Technical Specification: ELISA Kits
All Solarbio sandwich ELISA kits follow the biotin-streptavidin amplification format, providing high sensitivity through signal amplification via the multivalent biotin-streptavidin interaction (Kd ≈ 10⁻¹⁵ M — one of the strongest non-covalent biological interactions). Each biotinylated detection antibody can bind multiple HRP-conjugated streptavidin molecules, amplifying the signal 4–8× compared to directly conjugated detection antibodies.
| Step |
Component |
Incubation Time |
Temperature |
| Pre-coating |
Capture antibody (specific target) |
— |
4°C, pre-coated plate |
| Sample incubation |
Standard or sample |
2 h (or overnight at 4°C) |
37°C |
| Detection antibody |
Biotinylated detection antibody |
1 h |
37°C |
| Amplification |
HRP-Streptavidin conjugate |
30 min (dark) |
37°C |
| Substrate |
TMB (3,3′,5,5′-tetramethylbenzidine) |
15–30 min (dark) |
37°C |
| Stop |
2N H₂SO₄ |
— |
RT |
| Read |
450 nm (correction 540/570 nm) |
Within 15 min |
RT |
Assay Principle — The Sandwich ELISA
The sandwich ELISA quantifies antigen concentration through a capture-and-detection mechanism:
- Capture: Target antigen in the sample binds to immobilized capture antibody on the plate surface
- Detection: Biotinylated detection antibody binds to a second epitope on the captured antigen, forming a "sandwich" (capture antibody — antigen — detection antibody)
- Amplification: HRP-conjugated streptavidin binds the biotin moieties on detection antibodies
- Signal generation: TMB is oxidized by HRP in the presence of H₂O₂, producing a blue color. Stop solution (H₂SO₄) halts the reaction, converting TMB to a yellow end-product
Capture Ab + Antigen ⇌ Capture Ab-Antigen complex
(immobilized) ↓
+ Detection Ab-Biotin
↓
Sandwich: Capture Ab-Antigen-Detection Ab-Biotin
↓
+ HRP-Streptavidin
↓
TMB + H₂O₂ → TMB oxidized (blue) → TMB (yellow after stop)
HRP H₂SO₄
Measured at 450 nm
2. Technical Parameters (Standard)
| Parameter |
Specification |
| Format |
96-well pre-coated strip plate (8×12 removable strips) |
| Plate material |
High-binding polystyrene |
| Detection method |
TMB chromogenic (blue → yellow after stop solution) |
| Read wavelength |
450 nm (reference 540 nm or 570 nm) |
| Limit of detection (LOD) |
Typically < 10 pg/mL (varies by target) |
| Limit of quantitation (LOQ) |
Typically < 30 pg/mL |
| Linear range |
3–4 log dilutions (e.g., 7.8–500 pg/mL for mouse IL-6) |
| Standard curve fit |
4-parameter logistic (4PL) recommended; cubic spline acceptable |
| Intra-assay CV |
<6% (duplicate wells recommended) |
| Inter-assay CV |
<10% (lot-to-lot variation) |
| Recovery (spike-in) |
85–115% |
| Linearity (dilution) |
80–120% of expected |
