Broadening the detection spectrum of small analytes using a two

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Broadening the detection spectrum of small analytes using a two-antibody-designed hybrid immunoassay Inna A Galvidis, Zhanhui Wang, Rinat I Nuriev, and Maksim A Burkin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00566 • Publication Date (Web): 13 Mar 2018 Downloaded from http://pubs.acs.org on March 14, 2018

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Analytical Chemistry

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Broadening the detection spectrum of small analytes using a two-antibodydesigned hybrid immunoassay. Inna A Galvidis1, Zhanhui Wang2, Rinat I Nuriev1,3, Maksim A Burkin1* 1- I. Mechnikov Research Institute for Vaccines and Sera, Moscow 105064 Russia; 2- College of Veterinary Medicine, China Agricultural University, Beijing Laboratory for Food Quality and Safety, Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety, Beijing 100193, China; 3- I.M. Sechenov First Moscow State Medical University, Moscow 119991, Russia ABSTRACT: The recognition spectrum of immunoassays developed on the basis of class-specific antibodies can include the several nearest analytes but rarely all of the desired representatives of the group. The situation may be sufficiently improved using a hybrid assay combining two antibodies with specificities that complement each other. Two monoclonal antibodies (mAb) with broad but different specificities towards sulfonamides were examined for their binding to a panel of hapten conjugates. MAb – hapten pairs without mutual cross-reactions were identified, and classical direct antigen-coated and mAb-coated ELISAs were developed as formats with referent specificities. Both interactions were combined in a single hybrid assay, which was designed as a one-step double-competitive sandwichELISA. For this assay, the intermediate bifunctional reagent mAb(1)-hapten(2) conjugate was synthesized and was able to simultaneously bind to hapten(1) and be bound by mAb(2). Formation of a two-mAbs sandwich complex was inhibited by competitors of interaction(1) as well as by competitors of interaction(2). Thus, due to the summation effect, simultaneous determination of analytes recognized by both mAbs was achieved. The hybrid assay can be performed in two reversed arrangements using a coating antigen or coating antibody, the characteristics of which were compared and found to be similar in sensitivity and extended specificity. The suitability of the developed test for the determination of 14 sulfonamides at their MRL concentration was demonstrated using the examples of turkey muscle and milk samples. INTRODUCTION Antibiotics are important representatives of biologically active low-molecular weight substances that are widely applied to treat and prevent infectious diseases. Separate groups of antibiotics may include tens or even hundreds of structural analogs, many of which are applied in human and veterinary medicine, livestock and horticulture 1, 2. However, the present scale of growth of microbial resistance requires paying special attention to the prudent administration, spreading and control of these analytes in foodstuffs and the environment using reliable methods 3-6. Among the effective means for screening various probes, there are known methods based on immunochemical detection of antibiotic residues. These methods meet the requirements of high sensitivity, specificity and high throughput, and are time-saving, unlike traditional microbiological tests. Besides, they are simple, not labor-intensive and inexpensive, in contrast to chromatographic methods. Screening procedures may be more effective when a broad analytical spectrum immunoassay is used, which allows any one of a number of structurally related analogs to be detected. Great progress has been achieved in the creation of assays with a broad recognition specificity that are capable of detecting multiple related analytes. By using various techniques and approaches, antibodies to generic haptens bearing common epitopes were generated for small analytes from many classes, including antibiotics of the β-lactam, macrolide, and amphenicol families 7-9, pesticides from the pyrethroid, organophosphorus, and triazine groups 10-12, mycotoxin analogs of zearalenone, fumonisin, and microcystin 13-15 and many others. Nevertheless, the recognition spectrum of one antibody with broad specificity can hardly include all of the desired analytes from large families, such as aminoglycosides, fluoroquinolones, sulfonamides, and so on. As a rule, specificity includes the limited number of representatives in a group. Therefore, a reliable approach for the development of assays with broad ACS Paragon1Plus Environment

