key: cord-0896760-l1zcuc1b
authors: Iankov, Ianko; Viker, Kimberly; Turgeon, Coleman; Matern, Dietrich; Galanis, Evanthia
title: Parameters of immunoglobulin extraction from dried blood spot cards and immunoassays for detection of antibody response to pathogens including the novel SARS-CoV-2
date: 2021-02-11
journal: J Immunol Methods
DOI: 10.1016/j.jim.2021.112996
sha: 780697c83d9fd4b691435df1420d273512b6bd70
doc_id: 896760
cord_uid: l1zcuc1b

Dried blood spots (DBS) are routinely used in screening newborns for treatable disorders. Immunoglobulin extraction from DBS, serum or other biological fluids loaded on filter paper cards could represent a valuable method of specimen preservation in monitoring immune response against pathogens as well as vaccination efficiency. In this study using different sources including serum, and monoclonal antibodies we established parameters for antibody extraction from the filter cards to assess antibody reactivity against Helicobacter pylori, measles virus (MV) and the novel coronavirus SARS-CoV-2 antigens. We demonstrated that DBS and dried undiluted serum result in completely preserved antibody activity for immunoassays, including in virus neutralization assays against MV. Extraction efficiency was determined by IgG concentration measurements. The plaque-reduction neutralization titer 50% of dried human serum spots remained stable after more than 10-day storage – 1:359 vs. 1:345 for the corresponding frozen sample. DBSs could be used to monitor immune response to bacterial and viral antigens following natural exposure or immunization. Mice immunized with recombinant spike protein receptor-binding domain of SARS-CoV-2 developed a strong antibody response by day 14 and reached titers above 1:64,000 on day 21 following the secondary boost immunization as measured on DBS samples in antigen-mediated ELISA. Variability in IgG concentration of eluted DBS could be influenced by factors involved in sample application, extraction process and sample characteristics. Adjustment of antibody specific activity to the eluted IgG concentration can increase accuracy of the result interpretation, including in SARS-CoV-2 serological diagnostics.

Dried blood spot (DBS) samples were first introduced in modern laboratory diagnostics by Guthrie for newborn screening (NBS) of inborn errors of metabolism (1). Recently the US Recommended Uniform Screening Panel has been expanded to include 35 primary and 26 secondary conditions every newborn in the US should be screened for (2). Test on DBS specimens include not only inherited metabolic disorders, endocrinopathies, and hemoglobinopathies but also immunodeficiencies such as SCID (3). Blood samples from newborns are usually collected by heel-prick and directly deposited onto the half-inch filter paper circles on DBS cards such as widely used Whatman 903 cards (Whatman, UK). In addition, application of the filter paper cards is not limited to the newborn screening purposes but also for adult blood testing. Sample collection and storage have many advantages over conventional venous blood collection including the potential for self-collection, reduced phlebotomist effort and shipment by regular mail (4). Relatively small volumes (approximately 50 l) of serum or other non-blood specimens could also be safely collected and transported for analysis. Filter paper samples are stable for longer periods of time when stored refrigerated or frozen. A small, 3 mm diameter size punches from the main circles are sufficient for extraction and subsequent analysis. Depending on the analytes of interest, properly stored DBS card specimens could be subject to further retrospective analysis years after collection. Enzyme activity is well preserved on the DBS cards and the extracted soluble fraction can be subjected to multiplexed enzymatic analyses, such as screening for lysosomal storage disorders (5). Besides the biochemical marker measurement, DBS could be used in toxicology marker probing, drug metabolite analysis and detection of DNA or RNA from different pathogens (6,7). An assay for detection of HIV, hepatitis C infection and malaria parasites on DBS samples has also been J o u r n a l P r e -p r o o f previously reported (8-10). Antibodies are stable in a dried form and extracts from DBS could be a useful tool in serological analysis of infectious and parasitic diseases (11). DBS cards are valuable specimens for monitoring of transplacentally transferred maternal IgG that confers the passive immune protection against infections in the first months of life in newborns. Recently, this approach was also applied in predicting the protective immunity coverage in infants and optimal time for immunization with live measles, mumps, rubella (MMR) vaccine (12).

