AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 6, Number 3, 1990 Mary Ann Liebert, Inc., Publishers
A Sensitive Detection and
Radioimmunoprecipitation Assay for the Quantitation of Antibodies to the Envelope Glycoprotein gpl20 of the Human Immunodeficiency Virus (HIV-1)
CATHERINE LUCAS,1 MICHAEL L. PETERSON,1 GREGORY L. BENNETT,1 STEVEN W. FRIE,1 PHILLIP W. BERMAN,2 and ANTHONY B. CHEN1
ABSTRACT
radioimmunoprecipitation (RIP) assay was developed to detect antibodies to the envelope glycoprotein gpl20 of the human immunodeficiency virus (HIV-1). The assay, which utilized recombinant gpl20 (rgpl20), was quantitative, reproducible, and specific for antibodies to rgpl20 or antibodies to native gpl20 resulting from natural infection with HIV. Polyethylene glycol-8000 (PEG), used in the assay at a final concentration of 10% to precipitate immune complexes, was demonstrated to be effective in titering sera from different animal species. Provided samples were diluted at least 1:100, antibody titers could be determined either by the classical dilution method or by interpolation from a calibration curve prepared with a positive serum. The humoral response of animals immunized with rgpl20 was monitored and a positive correlation was found between titers determined in the RIP assay and the ability of the sera to neutralize. In addition, RIP titers of HIV-positive human sera correlated very well with reactivity obtained in a commercial HIV immunoblot assay. The assay has the advantage of quantitation, fast turnaround time, and versatility. A
INTRODUCTION
virus,1"3 the envelope glycoproteins of the human virus type 1 (HIV-1), the retrovirus responsible for acquired immunodeficiency syndrome (AIDS),4"6 have been the subject of intensive research for vaccine development. Of these two proteins, gpl20 and gp41, gpl20 has been considered the protein of choice for vaccine development. It is located on the surface of the virus,1"3-7 possesses the domain by which HIV binds to CD4,89 appears to be essential for cell to cell transmission of HIV, ' °- " and elicits antibodies which neutralize HIV-1 infectivity in
Because immunodeficiency
of their location on the surface of the
vitro.12'13
Departments of 'Medicinal and Analytical Chemistry, and 2Molecular Biology, Genentech Inc., 460 Pt San Bruno Blvd, South San Francisco, CA 94080. 357
LUCAS ET AL. For these reasons, a method was devised to produce useful quantities of recombinant gpl20 (rgpl20) from the IIIB isolate of HI V-1 in a continuous Chinese hamster ovary (CHO) cell line.12 The purified protein, which is glycosylated and reacts with CD4 like native gpl20,14 was injected into animals of several species912'15 to examine its immunogenicity. A radioimmunoprecipitation assay (RIP) was developed to monitor the formation and determine the titer of antibodies to rgpl20. This assay provided a simple, quantitative and cost-effective alternative to the commercially available Western blots and allowed us to monitor specifically the immune response to gpl20. In this report, the RIP was evaluated for its precision and specificity, tracer stability, serum effects, and precipitation systems. The assay results were compared with viral neutralization results, as well as to results obtained with a commercial immunoblot assay, as well as with results obtained with the immunoblotting of recombinant gpl20.