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class specificity may be realized using a combination of several antibodies with distinct specificities in a single hybrid assay. The present study attempts to broaden immunoassay specificity by using two antibodies against sulfonamides as model analytes. EXPERIMENTAL SECTION Chemicals. Sulfanilamide (SAM), sulfacetamide (SAC), sulfaguanidine (SGN), asulam (ASU), sulfanylic acid (SAA), sulfisoxazole (SIZ), sulfamethoxazole (SMX), sulfaethidole (SET), sulfamethizole (SMT), sulfathiazole (STZ), phtalylsulfathiazole (PST), sulfanitran (SNT), sulfapyridine (SPY), sulfasalazine (SSZ), sulfachloropyridazine (SCP), sulfamethoxypyridazine (SMP), sulfadiazine (SDZ), sulfamerazine (SMR), sulfamethazine (SMZ), sulfadimethoxine (SDM), sulfamonomethoxine (SMM), sulfadoxine (SDX), sulfalene (SLE), and sulfaquinoxaline (SQX) were purchased from Chimmed (Moscow, Russia). Carboxylate derivatives of sulfonamides, BS (6-(4-aminobenzenesulfonylamino)butanoic acid), HS (6(6-aminobenzenesulfonylamino)hexanoic acid), TS (2-(2-(4-aminophenylsulfonamido)thiazol-5-yl)acetic acid), CS (4-(4-aminobenzenesulfonylamino)benzoic acid), and PB (SA10, 4-(4-(4aminophenylsulfonamido) phenyl)butanoic acid) were used as haptens for conjugate preparation as described previously 16. N-Hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), ethylendiamine (EDA), adipic acid dihydrazide (ADH), bovine serum albumin (BSA), and horseradish peroxidase (HRP) were purchased from Sigma (St. Louis, MO). Bovine fetuin (Fet), glutaraldehyde (GA), dimethylformamide (DMF) and dimethylsulfoxide (DMSO) were obtained from Serva (Heidelberg, Germany). Gelatin (Gel) was purchased from Bio-Rad (Hercules, CA, USA). Mouse monoclonal antibodies 4C7 (anti-TS) and 4D11 (anti-PB) were prepared in previous 16 work and used for hybrid assay development. Rabbit anti-mouse IgG conjugated with horseradish peroxidase (RAM-HRP) was from Imtek (Moscow, Russia). The coating buffer was 0.05M carbonatebicarbonate buffer (CBB, pH 9.5), and phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBS-T) was used for washing and sample dilution. One-component TMB substrate was a product of Biotest Systems (Moscow, Russia). The other chemicals were of analytical grade. Preparation of conjugated antigens based on carboxylate derivatives of sulfonamide. Protein conjugates were coated onto plates in both the classical assay and experimental hybrid assay. Gel and BSA were used as protein carriers by coupling their amines to the activated carboxyl groups of sulfonamide derivatives. The structures of these haptens have various substituents at the N1 atom. The derivatives BS and HS have aliphatic C4- and C6-length carboxyl-terminated chains, TS has a 5membered thiazolyl ring, and CS and PB have 6-membered aromatic rings with a carboxylic group at the end (Figure 1). After activation using a mixture of NHS/EDC in DMF, the above substances were dropwise added to proteins at different ratios and stirred for conjugation according to a previously detailed procedure 17. Preparation of tracers based on haptens conjugated to horseradish peroxidase. Carboxylatecontaining haptens (PB and TS) were linked to HRP directly or through a spacer arm with an ADHmodified enzyme using the NHS/EDC method. The prepared conjugates were universally applied as tracers in a direct coated antibody-based assay as well as in a hybrid assay format. For enzyme modification, 4 mg of HRP was first oxidized with 2 mg of sodium periodate for 20 min and then incubated overnight with a 100-fold excess of ADH at 4°C. Then, 0.1 ml of sodium borohydride (2 mg/ml) was added to reduce the Schiff base and stabilize the HRP-ADH product, and the mixture was allowed to stand for 30 min at room temperature. Afterwards, an exhausted dialysis against PBS (pH 7.2) was performed to remove the excess reagents. The molar ratio between the haptens and enzyme at synthesis did not exceed the range of 5-30 mol/mol. Preparation of HRP-labeled antibodies. Preparation of the HRP-labeled antibodies was conducted for development of both the classical and hybrid assay formats. The mAbs 4C7 and 4D11 isolated from ACS Paragon2Plus Environment

