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In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. www.rsc.org/analyst Page 1 of 6 Please do not adjust marginsAnalyst 1 2 3 4 Journal Name 5 6 7 8 ARTICLE 9 10 11 12 Combining Complement Fixation and Luminol Chemiluminescence 13 14 for Ultrasensitive Detection of Avian Influenza A rH7N9 15 Received 00th January 20xx, Man Li,a,b ZhuanZhuan Shi,a,b ChangMing Li a,b and Ling Yua,b* 16 Accepted 00th January 20xx 17 DOI: 10.1039/x0xx00000x 18 19 www.rsc.org/ The complement fixation test (CFT) is a serological test that can be used to detect the presence of either specific antibody 20 or antigen to diagnose infections. In a conventional CFT, the assay result is determined by observing the clarity of the 21 reaction solution or sediment of red cells by eyes. Although the assay condition is thereafter simplified, the sensitivity of 22 the assay Would be sacrificed due to the limitation of bulk observation. Inspired by the forensic scientist to exam blood at 23 a scene in criminal, We rationally argued that luminol chemiluminescence (CL) reaction could be applied in CFT to sense 24 physiological complement-mediate haemolytic phenomena for sensitive protein detection. The combination of CFT and 25 lumino CL system Was demonstrated in detection of rH7N9, a recombinant avian influenza virus protein. The testing can 26 be accomplished Within 2.5 h and the linear detection range covers 0.25 fg/mL to 25 ng/mL. The feasibility of the CL based 27 CFT in assaying real biopsy Was successfully demonstrated by specifically detection rH7N9 and carcinoembryonic antigen 28 (CEA) in human serum. This neW type of protein detection approach inherits the beauty of complement-mediate assay, 29 such as fast, no protein immobilization, blocking and Washing. In addition, the participation of luminol CL enables Manuscript 30 quantitatively analyse the intensity of a hameolysis process, ameliorating the limitation of bulk observation in traditional 31 CFT. It is anticipated the luminol CL-CFT assay Would be particularly suitable for small molecules, toxic, short peptide 32 investigation. 33 34 35 36 37 38 39 Accepted 40 appropriate for the analysis of small molecules, such as toxins 41 1. Introduction and short peptides. The complement fixation test (CFT) is a homogenous serology test. The fundamental principle of this 42 In clinical practise and epidemiology, serological techniques assay is that activation of the complement, a system of serum 43 play a critical role in various aspects of infection, vaccine 44 1 proteins, is initiated by complexes of Ags and IgG1, IgG2, IgG3, development, and evaluation. Serological tests, such as 45 and IgM antibodies (Abs).2, 3 When activation occurs on the cell immunoassays, are performed in homogenous and 46 surface, trans-membrane pores form, ultimately resulting in heterogeneous formats. The key difference betWeen these two 4 47 cell lysis. This biochemical property has been utilized to Analyst types of assay is that homogenous assays do not require an 48 develop the CFT for testing biomarkers of infectious and additional physical separation step, and are generally simpler 49 autoimmune diseases.5-7 As illustrated in Figure 1, the CFT to perform with a shorter assay time. Most importantly, unlike 50 assay includes tWo systems: the sampler and indicator systems. heterogeneous sandwich assays, Where an antigen (Ag) must 51 The complement is added to the sampler system, which possess tWo or more epitopes, homogeneous assays are 52 contains the unknoWn sample to be tested. Since the 53 complement can be fixed/consumed by the Ab-Ag complex, 54 this stage is also called the complex fixation stage. The 55 indicator system (haemolytic system) contains Ab-coated 56 erythrocytes (one type of Ag-Ab complex). If a target molecule 57 exists in the sampler system, the recognition/binding of Ag and 58 Ab leads to formation of a specific Ab-Ag complex that Will 59 fix/consume the complement (Fig. 1c). The residual 60 complement can be further fixed/consumed by the Ab- This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 1 Please do not adjust margins Please do not adjust marginsAnalyst Page 2 of 6 1 ARTICLE Journal Name 2 3 erythrocyte complex, leading to lysis of the erythrocytes lumino CL system Was demonstrated in detection of rH7N9, a 4 (haemolysis) (Fig. 1a,b). because the occurrence of haemolysis recombinant avian influenza virus protein. With the optimized 5 is dependent upon the non-activated complement remaining usage of luminol/H2O2, the sensitivity of the CFT assay for 6 in the sampler system, the intensity of haemolysis indirectly rH7N9 detection Was characterized. In addition, the assay is 7 represents the amount of Ab-Ag complex formed in the conducted With human serum sample to evaluate the 8 sampler system. The benefits of CFT compared to other feasibility for real biopsy analysis. 