JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1991, p. 2187-2192

Vol. 29, No. 10

0095-1137/91/102187-06$02.00/0 Copyright C 1991, American Society for Microbiology

Development of Simian Immunodeficiency Virus Isolation, Titration, and Neutralization Assays Which Use Whole Blood from Rhesus Monkeys and an Antigen Capture Enzyme-Linked Immunosorbent Assay BARBARA L. LOHMAN,l.2* JOANNE HIGGINS,2 MARTA L. MARTHAS,2 PRESTON A. MARX,3 AND NIELS C. PEDERSEN2 California Regional Primate Research Center' and Department of Medicine,2 School of Veterinary Medicine, University of California, Davis, California 95616, and New Mexico Regional Primate Research Laboratory, New Mexico State University, Holloman Air Force Base, New Mexico 883303 Received 7 March 1991/Accepted 12 July 1991

Assays that use rhesus macaque whole blood and an antigen capture enzyme-linked immunosorbent assay for the simian immunodeficiency virus (SIV) p27 core protein were developed for the isolation of SIV from the blood of infected animals, the titration of infectivity of SIV inocula, and the quantitation of virus neutralizing antibodies in serum. These assays required small amounts of whole blood, were adaptable to a microtiter format, and used substrates mainly of rhesus macaque origin.

Simian immunodeficiency virus (SIV) infection is an important animal model for research studies of human AIDS (3, 21). Improvements in the assays used to detect and titrate SIV would facilitate the use of the simian system. Current assay procedures use primary or continuous human lymphoblastoid cells as substrates for SIV propagation (17, 26). Although primary and continuous human cell lines are readily available and most SIV isolates propagate in them to a relatively high titer, the use of simian cells in routine assays would be biologically more relevant. Unfortunately, continuous rhesus monkey peripheral blood mononuclear cell (PBMC) cultures are difficult to maintain. Primary monkey PBMC cultures also require interleukin 2 (IL-2) and a constant replenishment with normal monkey blood. The small size of rhesus macaques precludes collection of the same number of normal PBMCs for tissue culture assays as is possible from humans. Leukophoresis, an accepted procedure for obtaining large numbers of human PBMCs without substantial loss of blood volume, is difficult to perform on rhesus monkeys. The inability to harvest large amounts of blood from monkeys results in fewer tests on the same experimental animal. The aim of this study was to develop a series of assay procedures that would be applicable to SIV vaccine research, make maximal use of small amounts of blood, be adaptable to a microtiter format, and use substrates mainly of rhesus macaque origin. The basis of this method was (i) the use of whole blood as a substitute for purified PBMCs or CEM x 174 cells and (ii) the use of a sensitive SIV p27 antigen capture enzyme-linked immunosorbent assay (ELISA) to detect virus replication in microtiter culture wells.

used as a source of infected blood (12, 18, 24). These monkeys were infected with either uncloned SIVmac or molecularly cloned SIVmac239 isolates from the New England Regional Primate Research Center (12, 24). All animals were housed in facilities of the California Regional Primate Research Center, University of California, Davis. Blood collection and preparation. Blood (2.5 ml) was collected directly into sterile evacuated tubes (13 by 75 mm) containing either no anticoagulants or sodium heparin. Tubes without anticoagulants were preloaded with 20 sterile 4-mm-diameter glass beads for defibrination. These tubes were rocked gently for 30 min, to allow clots to form around the beads (7, 25). Virus and cell stocks. SIVmac was obtained originally from the New England Regional Primate Research Center and was propagated in HUT 78 human T-lymphoblastoid cells (1). Cell culture supernatants that contained high levels of viral reverse transcriptase were pooled and frozen at -70°C in 1-ml aliquots. The CEM x 174 cell line, a human B- and T-lymphoblastoid somatic hybrid, was kindly provided by James A. Hoxie (University of Pennsylvania, Philadelphia). All cell cultures used in this study were maintained in RPMI 1640 medium supplemented with 1% penicillin-streptomycin and 1% L-glutamine (GIBCO, Grand Island, N.Y.) and 10% fetal bovine serum (Hyclone Labs, Logan, Utah) and were incubated at 37°C in humidified air containing 5% CO2. SIV p27 monoclonal antibody production. Mouse monoclonal antibodies to the SIV p27 major core protein were produced by methods that have been described previously for the production of monoclonal antibodies to human immunodeficiency virus (HIV) (10). The characterization of these monoclonal antibodies is reported elsewhere (9). On the basis of its high avidity for SIV p27, one monoclonal antibody, 55-2F, was selected for use in the antigen capture ELISA. This monoclonal antibody was of the immunoglobulin G2b (IgG2b) isotype, which is specific only for isolates of SIVmac, e.g., SIVmac239, SIVmac251, and SIVmaclAll, and was very efficient for antigen capture. Monoclonal

