 |
7.65 |
6.99 |
%20and%20standard%20curve%20(right)%20from%202022%20to%202025.png)
Recovery
The Elabscience® Uncoated Human IL-6 ELISA Kit (Cat. No. E-UNEL-H0097):
Table 3. Recovery analysis of the Uncoated Human IL-6 ELISA Kit
|
Sample Type |
Range % |
Average Recovery % |
|
Serum (n=8) |
88-101 |
93 |
|
EDTA Plasma (n=8) |
93-103 |
98 |
|
Cell Culture Media (n=8) |
92-104 |
98 |
04 Application Scenarios of Uncoated ELISA Kits
Application
Operators who have limited budget, butskilled in establishing ELISA systems by themselves.
Operators who have detection needs for large amount of samples.
Experiments that require to pair special sample type with unique buffer system.
Recommended Elabscience® Uncoated ELISA Kits
The research areas covered by the Uncoated ELISA Kits include cancer, immunology, Cardiovascular, Signaling Transduction, Metabolism, Developmental Biology, etc.
Table 4. Products in the field of cancer research
|
Cat. No. |
Product Name |
Size |
|
E-UNEL-H0044 |
Uncoated Human EPO(Erythropoietin) ELISA Kit |
96T*5/96T*15 |
|
E-UNEL-R0055 |
Uncoated Rat TIMP-1(Tissue Inhibitors of Metalloproteinase 1) ELISA Kit |
96T*5/96T*15 |
|
E-UNEL-H0171 |
Uncoated Human TIMP-1(Tissue Inhibitors of Metalloproteinase 1) ELISA Kit |
96T*5/96T*15 |
|
E-UNEL-M0186 |
Uncoated Mouse αFP(Alpha-Fetoprotein) ELISA Kit |
96T*5/96T*15 |
Table 5. Products in the field of immunology research
|
Cat. No. |
Product Name |
Size |
|
E-UNEL-H0175 |
Uncoated Human TNF-α ELISA Kit (Human TNF alpha uncoated ELISA Kit) |
96T*5/96T*15 |
|
E-UNEL-M0044 |
Uncoated Mouse IgA ELISA Kit (IgA Mouse uncoated ELISA Kit) |
96T*5/96T*15 |
|
E-UNEL-H0097 |
Uncoated Human IL-6(Interleukin 6) ELISA Kit |
96T*5/96T*15 |
|
E-UNEL-M0070 |
Uncoated Mouse IL-6(Interleukin 6) ELISA Kit |
96T*5/96T*15 |
|
E-UNEL-R0030 |
Uncoated Rat IL-6(Interleukin 6) ELISA Kit |
96T*5/96T*15 |
|
E-UNEL-H0069 |
Uncoated Human IFN-γ(Interferon Gamma) ELISA Kit |
96T*5/96T*15 |
|
E-UNEL-M0043 |
Uncoated Mouse IFN-γ(Interferon Gamma) ELISA Kit |
96T*5/96T*15 |
|
E-UNEL-H0091 |
Uncoated Human IL-1β(Interleukin 1 Beta) ELISA Kit |
96T*5/96T*15 |
|
E-UNEL-M0064 |
Uncoated Mouse IL-1β(Interleukin 1 Beta) ELISA Kit |
96T*5/96T*15 |
|
E-UNEL-H0096 |
Uncoated Human IL-4(Interleukin 4) ELISA Kit |
96T*5/96T*15 |
|
E-UNEL-H0099 |
Uncoated Human IL-8(Interleukin 8) ELISA Kit |
96T*5/96T*15 |
Table 6. Products in the field of Cardiovascular research
|
Cat. No. |
Product Name |
Size |
|
E-UNEL-H0134 |
Uncoated Human PCSK9(Proprotein Convertase Subtilisin/Kexin Type 9) ELISA Kit |
96T*5/96T*15 |
|
E-UNEL-H0003 |
Uncoated Human ADP/Acrp30(Adiponectin) ELISA Kit |
96T*5/96T*15 |
|
E-UNEL-H0013 |
Uncoated Human TNNI3/cTn-I(Troponin I Type 3, Cardiac) ELISA Kit |
96T*5/96T*15 |
|
E-UNEL-H0047 |
Uncoated Human cTnT/TNNT2(Troponin T Type 2, Cardiac) ELISA Kit |
96T*5/96T*15 |
|
E-UNEL-M0194 |
Uncoated Mouse TFPI(Tissue Factor Pathway Inhibitor) ELISA Kit |
96T*5/96T*15 |
Table 7. Products in the field of Signaling Transduction research
|
Cat. No. |
Product Name |
Size |
|
E-UNEL-H0041 |
Uncoated Human EGFR(Epidermal Growth Factor Receptor) ELISA Kit |
96T*5/96T*15 |
|
E-UNEL-H0143 |
Uncoated Human FAS/CD95(Factor Related Apoptosis) ELISA Kit |
96T*5/96T*15 |
Table 8. Products in the field of Metabolism research
|
Cat. No. |
Product Name |
Size |
|
E-UNEL-H0101 |
Uncoated Human INS(Insulin) ELISA Kit |
96T*5/96T*15 |
|
E-UNEL-H0105 |
Uncoated Human LEP(Leptin) ELISA Kit |
96T*5/96T*15 |
|
E-UNEL-H0121 |
Uncoated Human OC/BGP(Osteocalcin) ELISA Kit |
96T*5/96T*15 |
|
E-UNEL-H0130 |
Uncoated Human OPG(Osteoprotegerin) ELISA Kit |
96T*5/96T*15 |
|
E-UNEL-H0185 |
Uncoated Human FGF21(Fibroblast Growth Factor 21) ELISA Kit |
96T*5/96T*15 |
|