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ascitic fluids using ammonium persulfate precipitation were conjugated with HRP using a reductive amination method 18. The reactive aldehyde groups in the HRP were formed after 20 min of stirring with sodium periodate, as described above. Fast removal of the excess periodate was achieved by ultrafiltration using a 30-kDa-cutoff membrane (Ultracent-30 Toyosoda). The retentate was adjusted to the initial volume with water and centrifuged at 4000 rpm for 20 min, and this procedure was performed four times. Monoclonal antibodies in CBB (pH 9.6) were mixed with oxidized HRP at a ratio of 1 to 8 mol/mol and stirred for 2 h. Then, the mixtures were supplemented with 0.1 ml of sodium borohydride (2 mg/ml), and 1 h later, the solution was extensively dialyzed against PBS, pH 7.4. The prepared conjugates were stored at –20°C as 50% glycerol solutions stabilized with BSA (5 mg/ml). Preparation of antibody-hapten conjugates (AHC). This original reagent served as an intermediate combining element in the sandwich-type hybrid assay. The mAbs 4С7 and 4D11 were precipitated from ascitic fluids at 50% ammonium sulfate saturation, and after centrifugation, the pellet was dissolved in CBB to final concentration of 1.5 mg/ml. The haptens PB and TS dissolved in DMF (0.2 mg, 0.6 µmol) were incubated with the EDC and NHS mixture (both at 2 µmol) in DMF with stirring using a magnet stirrer for 2 h at room temperature. After activation, the haptens were dropwise added with vigorous stirring to solutions of noncomplementary antibodies namely, PB to 4C7 and TS to 4D11. The molar ratios between the mAb and haptens in the reaction mixtures ranged from 1/20 to 1/100. After a 2-h coupling, the unbound haptens were dialyzed out and the resultant AHC were preserved with glycerol and stored at –20°C until usage. Competitive ELISA procedure. A classical competitive coating antigen-based enzyme-linked immunosorbent assay (ELISA) was initially developed for selection of the preferred antibody-hapten pairs. Then, a direct assay format (coating antibody-based ELISA) was developed using the selected antibody-hapten pairs. The detailed description is presented in the Supporting Information section. Hybrid double-antibody based competitive ELISA procedure. A hybrid assay based on two mAbs with different spectra of sulfonamide recognition was engineered in sandwich configuration using antibody-hapten conjugates (AHC). This intermediate bifunctional immunoreagent bound to hapten-1 and was able to be bound by a mAb to hapten-2. Moreover, such interactions within the heterogeneous immunoassay were arranged oppositely in two formats, antigen- and antibody-immobilized. Immobilization of the selected protein-hapten conjugates or antibodies was carried out according to the usual adsorption procedure in antigen-coated or antibody-coated ELISAs. After the coating and washing step, the reagents AHC and mAb-HRP (50 µL, in 1% BSA-PBS-T) and 100 µL of the standard or sample in PBS-T were added into the wells containing the immobilized protein-hapten conjugate. In the case of the antibody-coated assay format, the wells were filled with 50 µL solutions of AHC and hapten-HRP in 1% BSA-PBS-T and 100 µL of the standard or sample in PBS-T. The incubation lasted for 2 h at 25°C. Completion of the enzymatic reaction and data registration did not differ from the above description. Examination of the assay recognition spectrum. Numerous representatives of the SA family were prepared as individual standard solutions in a concentration range of 10000 – 0.01 ng/ml and were analyzed in each assay format. The inhibitory activity of each concentration of analyte is expressed as the relative antibody binding (B/B0 ×100) and is presented in the form of sigmoid standard curves using OriginPro 8.0 software. For assessment of the recognition spectrum of the experimental hybrid assay, the IC50 values for each analyte were calculated from the standard curves and compared with those obtained in classical formats. Sample preparation, matrix effect estimation and recovery experiments. Milk and turkey breast muscle samples purchased from a private organic farm were used as blank matrices to estimate the matrix effect and for recovery experiments. Milk samples were ten-fold diluted with assay buffer. The ACS Paragon3Plus Environment