MATERIALS AND METHODS Experimental animals. Adult rhesus macaques from previous and ongoing SIV pathogenesis and vaccine studies were *

Corresponding author. 2187

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LOHMAN ET AL.

antibody 55-2F failed to react with SIVagm, SIVsmmI SIVstm, HIV type 1 (HIV-1), or HIV-2. SIV p27 polyclonal antibody production. A polyclonal antibody specific to SIV p27 was raised in rabbits by immunizing them with SIV p27 that was purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (13). Five hundred micrograms of gradient-purified SIVmac251 was electrophoresed on a 10% polyacrylamide gel (12 by 16 cm). The entire slab gel was sliced horizontally, and the slices were placed into tubes. After three rinses with distilled water, 2 ml of 0.01 Tris-0.1 M NaCl buffer (pH 7.4) was added, and the gel and buffer were dispersed into a slurry. The gel fractions containing the SIV p27 major gag protein were identified by ELISA by the procedure of Lutz and coworkers (15). One-fourth milliliter of the slurry was emulsified with an equal volume of Freund complete adjuvant and was injected intramuscularly into each of three New Zealand White rabbits. Second and third immunizations with an equal amount of immunogen in Freund incomplete adjuvant were given at 3- to 4-week intervals. Postiinmunization serum was screened by Western blotting (immunoblotting) against gradient-purified whole SIV and was found to be highly specific for SIV p27 (data not shown). SIV p27 antigen capture ELISA. The antigen-capturing ability of mouse monoclonal antibodies is compromised to various degrees by direct binding to plastic (23), which was the case with the 55-2F monoclonal antibody (see Fig. 1). In order to overcome this problem, 55-2F IgG was attached to ELISA plates via a biotin-avidin "bridge." Mouse IgG monoclonal antibody purified by ammonium sulfate precipitation was biotinylated with (+)-biotin-N-hydroxysuccinimide ester (Calbiochem-Behring, La Jolla, Calif.) that was dissolved in dimethyl sulfoxide to a concentration of 1 mglml (8). Twenty-five micrograms of biotin-NHS was added to 1 mg of IgG in 1 ml of 0.1 M NaHCO3 (pH 8.0). After a 4-h incubation at room temperature, the biotinylated IgG was purified on a G-25 Sephadex PD10 column (Pharmacia, Piscataway, N.J.) that was equilibrated with phosphatebuffered saline. Canine IgG (Sigma, St. Louis, Mo.) was similarly biotinylated (100 p.g of biotin per mg of canine IgG) and coated onto ELISA Immulon 2 plates (Dynatech, Chantilly, Va.) at a concentration of 200 ng of IgG per well, and the plates were incubated overnight at room temperature. The volume used for coating the plates, as for all other steps of the antigen capture ELISA, was 200 RI per well. After washing out excess canine IgG, the plates were incubated overnight at 4°C with 2 jxg of avidin-D (Vector Laboratories, Burlingame, Calif.) per well and were stored at 4°C at this step. Plates were washed prior to the addition of monoclonal antibody. One microgram of biotinylated IgG monoclonal antibody 55-2F was added to, each well and was allowed to bind to the biotin-avidin bridge for 2 h at 37°C. The plates were then washed and were ready to be used for antigen capture. Cell culture supernatants of SlVmac-infected HUT 78 cells were diluted twofold in ELISA dilution buffer containing 0.1% Tween 20 (Sigma) and were tested in the antigen capture ELISA. The quantity of SIV p27, in terms of whole virus, was determined from a standard curve that was generated by using sucrose density gradient-purified SIVmac. ELISA plates were incubated for 1 h at 370C and washed, and 200 RI of a 1:4,000 dilution of polyclonal rabbit anti-SIV p27 serum was added to each well and incubated for 1 h at 37°C. The plates were washed, and 200 RIu of a 1:4,000 dilution of goat anti-rabbit IgG conjugated to horseradish peroxidase (Bio-Rad, Inc., Richmond, Calif.)