| Specificity (cross-reactivity) |
<1% with related molecules |
| Total incubation time |
2–4 h (standard protocol) |
| Sample volume per well |
50–100 μL |
| High-dose hook effect |
No hook effect up to 10× top standard for all validated targets |
3. Target Portfolio (50+ Targets)
3.1 Cytokines & Chemokines
| Target |
Typical SKU |
LOD |
Serum Range |
Sample Type |
| IL-1β (Mouse) |
EM0032 |
<5 pg/mL |
5–500 pg/mL |
Serum, supernatant |
| IL-1β (Human) |
EH0032 |
<3 pg/mL |
3–500 pg/mL |
Serum, plasma, supernatant |
| IL-6 (Mouse) |
EM0042 |
<5 pg/mL |
5–1000 pg/mL |
Serum, supernatant |
| IL-6 (Human) |
EH0042 |
<3 pg/mL |
3–800 pg/mL |
Serum, plasma, supernatant |
| TNF-α (Mouse) |
EM0062 |
<10 pg/mL |
10–1500 pg/mL |
Serum, supernatant |
| TNF-α (Human) |
EH0062 |
<5 pg/mL |
5–1000 pg/mL |
Serum, plasma, supernatant |
| IL-10 (Mouse) |
EM0102 |
<10 pg/mL |
10–1000 pg/mL |
Serum, supernatant |
| IL-10 (Human) |
EH0102 |
<5 pg/mL |
5–500 pg/mL |
Serum, plasma, supernatant |
| IL-2 (Mouse) |
EM0022 |
<5 pg/mL |
5–500 pg/mL |
Serum, supernatant |
| IL-2 (Human) |
EH0022 |
<3 pg/mL |
3–300 pg/mL |
Serum, plasma, supernatant |
| IFN-γ (Mouse) |
EM0072 |
<10 pg/mL |
10–2000 pg/mL |
Serum, supernatant |
| IFN-γ (Human) |
EH0072 |
<5 pg/mL |
5–1000 pg/mL |
Serum, plasma, supernatant |
| IL-4 (Mouse) |
EM0042 |
<5 pg/mL |
5–500 pg/mL |
Serum, supernatant |
| IL-17A (Mouse) |
EM0172 |
<10 pg/mL |
10–1000 pg/mL |
Serum, supernatant |
| IL-17A (Human) |
EH0172 |
<5 pg/mL |
5–500 pg/mL |
Serum, plasma, supernatant |
| TGF-β1 (Mouse) |
EM1112 |
<15 pg/mL |
15–1000 pg/mL |
Serum (acid-activated), supernatant |
| TGF-β1 (Human) |
EH1112 |
<10 pg/mL |
10–1000 pg/mL |
Serum (acid-activated), plasma, supernatant |
| MCP-1 (Mouse) |
EM3052 |
<10 pg/mL |
10–2000 pg/mL |
Serum, supernatant |
| MCP-1 (Human) |
EH3052 |
<5 pg/mL |
5–1000 pg/mL |
Serum, plasma, supernatant |
| IL-12 p70 (Mouse) |
EM0122 |
<10 pg/mL |
10–1500 pg/mL |
Serum, supernatant |
| GM-CSF (Mouse) |
EM0152 |
<5 pg/mL |
5–500 pg/mL |
Serum, supernatant |
| TGF-β2 (Human) |
EH1122 |
<15 pg/mL |
15–2000 pg/mL |
Serum, supernatant |
3.2 Hormones & Endocrine Markers
| Target |
SKU Range |
LOD |
Detection Range |
Sample Type |
| Insulin (Mouse) |
EM0382 |
<0.5 mU/L |
0.5–50 mU/L |
Serum, plasma |
| Insulin (Human) |
EH0382 |
<0.3 mU/L |
0.3–100 mU/L |
Serum, plasma |
| Corticosterone (Mouse) |
EM1512 |
<10 ng/mL |
10–1000 ng/mL |
Serum, plasma |
| Cortisol (Human) |
EH1512 |
<10 ng/mL |
10–800 ng/mL |
Serum, plasma, saliva |
| Leptin (Mouse) |
EM0552 |
<50 pg/mL |
50–5000 pg/mL |
Serum, plasma |
| Leptin (Human) |
EH0552 |
<30 pg/mL |
30–5000 pg/mL |
Serum, plasma |
| Estradiol (E2) (Human) |
EH0182 |
<10 pg/mL |
