Southeast Asian-type α‑thalassemia Zeta-chain protein assay kit (ELISA)

Zeta-chain protein in Southeast Asian deletion-type alpha-thalassemia
Instruction Manual for the Test Kit (Enzyme-Linked Immunosorbent Assay)
[Product Name]
Generic Name: Southeast Asian Deletion-Type Alpha-Thalassemia Zeta-Chain Protein Detection Kit (ELISA)
[Packaging Specifications]
96 servings per box, 48 servings per box.
[Intended Use]
This kit is intended for the in vitro qualitative detection of Zeta-chain protein in human blood.
This kit is intended for the auxiliary diagnosis of Southeast Asian deletion-type α‑thalassemia in individuals aged 0.5 years and older.
Thalassemia is a hemolytic anemia caused by autosomal genetic defects that disrupt the synthesis of globin chains, leading to insufficient or complete absence of one or more globin polypeptides. This prevents the formation of normal hemoglobin, resulting in the premature destruction of red blood cells and a shortened lifespan. Thalassemia exhibits considerable genotypic and phenotypic heterogeneity; depending on the specific gene(s) affected, it is classified into α‑thalassemia and β‑thalassemia.
Among α‑thalassemias, the Southeast Asian deletion type (–/–SEA) is the most prevalent in Southeast Asia and southern China, representing the primary form of potential clinical concern. Although carriers of the (–/–SEA) genotype are largely asymptomatic, when two carriers marry, their offspring may develop hydrops fetalis or Hb H disease, imposing a substantial burden on families and society.
It is known that the hemoglobin zeta chain is present in the fetal stage of normal individuals but becomes undetectable by three months after birth; however, it persists throughout life in the blood of carriers of the Southeast Asian deletion-type α‑thalassemia. Consequently, the zeta chain can serve as a biomarker to aid in the diagnosis of this condition.
[Principle of Assay]
Principle of the sandwich ELISA: A microtiter plate is coated with a purified anti‑Zeta chain monoclonal antibody. Lysed blood samples to be tested are added; any Zeta chain present in the sample binds to the coating antibody, forming an antigen–antibody complex. A horseradish peroxidase (HRP)-labeled monoclonal antibody specific for a different epitope on the Zeta chain is then added, resulting in an immune complex comprising antibody–antigen–secondary antibody–enzyme conjugate. The plate is washed to remove unbound components, followed by the addition of a chromogenic substrate that generates a colored product. The color development is measured using a microplate reader, and the results are interpreted qualitatively based on the intensity of the signal.
[Main Ingredients]
1. Components included in this kit
Component Name Main Ingredient Usage Instructions Package Quantity
96 servings per box, 48 servings per box
1 Monoclonal Antibody Zeta Chain Coated Plate: Microtiter plate pre-coated with mouse-derived anti‑Zeta chain monoclonal antibody; can be separated into individual wells for direct use; available in 96‑well and 48‑well formats.
2 Monoclonal antibody Zeta-chain conjugate: horseradish peroxidase (HRP)-labeled anti‑Zeta-chain monoclonal antibody (mouse‑derived); borate buffer containing 0.1% Proclin‑300, 0.002% phenol red, and a protein stabilizer. Ready to use. 6 mL × 2 vials; 6 mL × 1 vial.
3 Zeta-chain protein positive control: blood from a carrier of α‑thalassemia, containing 40% glycerol and 0.02% thimerosal. Add 25 µL to 200 µL of sample lysis buffer and vigorously vortex for 60 seconds to ensure complete hemolysis of red blood cells. 0.5 mL × 1 vial; 0.5 mL × 1 vial.
4 Zeta-chain protein negative control: Normal human blood containing 40% glycerol and 0.02% thimerosal. Add 25 µL to 200 µL of sample lysis buffer, and vigorously vortex for 60 seconds to ensure complete hemolysis. 0.5 mL × 1 vial; 0.5 mL × 1 vial.
5 Zeta-chain protein critical control: Recombinant Zeta-chain protein, 40 ng/mL; borate buffer containing 40% glycerol, 0.2% thimerosal, and a protein stabilizer. For direct use. 1.5 mL × 1 vial, 1.5 mL × 1 vial.
6 Color-developing solution A contains citric acid and hydrogen peroxide; use directly. 6.5 mL × 1 bottle, 3.5 mL × 1 bottle.
7 Color Development Solution B contains citric acid and tetramethylbenzidine (TMB). Ready to use. 6.5 mL × 1 bottle, 3.5 mL × 1 bottle.