J. CLIN. MICROBIOL.

was added to each well. After 30 min of incubation at 37°C, the plates were washed and a substrate solution consisting of tetramethylbenzidine (Sigma) in 0.05 M citric acid (pH 4.0) and 0.01% H202 was added to each well. Color development was stopped with 1 N H2SO4 after 5 to 7 min. Plates were read at 450 nm with a reference wavelength of 570 nm. Virus isolation. Virus was isolated from the blood of SIV-infected rhesus monkeys by using a standard method with purified PBMC (16, 24). PBMCs were purified from heparinized blood by Ficoll gradient separation. A total of 3 x 106 to 5 x 106 PBMCs were cocultivated with 106 CEM x 174 cells in a 25-cm2 disposable culture flask. Cultures were incubated, and half of the cell suspension and spent medium was replaced with fresh medium biweekly. Samples for SIV antigen capture ELISA were saved biweekly. Negative cultures were maintained for 8 weeks before being discarded. Virus was isolated from infected whole blood by a modification of the procedure described above. Two hundred fifty microliters of heparinized blood, containing approximately 1.5 x 106 to 2.6 x 106 leukocytes, was washed one time in phosphate-buffered saline, diluted in 5 ml of tissue culture medium, and incubated in a 25-cm2 disposable culture flask with 106 CEM x 174 cells. Spent medium was replaced with fresh medium during the first week of culture. One-half of the cell suspension and associated medium was replaced with fresh medium biweekly thereafter. Samples of culture medium for SIV antigen capture ELISA were also harvested biweekly. To increase the number of leukocytes in the coculture, 500 ,ul of heparinized blood was also used in the procedure described above. No deleterious effects were observed with the increased blood volume. Virus titration. An aliquot of stock SIV inoculum was thawed and titrated for viral infectivity by using either CEM x 174 cells or normal whole blood in 96-well microtiter culture plates (Falcon from Fisher, Santa Clara, Calif.). One hundred microliters of serially twofold diluted stock culture supernatant was added in sextuplet to wells containing 104 CEM x 174 cells in 100 ,ul of culture medium. Six control wells containing 104 CEM x 174 cells and 200 pI of culture medium were included in each plate. The plates were then incubated, and 150 RI of medium was harvested at day 5 and assayed for the presence of SIV p27 antigen. The mean background optical density values of the control wells were averaged and subtracted from the test values. The 50% tissue culture infectious dose (TCID50) was calculated by the method of Reed and Meunch (19). A similar titration of viral infectivity was developed with whole blood. Defibrinated whole blood was diluted 1:20 with tissue culture medium containing 0.5 ,ug of Staphylococcus enterotoxin A (SEA; Toxin Technology, Madison, Wis.) and 100 U of human recombinant IL-2 (courtesy of Cetus Corp., Emeryville, Calif.) per ml. Two hundred microliters of the diluted blood mixture was then added to each well of a 96-well microtiter plate. After 3 days of incubation, 100 ,ul of spent medium was removed from the top of each well. The virus stock was then diluted and added to each well as described above for the conventional CEM x 174 cell assay. SIV antigen levels were measured on culture supernatants taken on day 7 of incubation, and the TCID50 was calculated. Virus neutralizing antibody assay. The neutralizing antibody titers in assays using whole blood as target cells were compared with those in assays using CEM x 174 as target cells. A standard virus neutralizing assay with CEM x 174 cells as a target was used (24), with the exception that the endpoint was determined by an SIV p27 antigen capture ELISA rather than cell death, as measured by 3-(4,5-dimeth-

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TABLE 1. Virus isolation from whole blood or purified PBMC cultures from SIV-infected monkeys

0.6 -

No. of weeks in culturea

Animal no.

