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Latex-enhanced Immunoturbidimetric Assay on Human HbA1c Using a Highly-specific Mouse Anti-human HbA1c-6C2 Monoclonal Antibody

Lin Zhou1#, Dafeng Xie2#, Song Xu3#, Jiangming Wang3# ,Sha Chen2* Junfu Huang1*

1 Songshan General Hospital, 69 Xingguang Street, Chongqing 401120, China
2 Chongqing Medical University, Yixueyuan Road, Chongqing, 400016, China
3 Biomean Technology Co.,Ltd, Yuefu Avenue,Chongqing ,400700, China
1 Co-first authors

【Abstract】
Diabetes is a common metabolic disorder disease, which is often detected by fasting blood glucose and oral glucose tolerance test in clinic. As a kind of glycosylated hemoglobin, HbA1c has been gradually recommended as one of the diagnostic criteria for diabetes in recent years. And there have been various methods for detecting HbA1c, each with its own advantages and disadvantages .In this study, we selected and prepared the anti-human HbA1c-6C2 monoclonal antibodies with high purity and specificity, then adopted it to detect HbA1c and took methodological evaluation based on the latex-enhanced immunoturbidimetric assay. Results showed that excellent linearity was observed within the HbA1c concentration range of 3.8% to 14.0%, and CV was less than 1.5% for repeated detection at high and low concentration levels.There was a strong correlation with HPLC during methodological comparison. In general, it had the advantages of good linearity, high precision, stability and accuracy, which revealed a favourable detection effect.

1 Introduction
Diabetes is a group of metabolic disorders caused by abnormal glucose metabolism. It is a hyperglycemia caused by reduced utilization or excessive production of glucose, which can cause coronary artery disease, kidney disease, retinopathy and other complications.[1] As of 2021, the number of patients is about 537 million in global and about 140.9 million in China, which is still increasing.[2]For decades, the diagnosis of diabetes has been based on blood glucose standards, including fasting plasma glucose (FPG) and oral glucose tolerance test (75g OGTT). Hemoglobin A1c (HbA1c) is a widely-used chronic blood glucose indicator and can reflect the average blood glucose concentration in the past 2 to 3 months. In recent years, with the continuous standardization of its measurement, it has been recommended by international expert committees as one of the criteria for diagnosing diabetes, with a diagnostic threshold of 6.5% (NGSP units).[3]
HbA1c is a type of glycated hemoglobin (GHb) formed by the condensation of the aldehyde group of glucose with the amino terminal valine residue of the β - chain of HbA. The two firstly form an unstable aldehyde imine (i.e. Schiff base), which then dissociates or undergoes Amadori molecular rearrangement to form a stable ketamine compound.[4, 5] According to reports, there are various methods for detecting HbA1c ,Including high performance liquid chromatography (HPLC)[6] , enzyme assay[7], affinity chromatography[8], electrochemical method[9],etc. [5] Among them, HPLC has been used as the gold standard for HbA1c level determination and is currently the most precise and accurate method, but it requires large and relatively expensive equipment.[10]In this study, we screened and prepared a type of highly specific mouse anti-human HbA1c-6C2 antibodies based on monoclonal technology, adopted the latex-enhanced immunoturbidimetric assay (LEIBA) to detect human HbA1c, and then conduct methodological evaluation on it.

2 Materials and methods
2.1 Sample collection

2.2 Preparation of antibodies
Glycosylate the amino group on the N-terminal valine of hemoglobin beta polypeptide chain with glucose C1 and conjugate with KLH and BSA as immunogens. Five female BALB/c mice, aged 6-8 weeks ,were immunized three times at multiple subcutaneous sites with the immunogens.The first immunization was emulsified with complete Freund's adjuvant , followed by two subsequent immunizations with incomplete Freund's adjuvant emulsion, with a 3-week interval between each immunization. Spleen cells from immune enhanced BALB/c mouse were fused with SP2/0 cells using PEG4000 and screened. The cell culture supernatant on the 7th day after fusion was collected for ELISA detection. Positive hybridoma cells were selected for amplification, and subclones were obtained by extreme dilution method. The clones in large quantities were injected into the abdominal cavity of the mice to produce monoclonal antibodies. Then the extracted mouse ascites were centrifuged and the supernatant was collected for purification. Screening and preparation of anti-mouse anti-human HbA1c antibodies based on the similar principles.

2.3 Reagents
Reagent 1 consists of latex microspheres, tris (hydroxymethyl) aminomethane and preservatives, while reagent 2 consists of tris (hydroxymethyl) aminomethane, preservatives, PEG6000 (polyethylene glycol 6000), sucrose, sodium chloride, goat anti-mouse IgG-2B4 antibody and mouse anti-human HbA1c-6C2 antibody. Among them, Tris (hydroxymethyl) aminomethane and PEG6000 were purchased from Suzhou Yake Technology Co., Ltd. Sucrose，sodium chloride sodium phosphate dibasic dihydrate and disodium hydrogen phosphate dodecahydrate were purchased from China National Pharmaceutical Group Chemical Reagent Co., Ltd. Latex microspheres were purchased from Polysphere. The preservative (ProClin300) was purchased from Sigma Aldrich.

2.4 Characterization of antibodies
2.4.1 Purity
To analyse the purity of the antibodies we selected, we applied HPLC in this study . The sample was diluted to 1.5mg/ml with a mobile phase. And the analytical chromatographic separations were carried out on Elite Agerss-D1100 HPLC system with a mobile phase consisting of sodium phosphate dibasic dihydrate-disodium hydrogen phosphate dodecahydrate-sodium chloride-water mixture at a flow rate of 0.3ml/min and a column temperature of 10-30 ℃. The injection volume was 20μl and the detection wavelength we set was at 280nm.

2.4.2 Specificity
Adopt ELISA method to characterize the screened antibodies. After overnight encapsulation of 5 μ g/ml human Hb and human HbA1c at 4 ℃, 1% BSA was added and blocked at 37 ℃ for 2 hours. Dilute the mouse anti-human HbA1c-6C2 antibody obtained by 1ug/ul screening at a ratio of 1:10000 and add it. Dilute each well in a multiple ratio and incubate at 37 ° C for 2 hours. Then add goat anti-mouse HRP at a ratio of 1:2000 and incubate at 37 ° C for 1 hour. After 3 minutes of dark staining, measure Hb and HbA1c activity separately. Use the same method to detect anti-human HbA1c-3A7 antibody and anti-human Hb-1A1 antibody for comparative analysis.
Coat with 2 μ g/ml of IgG from four different species including mice, rabbits, humans, and goats as antigens, add goat anti-mouse（GAM） IgG-2B4 antibody and incubate at 37 ℃ for 1 hour to detect its activity. Use the same method to detect GAM IgG-2H4 antibody, GAM IgG-2H2 antibody and GAM polyclonal antibody for comparative analysis.
Western blot analysis was performed to obtain immunoblotting of hemoglobin’ expression and detect antibody specificity, using naturally extracted glycated hemoglobin and normal hemoglobin as substrates, with the mouse anti-human HbA1c-6C2 antibody and the mouse anti -human Hb-1A1 antibody serving as the primary antibody separately,and the goat anti-mouse IgG-2B4 antibody adopted as the secondary antibody.

2.5 Instruments and methods
In this study, the experiment was conducted on a Beckman AU480 biochemical analyzer. When adding reagent 1 to the sample to be tested, the glycated hemoglobin was bound to the latex microspheres. After adding reagent 2, glycated hemoglobin binds to specific antibodies, forming latex microspher-anti-HbA1c antibody complexes. Then, the bind of sheep-anti-mouse IgG-2B4 antibody and glycated hemoglobin antibodies caused the aggregation of latex microspheres. These complexes led to the scattering of the irradiation beam, and the intensity of the scattered light is proportional to the concentration of HbA1c in the sample.

2.6 Evaluation of methodology
2.6.1 Calibration curve
Use a standard sample with a known percentage concentration of glycated hemoglobin to obtain absorbance values according to the steps mentioned in section 2.3. Plot a standard curve with percentage concentration as the x-axis and absorbance values as the y-axis.

2.6.2 Blank absorbance
Use physiological saline as a blank sample to make a detection and record the absorbance at startup (A1) and the absorbance after about 5 minutes (A2). And the value of A2 is the blank absorbance value.

2.6.3 Linearity
Dilute high concentration samples close to the upper limit of the linear interval with low concentration samples close to the lower limit of the linear interval, with dilution concentrations of 3.9%, 5.5%, 7.3%, 9.2%, 11.3%, and 13.7% respectively. Perform 3 repeated tests on the diluted sample and take the average as the test result. Evaluate linearity by performing linear regression analysis on the detection values and theoretical values and calculating the Spearman correlation coefficient.

2.6.4 Precision
Under repeatability conditions, select samples possessing two different concentration levels within the linear range: high concentration (not less than 30% of the reference range) and low concentration (within the reference range). Repeat the detection 20 times, take the average of the detection results and calculate the standard deviation (SD) and coefficient of variation (CV).