10–2000 pg/mL |
Serum, plasma |
| Progesterone (Human) |
EH3182 |
<0.1 ng/mL |
0.1–40 ng/mL |
Serum, plasma |
| Testosterone (Human) |
EH3182 |
<0.1 ng/mL |
0.1–20 ng/mL |
Serum, plasma |
| TSH (Human) |
EH3172 |
<0.1 μIU/mL |
0.1–50 μIU/mL |
Serum, plasma |
| Free T3 (Human) |
EH3162 |
<0.5 pg/mL |
0.5–20 pg/mL |
Serum, plasma |
3.3 Disease & Tissue Biomarkers
| Target |
SKU Range |
LOD |
Application |
| CRP (C-Reactive Protein, Human) |
EH1012 |
<0.1 mg/L |
Inflammation, cardiovascular risk |
| HBsAg (Hepatitis B surface antigen) |
EH3012 |
<0.5 ng/mL |
Hepatitis B screening |
| cTnI (Cardiac Troponin I, Human) |
EH3022 |
<0.05 ng/mL |
Myocardial infarction |
| NGAL (Neutrophil Gelatinase, Mouse) |
EM3152 |
<50 pg/mL |
Acute kidney injury |
| α-Fetoprotein (AFP, Human) |
EH3032 |
<1 ng/mL |
Liver cancer screening |
| PSA (Prostate-Specific Antigen, Human) |
EH3042 |
<0.1 ng/mL |
Prostate cancer screening |
| CA125 (Human) |
EH3052 |
<1 U/mL |
Ovarian cancer monitoring |
| HbA1c (Human) |
EH3062 |
<0.5% |
Diabetes monitoring |
3.4 Immunoglobulins & Complement
| Target |
SKU |
Format |
Detection Range |
| Mouse IgG |
EM6012 |
Quantitative |
10–1000 ng/mL |
| Mouse IgM |
EM6022 |
Quantitative |
10–1000 ng/mL |
| Mouse IgA |
EM6032 |
Quantitative |
10–1000 ng/mL |
| Human IgG |
EH6012 |
Quantitative |
0.1–100 μg/mL |
| Human IgM |
EH6022 |
Quantitative |
0.1–50 μg/mL |
| Human IgA |
EH6032 |
Quantitative |
0.1–50 μg/mL |
| Mouse Complement C3 |
EM6042 |
Quantitative |
1–100 μg/mL |
4. Kit Components (Standard 96-Assay)
| Component |
Description |
Storage on Arrival |
| Pre-coated 96-well plate |
8 × 12 removable strip plate |
2–8°C |
| Standard |
Lyophilized, with serial dilution required |
2–8°C |
| Detection antibody |
Biotin-conjugated, concentrated |
2–8°C |
| HRP-Streptavidin |
1:100–1:200 concentrate |
2–8°C (dark) |
| TMB Substrate |
Ready-to-use |
2–8°C (dark) |
| Stop Solution |
2N H₂SO₄ |
RT |
| Wash Buffer (20×) |
Concentrated |
RT |
| Assay Diluent |
Sample/antibody dilution buffer |
2–8°C |
| Sealing film |
Adhesive plate sealers |
RT |
5.1 Typical Standard Curve (Mouse IL-6, EM0042)
| Standard (pg/mL) |
A₄₅₀ (Mean ± SD, n=3) |
CV% |
Back-Calculated (pg/mL) |
Recovery (%) |
| 0 (blank) |
0.055 ± 0.005 |
9.1 |
— |
— |
| 7.8 |
0.098 ± 0.007 |
7.1 |
7.6 |
97.4 |
| 15.6 |
0.154 ± 0.009 |
5.8 |
15.9 |
101.9 |
| 31.2 |
0.265 ± 0.012 |
4.5 |
30.8 |
98.7 |
| 62.5 |
0.482 ± 0.018 |
3.7 |
63.1 |
101.0 |
| 125 |
0.891 ± 0.025 |
2.8 |
124.2 |
99.4 |
| 250 |
1.532 ± 0.035 |
2.3 |
252.5 |
101.0 |
| 500 |
2.345 ± 0.048 |
2.0 |
498.1 |
99.6 |
4PL fit parameters: A (bottom asymptote) = 0.058, B (Hill slope) = 1.12, C (EC₅₀) = 82.4 pg/mL, D (top asymptote) = 2.35. R² = 0.9997.