0.4

PBMC, CEM x 174

Blood,

Blood,

CEM x 174

SEA-IL-2

Neg 4.0 1.0 2.0 2.5 Neg Neg Neg 5.5 1.5 2.5 4.0 2.0 3.0 6.0 4.0 7.0

ND ND ND ND 2.5 Neg Neg Neg 2.5 2.5 3.5 2.0 4.0 3.0 2.0 Neg

Blood, CEM x 174,

SEA-IL-2

0.3 0.2bridge

00.1 -

0.0

4 0

format

direct format

, * IS* 10

30

20

ng

40

50

SlVmac-p27

FIG. 1. Sensitivity of the SIV p27 antigen capture ELISA. Mean optical density levels were calculated from the direct and bridge format assays against concentrations of gradient-purified SIVmac. Plates were blanked on wells containing diluent.

ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Serial 10-fold dilutions (100 RI1) of heat-inactivated test serum in tissue culture medium were incubated for 1 h with 100 RId of culture medium containing 4 TCID50s of SIVmac in a 24-well tissue culture plate. After incubation, 200 ,u1 of fresh medium was added to each well containing serum and virus. One hundred microliters of the diluted serum-virus mixture was then added to triplicate wells of a 96-well microtiter culture plate that contained 104 CEM x 174 cells in 100 ,u1 of culture medium per well. Known SIV antibody-negative and -positive serum samples were included with each test run. Culture plates were incubated for 5 days, and at the end of incubation, 150 of culture supematant was removed for testing by SIV antigen capture ELISA. The virus neutralizing antibody titers were defined as the inverse of the serum dilution that gave a 50% reduction of SIV antigen expression when it was compared with that in control wells containing SIV antibody-negative serum. The assay described above was performed with rhesus monkey whole blood in place of CEM x 174 cells. Defibrinated blood was diluted and SEA-IL-2 was stimulated in the same manner as described above for the viral infectivity titration. Three-day-old whole-blood cultures were then substituted for CEM x 174 cells in the virus neutralizing antibody assay described above, and the remainder of the of assay was identical, with three exceptions. First, 100 of the spent medium was removed prior to adding 100 serum-virus mixture. Second, the serum-virus mixture contained 10-fold higher levels of input virus, i.e., 40 TCID50s compared with the 4 TCID50s used in the CEM x 174 cell assay. Third, the cultures were incubated for 7 days rather than for the 5 days used in the CEM x 174 cell assay. RESULTS

Antigen capture ELISA. Various concentrations of gradient-purified whole SIVmac were tested by an SIV p27 antigen capture ELISA. The bridge assay was capable of detecting as

little

as

10 ng of whole SIV per ml of tissue culture

supernatants (Fig. 1). The sensitivity of the assay was greater in the bridge than in the nonbridge format. Virus isolation. Virus isolation from the blood of 16 chronically SIV-infected rhesus monkeys was attempted by using both conventional and whole-blood techniques. Three

17002 19542 20158 17133 8233 16622 18012 19090 19690 21346 22136 23058 23065 23383 24044 22993 22993

5b

ND

Neg

ND

4.0

ND ND Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg

1.0 6.0 3.5 Neg Neg Neg 2.5 2.5 2.5 2.0 2.0 4.0 2.0 Neg ND

Neg ND

I The PBMC fraction was cocultivated with CEM x 174 cells without SEA-IL-2 stimulation (PBMC, CEM x 174). Heparinized blood (250 1,u) was cocultivated with CEM x 174 (blood, CEM x 174), stimulated with SEA-IL-2 and not cocultivated (blood, SEA-IL-2), or stimulated with SEA-IL-2 and cocultivated with CEM x 174 cells (blood, CEM x 174, SEA-IL-2). Values represent the number of weeks in culture prior to the detection of SIV p27 antigen in culture supernatants. Neg, negative; ND, not done. b 500 RI of blood.