2.6.5 Stability
Store the reagents in a refrigerator at 2-8 ℃ for at least 30 days, and take samples at 1, 4, 7, 10, 15, 20, 25, 30, 40, and 45 days from the date of storage. Follow the reference detection method for testing and record the results. After each sampling, store the reagents back at 2-8 ℃.

2.6.6 Methodology comparison
Select 50 blood samples, which cover a wide range of glycated hemoglobin concentrations. The samples will be tested three times on the Beckman AU480 biochemical analyzer and the Elite Agerss-D1100 HPLC system respectively, and the average value will be taken for comparative analysis. Correlation analysis was conducted between the results obtained by latex enhanced immunoturbidimetry and HPLC to obtain linear correlation coefficients and linear regression equations, in order to evaluate their comparability.

3 Results
3.1 Preparation of the antibodies.
Following the steps in section 2.2, after four subclone screening, one positive monoclonal hybridoma was selected and named 6C2. It was injected into the peritoneal cavity of mice to obtain ascites. Meanwhile, purified cell culture supernatant and ascites were used to obtain mouse anti-human HbA1c-6C2 monoclonal antibody (mAb). Further identification revealed that the monoclonal antibody subtype was IgG1. Similarly, using the aforementioned antibody as an immunogen, a goat anti-mouse（GAM） IgG-2B4 monoclonal antibody was obtained.

3.2 Characterization of antibodies
3.2.1 Purity
By using HPLC method to analyze the purity of the prepared antibodies, as shown in Fig.1, only one main peak was observed for both mouse anti-human HbA1c-6C2 antibody and goat anti-mouse IgG-2B4 antibody, indicating that they are intact antibodies. The peak areas were 8136 mAU. s and 7142 mAU. s, respectively, with peak area ratios of 95.57% and 96.89%, indicating that both have high purity.

3.2.2 Specificity
As shown in the Fig.2, the anti-human HbA1c-3A7 antibody has a cross reaction with Hb and the anti-human Hb-1A1 antibody binds to both HbA1c and Hb, while the anti-human HbA1c-6C2 antibody can bind well to HbA1c instead of Hb. With increasing dilution ratio, the HbA1c activity measured relatively stable, with a potency of 256w. This proves that the anti-human HbA1c-6C2 antibody has high specificity for HbA1c and can eliminate interference from Hb in its detection.
When characterizing the goat anti-mouse IgG-2B4 antibody, the results showed that the antibody could specifically bind to mouse IgG and did not cross react with other species (Fig.3A). Although the GAM IgG-2H4 antibody and GAM IgG-2H2 antibody didn’t bind to IgG antibodies of rabbit, human, sheep and other species and have good specificity, their sensitivity is not as good as the GAM IgG-2B4 antibody (Fig.3B and C). The GAM polyclonal antibody has cross reaction with IgG antibodies of multiple species (Fig.3D). Therefore, it can be seen that the goat anti-mouse IgG-2B4 antibody has high specificity and sensitivity, which can avoid interference caused by cross reaction with other species’ IgG antibodies.
In addition, Western Blot analysis showed that when the secondary antibody was goat anti-mouse IgG-2B4 antibody and remained unchanged, if the primary antibody was mouse anti-human Hb-1A1 antibody, both Hb and HbA1c bands appeared simultaneously, while the primary antibody was mouse anti-human HbA1c-6C2 antibody, only HbA1c bands appeared (Fig.4A and B).It further proved that the mouse anti-human HbA1c-6C2 antibody does not bind to Hb and has high specificity for HbA1c.

3.3 Evaluation of methodology
The blank absorbance of the reagent at a wavelength of 660nm is 0.3768, which is less than 1.5. The linearity shows a strong correlation (=0.99) within the concentration range of 3.8% to 14.0%, with linear absolute deviations not exceeding ± 0.5% and linear relative deviations not exceeding ± 7% (NGSP units) (Fig.5B). The detection CV at low concentration levels is 1.438% and the one at high concentration levels is 0.686%, indicating that it has good precision (Table 1). As shown in the Fig.5C, this method has good stability and the detection results are still relatively stable even after the reagents are stored for 30 days under the same conditions. The correlation and regression studies used for method comparison showed a good linear relationship between latex-enhanced immunoturbidimetric assay and HPLC(=0.99), with an intercept of 0.07075 and a slope of 0.9895 (as shown in Fig.5D).

4 Discussion
Diabetes is a group of hyperglycemia caused by abnormal glucose metabolism, which can lead to a variety of complications. FPG and OGTT are often used to clinical diagnose diabetes. In recent years, with the standardization of HbA1c detection, HbA1c has gradually been used in the diagnosis of diabetes, becoming an effective tool for monitoring long-term blood glucose control, with a diagnostic threshold of 6.5% (NGSP units).[11]
Currently, multiple methods have been used for the detection of HbA1c. The testing methods selected by clinical laboratories should not only meet the requirements of simplicity, speed, batch testing, and timely reporting, but also ensure the quality of the testing results. The long-term use of methods requires strict quality management, including laboratory certification, specialized internal control design, and participation in external quality assessment (EQA) programs.[12, 13] The American Diabetes Association (ADA) recommends that laboratories only use testing methods that are certified and traceable to DCCT GHB references. For methods performed in the laboratory, the CV should be less than 1.5% and ideally there should be no measurable deviation. [12]Based on this, it is of great significance to evaluate whether the analytical performance of immunoturbidimetry meets the requirements. In this study, we screened and prepared a kind of mouse anti-human HbA1c antibody possessing high purity and specificity. We had verified its performance and conducted methodological evaluation on the latex-enhanced immunoturbidimetric assay. HPLC, ELISA and WB were adopted to characterize the antibodies and the results proved that mouse anti-human HbA1c antibodies can specifically bind to HbA1c without reacting with Hb. During conducting methodological evaluation, the CV of indoor tests at high and low concentration levels were both less than 1.5% (NGSP units) , indicating good precision and meeting the precision requirements of medical diagnosis and clinical practice. At the same time, there is a strong correlation within 3.8%~14.0% concentration range, and the linear deviation meets the requirements. In addition, a strong linear relationship has been shown between this method and HPLC, with a slope of 0.9895, intercept of 0.07075, and correlation coefficient of 0.99, which reflected the good detection performance of this method. Besides,it also possessed good stability. After the reagent we made was maintained for 30 days under the suitable conditions, the detection results were still relatively accurate.
In the clinical detection of HbA1c, triglycerides, uric acid, ethanol, and other potential interfering substances, as well as hemoglobin variations such as HbE, may also interfere with its detection. [6, 14]However, we have not evaluated the interference of these substances on HbA1c’s detection, which is a limitation of our research and will be considered in the future.
Overall, this study prepared a highly specific and high-purity mouse anti-human HbA1c antibody, and used latex-enhanced immunoturbidimetric assay to detect human HbA1c. It has good linearity, precision, accuracy and stability, which can help clinical doctors better monitor patients' blood glucose levels.References

References
[1] BARBARO M, KU-CHULIM C, JOHNSTON F, et al. Evaluation of the performance of an immunoturbidimetric HbA1c reagent applied to the Siemens ADVIA 2400 automatic analyzer[J]. Clinical Biochemistry, 2015: 177-180.
[2] David B. Sacks, Mark Arnold, George L. Bakris, et al. Guidelines and Recommendations for Laboratory Analysis in the Diagnosis and Management of Diabetes Mellitus[J]. Clinical Chemistry, 2023,69: 808-868.
[3] Diagnosis and Classification of Diabetes Mellitus[J]. Diabetes Care, 2011,34(Supplement_1): S62-S69.
[4] MURALIDHARAN M, BHAT V, MANDAL A K. Structural analysis of glycated human hemoglobin using native mass spectrometry[J]. FEBS J, 2020,287(6): 1247-1254.
[5] MOLAZEMHOSSEINI A, MAGAGNIN L, VENA P, et al. Single-Use Disposable Electrochemical Label-Free Immunosensor for Detection of Glycated Hemoglobin (HbA1c) Using Differential Pulse Voltammetry (DPV)[J]. Sensors (Basel), 2016,16(7).
[6] YU Y, ZHANG X, LIN K. Analytical performance evaluation of hemoglobin A1c on an ARKRAY HA-8160 analyzer with newly-developed mobile phase buffer [J]. Practical Laboratory Medicine.
[7] JAISSON S, DESMONS A, RENARD B, et al. Analytical performances of a new enzymatic assay for hemoglobin A1c[J]. Clinica chimica acta, 2014,434: 48-52.
[8] KOSKINEN L K. Specificity of hemoglobin A1c measurement cation exchange liquid chromatography. [J]. Clinica Chimica Acta, 1996: 159-169.
[9] Mukesh Thapa, HEO Y S. Label-free electrochemical detection of glucose and glycated hemoglobin (HbA1c)[J]. Biosensors and Bioelectronics: X, 2023.
[10] CHANG K, JINGLUN L, CHING-HSUAN Y, et al. An integrated microfluidic system for measurement of glycated hemoglobin Levels by using an aptamer–antibody assay on magnetic beads[J]. Biosensors and Bioelectronics, 2015: 397-403.
[11] DEVI R, CHAUHAN A, YADAV S, et al. Development of aptamer based colorimetric assay for whole blood glycated hemoglobin (HbA1c) estimation using gold nanoparticles[J]. Biosensors and bioelectronics. X, 2023,13: 100304.
[12] David B. Sacks, Mark Arnold, George L. Bakris, et al. Guidelines and Recommendations for Laboratory Analysis in the Diagnosis and Management of Diabetes Mellitus[J]. Diabetes Care, 2023: e151-e199.
[13] WEYKAMP C. HbA1c: A Review of Analytical and Clinical Aspects[J]. Annals of Laboratory Medicine, 2013,33(6): 393-400.
[14] LOH T P, CHENG W L, KAO S L, et al. Effects of haemoglobin E traits on HbA1c measurement by two cation-exchange HPLC and two immunoturbidimetric methods[J]. Pathology, 2014.