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latter was also used to prepare the tissue extract. One gram portions of muscle homogenate were placed into tubes and extensively stirred with 4 ml of PBST. After debris sedimentation as a result of centrifugation at 3000 rpm for 10 min, the supernatant was appropriately diluted with assay buffer. The extent of the matrix interference on the absorbance level was estimated using the dilution method and was expressed as relative antibody binding (BMATRIX/BPBST) 17. The samples of milk and turkey muscle known to be antibiotic free were fortified with some of the SAs at the established maximum residue level (MRL), 100 µg/kg 19, and then, they were pretreated as described above and tested in the hybrid ELISA. The recovery rate of the SAs was determined using the standard curves for the corresponding analytes, and the ratios between the spiked and measured concentrations are expressed as a percent. The spectrum of detectable sulfonamides was judged by the number of analogs that could be registered at their MRL concentration in the matrix using the developed hybrid test. For each studied matrix, a cut-off level was accepted and determined as a relative absorbance value (Bo – 3×SD) ×100 /Bo. RESULTS AND DISCUSSION A combination of variously targeted antibodies within a hybrid immunoassay was used with the aim of broadening the resulting analytical spectrum of recognition. It is extraordinarily rare that a single classspecific antibody can identify all of the desired representatives of a class with satisfactory sensitivity. Although this initiative seems Utopian and was called so earlier 20, undoubted progress in this area can be made by using alternative approaches that involve bispecific antibodies 21 or hybrid-type assays, the latter of which is the object of the present study. The design of the hybrid assay studied here can be considered to be a fusion of elements and interactions from classical ELISA formats (Table 1, scheme). Classical direct antigen-coated and antibody-coated ELISAs were initially developed to select and test the immunoreagents suitable for the hybrid assay, and then, they served as referent assays in comparative examinations. The maximal effect of the specificity summation (hybridization) was expected when the original antibodies were of different, and preferably of non-overlapped, specificity. Therefore, the first objective in the development of the hybrid assay was to choose a suitable pair of antibodies. Another task and indispensable requirement for studied hybrid assay design was to select antibody-hapten pairs that did not have mutual cross-reactions. Selection of the antibody–hapten pairs for the hybrid assay. A panel of carboxylate SA derivatives, BS, HS, TS, CS and PB (Figure 1), were conjugated to Gel and BSA using different hapten loads. The effectiveness of the coupling was confirmed by the UV spectral characteristics of the synthesized conjugates, which combined the features of both the carrier and the corresponding hapten (Figure S-1). We often apply coating antigens based on Gel as a carrier because of its relatively poor immunogenicity and lack of undesirable background immune reactions. The Gel-based conjugates were prepared using lower hapten loads. It has been repeatedly observed that lower hapten loads promoted better assay sensitivity 8, 22. Here, we found that the lowest ratio between hapten and protein (10 mol/mol) was preferable for almost all of the prepared conjugates that were selected as the best coating antigens. Their interactions with mAb 4C7 and mAb 4D11 were examined, and a preliminary sensitivity level was determined for the studied SAs (Tables S-1, S-2). The recognition spectra of the SAs and haptens for both mAbs are summarized in Figure 1. It can be seen from the figure and table data that anti-TS mAb 4C7 was capable of binding to the homologous TS-conjugate and heterologous CS-based conjugate. The latter interaction, however, required a higher antibody concentration (1/500) and did not exhibit a noticeably modified specificity. The spectrum of recognition included SAs with a 5-membered ring N1substituent, several six-atom ring-containing SAs, and SAC. MAb 4D11 failed to interact with the TS-based antigen but was able to bind with the other 4 conjugates, including CS-based ones. MAb 4D11 mainly recognized SAs with a six-atom-ring at the N1 position, with SMX as an exception (Table S-2). The heterology of the coating hapten revealed no advantage for detection sensitivity. ACS Paragon4Plus Environment

