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**Human Mann's Split-Head Antibody (SM Ab) Enzyme-Linked Immunosorbent Assay (ELISA)**
**Purpose:**
This ELISA kit is designed to detect the presence of Human Mann’s Split-Head Antibody (SM Ab) in human serum, plasma, and other biological fluids. It provides a reliable and sensitive method for screening and quantifying SM Ab levels, which may be associated with specific autoimmune or inflammatory conditions.
**Principle of Operation:**
The assay follows a double-antigen sandwich immunoassay approach. The microtiter plate is pre-coated with purified antigens that specifically bind to SM Ab present in the sample. After incubation, unbound components are washed away. A horseradish peroxidase (HRP)-labeled secondary antigen is then added, forming an antigen-antibody-enzyme complex. Following another washing step, the substrate TMB is introduced, leading to a color change from blue to yellow in the presence of HRP. The optical density (OD) at 450 nm is measured using a microplate reader, and results are interpreted against a calculated cutoff value to determine the presence or absence of SM Ab in the sample.
**Sample Preparation and Storage:**
It is recommended to process samples as soon as possible after collection. If testing cannot be performed immediately, specimens should be stored at -20°C. Repeated freeze-thaw cycles should be avoided to maintain sample integrity. Note that samples containing sodium azide (NaN3) are not suitable for this test, as NaN3 can inhibit HRP activity and interfere with the enzymatic reaction.
**Procedure Overview:**
1. **Labeling:** Assign unique numbers to each well on the microtiter plate. Include 2 negative control wells, 2 positive control wells, and 1 blank control well.
2. **Loading:** Add 50 µL of negative and positive controls to their respective wells. For the test samples, add 40 µL of sample diluent followed by 10 µL of the actual sample into each sample well. Ensure gentle mixing without touching the sides of the wells.
3. **Incubation:** Cover the plate with a sealing film and incubate at 37°C for 30 minutes.
4. **Washing:** Prepare a 30x diluted washing solution using distilled water. Wash the plate 5 times, ensuring each well is filled, allowed to stand for 30 seconds, and then discarded.
5. **Enzyme Addition:** Add 50 µL of HRP-conjugated reagent to all wells except the blank control.
6. **Second Incubation:** Repeat the incubation step at 37°C for 30 minutes.
7. **Second Washing:** Perform the same washing procedure as above.
8. **Color Development:** Add 50 µL of TMB solution A and B to each well. Mix gently and incubate at 37°C for 15 minutes in the dark.
9. **Stop Reaction:** Add 50 µL of stop solution to each well to halt the reaction. The color will turn from blue to yellow.
10. **Measurement:** Read the OD values at 450 nm within 15 minutes of adding the stop solution using a microplate reader.
**Data Interpretation:**
- **Validity Check:**
- Positive Control Average ≥ 1.00
- Negative Control Average ≤ 0.10
- **Cutoff Value Calculation:**
Cutoff = Negative Control Average + 0.15
- **Result Determination:**
If the sample OD value is below the cutoff, it is considered negative. If it is above the cutoff, it is considered positive.
**Notes:**
For optimal results, follow all instructions carefully. Always use fresh reagents and ensure proper calibration of equipment. This kit is intended for research or diagnostic use only and should not be used for clinical diagnosis without validation.
**Download:**
[Human Mann's Split-Head Antibody (SM Ab) Kit Instructions.rar](#)
**Company Information:**
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**Disclaimer:**
All content provided is for informational purposes only. The accuracy and completeness of the information are not guaranteed. Users are encouraged to consult official documentation and professional guidance when interpreting results.
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