whole-blood culture conditions were tested: (i) SEA-IL-2 stimulated whole blood cocultivated with CEM x 174 cells, (ii) SEA-IL-2 stimulated whole blood cultured by itself, and (iii) unstimulated whole blood cocultivated with CEM x 174 cells. The correlation between PBMCs and CEM x 174 cells and unstimulated whole blood and CEM x 174 cell cocultures was extremely good (Table 1). In three animals, whole blood and CEM x 174 cocultures were positive earlier than the corresponding PBMC and CEM x 174 cocultures, four were slower, and two tested positive at the same time. Only one discordant result was observed: one culture that was negative by the whole-blood technique was positive with PBMCs. The whole blood and CEM x 174 assay was positive for viral antigen production when it was retested with 500 1±l rather than 250 pul of blood. Stimulation of whole blood with SEA and IL-2 did not enhance virus recovery over that when unstimulated blood was used. The average length of time for the appearance of SIV antigen was 2.75 weeks for unstimulated whole blood and CEM x 174 cocultures and 2.63 weeks for stimulated cocultures. No SIV antigen was detected in SEA-IL-2stimulated whole-blood cultures that were not cocultivated with CEM x 174 cells. Virus infectivity titrations. A stock SIV inoculum was titrated for infectious virus by conventional and whole-blood procedures. Although virus replication was greater in the assay with CEM x 174 cells, as witnessed by SIV p27 antigen expression, the actual TCID50 was similar for both tests (Fig. 2). The TCID50 of the stock inoculum was 10-3.21 when the inoculum was titrated on human CEM x 174 cells and 10-3-02 when the inoculum was titrated on defibrinated rhesus monkey blood. Heparinized whole blood washed once in phosphate-buffered saline worked equally well as defibrinated blood in virus infectivity assays (Fig. 3).

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2190

A

30

25

A0. Cu

20

N N1-

15 CD

0) Cu

c

co

30J)O 01N~~~~ C~ 10

~ cq

~

Ca

q

105

N

SJ

0)

CD

CD

CD

reciprocal of serum dilution

reciprocal dilution of SlVmac

B

3025 -

B 20 -

co

Cu 0C-

10

C

E

0)

1

C

20 15 10

-

c

co

50% endpoint

5.

Ae 10 U

. N

v

X)

ZD

.

. N

U)

C

. -

s CQ

CD

ND"

CD

Cma)

)

_

U)

csl a reciprocal dilution of SlVmac titration by CEM x 174 or defibrinated blood.

FIG. 2. Virus Solid bars represent the mean levels of SIV p27 antigen produced by microtiter cultures of either CEM x 174 (A) or defibrinated blood (B) following 5 or 7 days of incubation, respectively, with serial dilutions of SIVmac. The TCID50 calculated from CEM x 174 was 10-3 21. The TCID50 calculated from defibrinated blood was 10-3-01. Hatched bars represent mean input levels of SIV p27 antigen.

The effect of residual input virus on the amount of SIV p27 antigen that was detected after 5 to 7 days of incubation was negligible in both types of assays (Fig. 2). This was because of (i) the relatively small amount of input virus and (ii) the almost complete degradation of the input p27 antigen during the incubation period (data not shown). Virus neutralizing antibody titrations. The virus neutralizing antibody titer of known positive monkey sera was determined by both CEM x 174 cell and defibrinated rhesus blood assays. A rhesus monkey serum sample that had a SIV neutralizing antibody titer of 10-2.5 in the CEM x 174 cell 5 4 3

-

-

CD

2/

co

1

/

defibrinated blood heparinized blood

0

'.,I,.......... 0

2

8 4 6 10 days post SIV infection

12

14

FIG. 3. Time course for SIV infection of whole blood. Values are the mean levels of SIV p27 antigen produced by cultures of either defibrinated or heparinized blood grown in 25-cm2 disposable culture flasks.

3 1. 4 1 o2. reciprocal of serum dilution

FIG. 4. Virus neutralization titers by CEM x 174 or defibrinated blood assays. Serum mediated inhibition of SIV infectivity in microtiter cultures with either CEM x 174 human lymphoblastoid cells (A) or defibrinated rhesus macaque blood (B). Identical standard SIV immune (closed circles) or nonimmune (open circles) rhesus macaque serum was used for both assays. The dotted line indicates the values obtained when virus but no serum was added to either CEM x 174 cells or rhesus macaque whole blood. The same immune serum had a higher mean inhibitory titer (50% inhibition of antigen expression) when it was tested with CEM x 174 cells than it did with rhesus macaque whole blood.