MATERIALS AND METHODS
Preparation of antigen Recombinant gpl20 (rgpl20) was produced in a continuous Chinese hamster ovary cell line as described previously,12 and consisted of the N-terminal 25 amino acids from the mature form of the herpes simplex virus 1 glycoprotein D (GD) fused to residue 31 of the mature form of gpl20 from the HIV-1 IIIB isolate.12 The protein was purified by immunoaffinity chromatography using a monoclonal antibody prepared against rgpl20,15 and was approximately 95% pure as judged by silver staining of sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE).1516
Polyacrylamide gel electrophoresis Slab gel electrophoresis was performed in 8-12% gradient gels under dissociating, but nonreducing conditions (0.1% SDS) in 0.05 M Tris-HCl (pH 8.4) according to the method of Laemmli.16 Approximately 10 p.g protein were electrophoresed in each lane and a current of 22 mA per gel was applied. Gels were stained by the silver staining method of Morrissey.17
Preparation of tracers Purified preparations of rgpl20 were radiolabeled with 125I by a lactoperoxidase method similar to that described by Thorell and Johansson.18 Briefly, 100 p.g of rgpl20 diluted in 100 pJ of phosphate-buffered saline (PBS) pH 7.4, was iodinated for 15 min at ambient temperature with 1 mCi (10 p.1) of Na125I (Amersham, Arlington Heights, IL), 7 p,g (20 pJ) of lactoperoxidase (Sigma Chem. Co., St. Louis, MO), and 20 p.1 of 1 raM H202 (Kodak, Rochester, NY). Next, 20 p,l of 1 mM ß-mercaptoethanol (BioRad, Richmond, CA) were added for 2 min to stop the reaction, followed by 20 p.1 of 0.5 M potassium iodide (Sigma) and 50 p.1 of tracer diluent (PBS containing 0.5% bovine serum albumin (BSA) and 0.01 % thimerosal). Radiolabeled rgpl20 was separated from free 125I by filtration over Sephadex G-100 (Pharmacia, Piscataway, NJ) column equilibrated with tracer diluent. Fractions of 0.5 ml were collected and measured for radioactivity in an Apex automatic gamma counter (Micromedic Systems Inc., Horsham, PA). The radioactive protein peak fractions were pooled and precipitability with 10% trichloroacetic acid (TCA) was determined. The tracer was stored for two months at +4°C. Autoradiograms of rgpl20 tracers were obtained by electrophoresing 1000 cpm (60-90 pg) by the SDS-PAGE method of Laemmli16 and exposing the resulting gel to a Kodak X-Omat XAR-2 X ray film for approximately 2 days.
Preparation of antibodies Antisera to the recombinant surface antigen of the hepatitis B virus (rHBs Ag), to herpes simplex virus 2, to Chinese hamster ovary proteins (CHOPS), and to rgpl20 were produced in rabbits or guinea pigs following
358
RADIOIMMUNO ASS AY FOR ANTIBODIES TO
gpl20
standard immunization procedures. '9 The affinity of the rabbit anti rgp 120-positive control used in the assay was determined by Scatchard analysis to be 2 x 109 1/M.
Radioimmunoprecipitation procedure The RIP method for antibody detection described in this report is essentially a modification of the method developed by Farr20 to study interactions of antibody with antigen and described previously by Chen et al.21 Serum samples (100 pJ, prediluted 1:50) were assayed as a threefold dilution series in assay diluent (PBS, 0.5% BSA, 0.01% thimerosal) in duplicate, incubated overnight at +4°C with a fixed amount of [l25I]rgpl20 tracer (2 ng, 20,000 cpm in 100 pJ of assay diluent). Immune complexes were then precipitated by adding 50 pJ of 1% bovine gamma globulins (Pel Freez, Rogers, AR) in PBS containing 0.01% thimerosal and 1 ml of 12.5% polyethylene glycol (PEG) 8000 (J.T. Baker, Phillipsburg, NJ) to each tube for 30 min at ambient temperature (the final concentration of PEG was 10%). In some experiments, immune complexes were precipitated by adding 50-100 pJ of specific antispecies antiserum. After centrifugation at 2000 x g for 20 min in a Sorvall RC-3B centrifuge, supernatants were decanted and pellets were counted for 1 min each for 125I activity. To keep background levels to a minimum, pellets were washed with assay diluent prior to counting in the gamma counter. The mean nonspecific binding (NSB) was determined by averaging the counts per minute (cpm) of four NSB tubes which contained assay buffer in place of sample. For all the other tubes, the percent B/T (B binding cpm of the sample; T total counts mean cpm of 2 tubes containing 100 pJ tracer) was calculated. Sera were first screened at 1:100 final dilution and determined to be antibody positive if the counts in the pellets amounted to at least twice the amount of counts given by an equivalent dilution of a species-specific negative control serum. Subsequently, titers of positive sera were determined as the log10 of the reciprocal of the dilution equal to 2 X NSB. Titers falling between dilution points were interpolated from a point to point curve-fitting program. In an attempt to simplify the format of the assay, the suitability of calibrating the binding titer relationship, rather than directly titering samples to endpoint, was examined. For this purpose, experimental data were normalized and reduced with a logit-log procedure against a standard curve prepared with representative binding data from 6 serial dilutions of the rabbit anti-rgpl20-positi ve serum. =