8 Concentrated Detergent (10×) Phosphate-buffered solution, containing Tween‑20; 10× concentrated. Dilute with distilled or deionized water to 1× before use. 50 mL × 1 bottle 50 mL × 1 bottle
9 Sample Lysis Buffer, containing 0.83% ammonium chloride, for direct use, 30 mL × 1 bottle, 30 mL × 1 bottle
10 Stop solution: 0.5 mol/L H2SO4 solution; ready to use; 6.5 mL × 1 bottle; 3.5 mL × 1 bottle
11 Sealed bags, used for sealing slats, 1 piece, 1 piece
12 sealing films, used to seal the slats, 4 pieces, 4 pieces
13 Record sheet for test records, 1 copy, 1 copy
Note: To ensure optimal assay performance, all reagents must be sourced from this company; do not mix with products from other manufacturers. Components from different lot numbers must not be intermixed.
2. Materials required but not yet provided:
Distilled water or deionized water
Microplate washer
10 μL to 100 μL, 1 mL pipettes with disposable tips
37°C constant-temperature equipment
Micro-pore oscillator
A timer with a duration of 60 minutes or longer
[Storage Conditions and Shelf Life]
Store protected from light at 2°C to 8°C; shelf life is 12 months. See label for the date of manufacture and the expiration date.
After opening, any unused microtiter plates should be stored in a sealed bag at 2°C to 8°C. Liquid components should be tightly capped and stored at 2°C to 8°C. All components must be used within one month of opening.
[Applicable Instruments]
A microplate reader with a 450 nm wavelength
[Sample Requirements]
Sterile, anticoagulated venous blood is used. If whole-blood samples cannot be tested immediately, they may be stored at 4°C for up to one week, at −20°C for one month, or at −70°C for up to three years. This assay is suitable for samples containing EDTA, sodium citrate, sodium heparin, sodium oxalate, or double‑oxalate anticoagulants; however, no experimental data are currently available regarding the suitability of samples containing other types of anticoagulants. Do not use specimens that are contaminated with microorganisms. Hemolysis does not affect the assay results. For samples from patients with severe anemia, adjust the plasma-to‑red blood cell ratio to 1:1 before performing the required dilutions.
[Testing Method]
1. Precautions During the Experiment
1.1 When the kit components are removed from refrigeration, they should be allowed to equilibrate at room temperature (20°C–25°C) until no condensation is present before use. Any unused microtiter strips after opening should be stored in a sealed bag.
1.2 During storage and incubation, direct exposure to strong light should be avoided. All reagent vials must be tightly capped to prevent evaporation and contamination.
1.3 To prevent microbial contamination and cross-contamination of reagents or samples, disposable pipette tips should be used and not reused.
1.4 Before aliquoting the standard and enzyme‑labeled reagents, gently shake them to ensure thorough mixing. Prior to sample addition, use a microplate shaker to thoroughly mix the diluted samples.
1.5 Strictly adhere to the specified incubation time and temperature. If the interval between adding samples to the first well and the last well is too long, it will result in varying “pre-incubation” times, thereby compromising the accuracy and reproducibility of the measurements.
1.6 At the end of the washing step, invert the microplate onto a paper towel and gently tap it to ensure that no residual wash solution remains in any well, thereby preventing foam formation. If using an automated plate washer, be sure to operate it correctly.
2. Experimental Preparation
2.1 Preparation of Reagent Kit Reaction Reagents
2.1.1 Microporous Strip
Secure the required number of microplate strips to the plate holder, and store the remaining strips in a sealed bag.
2.1.2 Washing Solution
Dilute the concentrated detergent in a clean graduated cylinder. Add 50 mL of concentrated detergent to distilled or deionized water up to a final volume of 500 mL, and mix thoroughly.
2.1.3 Other Liquid Components
Use directly; shake gently before use to ensure thorough mixing.
2.2 Sample Preparation
Add 25 µL each of thoroughly mixed whole blood samples to be tested, positive control, and negative control into 200 µL of sample lysis buffer, and vigorously vortex for 60 seconds to ensure complete lysis of red blood cells.
3. Testing Procedure
3.1 For each assay, include one blank well per plate (no blank well is required when using dual-wavelength detection), one positive control well, one negative control well, and three critical‑control wells. The blank well contains no sample or enzyme conjugate; add only wash solution, developing reagent A, developing reagent B, and stop solution. Use the critical‑control samples directly, at 100 µL per well. Add 100 µL of lysed sample to all other wells. Seal the plate with a film and incubate at 37°C ± 1°C for 60 minutes.
3.2 Discard the liquid from the wells, then fill each well with washing solution (approximately 300 μL per well). After allowing it to stand for 60 seconds, discard the solution and blot dry; repeat this process three times. Except for the blank well, add 100 μL (2 drops) of enzyme‑labeled reagent to each well, seal with a plate cover, and incubate at 37°C ± 1°C for 60 minutes.
3.3 Discard the liquid from the wells, wash the plate three times as described above, and after blotting dry, add 50 μL of developing reagent A and 50 μL of developing reagent B to each well. Incubate at 37°C in the dark for 15 minutes, then add 50 μL of stop solution to each well and gently shake the microplate to ensure thorough mixing.
3.4 Single-wavelength detection: Zero the instrument using the blank well, then measure the absorbance (A) at 450 nm with a microplate reader.