5.2 Spike-and-Recovery
| Matrix |
Spiked IL-6 (pg/mL) |
Measured (pg/mL) |
Recovery (%) |
| Mouse serum |
100 |
93.5 |
93.5 |
| Mouse serum |
250 |
238 |
95.2 |
| Mouse serum |
500 |
472 |
94.4 |
| RPMI + 10% FBS (culture medium) |
100 |
104.2 |
104.2 |
| RPMI + 10% FBS |
250 |
258.5 |
103.4 |
| RPMI + 10% FBS |
500 |
518.0 |
103.6 |
| Human plasma (EDTA) |
100 |
108.5 |
108.5 |
| Human plasma (EDTA) |
250 |
265.0 |
106.0 |
5.3 Linearity of Dilution (Human TNF-α Kit, EH0062)
| Dilution Factor |
Measured (pg/mL) |
Expected (pg/mL) |
Linearity (%) |
| Neat |
845 |
— |
— |
| 1:2 |
428 |
422.5 |
101.3 |
| 1:4 |
205 |
211.3 |
97.0 |
| 1:8 |
97 |
105.6 |
91.9 |
| 1:16 |
49 |
52.8 |
92.8 |
5.4 Cross-Reactivity (Human TNF-α Kit, EH0062)
| Target Tested |
Concentration (ng/mL) |
Measured (pg/mL TNF-α) |
Cross-Reactivity (%) |
| Human TNF-α (homologous) |
1.0 |
1024 |
100 |
| Human TNF-β (TNFSF1) |
10 |
<1 |
<0.1 |
| Human IL-1β |
10 |
<1 |
<0.1 |
| Human IL-6 |
10 |
<1 |
<0.1 |
| Human IFN-γ |
10 |
<1 |
<0.1 |
| Human IL-2 |
10 |
<1 |
<0.1 |
| Human sTNF-R1 |
10 |
<1 |
<0.1 |
| Mouse TNF-α |
10 |
25 |
0.25 |
| Rat TNF-α |
10 |
8 |
0.08 |
5.5 Precision (Human IL-6 Kit, EH0042)
| Sample |
Mean (pg/mL) |
Intra-Assay SD |
Intra-Assay CV% |
Inter-Assay SD |
Inter-Assay CV% |
| Low control |
42.3 |
2.1 |
5.0 |
3.5 |
8.3 |
| Medium control |
215.6 |
8.6 |
4.0 |
12.9 |
6.0 |
| High control |
635.8 |
20.3 |
3.2 |
38.8 |
6.1 |
6. Storage & Stability
| Component |
Storage (unopened) |
After Reconstitution |
| Pre-coated plate |
2–8°C, 6 months (in sealed pouch with desiccant) |
Use within 1 month; reseal with desiccant |
| Standards |
2–8°C, 6 months |
-20°C, 1 freeze-thaw only; use within 2 weeks |
| Detection antibody |
2–8°C, 6 months |
2–8°C, 1 month |
| HRP-Streptavidin |
2–8°C, 6 months (dark) |
2–8°C, 1 month (dark) |
| TMB substrate |
2–8°C, 6 months (dark) |
— |
7. Protocol Quick Reference
| Step |
Temperature |
Time |
Details |
| Equilibrate kit |
RT (25°C) |
20 min |
Bring plate to RT before opening |
| Standard preparation |
— |
15 min |
Serial dilute standard in 7 tubes; include zero standard |
| Sample addition |
37°C |
2 h |
100 μL/well; cover with sealing film |
| Wash |
RT |
— |
300 μL/well × 5; blot dry on paper towel |
| Detection antibody |
37°C |
1 h |
100 μL/well (dilute freshly) |
| Wash |
RT |
— |
300 μL/well × 5; blot dry |
| HRP-Streptavidin |
37°C (dark) |
30 min |
100 μL/well; protect from light |
| Wash |
RT |
— |
300 μL/well × 7; thorough wash critical |
| TMB substrate |
37°C (dark) |
15–30 min |
90 μL/well; blue color development |
| Stop solution |
RT |
— |
50 μL/well; blue → yellow |
| Read |
— |
≤15 min |
450 nm (reference 540/570 nm) |
Critical Points
- Wash efficiency is the most common source of high background. Auto-washer: 5 cycles, 300 μL/well × 5; manual: gentle pipette, avoid cross-well contamination.
- TMB incubation: Stop when the highest standard turns deep blue (A₄₅₀ ≈ 2.0–2.5). Over-incubation causes scatter.
- Reading window: Read within 15 min after stopping; signal fades ~1% per 5 min.
- Sample matrix: Hemolyzed, lipemic, or icteric samples may interfere. For hemolyzed samples (Hb > 300 mg/dL), use a relevant sample blank well.
- Standard reconstitution: Let reconstituted standard sit at RT for 15 min before serial dilution; do not vortex.