=
=
=
Immunoblotting procedures The presence of antibodies to HIV-1 proteins was monitored with commercially available reagents (BioRad Laboratories) following the manufacturer's recommendations. Titration of anti-gpl20 antibodies by immu-
noblotting was performed on 25 p-g of rgp 120 resolved by SDS-PAGE as described above and transferred to nitrocellulose (BioRad Laboratories) according to the method of Towbin et al.22 Electroelution from the gel to nitrocellulose was conducted for 35 min at 1 A. After blocking excess protein binding sites with blocking buffer (50 mM Tris, 5 mM EDTA, 150 mM NaCl, 0.05% Triton X-100, and 0.25% gelatin), the nitrocellulose sheet was cut into 2-mm-wide strips. Individual strips were incubated for 2 h at room temperature with threefold serial dilutions (~3 ml/strip) of the test sera in negative control serum prediluted 1:50 in blocking buffer. Initial dilutions were 1:50 for patient samples and negative controls, or 1:10,000 for the rabbit anti-rgpl20 hyperimmune serum. After rinsing the strips three times with PBS containing 0.05% Tween 20 (Sigma Chem. Co.), HRP-conjugated anti-human IgG (CalTag, South San Francisco, CA) diluted 1:5000 or antirabbit IgG (Tago, Burlingame, CA) diluted 1:3000 in blocking buffer was added for 1 h at room temperature. Nitrocellulose strips were washed again three times with PBS-Tween, and exposed to substrate solution as described by Adams.23 The reaction was stopped with tap water. Animal and human studies Animal sera examined in the RIP were obtained from immunization studies conducted in either guinea pigs, or chimpanzees.15 Additional sera were obtained from mice, goats, or rabbits immunized with 120 for the purpose of generating antibodies. Also, human HIV-seropositive sera were obtained from the rgp of laboratory Dr. J. Groopman.
baboons,
359
LUCAS ET AL.
RESULTS Characterization
of the 125I-rgpl20
tracer
As described in Materials and Methods, the RIP assay is based on the binding of antibodies in test sera to rgpl20 iodinated by lacroperoxidase. It was therefore important to characterize the tracer for use in the assay. Reproducibility of the lactoperoxidase method of radiolabeling rgpl20 was demonstrated by the consistent specific activity of tracers obtained from different lots of rgpl20, ranging from 6 to 8 p.Ci/p.g (720-960 mCi/p,M), for an average labeling of 1 molecule of 125I for every 2-3 molecules of protein (data not shown). The tracer chromatographed as a single radioactive protein peak by filtration over a Sephadex G-100 column (Pharmacia, Piscataway, NJ) equilibrated in PBS and migrated as a single band at 120 kD as detected by autoradiography of SDS polyacrylamide gels (data not shown). This tracer was used for binding studies with a recombinant, soluble form of the CD4 receptor of T-helper cells14 and binding of rgpl20 to CD4 was not altered by the radiolabeling.9 Iodinated rgp 120 bound to anti-gpl20 antibodies, showing a typical binding curve with 92-25% binding between 2 x 102 and 2 x 104 dilutions of a rabbit hyperimmune serum to gp 120 (data not shown). Nonspecific binding by control sera (normal rabbit serum or rabbit antibody to herpes simplex virus type 2) was eliminated at a dilution of approximately 1:500. Subsequent screening of other rabbit negative sera showed variable amount of nonspecific binding, many sera displaying much lower background (data not shown). To verify that iodination did not alter the conformation of rgp 120, competitive
120
100
o
CÛ
m
Unlabeled Protein
(|ig/ml)
'
Displacement of [ 25I]-rgp 120 by unlabeled rgp 120 (•), or the GD protein of herpes simplex type I (P. Berman, Genentech) (O). In this experiment, the rabbit anti-rgpl20 hyperimmune serum at 1:4500 dilution was incubated overnight at +4°C with varying concentrations (0.1-100 pg/ml) of the two proteins. Immune complexes were FIG. 1.
precipitated with goat antirabbit IgG and pellets were counted for radioactivity in an automatic gamma counter. Sixty percent of input counts were precipitated at this dilution of rabbit antiserum. Each data point represents the mean of two
determinations.