Dual-wavelength detection: Read the absorbance values at 450 nm and 630 nm using a microplate reader, and calculate the net absorbance as A450nm – A630nm.
4. Reference Range
The negative control A-value must be ≤0.15; the average A-value of the cutoff control must be between 0.20 and 0.80, with an A-value ratio of cutoff control to negative control ≥2.0; and the positive control A-value must exceed the average A-value of the cutoff control. Only under these conditions is the assay considered valid. Otherwise, the results are invalid and the assay must be repeated.
5. Result Interpretation
Calculate the S/CO value as follows: S/CO = Sample A value / Mean of the critical control A values.
Reactivity (R): S/CO ≥ 1.15
Suspicious (S): 0.85 < S/CO < 1.15
Negative (N): S/CO ≤ 0.85
[Interpretation of Test Results]
The result was reactive, indicating that the individual is a carrier of the Southeast Asian deletion-type α‑thalassemia gene. Samples with suspicious results should be retested; if the repeat test remains inconclusive, genetic testing should be performed for definitive diagnosis.
[Limitations of the Analytical Method]
1. Sample or reagent contamination by microorganisms, as well as cross-contamination among reagents, may lead to erroneous results.
2. This kit is not intended for screening Southeast Asian deletion-type α‑thalassemia in individuals under 0.5 years of age, nor is it suitable for quantitative analysis of Zeta-chain protein.
3. This product should be used by qualified professionals, and the results are intended for clinical reference only; clinicians should interpret and diagnose in conjunction with the patient’s medical history.
[Product Performance Specifications]
1. Sensitivity, specificity
In a clinical study involving 1,034 cases, compared with genetic diagnostic methods, the assay demonstrated 100% sensitivity and 100% specificity.
2. Precision
The within-run coefficient of variation (CV, %) shall not exceed 15% (n = 10).
3. Cross-reactivity
No cross-reactivity was observed between this product and the blood of beta‑thalassemia gene carriers. No experimental data are currently available regarding cross-reactivity with other types of thalassemia gene carriers.
4. Interference Test
4.1 Anticoagulants that come into contact with the sample (EDTA, sodium citrate, sodium heparin, sodium oxalate, and double oxalate) do not significantly interfere with the assay results of this product.
4.2 Endogenous substances within a certain concentration range (bilirubin 0.1–0.4 mg/mL, hemoglobin 5–50 mg/mL, triglycerides 2–8 mg/mL) do not significantly interfere with the assay results of this product.
[Precautions]
1. This product is intended for in vitro diagnostic use only and is for single-use.
2. The blood used to prepare the negative and positive controls for this product has tested negative for HBsAg, HCV antibodies, Treponema pallidum antibodies, and HIV antibodies, and all other components included in the kit are non‑infectious. However, since it cannot be guaranteed that all potential infectious agents have been eliminated from each component, this product should be treated as potentially infectious material, and all personnel handling the kit must follow standard laboratory procedures.
3. All clinical samples should be treated as infectious materials.
4. Samples awaiting testing and other potentially infectious materials shall be disinfected after testing.
5. Take care to avoid direct contact between the reagents or samples and skin or mucous membranes. The chromogenic solution contains TMB, and the stop solution contains sulfuric acid. Reagents containing TMB may trigger unintended reactions during the assay, while those containing sulfuric acid are corrosive. If these liquids come into contact with the skin or eyes, rinse thoroughly with water immediately.
[Explanation of the Logo]
Packaging label
The logo
Meaning of the label: Temperature limits; In vitro diagnostic medical device; Refer to the instructions for use; Do not reuse.
[References]
[1]Chui DH, Wong SC, Chung SW, Embryonic Zeta-globin chains in adults: a marker for alpha-thalassemia-1 haplotype due to a greater than 17.5-kb deletion. N Engl J Med. 1986 Jan 9;314(2):76-9
[2]Lebo RV, Saiki RK, Swanson K et al. Prenatal diagnosis of alpha-thalassemia by polymerase chain reaction and dual restriction enzyme analysis. Hum Genet. 1990
Aug;85(3):293-9.
[3]Kutlar F, Moscoso H, Kiefer CR et al. Quantities of adult, fetal and embryonic globin chains in the blood of eighteen- to twenty-week-old human fetuses. J Chromatogr. 1991 Jul 5;567(2):359-68.
[Basic Information]
Registrant/Manufacturer Name: Zhuhai Special Economic Zone Haitai Biopharmaceutical Co., Ltd.
Address: No. 18, Innovation Road No. 4, Gangwan Avenue, Tangjiawan Town, Zhuhai City
Contact Information:
Name of After-Sales Service Provider:
Contact Information:
Production Address: No. 18, Innovation Road 4, Gangwan Avenue, Tangjiawan Town, Zhuhai City
Production License No.:
[Medical Device Registration Certificate Number/Product Technical Requirement Number]
[Date of Approval and Date of Amendment of the Instruction Manual]