assay had an antibody titer of 10-1.5 in the rhesus monkey blood assay (Fig. 4). This identical difference in titer between CEM x 174 and whole blood was observed upon one repetition of the assay by using the identical SIV immune serum. DISCUSSION This study demonstrated the usefulness of rhesus monkey whole blood as a substrate for isolation, titration, and neutralization antibody assays. SIV was isolated equally well from infected whole blood as it was from infected gradient-purified PBMCs. The infectivity titer of tissue culture-propagated SIV stocks was similar whether the virusreplicating cells were from normal monkey whole blood or continuous human lymphoblastoid cell cultures. Virus neutralizing antibodies could also be titrated as readily with normal monkey whole blood as it could with human cells, albeit at lower relative titers and with an incubation period that was 2 days longer. Although the results of the human and monkey cell assays were similar, the use of reagents entirely of rhesus origin may be attractive. Virus inhibition by such things as drugs, biologic response modifiers, immune cells, and antibodies may be more relevant if it is measured in a system that is allogeneic rather than xenogeneic. The use of whole blood for studies that require homologous cells is definitely more efficient than the use of PBMCs. Whole blood may also be a preferable substrate for field stations, eliminating the need for PBMC isolation.

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VOL. 29, 1991

The assay procedures described in this report were also designed to use as little blood as possible. Instead of the usual 3 to 5 ml of blood required when PBMCs were used as a substrate, virus isolation was just as readily accomplished with 500 ,l of whole blood. The virus isolation technique has application for pediatric studies in both the SIV and HIV systems. A similar method has been used successfully for the isolation of HIV-1 from the blood of infected human subjects (4). For the titration and neutralization assays, only a fraction of the amount of whole blood than would have been required to purify PBMCs for the same procedures was used. As little as a 10-,u equivalent of whole blood per well in a 96-well culture plate was required for these assays, compared with 0.5 million to 1 million purified PBMCs derived from 500 to 1,000 pul of whole blood for more conventional assays. Such economy may be particularly useful for studies of SIV infection in infant monkeys. Virus isolation was the only assay procedure in which human cells could not be replaced entirely with primate cells. Whole blood cell cultures from SIV-infected animals did not yield viral antigens, even when they were stimulated with SEA-IL-2, unless they were cocultivated with human lymphoid cells. The reason for this finding was paradoxical, because normal rhesus whole blood cells were very good substrates for SIV replication in the infectivity titration assay. It is possible that the blood of SIV-infected monkeys contains inhibitory factors, such as the viral suppressive CD8+ cells that have been previously recognized in SIVinfected rhesus and sooty mangabey monkeys (11). If this were so, then the CEM x 174 substrate cells were not inhibited in the same manner as monkey leukocytes were. The use of a sensitive SIV p27 antigen capture ELISA also contributed greatly to the miniaturization and economy of the virus isolation, virus titration, and virus neutralization assays described here. Antigen could readily be detected in 100 pI of tissue culture supernatant, while conventional tests with reverse transcriptase activity require larger volumes of culture fluid, cannot be readily miniaturized, and are much more expensive to conduct. The use of antigen capture ELISAs for tests such as these is certainly not novel (20, 26). However, it does have advantages over other more indirect measures of SIV or HIV replication, such as cell death assays based on thymidine incorporation (2, 26), syncytium production (5, 6, 17, 22), and MTT incorporation (16, 24) or on reverse transcriptase elaboration (12, 22, 24). The rationale for using defibrinated blood in this study was to avoid the contamination of cultures with anticoagulants and to prevent clot formation in the cultures. Anticoagulants such as heparin may contain viral inhibitors (14). Although there was a theoretical advantage in using defibrinated blood, there was no significant difference between heparinized and defibrinated blood in the assay procedures that we studied. If the assays would have required greater amounts of blood per test well, then it may have been preferable to use defibrinated blood. ACKNOWLEDGMENTS Funds for this study were provided by a base grant to the California Primate Research Center (Public Health Service grant RR00169 from NIH-NCRR) and grants A129207 and A162559 from the National Institutes of Health. Human recombinant IL-2 was generously provided by Cetus Corp. REFERENCES 1. Daniel, M. D., N. L. Letvin, N. W. King, M. Kannagi, P. K. Sehgal, P. K. Kanki, M. Essex, and R. C. Desrosiers. 1985.

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Development of simian immunodeficiency virus isolation, titration, and neutralization assays which use whole blood from rhesus monkeys and an antigen capture enzyme-linked immunosorbent assay.

Assays that use rhesus macaque whole blood and an antigen capture enzyme-linked immunosorbent assay for the simian immunodeficiency virus (SIV) p27 co...
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