360
RADIOIMMUNOASSAY FOR ANTIBODIES TO
gpl20
Table 1. Interassay Precision of the RIP Assay for Detection of Antibodies to rgp 120a
Tracer lot# 1
2
3
4
5
Tracer age assay
at
Rabbit
days)
32 35 41 16 24 25 30 38 45 51 59 1 3 9 14 18 21 1 5 6 15 19 1
Control serum titer0 Baboon
Goat
4.1
4.0 4.1 4.2 4.2 4.1 4.0 4.2
4.4 4.4
4.2 4.2
4.3
4.3
4.2
4.4
4.2
4.4
4.2 4.3
4.4 4.6
4.3 0.11 2.6 7
4.4 0.12
4.5 4.5 4.1 4.2 4.2 4.4 4.3 4.3 4.4 4.2 4.1 4.2 0.16 3.8 19
X a
CV(%) n
2.8 7
aTo titer antibodies to rgp 120, sera were prediluted 1:50 in phosphate-buffered saline containing 0.5% BSA and 0.01% thimerosal, and assayed as a three fold dilution series in this buffer by overnight incubation at+4°C with a fixed amount (20,000 cpm, 2 ng)of [l25I] rgp 120 tracer. Immune complexes were precipitated by adding 50 p\ of 1 % boving gammaglobulins and 1 ml of 12.5% polyethylene glycol for 30 min at ambient temperature. Pellets were measured for in an automatic gamma counter. radioactivity b Titers were determined as the logio of the reciprocal of the dilution equal to 2 X the mean nonspecific binding (NSB, determined with tubes containing assay buffer instead of serum).
binding with unlabeled rgpl20 was examined. The displacement of the tracer by unlabeled rgpl20 is shown in Figure 1 (circles). Displacement curves with increasing ratios of unlabeled to radiolabeled rgp 120 were indistinguishable from the curve shown in Figure 1 and indicated that the radiolabeling did not modify the behavior of the protein in the assay. Precision
of the RIP assay
The precision of the assay was determined by examining the interassay coefficient of variation of titers obtained for positive control sera from three species. Data in Table 1 were accumulated over a period of two months, using five different preparations of tracer representing three lots of purified rgp 120, from 19 assays for the rabbit positive control serum and 7 assays for the baboon and goat control sera. Data from this table show that the standard deviation from a mean of 4.3 was less than 0.2 and that the coefficients of variation were 3.8, 2.6, and 2.8% for the three control sera tested (Table 1). This indicates a high level of reproducibility for the assay and confirms the reproducibility of the lactoperoxidase method of radiolabeling 361
LUCAS ET AL.
rgpl20. Good tracer reproducibility was also shown, since no significant difference in titer results obtained with different tracer lots or with the use of tracers over a period of two months.
was
Specificity of the RIP assay Specificity was examined by assaying a rabbit antiserum to herpes simplex virus type 2 and indicated no reactivity with rgp 120 at dilutions greater than 1:10. Normal rabbit serum gave somewhat higher background. In addition, an antiserum to proteins originating from the CHO cell line used to produce rgpl20, and two antisera to rHBsAg did not precipitate rgp 120 tracer, when tested at 1:100 dilution (data not shown). A radioimmunoassay formatted with a 1:4500 dilution of the rabbit positive control (allowing the precipitation of 60% of the total counts) assayed against several dilutions of the GD molecule of the herpes simplex type 1 virus resulted in no displacement of the rgp 120 tracer (Fig. 1), also indicating good specificity of the anti-rgpl20 antiserum. cross