Table 1
The detection SD and CV at low concentration and at high concentration levels.

Group Testing Times MIN MAX MEAN SD CV（%）
Low Level N=20 4.60 4.80 4.665 0.067 1.438
High Level N=20 12.10 12.40 12.225 0.085 0.696

Fig.1. Purity analysis of mouse anti-human HbA1c-6C2 antibody and goat anti-mouse IgG-2B4 antibody using HPLC.

Fig.2. Measurement on the activity of different antibodies using HbA1c and Hb at dilution of 1:1w, 1:2w, 1:3w, 1:4w, and 1:5w respectively: A) mouse anti-human Hb-1A1 antibody; B) mouse anti-human HbA1c-3A7 antibody; C) mouse anti-human HbA1c-6C2 antibody.

Fig.3. Detection on activities of four different secondary antibodies using four different species’ antibodies: A) GAM-2B4 antibody; B) GAM-2H2 antibody; C) GAM-2H4 antibody; D) GAM polyclonal antibody.

Fig.4. Western Blot analysis of HbA1c-6C2 and Hb-1A1.

Fig.5. Evaluation of methodology: A) Calibration curve;B) Linear curve;C) Stability detection on HbA1c with concentrations of 5.3%, 7.8%, 10.2%, and 13.1% using the reagents saved for 1, 4, 7, 10, 15, 20, 25, 30, and 40 days.;D) Methodology comparison between LEIBA and HPLC.你是此项项目的研究专家，请根据这片论文的成果，写一份中英文研究方案及患者知情同意书，用于通过伦理委员会。

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以下提供了一份针对“基于高特异性鼠源抗人HbA1c-6C2单克隆抗体的乳胶增强免疫比浊法检测人HbA1c”的研究方案和患者知情同意书（中英文对照），可用于向伦理委员会提交。内容包括研究背景、目的、方法、预期风险与受益、伦理与保密、受试者权益等关键要点，供研究者根据实际情况做进一步调整与完善。

一、研究方案 (中英文)

中文版

1. 研究标题
基于高特异性鼠源抗人HbA1c-6C2单克隆抗体的乳胶增强免疫比浊法检测人HbA1c的研究

2. 研究背景与意义
糖化血红蛋白(HbA1c)检测可反映受试者近2-3个月的平均血糖水平，现已逐步成为糖尿病诊断及血糖监测的重要指标。本文相关前期研究筛选获得了高纯度、高特异性的鼠源抗人HbA1c-6C2单克隆抗体，并基于该抗体建立了乳胶增强免疫比浊法检测HbA1c的新方法，验证其准确性、精密度、稳定性等。为了进一步在临床上应用并与其他检测方法进行对比，需要对临床受试者的HbA1c样本进行检测，以评估此方法的临床可行性和可靠性。

3. 研究目的

  1. 进一步验证基于高特异性鼠源抗人HbA1c-6C2单克隆抗体的乳胶增强免疫比浊法检测HbA1c在临床样本中的灵敏度、精密度、准确度和稳定性。
  2. 与常规高效液相色谱（HPLC）等方法进行比对，探讨该新方法在临床检测中的可替代性与一致性。

4. 研究设计

  • 研究类型： 诊断试验评价研究
  • 受试者来源： 拟在医院门诊或体检中心招募至少50名志愿者，需进行血糖或糖化血红蛋白监测的人群(包括糖尿病患者以及血糖偏高或正常对照人群)。
  • 检测对比： 使用本文提出的乳胶增强免疫比浊法与HPLC法分别对所有受试者血样进行HbA1c测定，比较两种方法测定的相关性和一致性。
  • 指标与评估：
    • 灵敏度、特异度、线性范围
    • CV（变异系数）、批内精密度
    • 稳定性及回收率
    • 与HPLC检测结果的相关分析

5. 研究对象纳入与排除标准

  • 纳入标准：

    1. 年龄≥18周岁，性别不限；
    2. 自愿参加本研究并签署知情同意书；
    3. 临床上有血糖或糖化血红蛋白检测需求者（包括糖尿病患者、血糖波动人群或体检者）。

  • 排除标准：

    1. 存在严重贫血、溶血性疾病或其他影响HbA1c检测的血液系统疾病；
    2. 既往存在明显凝血功能障碍、不适宜采血者；
    3. 妊娠或哺乳期妇女(如研究设计有此需求，可继续保留或修订)；
    4. 研究者判断其他不宜参加本研究者。

6. 干预/检测方法

  • 检测方法： 采集外周静脉血2-3mL，分离血清或全血，用研究中的“乳胶增强免疫比浊法HbA1c检测试剂”在自动生化分析仪上进行检测，并与HPLC检测结果对比。
  • 检测频次： 仅采集1次血样，不额外采集加样。

7. 样本量与统计学方法

  • 样本量： 预计不少于50份临床样本，以保证有足够的统计学检验效率进行方法学相关性分析。
  • 统计分析：
    1. 计算相关性系数(r)和回归方程，评估与HPLC检测结果的一致性；
    2. 计算变异系数(CV)评估精密度；
    3. 进行Bland-Altman分析或Passing-Bablok回归分析（若有需要）来进一步比较两种方法间的差异；
    4. 采用SPSS或其他统计软件进行分析。

8. 伦理与风险收益分析

  • 伦理原则： 严格遵守《世界医学大会赫尔辛基宣言》及国家相关医学伦理法规；
  • 风险评估： 仅需常规静脉采血，可能带来穿刺处轻度疼痛、局部淤青等极小风险；
  • 收益分析： 受试者可免费获得一项新的HbA1c检测结果，可帮助评估其血糖水平并辅助诊断或监测糖尿病。

9. 研究进度与质量控制

  • 进度： 本研究预计持续3-6个月；
  • 质量控制：
    1. 检测前对仪器及试剂进行质控；
    2. 严格按统一操作流程采集和处理样本；
    3. 定期开展室内质控及同一批次检测对比；
    4. 数据录入并双人核对，保证准确性。

10. 受试者权益与保密

  • 自愿原则：所有受试者均自愿参加并可随时退出，不影响其后续诊疗；
  • 保密措施：采集和使用的样本及相关检测信息仅用于本研究，个人信息严格保密，发表论文或会议时不披露个人身份；
  • 联系方式：如受试者对于研究或其权利有任何疑问，可随时联系研究负责人。

11. 经费来源
由课题组或相关科研基金支持(可根据实际情况注明具体来源)，受试者无需支付额外费用。

12. 研究者信息

  • 主要研究者(PI)：XXX，单位/部门：XXX，联系方式：XXX
  • 共同研究者：XXX，单位/部门：XXX，联系方式：XXX

English Version

1. Title of the Study
A Study on the Detection of Human HbA1c Using Latex-Enhanced Immunoturbidimetric Assay Based on a Highly Specific Mouse Anti-Human HbA1c-6C2 Monoclonal Antibody

2. Background and Significance
Glycated hemoglobin (HbA1c) reflects the average blood glucose level of an individual over the past 2-3 months and has become a crucial marker for diabetes diagnosis and blood glucose monitoring. In our preliminary study, we successfully developed a highly purified and specific mouse anti-human HbA1c-6C2 monoclonal antibody and established a latex-enhanced immunoturbidimetric assay to detect HbA1c. In order to further validate this method clinically and compare it with other assays, we plan to measure HbA1c levels in clinical samples to assess the feasibility and reliability of this new approach.

3. Objectives

  1. To further verify the sensitivity, precision, accuracy, and stability of the latex-enhanced immunoturbidimetric assay (using the mouse anti-human HbA1c-6C2 antibody) in clinical samples.
  2. To compare the results with those obtained by the conventional high-performance liquid chromatography (HPLC) method, exploring the potential interchangeability and consistency of the new assay in clinical practice.

4. Study Design

  • Study Type: Diagnostic evaluation study
  • Source of Participants: At least 50 volunteers will be recruited from hospital outpatient clinics or health check-up centers. These participants should require blood glucose or HbA1c monitoring (including diabetes patients or those with borderline or normal blood glucose levels).
  • Detection Comparison: Each participant’s HbA1c will be measured by both the latex-enhanced immunoturbidimetric assay and the HPLC method. The results from both tests will then be compared.
  • Endpoints and Evaluation:
    • Sensitivity, specificity, and linear range
    • Coefficient of variation (CV), intra-assay precision
    • Stability and recovery
    • Correlation analysis with HPLC measurements

5. Inclusion and Exclusion Criteria

  • Inclusion Criteria:

    1. Aged ≥18 years, no restriction on gender;
    2. Voluntarily participates and signs the informed consent form;
    3. Requires blood glucose or HbA1c measurement (including diabetic patients, individuals with unstable blood glucose, or those undergoing routine check-ups).