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mAb 4C7

H 2N

O S O

O H2N

S

O

NH

N

S

CH 2 COOH

N

O S O

H2N

N

Sulfamethizole (SMT)

O S O

NH

NH

Cl N

O S NH O

H 2N N

Sulfathiazole (STZ)

CS

N

N

Sulfachlorpyridazine (SCP)

S

O S NH O

H2N

COOH

N NH

Sulfaquinoxaline (SQX)

S

O S NH O

H2N

TS

H2N

N

O S O

H2N

Sulfaethidole (SET) N

O S O

NH

O

Asulam (ASU)

H2N

S

O

NH

OCH3 N

N

Sulfamethoxypyridazine (SMP)

O H2N

S O

NH

(CH 2 ) 3COOH

O S NH O

O H 2N

S

NH N

O

PB

H2N

O

Sulfamethoxazole (SMX)

N N

Sulfamerazine (SMR)

O H2N

S

(CH 2 ) 3COOH

NH

O

O S

H2N

BS

H 2N

NH O

O O H2N

S

N

HS

S NH2 O

O S O

H 2N O H 2N

S

OCH3

O S NH O

H 2N

OCH3 N

N

Sulfapyridine (SPY)

NH N

O S NH O

O NH

Sulfalene (SLE)

O

N

Sulfamonomethoxine (SMM)

N

O NH

N NH

N NH

Sulfadoxine (SDX)

Sulfanilamide (SAM)

N

OCH3

H 3CO

O H 2N

O S O

H2N O S O

OCH3

OCH3

O

H 2N

N

Sulfadimethoxine (SDM)

Sulfisoxazole (SIZ)

(CH 2 ) 5 COOH

NH

O S NH O

Sulfacetamide (SAC)

NO2

Sulfanitran (SNT) COOH

H 2N

O S N O

NH2

NH

NH2

O

O S NH

S

O

N

H2N

Phthalylsulfathiazole (PST)

Sulfaguanidine (SGN)

O S NH O

N N

Sulfadiazine (SDZ)

COOH H 2N

O S OH O

Sulfanilic acid (SAA)

O

N NH

O S NH O

H2N N

Sulfasalazine (SSZ)

O S NH O

N N

Sulfamethazine (SMZ)

mAb 4D11 209 210 211 212 213 214

Figure 1. Chemical structures of haptens and sulfonamides and their recognition by mAbs. The immunizing haptens for the corresponding mAbs are framed. Compounds recognizable by mAb 4C7 are marked with red; compounds recognizable by mAb 4D11 are marked with blue. Violethighlighted names indicate structures that are recognizable by both antibodies.

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Thus, TS, unlike CS, was a specific hapten for mAb 4C7 and was indifferent for mAb 4D11. PB was a homologous hapten for mAb 4D11 and was chosen as the most reactive in comparison with the other candidates (BS, HS) that demonstrated no cross-reaction with mAb 4C7. Thus, using a panel of hapten conjugates, the binding characteristics of two anti-SA mAbs were examined. Two pairs of immunoreagents, mAb 4D11–PB and mAb 4C7–TS, free of mutual crossreactions were identified, and the conditions for the highest sensitivity together with a broad specificity were found. As they were generated against different immunizing haptens, these mAbs differed in specificity but still had some overlap (8 SAs) (Figure 1). Nevertheless, recognition of a number of analytes, such as ASU, SET, SMT, STZ, SNT, SDZ, and SMZ, differed critically depending on the mAb type. Thus, it was expected that the summation of the antibody properties in the hybrid assay would allow inclusion of these 7 analytes in the analytical spectrum of the developed test. Preparation of haptens and antibodies conjugated to horseradish peroxidase. The design of the two-antibody-based hybrid assay stipulated the involvement of a tracer or antibody labeled with the enzymes as detector agents. Once the suitable pairs (antibody–hapten) were identified, we synthesized peroxidase-labelled hapten conjugates (TS-ADH-HPR and PB-HPR) and peroxidase-labelled mAbs (4C7-HRP and 4D11-HRP) and optimized the parameters of their application in a classical direct ELISA (Table S-3). Preparation of antibody-hapten conjugates (AHC) and development of hybrid assay formats. Examination of specificity revealed a broad recognition spectrum of SAs for the anti-TS mAb 4C7 that was not able to bind to PB-based antigens (Table S-1). Anti-PB mAb 4D11 displayed the other specificity, and TS-antigen was the only conjugate unrecognizable by this antibody (Table S-2). Thus, for preparation of the AHCs, non-complementary pairs of immunoreagents that lacked immunobinding, namely, 4C7 - PB and 4D11 - TS, were chosen. The AHCs, 4C7-PB and 4D11-TS, were prepared using two molar ratios of ingredients 1:20 and 1:100. To confirm the formation of conjugates, the prepared AHCs were adsorbed onto plates and were allowed to interact with 4D11-HRP and 4C7-HRP. The PB-determinant on 4C7 was shown to remain intact after coupling and demonstrated good immunochemical activity, unlike 4D11-TS. It is highly probable that the TS-determinant lost the ability to bind to 4C7 after conjugation due to its short spacer arm, similar to the coupling problem seen with HRP mentioned above (SI). The optimal hapten load in 4C7-PB was judged in a competitive ELISA of SAs with five- and six-atom ring substituents at N1, SMX and SDM (Figure 2). 100