Efficiency of PEG precipitation The assay was first optimized with rabbit antiserum and 50 pi of 1 % bovine gammaglobulin added as a carrier for precipitation. The efficiency of precipitating immune complexes, using PEG-8000 at several concentrations, was then assessed with sera from four animal species and compared with precipitations with specific antispecies second antibody. Results in Table 2 showed that for all species, PEG-8000 at 9-10% allowed precipitation of 69-86% of the precipitable counts (corrected for NSB). This compared favorably with 81-91% precipitation using specific second antibody, considering that the use of a PEG precipitation system allowed the handling of sera from several animal species with a single, common, precipitating agent. Since sera are first screened by comparison to a species-specific negative control serum to distinguish positive from negative sera, the effect of serum in the assay was examined by testing negative control sera at several dilutions and comparing the resulting background level to NSB obtained with assay buffer. Table 3 shows that the effect varied with the animal species tested. Background obtained with serum from three animal species (guinea pig, goat, and chimpanzee) was comparable to that obtained with assay buffer (15% B/T or less), whereas rabbit serum had to be diluted at least 1:200 and human serum 1:100. In general, for most animal species, the assay was amenable to a PEG precipitation system provided the sera were diluted 1:100. We also compared endpoint titers obtained when sera were diluted in assay diluent with endpoint titers obtained when the sera were diluted in a constant level of species-specific negative serum (to maintain constant serum concentration throughout the dilution curve). Endpoint titers derived from the two dilution methods were identical (data not shown), thereby validating the derivation of titers from dilution curves prepared in assay diluent. Table 2. Radioimmunoprecipitation Assay for Antibodies to rgp 120: Efficiency of Polyethylene Glycol (PEG) for Precipitating Immune Complexes in Sera from Several Animal Species'1
Final concentration
of PEG-8000 (%)b
-
Animal species Rabbit Guinea
pig Chimpanzee
Baboon
7
9
10
11
12
75 88 66 87
76 86 66 86
72 NDC 69 ND
61 73 ND 81
59 ND
46 ND
Second antibody
precipitation0 81 ND 86 91
Results are presented as percent of counts percipitated, corrected for nonspecific binding (NSB). bIn this experiment, sera from the different animal species were tested at a dilution allowing maximal binding of [125I] rgp 120(1:50 for the primate sera to 1:500 for the rabbit hyperimmune serum). They were incubated overnight a
with a fixed quantity of [l25]rgp 120 (20,000 cpm, 2 ng). Immune complexes were precipitated by adding 50 /il of 1% bovine gamma globulins and 1 ml of varying concentrations of PEG for 30 min at ambient temperature, or 100 /il of specific antispecies antiserum. at +4°C
CND: not determined.
362
RADIOIMMUNOASSAY FOR ANTIBODIES TO
gpl20
Table 3. Immunoprecipitation Assay for Detection of Antibodies to rgp 120: Background levels of Negative Sera from Different Animal Species
Animal species* Rabbit
pool
(PelFreez)
Rabbit (X of 12) Individual sera) Guinea pig (1 individual serum)
Goat (X of 4 assays; 1 individual serum) Chimpanzee (X of 6 bleeds from 1 animal) Human pool
Dilution of the sera
%Binding by the buffer (NSB)h
%Binding by the negative sera0
1:25 1:50 1:75 1:100 1:225 1:675 1:2025 1:50
9.3 11.3 9.3 7.9 9.3 9.3 9.3 11.4
21.0 35.6 29.6 19.8 10.1 9.6 9.6 33.6 ± 3.2
1:25 1:75 1:225 1:25
9.3 9.3 9.3 13.0
10.2 8.7 9.0 13.6 ± 1.1
1:50 1:100 1:25 1:75 1:225
11.4 10.8 15.0 15.0 15.0
13.4 10.0+1.0 24.3 18.2 15.3
aIn this experiment, sera from the different animal species were tested as a series of dilutions with the tracer, to determine which dilution reduced the background to a level equivalent to that obtained with buffer alone. After overnight incubation of the serum dilutions with the tracer at +4°C, immune complexes were with 50 pi of 1% bovine gamma globulins and 1 ml of 12.5% polythylene glycol. preciptated b Results are presented as percent of counts precipitated.