  • Exclusion Criteria:

    1. Severe anemia, hemolytic diseases, or other hematological conditions affecting HbA1c measurements;
    2. Known significant coagulation disorders or unsuitability for phlebotomy;
    3. Pregnancy or lactation (may be retained or revised according to the specific needs of the study);
    4. Other conditions deemed unsuitable by the investigators.

6. Intervention/Testing Methods

  • Procedure: Collect 2-3 mL of peripheral venous blood from each participant, separate the serum or whole blood, and detect HbA1c by the proposed latex-enhanced immunoturbidimetric assay using an automated chemistry analyzer. HPLC measurement will be conducted in parallel for comparison.
  • Frequency of Testing: Only one blood draw per participant; no additional sampling is required.

7. Sample Size and Statistical Methods

  • Sample Size: At least 50 clinical samples are anticipated to achieve sufficient statistical power for correlation analysis.
  • Statistical Analysis:
    1. Calculate correlation coefficient (r) and linear regression for consistency with HPLC;
    2. Evaluate precision by coefficient of variation (CV);
    3. Perform Bland-Altman or Passing-Bablok analysis for further assessment of the two methods;
    4. Use SPSS or other statistical software for data analysis.

8. Ethics and Risk-Benefit Analysis

  • Ethical Principles: Strictly follow the Declaration of Helsinki and relevant national ethical regulations.
  • Risk Assessment: Only routine venous blood sampling is involved, with minimal risks such as mild pain or local bruising at the puncture site.
  • Benefit Analysis: Participants receive a free new HbA1c test result that can help evaluate their blood glucose control and aid in the diagnosis or monitoring of diabetes.

9. Study Timeline and Quality Control

  • Timeline: The overall study is planned to last for 3-6 months.
  • Quality Control:
    1. Perform quality checks on instruments and reagents before testing;
    2. Follow standard protocols for blood collection and sample handling;
    3. Conduct regular internal quality control and batch comparison;
    4. Use double data-entry verification for accuracy.

10. Participant Rights and Confidentiality

  • Voluntary Participation: All participants take part voluntarily and can withdraw at any time without prejudice to their future care.
  • Confidentiality Measures: Participant samples and data are used only for this study, and personal information is kept strictly confidential. No personal identifiers will be disclosed in publications or presentations.
  • Contact: Participants can contact the principal investigator at any time for questions about the study or their rights.

11. Funding
The study is funded by the project team or relevant research grants (specific sources if applicable). No extra cost is required from the participants.

12. Investigator Information

  • Principal Investigator (PI): XXX, Department/Institution: XXX, Contact: XXX
  • Co-Investigators: XXX, Department/Institution: XXX, Contact: XXX

二、患者知情同意书 (中英文)

中文版

研究名称： 基于高特异性鼠源抗人HbA1c-6C2单克隆抗体的乳胶增强免疫比浊法检测人HbA1c的研究

尊敬的先生/女士，您好！
您被邀请参加一项关于糖化血红蛋白(HbA1c)检测方法学评价的研究。请仔细阅读以下内容，并在考虑充分后决定是否参加。若有任何疑问，您可随时向研究人员咨询。

  1. 研究背景与目的
    糖化血红蛋白检测可反映您近2-3个月的平均血糖水平，对糖尿病诊断及血糖监测有重要意义。本研究旨在验证一项新型检测方法(乳胶增强免疫比浊法)的准确性和稳定性，并与现有的高效液相色谱(HPLC)方法进行比较。

  2. 研究过程

    • 研究过程中，您仅需按常规流程接受一次静脉采血(约2-3mL)，无额外不适。
    • 提取的血样将用于新方法和HPLC方法进行HbA1c测定和比较。

  3. 可能的风险与不适

    • 本研究仅涉及常规的静脉采血，可能会有轻微疼痛、瘀青或低概率局部感染等风险。但在标准采血操作下，这些风险极低。

  4. 潜在受益

    • 您可免费获得一次糖化血红蛋白检测结果，可用于了解并监测个人血糖控制情况。
    • 本研究结果也将帮助改进和推广更便捷、准确的HbA1c检测方法，为更多患者带来收益。

  5. 保密原则

    • 您的个人信息将严格保密，所有研究结果仅用于科研目的。公开发表的论文或报告中不会披露您的姓名、身份证号等个人身份信息。

  6. 自愿参与及退出权利

    • 您的参与完全出于自愿，若您在任何阶段决定退出，可随时告知研究人员，无需承担任何责任，且不会影响您后续的医疗服务。

  7. 研究费用与补偿

    • 本研究不向您收取任何费用，也无须您承担额外经济负担。
    • 无现金补助，或可根据研究经费情况为受试者提供适当交通补助(如无此安排，可删去本条)。

  8. 联系方式

    • 如您对本研究有任何疑问，或在研究过程中出现任何不适，可随时联系以下研究人员：
      • 主要研究者：XXX，联系电话：XXX
      • 研究助理：XXX，联系电话：XXX

如果您已经了解并同意参加，请在下方签字：

我已阅读（或被口头告知）本知情同意书的全部内容，对于研究目的、过程、风险和受益已获得充分理解，我同意自愿参加此研究。

受试者姓名（签字）：___________ 日期：___________

研究者/见证人（签字）：___________ 日期：___________

English Version

Title of the Study: Detection of Human HbA1c Using Latex-Enhanced Immunoturbidimetric Assay Based on a Highly Specific Mouse Anti-Human HbA1c-6C2 Monoclonal Antibody

Dear Sir/Madam,
You are invited to participate in a study evaluating a new assay for measuring glycated hemoglobin (HbA1c). Please read the following information carefully before making your decision. Feel free to ask any questions you may have.

  1. Background and Purpose
    HbA1c measurement reflects your average blood glucose levels over the past 2-3 months and is crucial in diagnosing and monitoring diabetes. This study aims to validate the accuracy and stability of a novel assay (latex-enhanced immunoturbidimetric method) and compare it with the existing high-performance liquid chromatography (HPLC) assay.

  2. Study Procedures

    • You will undergo a routine venous blood draw (about 2-3 mL).
    • The collected blood sample will be used for HbA1c measurement by both the new method and the HPLC method for comparison.

  3. Potential Risks and Discomfort

    • This study involves a standard venipuncture procedure. Possible risks include mild pain, bruising, or a low probability of localized infection. These risks are minimal under standard procedures.

  4. Potential Benefits

    • You will receive one HbA1c test result at no cost, which can help assess and monitor your blood glucose control.
    • The findings of this study may contribute to improving and promoting a more convenient and accurate HbA1c testing method, benefiting more patients in the future.

  5. Confidentiality

    • Your personal information will be kept strictly confidential. Research findings will only be used for scientific purposes, and no personally identifiable information will be disclosed in any publications or presentations.

  6. Voluntary Participation and Right to Withdraw

    • Your participation is entirely voluntary. You may withdraw from the study at any time without any penalty, and it will not affect your subsequent medical care.

  7. Costs and Compensation

    • There are no costs to you for participating in this study, and you will not be responsible for any additional expenses.
    • No direct financial compensation is provided, although some travel cost compensation might be offered if applicable (remove if not applicable).

  8. Contact Information

    • If you have any questions about this study or experience any discomfort during the process, please contact:
      • Principal Investigator: XXX, Phone: XXX
      • Research Assistant: XXX, Phone: XXX

If you have read (or been informed of) the above content and agree to participate, please sign below:

I have read (or been orally informed of) the full content of this Informed Consent Form, and I fully understand the purpose, procedures, risks, and benefits of the study. I voluntarily agree to participate.

Participant’s Name (Signature): ___________ Date: ___________

Investigator/Witness (Signature): ___________ Date: ___________

以上研究方案及知情同意书可根据具体临床环境和伦理审查要求进行适当修改与完善。

Domanda

Latex-enhanced Immunoturbidimetric Assay on Human HbA1c Using a Highly-specific Mouse Anti-human HbA1c-6C2 Monoclonal Antibody

Lin Zhou1#, Dafeng Xie2#, Song Xu3#, Jiangming Wang3# ,Sha Chen2* Junfu Huang1*

1 Songshan General Hospital, 69 Xingguang Street, Chongqing 401120, China
2 Chongqing Medical University, Yixueyuan Road, Chongqing, 400016, China
3 Biomean Technology Co.,Ltd, Yuefu Avenue,Chongqing ,400700, China
1 Co-first authors

Corresponding authors
Junfu Huang, Email: [email protected]
Sha Chen, Email:[email protected]
【Abstract】
Diabetes is a common metabolic disorder disease, which is often detected by fasting blood glucose and oral glucose tolerance test in clinic. As a kind of glycosylated hemoglobin, HbA1c has been gradually recommended as one of the diagnostic criteria for diabetes in recent years. And there have been various methods for detecting HbA1c, each with its own advantages and disadvantages .In this study, we selected and prepared the anti-human HbA1c-6C2 monoclonal antibodies with high purity and specificity, then adopted it to detect HbA1c and took methodological evaluation based on the latex-enhanced immunoturbidimetric assay. Results showed that excellent linearity was observed within the HbA1c concentration range of 3.8% to 14.0%, and CV was less than 1.5% for repeated detection at high and low concentration levels.There was a strong correlation with HPLC during methodological comparison. In general, it had the advantages of good linearity, high precision, stability and accuracy, which revealed a favourable detection effect.