9 serological tests, such as enzyme-linked immunosorbent assay 10 (ELISA) and immunofluorescence (IF), is that there is no need 11 for costly conjugates, and the assay time is shorter as it does 12 not include protein immobilization, Washing, and blocking 13 steps.6, 8, 9 14 15 16 17 18 19 20 21 22 Fig. 2 Principle of luminol CL detection of haemolysis. 23 24 2. Experiment 25 2.1 Materials 26 Complement (purified from from guinea pig serum), erythrocyte 27 (sheep red blood cell, SRbC) and its antibody (anti-SRbC antibody, 28 haemolysin) Were obtained from Nanfang reagent Inc., Shanghai, 29 Manuscript China. Carcinoembryonic antigen (CEA) and anti-CEA antibody Was 30 31 purchased from Sigma. Recombinant protein of avian influenza A 32 Fig. 1 Principle of the complement fixation test (CFT). There are tWo reaction (rH7N9) virus (A/Anhui/1/2013) and its specific antibody (anti- systems in the CFT: Sampler system and Indicator system. a. No target antigen 33 rH7N9 antibody) Were ordered from Sino biological Inc., China. (Ag) in the sample system. Free complement was activated by antibody (Ab)- Commerical human serum was from Gibco Scientific Technology 34 coated erythrocytes, leading to haemolysis; b. No target Ab in the sample system. 35 Free complement was activated by Ab-coated erythrocytes, leading to Co.LTD. Fresh, non-fasting serum samples were isolated from blood 36 haemolysis; c. There is target Ag-Ab in the sample system. The Ag-Ab complex of a healthy volunteer. The blood Was collected according to was activated/consumed by the complement, resulting in no available routine venipuncture protocols by hospitcal of SouthWest Unviersity, 37 complement for lysis of the erythrocytes in the indicator system. 38 and serum Was separated from blood by centrifugation according to 39 In the conventional CFT, an assay result is normally routine protocols. The samples Were analyzed Within 12 h of Accepted 40 determined by observing the clarity of the reaction solution or collection, therefore, these samples Were considered to be “fresh.” 41 the sediment of red blood cells (RbCs) with the naked eye (Fig. Luminol Was purchased from Aladin, China. It is dissolved in 0.1 M 42 1). Although the assay condition has been simplified, its NaOH to a concentration of 1.77 mg/mL. Hydrogen peroxide (H2O2) 43 sensitivity may be compromised due to the limitation of bulk was from Sinopharm Chemical Reagent Co. Ltd, China. 44 observation. Since haemoglobin leaks during haemolysis, We All other chemicals Were bought from Sigma-Aldrich and used 45 rationalized a sensitive analytical method to characterize the without further purification unless otherwise indicated. All solution 46 haemolysis process would enhance the benefit of CFT. was prepared with deionized (DI) water produced by PURELAb flex 47 Luminol is a versatile chemical that can be oxidized to system, ELGA Corporation. Analyst 48 exhibit a striking blue gloW, a phenomenon knoWn as 49 10 2.2 Testing of physiological complement-mediated haemolysis by chemiluminescence (CL). It is able to detect blood by reacting 50 luminol/H O CL with the iron found in haemoglobin, a tetramer consisting of 2 2 51 To investigate if physiological complement-mediated cell lysis four polypeptide chains; the center of the tetramer contains 52 could be assayed by luminol chemistry, complement was mixed four iron-containing molecules.11 Previous studies have shoWn 53 with Ab-coated erythrocytes to induce haemolysis. Sheep RBCs that the luminol CL system is catalysed by the iron in 54 (erythrocyte) and the corresponding Ab (haemolysin) were used. haemoglobin, alloWing the sensitive detection of blood at 55 The complement Was purified from guinea pig serum (Nanfang 56 crime scenes, as Well as haemoglobin, myoglobin, and glycated haemoglobin.12-15 Inspired by the forensic scientist to exam Reagent). First, erythrocytes (4%) and haemolysin were mixed at 57 a ratio of 1:125 (v/v) according to the manufacturer’s 58 blood at a scene in criminal, We rationally argued that luminol instructions (Nanfang Reagent).