Calibration
of the RIP assay
We examined the possibility of calibrating the assay in order to further simplify it and determine antibody titers in test sera from a standard curve prepared with dilutions of a positive serum. For this, standards were prepared with dilutions of the rabbit anti-rgpl20 antiserum and percent of counts precipitated were plotted against the titer values for each dilution (titers were determined previously by the classical endpoint method). Titers of sera from several immunized animal species were determined from this rabbit calibration curve (Fig. 2) and compared with titers obtained experimentally by dilution to endpoint. Since the rabbit antiserum used for calibration was a hyperimmune serum with very high titer, it had to be diluted considerably so that its binding level matched that of the test animal sera. Provided the calibration curve was prepared at lower binding levels (Fig. 2), calibration-derived titers were consistent across several dilutions and correlated well with titers obtained by the endpoint method. The data in Table 4 show good agreement of titers obtained either with PEG or a specific second antibody precipitation system. The maximum disagreement between calibration-derived and endpoint titers ranged from 0.1 titer units for the majority of samples to 0.5 for one high-titered sample (baboon third bleed, Table 4). This approach provided equivalent results using only 1 to 3 eightfold dilutions of samples rather than the 6 or more threefold dilutions typically required for the endpoint method. Table 4 also shows that titers determined with a second antibody precipitation were somewhat higher than titers determined with PEG precipitation, mostly due to the differences in background with the two precipitation systems (the 2 x NSB criterion used for endpoint determination results in lower titers if the background is higher, as was the case with the PEG precipitation).
363
LUCAS ET AL.
Correlation with immunoblot results A panel of 20 HIV-positive human sera titered with the RIP assay were analyzed by immunoblot assay, using commercially available reagents. Figure 3 shows that antibodies to rgp 120 reacted with both native viral gpl60 and gpl20 by immunoblot assay. All samples negative in the RIP were also negative by immunoblot, even when exhibiting high background in this assay (sample # 2). Similarly, RIP positive samples were positive by immunoblot. Sera with higher RIP titers to rgp 120 generally had stronger reactivity in the immunoblots, although this increased reactivity was reflected more with the pl8, p24, p32, p41-43, p51, and p65 bands than with the gp 120-gp 160 bands. Generally, reactivity was stronger with gp 160 than with gp 120. To further investigate the correlation between the RIP assay and immunoblotting, recombinant gpl20 was electrophoresed by SDS-PAGE and transferred to nitrocellulose (see Materials and Methods). Two HIV-positive human sera titered in the RIP (# 18 and 7) and the rabbit positive control were also titered by blot assay on nitrocellulose-bound rgp 120. The results in Figure 4 show good correlation between titers obtained
in the two methods, and demonstrate that the RIP assay is at least as sensitive as the immunoblot assay. Human serum titers of 4.4 and 3.9 by blot assay correlated with RIP titers of 4.9 and 3.9, respectively. The rabbit positive control (RIP titer 4.1) was titered to 5.1 by visual examination of the freshly developed, wet immunoblot (in this method, endpoints are subjectively distinguished above background rather than over 2 x NSB as in the RIP). Relatively high background obtained with rabbit serum in the RIP (Table 3) may explain the lower titer achieved with this method for the rabbit positive control, as compared with immunoblot titer.
CD
2.0
2.2
2.4
2.6
2.8
3.0
3.2
Titer FIG. 2. Calibration curve for antibodies to rgp 120 in the RIP assay. A calibration curve was prepared by incubating 6 dilutions of a rabbit anti-rgp 120 hyperimmune serum with [ ' 25I]rgp 120 and plotting percent of counts precipitated against the titer values for each dilution (titers were previously determined by the classical end point method). After overnight incubation at +4CC, immune complexes were precipitated with 50 pi of 1% bovine gamma globulins and 1 ml of 12.5%
polyethylene glycol (PEG).