1 Introduction
Diabetes is a group of metabolic disorders caused by abnormal glucose metabolism. It is a hyperglycemia caused by reduced utilization or excessive production of glucose, which can cause coronary artery disease, kidney disease, retinopathy and other complications.[1] As of 2021, the number of patients is about 537 million in global and about 140.9 million in China, which is still increasing.[2]For decades, the diagnosis of diabetes has been based on blood glucose standards, including fasting plasma glucose (FPG) and oral glucose tolerance test (75g OGTT). Hemoglobin A1c (HbA1c) is a widely-used chronic blood glucose indicator and can reflect the average blood glucose concentration in the past 2 to 3 months. In recent years, with the continuous standardization of its measurement, it has been recommended by international expert committees as one of the criteria for diagnosing diabetes, with a diagnostic threshold of 6.5% (NGSP units).[3]
HbA1c is a type of glycated hemoglobin (GHb) formed by the condensation of the aldehyde group of glucose with the amino terminal valine residue of the β - chain of HbA. The two firstly form an unstable aldehyde imine (i.e. Schiff base), which then dissociates or undergoes Amadori molecular rearrangement to form a stable ketamine compound.[4, 5] According to reports, there are various methods for detecting HbA1c ,Including high performance liquid chromatography (HPLC)[6] , enzyme assay[7], affinity chromatography[8], electrochemical method[9],etc. [5] Among them, HPLC has been used as the gold standard for HbA1c level determination and is currently the most precise and accurate method, but it requires large and relatively expensive equipment.[10]In this study, we screened and prepared a type of highly specific mouse anti-human HbA1c-6C2 antibodies based on monoclonal technology, adopted the latex-enhanced immunoturbidimetric assay (LEIBA) to detect human HbA1c, and then conduct methodological evaluation on it.

2 Materials and methods
2.1 Sample collection

2.2 Preparation of antibodies
Glycosylate the amino group on the N-terminal valine of hemoglobin beta polypeptide chain with glucose C1 and conjugate with KLH and BSA as immunogens. Five female BALB/c mice, aged 6-8 weeks ,were immunized three times at multiple subcutaneous sites with the immunogens.The first immunization was emulsified with complete Freund's adjuvant , followed by two subsequent immunizations with incomplete Freund's adjuvant emulsion, with a 3-week interval between each immunization. Spleen cells from immune enhanced BALB/c mouse were fused with SP2/0 cells using PEG4000 and screened. The cell culture supernatant on the 7th day after fusion was collected for ELISA detection. Positive hybridoma cells were selected for amplification, and subclones were obtained by extreme dilution method. The clones in large quantities were injected into the abdominal cavity of the mice to produce monoclonal antibodies. Then the extracted mouse ascites were centrifuged and the supernatant was collected for purification. Screening and preparation of anti-mouse anti-human HbA1c antibodies based on the similar principles.

2.3 Reagents
Reagent 1 consists of latex microspheres, tris (hydroxymethyl) aminomethane and preservatives, while reagent 2 consists of tris (hydroxymethyl) aminomethane, preservatives, PEG6000 (polyethylene glycol 6000), sucrose, sodium chloride, goat anti-mouse IgG-2B4 antibody and mouse anti-human HbA1c-6C2 antibody. Among them, Tris (hydroxymethyl) aminomethane and PEG6000 were purchased from Suzhou Yake Technology Co., Ltd. Sucrose，sodium chloride sodium phosphate dibasic dihydrate and disodium hydrogen phosphate dodecahydrate were purchased from China National Pharmaceutical Group Chemical Reagent Co., Ltd. Latex microspheres were purchased from Polysphere. The preservative (ProClin300) was purchased from Sigma Aldrich.

2.4 Characterization of antibodies
2.4.1 Purity
To analyse the purity of the antibodies we selected, we applied HPLC in this study . The sample was diluted to 1.5mg/ml with a mobile phase. And the analytical chromatographic separations were carried out on Elite Agerss-D1100 HPLC system with a mobile phase consisting of sodium phosphate dibasic dihydrate-disodium hydrogen phosphate dodecahydrate-sodium chloride-water mixture at a flow rate of 0.3ml/min and a column temperature of 10-30 ℃. The injection volume was 20μl and the detection wavelength we set was at 280nm.

2.4.2 Specificity
Adopt ELISA method to characterize the screened antibodies. After overnight encapsulation of 5 μ g/ml human Hb and human HbA1c at 4 ℃, 1% BSA was added and blocked at 37 ℃ for 2 hours. Dilute the mouse anti-human HbA1c-6C2 antibody obtained by 1ug/ul screening at a ratio of 1:10000 and add it. Dilute each well in a multiple ratio and incubate at 37 ° C for 2 hours. Then add goat anti-mouse HRP at a ratio of 1:2000 and incubate at 37 ° C for 1 hour. After 3 minutes of dark staining, measure Hb and HbA1c activity separately. Use the same method to detect anti-human HbA1c-3A7 antibody and anti-human Hb-1A1 antibody for comparative analysis.
Coat with 2 μ g/ml of IgG from four different species including mice, rabbits, humans, and goats as antigens, add goat anti-mouse（GAM） IgG-2B4 antibody and incubate at 37 ℃ for 1 hour to detect its activity. Use the same method to detect GAM IgG-2H4 antibody, GAM IgG-2H2 antibody and GAM polyclonal antibody for comparative analysis.
Western blot analysis was performed to obtain immunoblotting of hemoglobin’ expression and detect antibody specificity, using naturally extracted glycated hemoglobin and normal hemoglobin as substrates, with the mouse anti-human HbA1c-6C2 antibody and the mouse anti -human Hb-1A1 antibody serving as the primary antibody separately,and the goat anti-mouse IgG-2B4 antibody adopted as the secondary antibody.

2.5 Instruments and methods
In this study, the experiment was conducted on a Beckman AU480 biochemical analyzer. When adding reagent 1 to the sample to be tested, the glycated hemoglobin was bound to the latex microspheres. After adding reagent 2, glycated hemoglobin binds to specific antibodies, forming latex microspher-anti-HbA1c antibody complexes. Then, the bind of sheep-anti-mouse IgG-2B4 antibody and glycated hemoglobin antibodies caused the aggregation of latex microspheres. These complexes led to the scattering of the irradiation beam, and the intensity of the scattered light is proportional to the concentration of HbA1c in the sample.

2.6 Evaluation of methodology
2.6.1 Calibration curve
Use a standard sample with a known percentage concentration of glycated hemoglobin to obtain absorbance values according to the steps mentioned in section 2.3. Plot a standard curve with percentage concentration as the x-axis and absorbance values as the y-axis.

2.6.2 Blank absorbance
Use physiological saline as a blank sample to make a detection and record the absorbance at startup (A1) and the absorbance after about 5 minutes (A2). And the value of A2 is the blank absorbance value.

2.6.3 Linearity
Dilute high concentration samples close to the upper limit of the linear interval with low concentration samples close to the lower limit of the linear interval, with dilution concentrations of 3.9%, 5.5%, 7.3%, 9.2%, 11.3%, and 13.7% respectively. Perform 3 repeated tests on the diluted sample and take the average as the test result. Evaluate linearity by performing linear regression analysis on the detection values and theoretical values and calculating the Spearman correlation coefficient.

2.6.4 Precision
Under repeatability conditions, select samples possessing two different concentration levels within the linear range: high concentration (not less than 30% of the reference range) and low concentration (within the reference range). Repeat the detection 20 times, take the average of the detection results and calculate the standard deviation (SD) and coefficient of variation (CV).

2.6.5 Stability
Store the reagents in a refrigerator at 2-8 ℃ for at least 30 days, and take samples at 1, 4, 7, 10, 15, 20, 25, 30, 40, and 45 days from the date of storage. Follow the reference detection method for testing and record the results. After each sampling, store the reagents back at 2-8 ℃.

2.6.6 Methodology comparison
Select 50 blood samples, which cover a wide range of glycated hemoglobin concentrations. The samples will be tested three times on the Beckman AU480 biochemical analyzer and the Elite Agerss-D1100 HPLC system respectively, and the average value will be taken for comparative analysis. Correlation analysis was conducted between the results obtained by latex enhanced immunoturbidimetry and HPLC to obtain linear correlation coefficients and linear regression equations, in order to evaluate their comparability.