300

1000

3000

4D11-HRP, titer SDM SMX

10

IC50, ng/ml

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1

0,1 0,3

248 249 250 251 252 253 254

1 4C7-PB, µg/ml

3

Figure 2. Competitive ELISA of SMX and SDM based on immobilized AHC with different hapten loads. The selected ratios of the immunoreagents are shown for 4C7-PB (lower X-axis) and 4D11-HPR (upper X-axis). The AHCs with 20-fold and 100-fold molar excesses of hapten over antibody are indicated as 4C7-PB×20 (dash line) and 4C7-PB×100 (solid line), respectively.


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This examination found a tendency of improved assay performance when the concentration of the coated 4C7-PB was higher while the 4D11-HRP concentration was decreased. The better sensitivity of SA determination was observed when using 4C7-PB×100, except in the case of the highest concentration of 4C7-PB×20. In the latter case, the SDM IC50 value remained the same while the sensitivity of SMX detection was improved almost two-fold. For this reason, 4C7-PB×20 was selected for use in development of the hybrid assay. This AHC could be installed in two inverse sandwich formats as an intermediate reagent. In the first hybrid format, it bound to coated Gel-TS (Table 1, Scheme C), and in the inverted version, 4C7-PB×20 was captured by immobilized mAb 4D11 (Table 1, Scheme F). Because simultaneous binding between three reagents in the presence of an analyte (doublecompetitive interaction) was complicated, the conditions of the hybrid assay procedure had to be optimized. Therefore, the temperature of incubation and its duration, as well as the conducive role of mixing during the interaction, were examined (Figure 3). A

20

Absorbance, 450 nm

1,2

o Binding 25 C SMZ STZ

1,0

18 16 14

0,8

12 10

0,6

8 0,4

IC50, ng/ml

6 4

0,2

2

0,0

0 0

30

60 90 Incubation, min

120

267

B

20

Absorbance, 450 nm

1,2

o

18

Binding 37 C SMZ STZ

1,0

16 14

0,8

12 10

0,6

8 0,4

IC50, ng/ml

6 4

0,2

2 0

0,0 0

30


120

268

C

20

Absorbance, 450 nm

1,2

o Binding 25 C shaker SMZ STZ

1,0

18 16 14

0,8

12 10

0,6

8 0,4

IC50, ng/ml

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6 4

0,2

2 0

0,0 0

30


120

269 270 271

Figure 3. Dependence of the hybrid assay (Gel-TS + [4C7-PB×20 + 4D11-HRP]) characteristics on the incubation parameters: duration, temperature, and shaking.