E-UNEL-H0006 |
Uncoated Human ALB(Albumin) ELISA Kit |
96T*5/96T*15 |
Table 9. Products in the field of Developmental Biology research
|
Cat. No. |
Product Name |
Size |
|
E-UNEL-H0179 |
Uncoated Human VEGF165(Vascular Endothelial Growth Factor 165) ELISA Kit |
96T*5/96T*15 |
|
E-UNEL-M0085 |
Uncoated Mouse OPG(Osteoprotegerin) ELISA Kit |
96T*5/96T*15 |
|
E-UNEL-H0289 |
Uncoated Human BMP-7(Bone Morphogenetic Protein 7) ELISA Kit |
96T*5/96T*15 |
|
E-UNEL-H0100 |
Uncoated Human INHB(Inhibin B) ELISA Kit |
96T*5/96T*15 |
05 Coated vs Uncoated ELISA Kits vs Antibody Pairs
When designing an ELISA experiment, uncoated kits, coated kits, and antibody pairs each offer varying levels of convenience and flexibility. To help you make an informed decision, the table below provides a detailed comparison of their key characteristics.
Table 10. Comparison of three products: Uncoated, Coated ELISA Kits, Antibody Pairs
|
Feature |
Uncoated ELISA Kit |
Coated ELISA Kit |
Antibody Pair |
|
Format |
Core reagents Plate(Uncoated) |
Core reagents Plate(Pre-coated) |
Capture antibodies Detection antibodies |
|
Coating |
Required |
Not required |
Required |
|
Flexibility |
++ |
+ |
+++ |
|
Convenience |
++ |
+++ |
+ |
|
Time |
++ |
+++ |
+ |
|
Cost |
++ |
+ |
+++ |
|
Technical Skill |
Requires experienced users for optimization. |
Suitable for all user levels, including beginners. |
Requires expert-level knowledge in assay development. |
|
Application |
Assay development |
Routine testing |
Building a completely bespoke assay from the ground up |
06 Common troubleshooting tips for Uncoated ELISA kits
Uncoated ELISA kits offer valuable flexibility, but the self-coating process introduces additional critical steps where any oversight can compromise your results. To facilitate efficient troubleshooting, the table below provides a systematic summary of common issues, their potential causes, and practical solutions to help you quickly pinpoint the root cause.
Table 11. Common troubleshooting tips
|
Problem |
Causes |
Solutions |
|
Poor standard curve |
Inaccurate pipetting |
Check pipettes. |
|
Improper standard dilution |
Ensure briefly spin the vial of standard and dissolve the powder thoroughly by gentle mixing. |
|
|
Wells are not completely aspirated |
Completely aspirate wells in between steps. |
|
|
Low signal |
Insufficient incubation time |
Ensure sufficient incubation time. |
|
Incorrect assay temperature |
Use recommended incubation temperature. Bring substrate to room temperature before use. |
|
|
Inadequate reagent volumes |
Check pipettes and ensure correct preparation. |
|
|
Improper dilution |
||
|
HRP conjugate inactive or TMB failure |
Mix HRP conjugate and TMB, rapid coloring. |
|
|
Deep color but low value |
Plate reader setting is not optimal |
Verify the wavelength and filter setting on the Microplate reader. |
|
Open the Microplate Reader ahead to pre-heat. |
||
|
Large CV |
Inaccurate pipetting |
Check pipettes. |
|
High background |
Concentration of target protein is too high |
Use recommended dilution factor. |
|
Plate is insufficiently washed |
Review the manual for proper wash. If using a plate washer, check that all ports are unobstructed. |
|
|
Contaminated wash buffer |
Prepare fresh wash buffer. |
|
|
Low sensitivity |
Improper storage of the ELISA kit |
All the reagents should be stored according to the instructions. |
|
Stop solution is not added |
Stop solution should be added to each well before measurement. |
For more valuable insights, stay tuned to Elabscience®!
References:
[1] Butler JE, Enzyme-linked immunosor- bent assay. J Immunoass 21(2-3) (2000) 165-209
[2] Hornbeck P, Enzyme-Linked Immunosorbent Assays. Curr Protoc Immunol 1 (1992) 2.1.1-2.1.22.