3 Results
3.1 Preparation of the antibodies.
Following the steps in section 2.2, after four subclone screening, one positive monoclonal hybridoma was selected and named 6C2. It was injected into the peritoneal cavity of mice to obtain ascites. Meanwhile, purified cell culture supernatant and ascites were used to obtain mouse anti-human HbA1c-6C2 monoclonal antibody (mAb). Further identification revealed that the monoclonal antibody subtype was IgG1. Similarly, using the aforementioned antibody as an immunogen, a goat anti-mouse（GAM） IgG-2B4 monoclonal antibody was obtained.

3.2 Characterization of antibodies
3.2.1 Purity
By using HPLC method to analyze the purity of the prepared antibodies, as shown in Fig.1, only one main peak was observed for both mouse anti-human HbA1c-6C2 antibody and goat anti-mouse IgG-2B4 antibody, indicating that they are intact antibodies. The peak areas were 8136 mAU. s and 7142 mAU. s, respectively, with peak area ratios of 95.57% and 96.89%, indicating that both have high purity.

3.2.2 Specificity
As shown in the Fig.2, the anti-human HbA1c-3A7 antibody has a cross reaction with Hb and the anti-human Hb-1A1 antibody binds to both HbA1c and Hb, while the anti-human HbA1c-6C2 antibody can bind well to HbA1c instead of Hb. With increasing dilution ratio, the HbA1c activity measured relatively stable, with a potency of 256w. This proves that the anti-human HbA1c-6C2 antibody has high specificity for HbA1c and can eliminate interference from Hb in its detection.
When characterizing the goat anti-mouse IgG-2B4 antibody, the results showed that the antibody could specifically bind to mouse IgG and did not cross react with other species (Fig.3A). Although the GAM IgG-2H4 antibody and GAM IgG-2H2 antibody didn’t bind to IgG antibodies of rabbit, human, sheep and other species and have good specificity, their sensitivity is not as good as the GAM IgG-2B4 antibody (Fig.3B and C). The GAM polyclonal antibody has cross reaction with IgG antibodies of multiple species (Fig.3D). Therefore, it can be seen that the goat anti-mouse IgG-2B4 antibody has high specificity and sensitivity, which can avoid interference caused by cross reaction with other species’ IgG antibodies.
In addition, Western Blot analysis showed that when the secondary antibody was goat anti-mouse IgG-2B4 antibody and remained unchanged, if the primary antibody was mouse anti-human Hb-1A1 antibody, both Hb and HbA1c bands appeared simultaneously, while the primary antibody was mouse anti-human HbA1c-6C2 antibody, only HbA1c bands appeared (Fig.4A and B).It further proved that the mouse anti-human HbA1c-6C2 antibody does not bind to Hb and has high specificity for HbA1c.

3.3 Evaluation of methodology
The blank absorbance of the reagent at a wavelength of 660nm is 0.3768, which is less than 1.5. The linearity shows a strong correlation (=0.99) within the concentration range of 3.8% to 14.0%, with linear absolute deviations not exceeding ± 0.5% and linear relative deviations not exceeding ± 7% (NGSP units) (Fig.5B). The detection CV at low concentration levels is 1.438% and the one at high concentration levels is 0.686%, indicating that it has good precision (Table 1). As shown in the Fig.5C, this method has good stability and the detection results are still relatively stable even after the reagents are stored for 30 days under the same conditions. The correlation and regression studies used for method comparison showed a good linear relationship between latex-enhanced immunoturbidimetric assay and HPLC(=0.99), with an intercept of 0.07075 and a slope of 0.9895 (as shown in Fig.5D).

4 Discussion
Diabetes is a group of hyperglycemia caused by abnormal glucose metabolism, which can lead to a variety of complications. FPG and OGTT are often used to clinical diagnose diabetes. In recent years, with the standardization of HbA1c detection, HbA1c has gradually been used in the diagnosis of diabetes, becoming an effective tool for monitoring long-term blood glucose control, with a diagnostic threshold of 6.5% (NGSP units).[11]
Currently, multiple methods have been used for the detection of HbA1c. The testing methods selected by clinical laboratories should not only meet the requirements of simplicity, speed, batch testing, and timely reporting, but also ensure the quality of the testing results. The long-term use of methods requires strict quality management, including laboratory certification, specialized internal control design, and participation in external quality assessment (EQA) programs.[12, 13] The American Diabetes Association (ADA) recommends that laboratories only use testing methods that are certified and traceable to DCCT GHB references. For methods performed in the laboratory, the CV should be less than 1.5% and ideally there should be no measurable deviation. [12]Based on this, it is of great significance to evaluate whether the analytical performance of immunoturbidimetry meets the requirements. In this study, we screened and prepared a kind of mouse anti-human HbA1c antibody possessing high purity and specificity. We had verified its performance and conducted methodological evaluation on the latex-enhanced immunoturbidimetric assay. HPLC, ELISA and WB were adopted to characterize the antibodies and the results proved that mouse anti-human HbA1c antibodies can specifically bind to HbA1c without reacting with Hb. During conducting methodological evaluation, the CV of indoor tests at high and low concentration levels were both less than 1.5% (NGSP units) , indicating good precision and meeting the precision requirements of medical diagnosis and clinical practice. At the same time, there is a strong correlation within 3.8%~14.0% concentration range, and the linear deviation meets the requirements. In addition, a strong linear relationship has been shown between this method and HPLC, with a slope of 0.9895, intercept of 0.07075, and correlation coefficient of 0.99, which reflected the good detection performance of this method. Besides,it also possessed good stability. After the reagent we made was maintained for 30 days under the suitable conditions, the detection results were still relatively accurate.
In the clinical detection of HbA1c, triglycerides, uric acid, ethanol, and other potential interfering substances, as well as hemoglobin variations such as HbE, may also interfere with its detection. [6, 14]However, we have not evaluated the interference of these substances on HbA1c’s detection, which is a limitation of our research and will be considered in the future.
Overall, this study prepared a highly specific and high-purity mouse anti-human HbA1c antibody, and used latex-enhanced immunoturbidimetric assay to detect human HbA1c. It has good linearity, precision, accuracy and stability, which can help clinical doctors better monitor patients' blood glucose levels.References

References
[1] BARBARO M, KU-CHULIM C, JOHNSTON F, et al. Evaluation of the performance of an immunoturbidimetric HbA1c reagent applied to the Siemens ADVIA 2400 automatic analyzer[J]. Clinical Biochemistry, 2015: 177-180.
[2] David B. Sacks, Mark Arnold, George L. Bakris, et al. Guidelines and Recommendations for Laboratory Analysis in the Diagnosis and Management of Diabetes Mellitus[J]. Clinical Chemistry, 2023,69: 808-868.
[3] Diagnosis and Classification of Diabetes Mellitus[J]. Diabetes Care, 2011,34(Supplement_1): S62-S69.
[4] MURALIDHARAN M, BHAT V, MANDAL A K. Structural analysis of glycated human hemoglobin using native mass spectrometry[J]. FEBS J, 2020,287(6): 1247-1254.
[5] MOLAZEMHOSSEINI A, MAGAGNIN L, VENA P, et al. Single-Use Disposable Electrochemical Label-Free Immunosensor for Detection of Glycated Hemoglobin (HbA1c) Using Differential Pulse Voltammetry (DPV)[J]. Sensors (Basel), 2016,16(7).
[6] YU Y, ZHANG X, LIN K. Analytical performance evaluation of hemoglobin A1c on an ARKRAY HA-8160 analyzer with newly-developed mobile phase buffer [J]. Practical Laboratory Medicine.
[7] JAISSON S, DESMONS A, RENARD B, et al. Analytical performances of a new enzymatic assay for hemoglobin A1c[J]. Clinica chimica acta, 2014,434: 48-52.
[8] KOSKINEN L K. Specificity of hemoglobin A1c measurement cation exchange liquid chromatography. [J]. Clinica Chimica Acta, 1996: 159-169.
[9] Mukesh Thapa, HEO Y S. Label-free electrochemical detection of glucose and glycated hemoglobin (HbA1c)[J]. Biosensors and Bioelectronics: X, 2023.
[10] CHANG K, JINGLUN L, CHING-HSUAN Y, et al. An integrated microfluidic system for measurement of glycated hemoglobin Levels by using an aptamer–antibody assay on magnetic beads[J]. Biosensors and Bioelectronics, 2015: 397-403.
[11] DEVI R, CHAUHAN A, YADAV S, et al. Development of aptamer based colorimetric assay for whole blood glycated hemoglobin (HbA1c) estimation using gold nanoparticles[J]. Biosensors and bioelectronics. X, 2023,13: 100304.
[12] David B. Sacks, Mark Arnold, George L. Bakris, et al. Guidelines and Recommendations for Laboratory Analysis in the Diagnosis and Management of Diabetes Mellitus[J]. Diabetes Care, 2023: e151-e199.
[13] WEYKAMP C. HbA1c: A Review of Analytical and Clinical Aspects[J]. Annals of Laboratory Medicine, 2013,33(6): 393-400.
[14] LOH T P, CHENG W L, KAO S L, et al. Effects of haemoglobin E traits on HbA1c measurement by two cation-exchange HPLC and two immunoturbidimetric methods[J]. Pathology, 2014.

Table 1
The detection SD and CV at low concentration and at high concentration levels.