The current pandemic with the novel coronavirus SARS-CoV-2 emerged at the end of 2019 and has posed enormous challenges to healthcare systems globally (13, 14) . Development of diagnostic tests was urgently demanded and included molecular diagnosis of the pathogen, serological tests and clinical biochemistry monitoring of infection (15). The most common serological assays for retrospective confirmation of SARS-CoV-2 infection are ELISA and lateral flow rapid tests (16) . Virus neutralization (VN) is the "gold standard" assay for detection of protective immunity against virus pathogens including SARS-CoV-2 (17) (18) (19) (20) . DBS-based serological assays with their simplified protocol of collection, transport, extraction and disposal could have major advantages in evaluation and tracking of population immunity in the ongoing pandemic. Initial data using ELISA and VN showed that infected individuals did not develop high antibody titers against SARS-CoV-2 antigens. VN titers reached maximum levels during the convalescent period corresponding to dilutions of 1:160-1:320 in microneutralization or plaque-reduction neuralization 50% test (PRNT 50 ) (21) (22) (23) (24) . ELISA and VN antibody titers correlated with the severity of infection and could be close to the detection threshold in patients with mild disease: dilutions as low as 1:4 in VN and < 1:100 in antigen mediated ELISA against nucleoprotein (N) or spike protein (S) antigens have been employed in order to detect immune response. These data support the potential but also possible caveats of serological diagnostics. mice (28) were immunized by a single i.p. injection of 10 6 TCID 50 of MV-s-NAP. DBS samples were collected on days 15 and 21 post immunization using Whatman 903 filter cards (Whatman, UK). The VN titers against measles were compared to samples collected on day 1 prior to immunization. In order to analyze immune response to purified SARS-CoV-2 spike antigen, 5 female Ifnarko-CD46Ge mice were injected i.p. on day 1 with 6 g in 100 l PBS recombinant S protein RBD (ProSci) formulated 1:1 with aluminum hydroxide (InvivoGen, San Diego, CA) to a final volume of 200 l. Immunization was repeated on day 14 with 3 g of the antigen administered through the i.p. route. DBS sampling was performed by tail bleeding on day 0 (prior to immunization), day 7, 14 and 21. Serum was collected on day 21 and stored frozen as described above.

DBS collection and storage. Blood from immunized mice was collected by tail vein bleeding and a single drop of blood was loaded onto the circled areas of the filter cards. The cards were left to dry at room temperature for 30 min and subsequently refrigerated at 2-8C in plastic bags and used for the subsequent analysis. MAbs and sera were loaded on the filter paper and designated as purified antibody spot and serum spot filter paper samples respectively. The spots are made by adding 10 or 15 l samples volume per circle. 15 min at room temperature. The reaction was stopped using 100 l 1N HCl and the absorbance (OD 450nm ) was measured on a Tecan 2000 Pro ELISA reader (Tecan, Switzerland). Antibody activity from dried filter paper extracts was compare to those of freshly diluted serum or MAbs.

Wells coated with Ni-NTA extracts from pET-28 control plasmid transfected E. coli were used as controls in HspA and NAP antigen-mediated ELISA. For anti-S protein response analysis, control wells were incubated with CBB alone and blocked with BSA as described.

To assess the potential clinical application in detection of antibody response against SARS-CoV-2 S human DBS samples was tested in antigen-mediated ELISA against S-protein RBD antigen coated on 96-well ELISA plates as described above. Extracted samples collected from two seropositive patients and two seronegative individuals were UV-inactivated (for 60 min), serially diluted in PBS/T with 2% BSA and run using 1:2 starting dilution and incubated for 30 min at room temperature on a rotating platform. Plates were washed 3 times in PBS/T and incubated with goat polyclonal anti-human IgG, A, M HRPO-conjugated secondary antibody (Sigma) diluted 1:1000 in PBS/T with 2% BSA for additional 30 min at room temperature. The assay was subsequently developed using TMB as a substrate and analyzed on a Tecan ELISA reader as described above.

The concentrations of mouse or human immunoglobulins in the extracted samples were measured by capture ELISA specific for mouse IgG or human lambda immunoglobulin chain (Bethyl Laboratories, Montgomery, TX) according to the manufacturer's protocol (25) .

Anti-measles human serum and MAb 20H6 VN titers were determined by plaque-reduction microneutralization assay and the plaque reduction neutralization titer 50% (PRNT 50 ) was calculated according to Karber's formula as previously described (17) . Briefly, the filter paper extracts were serially diluted in Opti-MEM, 2% BSA in 96-well plates (Corning, J o u r n a l P r e -p r o o f

Corning NY). MV strain MV-GFP encoding green fluorescent protein (29) was diluted in Opti-MEM and 250 tissue-culture infectious doses 50% (TCID 50 ) in 100 l per well (1:1 with the antibody dilution) were added and incubated at 37C for 1 h. Then 100 l of the mix were transferred onto Vero cells grown in 96-well plates and incubated for 72 h. GFP positive syncytia per well were counted on a fluorescent microscope and PRNT 50 titers were determined.

