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使用目的：

本试剂盒用于测定血清、血浆及相关液体样本中的指标含量。

实验原理

本试剂盒应用双抗体夹心法测定标本中指标水平。用纯化的抗体包被微孔板，制成固相抗体，与HRP标记的抗体结合，形成抗体-抗原-酶标抗体复合物，经过彻底洗涤后加底物TMB显色。TMB在HRP酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的指标呈正相关。用酶标仪在450nm波长下测定吸光度（OD值），通过标准曲线计算浓度。

试剂盒组成

1	30倍浓缩洗涤液	20ml×1瓶	7	终止液	6ml×1瓶
2	酶标试剂	6ml×1瓶	8	标准品	0.5ml×1瓶
3	酶标包被板	12孔×8条	9	标准品稀释液	1.5ml×1瓶
4	样品稀释液	6ml×1瓶	10	说明书	1份
5	显色剂A液	6ml×1瓶	11	封板膜	2张
6	显色剂B液	6ml×1/瓶	12	密封袋	1个

操作步骤

1. 标准品的稀释：本试剂盒提供原倍标准品一支，用户可在小试管中进行稀释。

2. 加样：分别设空白孔（空白对照孔不加样品及酶标试剂，其余各步操作相同）、标准孔、待测样品孔。在酶标包被板上标准品准确加样50μl，待测样品孔中先加样品稀释液40μl，然后再加待测样品10μl（样品最终稀释度为5倍）。加样将样品加于酶标板孔底部，尽量不触及孔壁，轻轻晃动混匀。

3. 温育：用封板膜封板后置37℃温育30分钟。   

4. 配液：将30倍浓缩洗涤液用蒸馏水30倍稀释后备用

5. 洗涤：小心揭掉封板膜，弃去液体，甩干，每孔加满洗涤液，静置30秒后弃去，如此重复5次，拍干。

6. 加酶：每孔加入酶标试剂50μl，空白孔除外。

7. 温育：操作同3。

8. 洗涤：操作同5。

9. 显色：每孔先加入显色剂A50μl，再加入显色剂B50μl，轻轻震荡混匀，37℃避光显色10分钟.

10. 终止：每孔加终止液50μl，终止反应（此时蓝色立转黄色）。

11. 测定：以空白孔调零，450nm波长依序测量各孔的吸光度（OD值）。 测定应在加终止液后15分钟以内进行。

文献参考：文章标题：Optimizing anti-PD-1/PD-L1 therapy efficacy and fecal microbiota transplantation donor selection through gut mycobiome-based enterotype

作者列表：Muni Hu, Xiaoqiang Zhu, Xiaowen Huang, Li Hua, Xiaolin Lin, Hangyu Zhang, Ye Hu, Tianying Tong, Lingxi Li, Baoqin Xuan, Ying Zhao, Yilu Zhou, Jinmei Ding, Yanru Ma, Yi Jiang, Lijun Ning, Yue Zhang, Zhenyu Wang, Jing-Yuan Fang, Youwei Zhang, Xiuying Xiao, Jie Hong, Huimin Chen, Jiantao Li, Haoyan Chen

发表时间：2025-4-20

期刊：Cell Reports

影响因子：6.9

DOI:10.1016/j.celrep.2025.115589

Abstract

Introduction

Immunotherapy has demonstrated significant success in treating various hematological and metastatic solid malignancies.1,2,3,4 The impact of the gut microbiome on therapeutic responses has been validated through numerous clinical and preclinical trials.5 Ongoing investigations in gut microbiome profiling have opened avenues for its potential as a therapeutic tool or target. The term “gut microbiome” refers to the complex and diverse community of microorganisms, including bacteria, fungi, viruses, and other microbes, that inhabit the gastrointestinal tract of humans. Given the predominant presence of intestinal bacteria, comprising over 99% of the total gut microbiome,6 it has been a subject of profound interest. Research focusing on intestinal bacteria and immunotherapy has expanded, fueled by preclinical experiments affirming the impact of intestinal bacteria and their metabolites on tumor immunity.7,8 Nevertheless, with increasing research, the role of the gut mycobiome in the occurrence, progression, and treatment of diseases is becoming increasingly apparent.9,10,11,12,13 A recent study14 revealed that the gut mycobiome, outperforming its bacterial counterpart, exhibits superior precision in predicting the efficacy of immunotherapy.

Serving as a valuable framework for the exploration of the gut microbiome and its relation to human diseases, enterotypes are defined as distinct clusters or groups of microbial communities present in the human gut microbiome. Introduced initially in 2011, this classification, based on the genus-level analysis of human intestinal bacteria, identified three primary types: Bacteroides, Prevotella, and Ruminococcus.15 Enterotypes are generally considered stable and closely tied to the host’s lifestyle or physiological state.16,17 The majority of research on enterotypes has centered on gut bacteria, but there has also been exploration of fungal and viral communities to enable the categorization of individuals based on specific host phenotypes. In recent times, researchers have classified the human gut mycobiome at the genus level using fecal internal transcribed spacer sequencing data. They identified distinct enterotypes based on the gut mycobiome, revealing correlations with specific diseases and age.18 Additionally, another study indicated that classification of the human gut virome is linked to the therapeutic success of patients with inflammatory bowel disease.19 Hence, the utilization of enterotypes has been instrumental in investigating potential connections between microbial patterns and diverse health conditions.

Here, we utilized a metagenomic analysis pipeline to profile fungus abundance in patients undergoing anti-PD1/PD-L1 immunotherapy following previous studies.20,21 Our initial analysis revealed the presence of two distinct gut mycobiome-based enterotypes (favorable type and unfavorable type) that correlated with the efficacy of immunotherapy. Favorable-type patients harbored higher cytotoxic CD8+ T cell proportions in the TME, as determined by multi-omics data analysis. Noteworthy is the finding that individuals undergoing fecal microbiota transplantation (FMT) from favorable-type donors exhibited an elevated response rate to anti-PD-1/PD-L1 therapy. Our study provides novel insights into the gut mycobiome-based enterotype, presenting possibilities for improving the efficacy of anti-PD-1/PD-L1 immunotherapy and aiding in the identification of optimal FMT donors.