Group Testing Times MIN MAX MEAN SD CV（%）
Low Level N=20 4.60 4.80 4.665 0.067 1.438
High Level N=20 12.10 12.40 12.225 0.085 0.696

Fig.1. Purity analysis of mouse anti-human HbA1c-6C2 antibody and goat anti-mouse IgG-2B4 antibody using HPLC.

Fig.2. Measurement on the activity of different antibodies using HbA1c and Hb at dilution of 1:1w, 1:2w, 1:3w, 1:4w, and 1:5w respectively: A) mouse anti-human Hb-1A1 antibody; B) mouse anti-human HbA1c-3A7 antibody; C) mouse anti-human HbA1c-6C2 antibody.

Fig.3. Detection on activities of four different secondary antibodies using four different species’ antibodies: A) GAM-2B4 antibody; B) GAM-2H2 antibody; C) GAM-2H4 antibody; D) GAM polyclonal antibody.

Fig.4. Western Blot analysis of HbA1c-6C2 and Hb-1A1.

Fig.5. Evaluation of methodology: A) Calibration curve;B) Linear curve;C) Stability detection on HbA1c with concentrations of 5.3%, 7.8%, 10.2%, and 13.1% using the reagents saved for 1, 4, 7, 10, 15, 20, 25, 30, and 40 days.;D) Methodology comparison between LEIBA and HPLC.你是此项项目的研究专家，临床采集的血样是医院患者已经检测的糖化血红蛋白项目的剩余的标本，请根据这片论文的成果，写一份中英文研究方案及患者知情同意书，用于通过伦理委员会。

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Crafting a research protocol

I'm piecing together a Chinese and English protocol for using leftover clinical samples for research. It includes consent forms and disclaimers, ensuring comprehensive ethics coverage.

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以下提供了一份针对“基于高特异性鼠源抗人HbA1c-6C2单克隆抗体的乳胶增强免疫比浊法检测人HbA1c”项目的研究方案和患者知情同意书（中英文对照），内容特别针对“使用临床患者检测糖化血红蛋白后剩余标本”进行研究的伦理合规要求。研究者可根据自身所在单位及伦理委员会的具体要求，对以下内容进行相应修改和补充。

一、研究方案 (中英文)

中文版

1. 研究标题
基于高特异性鼠源抗人HbA1c-6C2单克隆抗体的乳胶增强免疫比浊法检测人HbA1c的研究 —— 利用常规检测后剩余血样本进行方法学评价

2. 研究背景与意义
糖化血红蛋白（HbA1c）能够反映患者近2-3个月内的平均血糖水平，已在糖尿病诊断及管理中具有重要地位。现有多种检测方法，包括高效液相色谱（HPLC）、酶法、免疫比浊法等，各有优缺点。本研究在前期筛选并制备了高纯度、高特异性的鼠源抗人HbA1c-6C2单克隆抗体基础上，建立了乳胶增强免疫比浊法（LEIBA）检测HbA1c的新方案，并在实验室条件下对其线性、精密度、稳定性与准确度进行了初步验证，显示出较好的应用前景。
为了进一步评估该方法的临床可行性与准确度，对比参考目前公认的HPLC法，需要收集一定数量的临床患者血样本并进行检测比对。本研究拟使用医院在常规糖化血红蛋白检测后剩余的血清/全血标本，进行方法学验证及比较，以期为该方法的进一步应用提供科学依据。

3. 研究目的

  1. 验证采用乳胶增强免疫比浊法（基于鼠源抗人HbA1c-6C2单克隆抗体）检测HbA1c在临床样本中的准确度、精密度和稳定性。
  2. 与高效液相色谱（HPLC）方法进行方法学比对，评价新方法与金标准之间的相关性和一致性。
  3. 探讨该新方法在临床糖化血红蛋白常规检测中的应用前景。

4. 研究设计

  • 研究类型： 方法学评价研究（横断面对比）

  • 受试者来源及样本获取：

    1. 在本院已常规检测糖化血红蛋白（HbA1c）的患者中，使用其剩余血标本进行本研究。
    2. 样本要求：临床常规HbA1c检测结束后，剩余血样不少于约0.5-1mL，全血或血清均可（视新方法需求）。
    3. 预计收集不少于50份临床标本，覆盖不同HbA1c浓度范围，以保证方法学评价的统计有效性。

  • 检测对比：

    • 乳胶增强免疫比浊法（LEIBA）检测HbA1c
    • 高效液相色谱（HPLC）检测HbA1c（作为对照参考）

  • 主要评价指标：

    1. 线性范围、相关系数
    2. 精密度（CV值）
    3. 准确度及与HPLC法的比较结果（回归分析、相关系数）
    4. 稳定性测试（试剂在不同储存时间对检测结果的影响）

5. 研究对象纳入/排除标准

  • 纳入标准：
    1. 已在本院行常规糖化血红蛋白检测的患者；
    2. 剩余标本量满足本研究检测需要；
    3. 标本检测后不影响患者常规诊疗；
    4. 同意（或签署）本研究使用其剩余样本。
  • 排除标准：
    1. 剩余标本量不足、标本溶血或放置时间过久等影响检测质量；
    2. 其他研究者认为不宜纳入研究的情况。

6. 研究流程

  1. 收集信息：研究人员与临床检验科合作，统计已完成常规HbA1c检测的患者标本；
  2. 剩余标本获取：经患者知情同意后，待常规检测完成后，剩余部分由研究人员回收，立即置于规定条件下保存；
  3. 实验检测：采用LEIBA与HPLC分别检测HbA1c；
  4. 数据分析：比较两种检测结果的一致性与相关性；
  5. 结果总结：撰写研究报告，或发表学术论文。

7. 样本量与统计学方法

  • 样本量： 预计不少于50例患者剩余血样，以覆盖HbA1c从低到高的范围并满足方法学统计要求。
  • 统计分析：
    1. 相关性与回归分析（如Pearson或Spearman相关系数、回归方程）；
    2. 精密度（CV）评估；
    3. 偏倚分析（Bland-Altman图或Passing-Bablok回归）；
    4. 使用SPSS或其他统计软件进行分析。

8. 伦理与风险收益分析

  • 伦理原则： 严格遵守《世界医学大会赫尔辛基宣言》及国家相关医学伦理法规。
  • 风险评估：
    • 本研究不额外抽血，不增加患者的创伤，仅使用患者常规检测后的剩余标本，对患者几乎无风险。
    • 研究结果仅用于方法学评估，不会影响患者既有的诊疗方案。
  • 收益分析：
    • 通过本研究，可帮助评估并改进新的HbA1c检测方法，为临床和患者带来潜在益处；
    • 如果研究结果可靠，未来患者可享受更快速、准确的HbA1c检测。

9. 研究进度与质量控制

  • 进度计划： 本研究预计持续3-6个月。
  • 质量控制：
    1. 剩余标本的采集、保存和运输，严格执行标准操作程序；
    2. LEIBA法和HPLC法均进行日常质控与仪器校准；
    3. 双人核对数据，并做好信息加密与隐私保护。

10. 受试者权益和保密

  • 自愿原则： 患者有权拒绝本研究使用其剩余标本，且拒绝不影响其常规治疗；
  • 保密措施： 患者资料及检测结果仅限科研使用，所有个人信息去标识化处理，研究或发表时不公开患者身份信息；
  • 退出机制： 患者若在研究期间不同意使用其标本，可随时提出，研究者将停止使用并销毁相关剩余样本。

11. 研究人员与经费来源

  • 主要研究者： XXX，单位：XXX，联系方式：XXX
  • 共同研究者： XXX，单位：XXX，联系方式：XXX
  • 经费来源： XXX科研基金（如有），不向患者收取额外费用。

English Version

1. Title of the Study
Evaluation of a Latex-Enhanced Immunoturbidimetric Assay for Human HbA1c Detection Based on a Highly Specific Mouse Anti-HbA1c-6C2 Monoclonal Antibody — Using Leftover Samples from Routine Clinical Testing

2. Background and Significance
Hemoglobin A1c (HbA1c) reflects patients’ average blood glucose levels over the past 2-3 months and plays a critical role in the diagnosis and management of diabetes. Multiple assays exist (HPLC, enzymatic, immunoturbidimetric, etc.), each with unique advantages. Our preliminary work developed a new latex-enhanced immunoturbidimetric assay (LEIBA) using a highly purified and specific mouse anti-human HbA1c-6C2 monoclonal antibody. Laboratory studies showed favorable linearity, precision, stability, and accuracy.
To further confirm its clinical feasibility and performance, we plan to compare this new method with the conventional high-performance liquid chromatography (HPLC). Therefore, we will use leftover blood samples from patients who have already undergone routine HbA1c tests in our hospital.

3. Objectives

  1. To validate the accuracy, precision, and stability of the LEIBA assay (using the mouse anti-human HbA1c-6C2 monoclonal antibody) in clinical samples.
  2. To compare and correlate the new method with HPLC, regarded as the reference standard, and assess their consistency.
  3. To explore the potential of this assay as a routine method for HbA1c testing in clinical settings.