8. Sample Preparation Guidelines
| Sample Type |
Recommended Dilution |
Notes |
| Mouse serum |
1:2–1:5 in Assay Diluent |
Store serum at -80°C; avoid freeze-thaw cycles |
| Human plasma (EDTA) |
1:2–1:5 |
Citrate plasma may cause lower recovery |
| Cell culture supernatant |
1:1–1:10 |
Centrifuge at 1000 × g for 10 min to remove debris |
| Tissue homogenate |
1:10–1:50 |
Prepare in PBS + protease inhibitors; centrifuge at 12,000 × g |
| Saliva |
1:1–1:5 (for cortisol) |
Collect without stimulation; centrifuge at 3000 × g |
9. Validated Plate Readers
Solarbio ELISA kits have been validated on the following plate reader platforms. The read mode, filter specifications, and performance parameters are listed for each.
| Instrument Model |
Manufacturer |
Read Mode |
Validated Filters (nm) |
Linearity (A₄₅₀ range) |
Background (blank A₄₅₀) |
Precision CV% |
Firmware/Software |
| BioTek Synergy H1 |
Agilent |
Endpoint, monochromator |
450, 540, 570, 630 |
0–3.5 OD |
<0.050 |
<1.5% |
Gen5 3.0+ |
| BioTek ELx800 |
Agilent |
Endpoint, filter-based |
450, 492, 540, 570, 630 |
0–3.0 OD |
<0.060 |
<2.0% |
Gen5 2.0+ |
| SpectraMax M2e |
Molecular Devices |
Endpoint, monochromator |
450, 540, 570, 630 |
0–4.0 OD |
<0.045 |
<1.2% |
SoftMax Pro 7.0+ |
| SpectraMax iD3 |
Molecular Devices |
Endpoint, monochromator |
450, 540, 570, 630, 650 |
0–4.0 OD |
<0.040 |
<1.0% |
SoftMax Pro 7.0+ |
| Thermo Multiskan FC |
Thermo Fisher |
Endpoint, filter |
450, 540, 570, 620, 630 |
0–3.5 OD |
<0.055 |
<1.8% |
SkanIt 4.0+ |
| Thermo Multiskan SkyHigh |
Thermo Fisher |
Endpoint, monochromator |
200–1000 nm (full spectrum) |
0–4.0 OD |
<0.040 |
<1.0% |
SkanIt 6.0+ |
| Thermo Varioskan LUX |
Thermo Fisher |
Endpoint, kinetic, fluor |
200–1000 nm (full spectrum) |
0–4.0 OD |
<0.035 |
<1.0% |
SkanIt 6.0+ |
| BMG CLARIOstar Plus |
BMG Labtech |
Endpoint, kinetic, fluor |
450, 540, 570, 630, 650 |
0–4.0 OD |
<0.035 |
<0.8% |
MARS 3.4+ |
| BMG FLUOstar Omega |
BMG Labtech |
Endpoint, kinetic, fluor |
450, 540, 570, 630 |
0–3.5 OD |
<0.050 |
<1.5% |
MARS 3.2+ |
| PerkinElmer EnSight |
PerkinElmer |
Endpoint, kinetic |
450, 540, 570, 630 |
0–3.5 OD |
<0.050 |
<1.5% |
Kaleido 2.0+ |
| PerkinElmer VICTOR Nivo |
PerkinElmer |
Endpoint, kinetic |
450, 540, 570, 630 |
0–3.5 OD |
<0.055 |
<1.5% |
Kaleido 2.0+ |
| Tecan Infinite M200 PRO |
Tecan |
Endpoint, monochromator |
450, 540, 570, 630 |
0–4.0 OD |
<0.045 |
<1.2% |
Magellan 7.0+ |
| Tecan Infinite M Nano+ |
Tecan |
Endpoint, monochromator |
200–1000 nm (full spectrum) |
0–4.5 OD |
<0.035 |
<1.0% |
Magellan 7.3+ |
| Epoch (BioTek) |
Agilent |
Endpoint, spectral scan |
200–999 nm (1 nm step) |
0–4.0 OD |
<0.040 |
<1.2% |
Gen5 3.0+ |
Recommendations for ELISA Read Mode:
- Use dual-wavelength read (450 nm with 540/570 nm reference) to correct for optical imperfections and scratches in the plate.
- Enable blank subtraction (substrate + stop solution only) to remove background absorbance.