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The competition step of the reaction was carried out over 15-120 min time periods at 25°C (A), 37°C (B) and 25°C with stirring in a thermoshaker (C). Binding without the analyte is represented by the average absorbance values from three replicates, and the error is expressed as the standard deviation. Changes in the sensitivity (IC50) of sulfonamide determination are shown for SMZ and STZ. Using STZ and SMZ, the analytes recognizable by the different detectors of the hybrid assay - 4C7 and 4D11, respectively - it was demonstrated that the optimal absorbance rate of 0.8-1.0 could be reached after 2 h, independent of the incubation temperature. However, variation of the temperature provided more sensitive detection of SMZ at T=25°C, but had no influence on STZ detection (Figure 3 A vs B). Stirring of the reaction mixture using a thermoshaker also did not assist in reaching the binding equilibrium faster. (Figure 3 A vs C). Thus, for more sensitive detection of SAs in the hybrid assay, the optimized conditions were established as 2 h of incubation at T=25°C, wherein the resultant absorbance level was equalized in all comparable formats over the range of 0.8-1.0. Comparative examination of the recognition spectra of the developed assay formats. Halfinhibition concentrations (IC50) were calculated from the standard curves obtained for each analog to compare the recognition spectra of the SAs in the classical and hybrid assay formats (Table 1). Weak analytes, the IC50 values of which were higher than 100 ng/ml, are highlighted in grey, and the number of well-recognizable SAs is indicated at the bottom of the table. It should be noted that the hybrid assay inherited the capability of recognizing analytes from the composing detectors, the mAbs 4C7 and 4D11. Both detectors recognized several common analytes (SMX, SCP, SMP, SMR and SQX), but differed towards the others. For example, the 4C7-based assay (format A and D) showed poor sensitivity towards SDZ, SMZ, SDM, SMM, SNT, and SPY, unlike the 4D11-based assay. By contrast, SET, SMT, and STZ were weakly recognized in the 4D11-based assay (format B and E) and were well-recognized in the 4C7-based assay. The recognition spectrum of the hybrid formats (format C and F) included all of the mentioned substances. Classical antigen-coated and antibody-coated ELISAs showed some differences in regard to the sensitivity of analyte detection, especially compared to the 4C7-based (A and D) formats. Nevertheless, the hybrid assays with a mutually antithetic architecture (formats C and F) demonstrated very similar characteristics. Thus, the spatial assay design seemed to be an insignificant factor for sensitivity, and the recognition spectrum entirely depended on the properties of the applied reagents.


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Table 1. Comparative recognition of different SAs in classical and hybrid-format immunoassays, expressed in half-inhibition concentrations (IC50).

Immunoassay format

Sulfonamides

IC 50, ng/ml

327 328 329 330 331 332 333 334 335

ASU SAM SAC SGN SAA

17 >10000 168 468 173

125 3111 7009 1570 >10000

62 3090 500 1050 465

77.7 >10000 450 738 524

58.7 2253 4044 1043 >10000

55.7 3930 650 1500 472

SET SMT STZ SIZ SMX

0.45 0.4 0.35 1326 1.3

454 >10000 454 1130 4.2

2.1 1.9 1.4 1170 2.3

1.2 0.9 0.6 9080 3.5

275 >10000 176 790 2.7

3.5 2.6 2.2 2260 2.6

PST SSZ

50 >10000

>10000 3536

410 3615

182 >10000

7260 3604

425 5515

>10000 390 350 >10000 275 SDX >10000 454 500 >10000 316 SLE 19 2.1 3.3 57.2 2.1 SQX 2.2 16.4 5.4 9.3 8.4 SCP 0.9 1.2 1.2 2.0 0.9 SMP 0.9 17.8 15.4 48.5 13.3 SMR 38 0.4 0.9 117 0.3 SDM 35 3.4 4.7 129 2.4 SMM 23 2.0 3.3 129 1.9 SPY 138 25.3 26.7 216 17.8 SDZ >10000 6.9 9.6 >10000 4.4 SMZ >10000 0.3 0.6 >10000 0.2 SNT ng/ml Number of sulfonamides detected with sensitivity level 13 10 15 9 12

Broadening the detection spectrum of small analytes using a two

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