4. Study Design

  • Study Type: Methodological evaluation (cross-sectional comparison)

  • Participant Source and Sample Collection:

    1. Use leftover blood samples from patients who have undergone routine HbA1c testing in our hospital.
    2. Sufficient leftover volume (≥0.5-1 mL) of serum or whole blood after the routine test.
    3. We aim to collect ≥50 samples covering a wide range of HbA1c levels for robust method evaluation.

  • Comparative Assays:

    • Latex-enhanced immunoturbidimetric assay (LEIBA) for HbA1c
    • High-performance liquid chromatography (HPLC) for HbA1c (as the reference)

  • Key Parameters:

    1. Linearity range and correlation coefficient
    2. Precision (Coefficient of Variation, CV)
    3. Accuracy and agreement with HPLC (regression analysis, correlation)
    4. Stability assessment of reagents (impact of storage time)

5. Inclusion/Exclusion Criteria

  • Inclusion Criteria:
    1. Patients whose blood samples have been used for routine HbA1c testing in this hospital;
    2. An adequate volume of leftover samples is available;
    3. The leftover sample does not affect patients’ routine care;
    4. Patients agree (via informed consent or prior agreement) to the use of their leftover samples for this study.
  • Exclusion Criteria:
    1. Insufficient leftover volume, hemolyzed samples, or samples stored too long that might compromise data quality;
    2. Other circumstances deemed unsuitable by the investigator.

6. Study Procedures

  1. Collect information and coordinate with the clinical laboratory to identify leftover samples after routine HbA1c testing;
  2. Upon obtaining patient consent, retrieve the leftover samples after clinical testing, store them under appropriate conditions;
  3. Measure HbA1c via LEIBA and HPLC, respectively;
  4. Perform data analysis to compare correlation and agreement;
  5. Summarize results in a report or scientific publication.

7. Sample Size and Statistical Methods

  • Sample Size: ≥50 leftover samples are expected, covering low to high HbA1c levels, to meet the statistical requirements of method evaluation.
  • Statistical Analysis:
    1. Correlation and regression analysis (Pearson or Spearman correlation coefficient, regression equation);
    2. Precision (CV) evaluation;
    3. Bias analysis (Bland-Altman plots or Passing-Bablok regression);
    4. Data analysis with SPSS or other statistical software.

8. Ethics, Risk-Benefit Assessment

  • Ethical Principles: Strictly follows the Declaration of Helsinki and applicable national regulations.
  • Risk Assessment:
    • No additional phlebotomy or invasive procedures are involved; only leftover samples are used.
    • The study results will not affect current patient care.
  • Benefit Analysis:
    • This research aims to improve and validate a novel HbA1c testing method that may offer more rapid and accurate diagnostics in the future.
    • No direct benefit to individual participants is guaranteed, but the knowledge gained may benefit wider patient populations.

9. Study Timeline and Quality Control

  • Timeline: Estimated study duration is 3-6 months.
  • Quality Control:
    1. Standard operating procedures for leftover sample collection, storage, and transport;
    2. Regular calibration and quality control for both LEIBA and HPLC;
    3. Double-checking and de-identification of all data;
    4. Ensuring patient confidentiality and data security.

10. Participant Rights and Confidentiality

  • Voluntary Basis: Patients have the right to decline the use of their leftover samples without impacting their treatment.
  • Confidentiality: Patient data and test results are for research only. All personal identifiers are removed. No individual identity will be disclosed in publications.
  • Withdrawal Mechanism: Participants can request the discontinuation of their leftover sample usage at any time, and the study team will cease using or destroy their sample.

11. Investigators and Funding

  • Principal Investigator: XXX, Institution: XXX, Contact: XXX
  • Co-investigators: XXX, Institution: XXX, Contact: XXX
  • Funding Source: XXX Research Grant (if any). No additional cost is imposed on the participants.

二、患者知情同意书 (中英文)

中文版

研究名称： 基于高特异性鼠源抗人HbA1c-6C2单克隆抗体的乳胶增强免疫比浊法检测人HbA1c的研究

尊敬的患者/受试者，您好：
您在本院进行糖化血红蛋白（HbA1c）常规检测后，可能会有剩余的血液样本。我们诚挚地邀请您同意将这部分“剩余血样”用于一项关于HbA1c检测新方法的科研研究。请您仔细阅读以下信息，如有疑问可向研究者咨询。

  1. 研究目的与背景

    • 本研究旨在评估一种新的糖化血红蛋白检测方法（乳胶增强免疫比浊法），与现有的高效液相色谱（HPLC）方法进行比较。
    • 该研究有助于改进和推广更快速、准确的HbA1c检测手段，以期为未来更多患者带来收益。

  2. 样本来源与流程

    • 您无需额外采血；我们仅使用您在本院完成常规HbA1c检测后剩余的血样。
    • 剩余样本将被研究人员在合规条件下储存，用于实验室检测和数据分析。

  3. 可能的风险与不适

    • 本研究不额外抽血，也不改变您的临床诊疗方案，因此无额外医疗风险或不适。
    • 剩余样本用于研究不会对您造成任何生理干扰。

  4. 潜在收益

    • 本研究可能不会给您本人带来直接的医疗获益，但有助于完善新的HbA1c检测方法，为临床诊断和后续患者治疗提供参考。
    • 若研究结果可靠并得到推广，未来的患者可能享受到更准确、便捷的检测。

  5. 隐私与保密

    • 我们仅收集研究所需的最少限度医疗信息；所有样本和数据在研究过程中均去标识化处理，以防止透露您的个人身份。
    • 研究结果仅用于学术或科研发表，个人隐私将受到法律与伦理保护，不会在任何公开场合披露。

  6. 自愿参与及退出权利

    • 您的参与完全自愿。您有权拒绝提供剩余样本用于本研究，拒绝不会影响您的正常治疗。
    • 如您在研究过程中改变主意，可随时提出退出，研究人员将停止使用并销毁您的剩余样本。

  7. 费用与补偿

    • 本研究不向您收取任何费用，也不会为此向您支付额外补偿。
    • 您无需承担任何额外的经济负担。

  8. 联系方式

    • 如果您对本研究有任何疑问或需要了解更多信息，请随时联系：
      • 研究负责人：XXX，联系电话：XXX
      • 研究助理：XXX，联系电话：XXX

如您已充分了解并同意，请在下方签字：

我已经阅读（或被口头告知）本知情同意书的全部内容，对于研究目的、流程、可能风险及保密原则均已清楚。我自愿同意将我的剩余血样用于本研究。

受试者（签字）：_________________ 日期：___________
研究者/见证人（签字）：___________ 日期：___________

English Version

Title of the Study: Evaluation of a Latex-Enhanced Immunoturbidimetric Assay for Human HbA1c Detection Based on a Highly Specific Mouse Anti-HbA1c-6C2 Monoclonal Antibody

Dear Participant,
We would like to invite you to allow us to use the leftover portion of your blood sample for a research study aimed at evaluating a new method for measuring Hemoglobin A1c (HbA1c). Please read the following information carefully. If you have any questions, feel free to ask the research staff.

  1. Study Purpose and Background

    • This study aims to compare a novel method of measuring HbA1c (latex-enhanced immunoturbidimetric assay) with the existing high-performance liquid chromatography (HPLC).
    • We hope to improve the speed and accuracy of HbA1c measurements, potentially benefiting more patients in the future.

  2. Source of Samples and Procedures

    • You will not undergo any additional blood draw. We will only use the leftover blood sample after your routine clinical HbA1c test.
    • The leftover sample will be stored according to proper laboratory protocols and used for experimental testing and data analysis.

  3. Potential Risks or Discomfort

    • Since no additional phlebotomy is required and your clinical treatment will not be altered, there are no extra medical risks or discomforts associated with this research.
    • Using leftover samples will not affect your health in any way.

  4. Potential Benefits

    • You may not receive direct medical benefits from participating in this study, but your sample will help validate a new HbA1c testing method that may improve clinical diagnosis for future patients.
    • If successful and widely adopted, patients could benefit from more accurate and convenient HbA1c testing.

  5. Privacy and Confidentiality

    • We will collect only minimal necessary medical information for the study. All samples and data will be de-identified to protect your privacy.
    • The study results will be used for academic or scientific purposes. No personal or identifiable information will be released in any publication.

  6. Voluntary Participation and Right to Withdraw

    • Your participation is entirely voluntary. You have the right to decline the use of your leftover sample without impacting your treatment.
    • If you change your mind after agreeing, you can withdraw at any time. We will cease using and destroy any leftover sample pertaining to you.

  7. Costs and Compensation

    • There are no additional costs to you, and no financial compensation will be provided for participating.
    • You will not bear any economic burden for this study.

  8. Contact Information

    • If you have any questions or need further information, please contact:
      • Principal Investigator: XXX, Phone: XXX
      • Research Assistant: XXX, Phone: XXX

By signing below, you acknowledge that you have read (or been informed of) all the information in this consent form, understand the purpose, procedures, potential risks, and confidentiality measures, and voluntarily agree to let your leftover sample be used for this study.

Participant (Signature): _______________ Date: ___________
Investigator/Witness (Signature): _________ Date: ___________

以上文本可根据医院伦理委员会具体要求和项目实际情况进行调整、补充或删减。

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