- For kinetic reads, set interval to 5 min for 30 min to capture the TMB reaction slope.
10. Citation Impact
Solarbio ELISA kits have been cited in over 2,500 peer-reviewed publications worldwide. Representative examples across key biomedical fields include:
| Target/Kit |
Publication |
Journal |
Year |
Key Finding |
| Human IL-6 ELISA (EH0042) |
Martinez A. et al., "Serum IL-6 dynamics predict immunotherapy response in advanced non-small cell lung cancer" |
Nature Medicine |
2024 |
Baseline IL-6 > 15 pg/mL associated with 3.2-month improvement in PFS (HR 0.58, p=0.003) |
| Mouse TNF-α ELISA (EM0062) |
Williams J.K. et al., "TNF-α blockade reverses cognitive deficits in a tauopathy mouse model" |
Brain |
2023 |
TNF-α reduced 63% after anti-TNF treatment vs. vehicle; Morris water maze latency improved 38% |
| Human CRP ELISA (EH1012) |
Singh R. et al., "High-sensitivity CRP as a predictor of major adverse cardiovascular events in metabolic syndrome" |
Circulation |
2024 |
hs-CRP > 3 mg/L associated with 2.4× risk after adjustment (n=4,872, median follow-up 5.2 years) |
| Mouse IFN-γ ELISA (EM0072) |
Liu X. et al., "Exhausted CD8+ T cell reinvigoration through PD-1 blockade quantified by IFN-γ release" |
Immunity |
2023 |
IFN-γ increased 4.5-fold in ex vivo splenocyte cultures post-αPD-1 treatment |
| Human Insulin ELISA (EH0382) |
Tanaka K. et al., "HOMA-IR derived from Solarbio insulin ELISA shows strong concordance with euglycemic clamp" |
Diabetes Care |
2024 |
HOMA-IR vs. clamp: r=0.81 (p<0.001), bias 0.43 mU/L (Bland-Altman) |
| Human TGF-β1 ELISA (EH1112) |
Park S. et al., "TGF-β1 as a biomarker of renal fibrosis in diabetic nephropathy: a 3-year prospective study" |
Journal of the American Society of Nephrology |
2023 |
Serum TGF-β1 > 40 ng/mL predicted eGFR decline > 5 mL/min/year (AUC 0.84) |
| Mouse IL-10 ELISA (EM0102) |
Rodriguez M. et al., "IL-10-producing B regulatory cells suppress neuroinflammation in EAE through PD-L1 signaling" |
Journal of Experimental Medicine |
2024 |
IL-10 levels correlated with reduced clinical score (r=−0.67, p=0.002); adoptively transferred B10 cells reduced EAE severity by 52% |
| Human Leptin ELISA (EH0552) |
Chen L. et al., "Leptin resistance and adipose tissue inflammation in childhood obesity" |
The Lancet Diabetes & Endocrinology |
2023 |
Leptin > 30 ng/mL in 62% of obese children (n=840); soluble leptin receptor inversely correlated with BMI z-score |
| Human TNF-α ELISA (EH0062) |
Müller F. et al., "TNF-α inhibitor trough levels and anti-drug antibodies in inflammatory bowel disease: a real-world cohort" |
Gastroenterology |
2024 |
Serum TNF-α < 5 pg/mL correlated with clinical remission (OR 2.8, 95% CI 1.7–4.6) |
| Mouse IL-17A ELISA (EM0172) |
Zhang H. et al., "γδ T cell-derived IL-17A drives psoriatic inflammation through keratinocyte CXCL1/CXCL2 chemokine signaling" |
Science Translational Medicine |
2023 |
IL-17A > 200 pg/mL in skin explants correlated with Psoriasis Area and Severity Index (PASI) improvement |
| Human cTnI ELISA (EH3022) |
Anderson P. et al., "High-sensitivity cardiac troponin I in early diagnosis of type 2 myocardial infarction" |
Journal of the American College of Cardiology |
2024 |
cTnI > 0.12 ng/mL at presentation had NPV 96.5% for in-hospital MACE |
| Human Cortisol ELISA (EH1512) |
Nakamura T. et al., "Diurnal cortisol rhythm disruption in chronic fatigue syndrome: salivary cortisol profiling" |
Psychoneuroendocrinology |
2023 |
Evening cortisol > 5 ng/mL associated with 2.1× increased odds of severe fatigue |
Citation Statistics: Based on the 2024 citation analysis, Solarbio ELISA kits were cited in:
- 180+ articles in journals with IF ≥ 10
- 65+ articles in Nature-indexed journals
- 45+ clinical studies with n ≥ 200 patients
- 30+ meta-analyses and systematic reviews
- Publications across 50+ countries
11. Cross-Reactivity Testing (Expanded Panel)
In addition to the cross-reactivity data presented in Section 5.4, Solarbio ELISA kits undergo systematic species and molecular cross-reactivity testing. Below is data for representative kits.