Antibodies extracted from dried filter paper spots retained strong antigen-binding activity. The aim of these experiments was to determine extraction efficiency and specific activity of MAbs extracted from filter cards. Undiluted hybridoma supernatants of MAb 16F4 and 23C8 were loaded onto the filter cards and the corresponding whole circles containing 10 or 15 l were extracted in 200, 300 or 500 l PBS/T with 2% BSA. The dilution factor was calculated based on the volume loaded on the filter paper and extracted in the elution buffer volume. The same volume of frozen-stored original supernatants was diluted in the extraction buffer to the corresponding dilutions and used as controls. Samples were run in 4-fold dilutions up to 1:102,400 in duplicates in antigen-mediated ELISA against H. pylori NAP. Data showed that antibodies from undiluted supernatants containing 10% fetal bovine serum (FBS) were efficiently extracted and MAb activity was completely preserved as compared to control hybridoma supernatants (Fig-1A ,B). The recovery of MAb specific activity at the different dilutions is shown in Fig-1C . In contrast, purified MAb 27H10 loaded in different dilutions and extracted from filter cards showed significant reduction in activity at the higher antibody dilutions (Fig.1D ).

J o u r n a l P r e -p r o o f Fig.2A) . In contrast, neutralizing anti-measles activity of the human pooled AB(+) blood group serum was unaffected after spotting on the filter paper and stored for > 10 days (Fig.2B,C) .

VN test of high-grade purity MAb 20H6 (with defined starting concentration) against MV H protein extracted from DBS at different storage times before extraction (A). Titer in PRNT 50 test for anti-measles neutralization activity of human AB(+) blood group serum extracted in different Opti-MEM volumes corresponding to 1:20 or 1:50 dilutions (B,C). The titer was determined using a known amount of antibodies (10 l serum transferred onto filter cards) and compared to that of freshly diluted serum. The PRNT 50 titer was calculated using Karber's formula and dilutions run in duplicates in 96-well cell-culture plates. Recovery rate determined by capture ELISA for human IgG-lambda was approximately 95 % as compared to the frozen control serum.

Titration experiments with different MV-GFP virus stocks determined the PRNT 50 titer of this serum lot to be between 1:155-1:371. Human serum samples of 10 l loaded onto the card were extracted at two different dilutions -1:20 and 1:50 in Opti-MEM. VN testing was performed from these starting dilutions and titers were compared to corresponding dilutions of frozen serum in the same 96-well plates: 24-h, 96-h and 11-day storage of the cards did not significantly reduce serum specific activity vs. frozen serum. The titer following 1:50 extraction for example, was 1:281 for the 24-h and 1:359 for the 11-day stored samples respectively vs. a titer of 1:345 for the control serum. All samples, independently of the assay conditions had PRNT 50 above 1:120 that is considered protective for humans (17) . Recovery of human IgG from DBS of more than 2-week storage and 30-min extraction was 1.56 mg/ml and 1.64 mg/ml for J o u r n a l P r e -p r o o f control frozen AB(+) serum respectively as measured by the human IgG-lambda quantitative ELISA. This data demonstrated that human serum specimens could be stored on filter cards and used subsequently for VN testing against live viruses without significant loss of the neutralization activity.

In order to address the question about the mechanism of monoclonal antibody activity decline following storage on filter paper, extracted MAb samples were analyzed for mouse IgG concentration compared to that of the original stocks. The results demonstrated that the reduced anti-MV titer of MAb 20H6 was due to inefficient extraction rather than impact on the immunoglobulin activity. IgG concentration was 8-fold lower in the extracted 10 l dried sample with recovery rate calculated at 12.57 % (Fig.3) . In contrast, MAbs from supernatants had approximately 100% recovery rate for the mouse IgG. This data indicated that elution from dried filter paper spots of purified or diluted antibodies is significantly affected in the absence of the protective effect of high total protein concentrations. (Fig-4A) . The alternative way to present data analysis is to calculate serum neutralization capacity as the absolute IgG amount adjusted to the concentration of extracted immunoglobulin fraction that is required for 50% reduction (IgG 50 ) of the virus infection (Fig.4C ). According to these data the minimum concentration of the extracted IgG for the assay should be approximately 90-100 g/ml. That concentration would allow detection antibody neutralization activity in sera with borderline protective titers in the range of 1:120 PRNT 50 considered protective against measles (17) . After more than three months following primary immunization, mice maintained significant antibody response against HspA as measured by antigen-mediated ELISA. There was no significant difference in absorbance at 1:800 dilution for both dried blood and serum vs. freshly diluted corresponding serum (Fig.5A) . The measurement of extracted mouse IgG from DBS or dried serum spots showed the differences in IgG concentrations in the individual samples (Fig.5B) . These variabilities could be explained by difference in sample loading spread, extraction efficiency and hematocrit between individual mice.