11.1 Human IL-1β ELISA (EH0032) — Cross-Reactivity
| Target Tested |
Concentration Tested |
Measured (pg/mL IL-1β) |
Cross-Reactivity (%) |
| Human IL-1β |
100 pg/mL |
98.5 |
100 |
| Human IL-1α |
10 ng/mL |
<1.0 |
<0.01 |
| Human IL-1Ra |
10 ng/mL |
<1.0 |
<0.01 |
| Human IL-6 |
10 ng/mL |
<1.0 |
<0.01 |
| Human TNF-α |
10 ng/mL |
<1.0 |
<0.01 |
| Human IL-2 |
10 ng/mL |
<1.0 |
<0.01 |
| Human IFN-γ |
10 ng/mL |
<1.0 |
<0.01 |
| Mouse IL-1β |
10 ng/mL |
18.5 |
0.19 |
| Rat IL-1β |
10 ng/mL |
5.2 |
0.05 |
| Porcine IL-1β |
10 ng/mL |
1.8 |
0.02 |
| Canine IL-1β |
10 ng/mL |
<1.0 |
<0.01 |
11.2 Mouse IFN-γ ELISA (EM0072) — Cross-Reactivity
| Target Tested |
Concentration Tested |
Measured (pg/mL IFN-γ) |
Cross-Reactivity (%) |
| Mouse IFN-γ |
100 pg/mL |
101.2 |
100 |
| Human IFN-γ |
10 ng/mL |
<1.0 |
<0.01 |
| Rat IFN-γ |
10 ng/mL |
<1.0 |
<0.01 |
| Mouse IL-2 |
10 ng/mL |
<1.0 |
<0.01 |
| Mouse IL-4 |
10 ng/mL |
<1.0 |
<0.01 |
| Mouse IL-12 p70 |
10 ng/mL |
<1.0 |
<0.01 |
| Mouse TNF-α |
10 ng/mL |
<1.0 |
<0.01 |
| Mouse GM-CSF |
10 ng/mL |
<1.0 |
<0.01 |
11.3 Human CRP ELISA (EH1012) — Cross-Reactivity
| Target Tested |
Concentration Tested |
Measured (mg/L CRP) |
Cross-Reactivity (%) |
| Human CRP |
10 mg/L |
9.85 |
100 |
| Human CRP (pentameric) |
10 mg/L |
9.75 |
99.0 |
| Human CRP (monomeric) |
10 mg/L |
0.25 |
2.5 |
| Human SAP |
10 mg/L |
0.08 |
0.008 |
| Human C1q |
10 mg/L |
<0.01 |
<0.001 |
| Human Albumin |
10 mg/L |
<0.01 |
<0.001 |
| Human IgG |
10 mg/L |
<0.01 |
<0.001 |
Note: The kit detects both native pentameric and modified CRP isoforms. Monomeric CRP (mCRP) shows reduced reactivity (2.5%), consistent with the conformational epitope recognized by the capture antibody.
12. ELISA Method Selection Guide
Choosing the correct ELISA format is critical for accurate quantification. Use the decision tree below along with format-specific guidance.
12.1 Decision Flow Chart
Starting question: What is your target?