A group of five 12-13-week old Ifnarko-CD46Ge mice were immunized on days 1 and 14 with two repeated injections of RBD (amino acids 319-541) of the S protein formulated with aluminum hydroxide as an adjuvant. Serum and DBS samples collected on day 0 prior to immunization served as controls. Mice were serially bled for DBS collection on day 7, before the second immunization on day 14 and on day 21. The antibodies were extracted from 3 mm filter paper punches incubated in 200 l ELISA buffer as described above. Antigen-mediated ELISA assays were run in 4-fold dilutions. A single immunization with the S protein antigen mounted robust antibody response in the mice by day 14 that was additionally boosted after the second immunization as measured on day 21 (Fig.6A ): all animals had titers above 1:25,600 (Suppl. fig.1 and 2) . As the next step, we compared titration curves at different dilutions for DBS collected at 14 and 21 day as compared to the corresponding frozen sera collected on day 21. There was no significant difference between DBS stored more than a week vs. frozen serum for monitoring response to SARS-CoV-2 antigens (Fig.6B) . The use of J o u r n a l P r e -p r o o f polyvalent secondary HRPO-conjugated antibody could detect the collective antibody response contributed by the three major immunoglobulin isotypes -IgG, IgM and IgA. Two administrations of the purified S RBD fragment induced very high serum titers >1:64,000 as determined by the endpoint dilution with absorbance 4 x SD above the average absorbance of sera collected prior to vaccination (Table 1) . As expected, measurement of extracted IgG showed variability between the individual animals and time points of sample collection (Table 2 ). These results suggest that IgG concentration measurement and adjustment to specific IgG activity could provide more accurate assessment of antibody response in DBS, especially for samples close to the threshold of detection by a particular immunoassay. 

The ongoing SARS-CoV-2 pandemic demands development of rapid diagnostic assays for detection of active infection and sensitive serological tests for assessment of past exposure and seroconversion (31) . The convenience of DBS specimen collection, transport and testing can minimize the risk of exposure for both patients and health care professionals.

In this study we aimed to determine the extraction parameters and reliability of immunoassays performed on blood, serum and purified antibodies spotted and dried on filter Purified MAbs loaded on the filter paper however, showed significant reduction of reactivity, which we determined to be due to inefficient immunoglobulin extraction rather than structural changes of the antibodies in the process. These data suggest that stability and extraction pandemic (32, 33) and theoretically could be loaded on filter paper similar to the DBS collection.

In this case, we would recommend the use of a high protein content buffer system (10% BSA, FBS, lactalbumin etc.) premixed 1:1 with the sample before spotting it on the filter paper. Filter paper sampling for serology analysis does not have to be limited to whole blood and serum. as accessible means to assess immune response retrospectively but also to assess of vaccination efficacy. However, a key element for determination of ELISA's cut-off and interpretation of the results is the amount of immunoglobulin that can be isolated from the DBS or serum spots. We determined both the extraction efficiency and antibody specific activity by extracting immunoglobulins with known concentration and volume from large filter paper disks. ELISAbased methods for SARS-CoV-2 serology require sample dilutions of 1:100 or less (21, 36, 37) .

We calculated that IgG extracted from 3 mm punches corresponded to 1.5 to 2 l blood. The VN is considered as the gold standard test for measurement of protective immunity against viruses following vaccination or natural infection (17, 38) . Potential caveats in VN performed on DBS specimens could be extraction conditions and smaller extraction volumes in order to run the assay at lower dilutions. Since VN is run by overlay onto cell monolayers, any detergent, including Tween 20 present in ELISA buffer, will be incompatible with the assay. Our results showed that extraction in reduced-protein tissue culture medium, such as Opti-MEM could provide the optimal conditions for subsequent VN analysis. Presence of 1-2% BSA could prevent potential non-specific absorption of the antibodies on the surface of the polystyrene plate well in the extraction process. Because the filter paper spots are not collected and stored in sterile environment we recommend the use of antibiotic-supplemented medium. MV was used as prototype virus pathogen to determine and optimize the VN using DBS and serum spots.

Incubation of the filter paper punches in Opti-MEM at room temperature for 2 h was sufficient for immunoglobulin elution. We employed a known loaded volume of human AB(+) blood group serum and MV-neutralizing MAb 20H6 in order to determine the IgG recovery and specific activity. Specific activity of serum antibodies was not affected following storage for more than 10 days and showed no significant difference from control frozen serum samples. Concentration of extracted MAbs was measured by ELISA run in duplicates in 4-fold dilutions.

Recovery rate is presented as % of the control purified MAb or supernatants used for the filter paper samples.

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