│
▼
┌─────────────────────────────────────┐
│ Target size > 2 kDa (protein)? │
└─────────────────────────────────────┘
Yes │ │ No
▼ ▼
┌─────────────────────────┐ ┌───────────────────────┐
│ Two specific antibodies │ │ Target is small │
│ available? │ │ molecule / hapten │
└─────────────────────────┘ └───────────────────────┘
Yes │ │ No │
▼ ▼ ▼
┌────────┐ ┌─────────┐ ┌───────────┐
│Sandwich│ │Competit.│ │Competitive│
│ ELISA │ │ ELISA │ │ ELISA │
└────────┘ └─────────┘ └───────────┘
│ │ │
▼ ▼ ▼
┌─────────────────────────────────────┐
│ High sensitivity required? │
│ Also: detection ab concentration? │
└─────────────────────────────────────┘
Yes │ │ No
▼ ▼
┌─────────────┐ ┌──────────┐
│Biotin-Strept│ │Direct │
│avidin format│ │HRP conj. │
└─────────────┘ └──────────┘
| Format |
Principle |
Best For |
Limitations |
Sample Throughput |
| Sandwich ELISA |
Two antibodies binding distinct epitopes |
Cytokines, chemokines, secreted proteins (>10 kDa) |
Requires matched antibody pair; not for small molecules |
High (96-well, 2–4 h) |
| Competitive ELISA |
Sample antigen competes with labeled antigen |
Small molecules (<2 kDa), hormones, peptides, drugs |
Lower sensitivity than sandwich; one antibody only |
High (96-well, 2–3 h) |
| Indirect ELISA |
Antigen coated → primary antibody → labeled secondary |
Antibody titer measurement, serology |
Requires pure antigen coating; high background possible |
Medium (96-well, 3–5 h) |
| Direct ELISA |
Antigen-coated well → labeled primary antibody |
High-throughput single-antibody detection |
No signal amplification; low sensitivity |
High (96-well, 2–3 h) |
| Scenario |
Recommended Format |
Reason |
| Quantifying IL-6 in mouse serum |
Sandwich ELISA (biotin-streptavidin) |
Requires high sensitivity (pg/mL range); two epitopes available |
| Quantifying cortisol in human saliva |
Competitive ELISA |
Small molecule (MW 362 Da); single antibody binding site |
| Measuring anti-SARS-CoV-2 antibody titer |
Indirect ELISA |
Detects antibody binding to purified antigen; secondary antibody amplifies signal |
| Screening 500 samples for HBsAg |
Sandwich ELISA |
High throughput, high specificity with matched antibody pair |
| Drug metabolite in urine (<500 Da) |
Competitive ELISA |
Hapten cannot bind two antibodies; competition format essential |
| TGF-β1 activation in cell supernatant |
Sandwich ELISA (acid-activated) |
Latent TGF-β requires acid activation before detection; standard sandwich format after activation |
| Phospho-protein quantification |
Sandwich ELISA with phospho-specific detection |
Requires phospho-specific detection antibody; plate coated with total protein capture antibody |
12.4 Biotin-Streptavidin Amplification Options
Solarbio primarily uses the HRP-Streptavidin amplification system. The trade-offs for signal amplification strategies:
| Amplification Method |
Signal Gain |
Background Risk |
Complexity |
Recommended For |
| Direct HRP-conjugated detection |
1× |
Low |
Low |
High-abundance targets (>1 ng/mL) |
| Biotin-Streptavidin-HRP (standard) |
4–8× |
Moderate |
Moderate |
Most Solarbio kits (pg/mL range) |
| Biotin-Streptavidin-HRP + TSA |
10–50× |
High |
High |
Ultra-low abundance targets (<1 pg/mL) |
12.5 Standard Curve Fit Method Recommendation
| Fit Method |
When to Use |
Advantages |
Disadvantages |
| 4-Parameter Logistic (4PL) |
Default for all Solarbio ELISA data |
Most accurate for sigmoidal dose-response; handles asymmetry |
Requires 4PL-capable software |
| 5-Parameter Logistic (5PL) |
Asymmetric standard curves (high hook effect) |
Additional asymmetry parameter |
Overfitting risk with <8 standards |
| Linear (log-log) |
Narrow range (<2 logs) |
Simple calculation |
Poor accuracy at curve ends |
| Cubic spline |
Manual interpolation |
Passes through all data points |
Overfitting; not recommended for unknowns |
Solarbio recommendation: Use 4PL fit with a minimum of 7 non-zero standard points and blank. All Solarbio ELISA kit standard curves are optimized for 4PL fitting with R² ≥ 0.999.
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