364
RADIOIMMUNOASSAY FOR ANTIBODIES TO
gpl20
Table 4. RIP Assay for Detection of Antibodies to rgp 120: Comparison of Endpoint to Calibration-Derived Titers
2nd antibody precip. °
PEG precipitation"
Dilution
factor
Sample Baboon 1st bleed
Titer by calibration0
100 300 900 X
Baboon 2nd bleed
300 900 2700 8100 X 900 2700
Baboon 3rd bleed
8100 24300 X
Chimpanzee
1
100 300 900 2700 X
Chimpanzee
2
100 300 X
Goat
100 300 900 2700 X
Guinea
pig
1350 4050 12150 X
Endpoint titer4
3.1 3.2 3.2 3.1 3.8 4.0 4.1 4.1 4.0 4.3 4.4 4.4 4.4 4.4 3.6 3.7 3.7 3.6 3.6 2.4 2.4 2.4 3.4 3.6 3.5 3.4 3.5 4.6
2.9
3.7
3.9
Titer by calibration0
Endpoint titer4
2.6 3.5 3.4 3.1 4.8 4.4 4.4 4.3 4.5 5.1 4.9 4.8 4.6 4.8
4.8
4.2 4.1 3.9 3.8 4.0
4.2
3.4
4.4
3.6 2.5
3.1
4.4 4.4 4.5
4.4
In this assay, dilutions of sera were incubated overnight at +4°C with [ l25I] rgp 120 tracer. Inmmune complexes precipitated with 50 p\ of 1% bovine gamma globulins and 1 ml of 12.5% polyethylene glycol. bIn this assay, immune complexes were precipitated by adding 50-100 pi of undiluted antispecies antiserum. c For calibration, a curve was prepared by incubating 5 dilutions of rabbit anti-rgp 120 hyperimmune serum with the [ 125I] rgp 120 tracer. Percent of counts precipitated were plotted against the titer values for each dilution. Animal sera were tested at several dilutions with [ ' 25I] rgp 120 and titers for each dilution were extrapolated from the rabbit calibration curve subsequently corrected for dilution. d For endpoint titration, animal sera were assayed as a threefold dilution series and titers were defined as the log i o of the reciprocal of the dilution equal to 2 X the mean nonspecific binding (NSB. determined with tubes containing assay buffer instead of serum). a
were
Correlation with neutralization results
Finally, we examined sera from two immunization studies conducted with rgp 120 in chimpanzees15 or baboons. Neutralization of viral infectivity by the sera was measured by in vitro inhibition of HIV-1-specific reverse transcriptase.1215'24 or by measuring the reduction in HIV-1 antigen production by HIV-1-infected MT2 cells as a result of incubating the viral inoculum with serum prior to infection. A positive correlation was found between titers determined in the RIP assay and the ability of the sera to neutralize where 86-88% of 365
1 2 3
4 5
6 7
8
9 10 11 12 13 14 15 16 17 18 19 20
kD 160 120 -65 —55 Il 41-43
-32 -24 -18
RIP Titers: 0 0 0 0
0 3.8'—3.9—'4.0^4.3—' 4.4 4.5 L 4.6 J 4.95.2 5.3
FIG. 3. Immunoblotting of HIV proteins with several human sera. A commercial kit (BioRad, Richmond, CA) was used and the experiment was performed following the manufacturer's recommendations. Molecular weight markers are shown to the right, serum identification numbers at the top of each immunoblot and titers obtained in the RIP assay at the bottom of each blot. These titers are expressed as the log10 of the reciprocal of the last dilution equal to or greater than 2 x the mean nonspecific binding (NSB).
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2
3
4
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8 9 10 11 12 13
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A Sensitive Detection and
Radioimmunoprecipitation Assay for the Quantitation of Antibodies to the Envelope Glycoprotein gpl20 of the Human Immunodeficiency Virus (HIV-1)
CATHERINE LUCAS,1 MICHAEL L. PETERSON,1 GREGORY L. BENNETT,1 STEVEN W. FRIE,1 PHILLIP W. BERMAN,2 and ANTHONY B. CHEN1
ABSTRACT
radioimmunoprecipitation (RIP) assay was developed to detect antibodies to the envelope glycoprotein gpl20 of the human immunodeficiency virus (HIV-1). The assay, which utilized recombinant gpl20 (rgpl20), was quantitative, reproducible, and specific for antibodies to rgpl20 or antibodies to native gpl20 resulting from natural infection with HIV. Polyethylene glycol-8000 (PEG), used in the assay at a final concentration of 10% to precipitate immune complexes, was demonstrated to be effective in titering sera from different animal species. Provided samples were diluted at least 1:100, antibody titers could be determined either by the classical dilution method or by interpolation from a calibration curve prepared with a positive serum. The humoral response of animals immunized with rgpl20 was monitored and a positive correlation was found between titers determined in the RIP assay and the ability of the sera to neutralize. In addition, RIP titers of HIV-positive human sera correlated very well with reactivity obtained in a commercial HIV immunoblot assay. The assay has the advantage of quantitation, fast turnaround time, and versatility. A
INTRODUCTION
virus,1"3 the envelope glycoproteins of the human virus type 1 (HIV-1), the retrovirus responsible for acquired immunodeficiency syndrome (AIDS),4"6 have been the subject of intensive research for vaccine development. Of these two proteins, gpl20 and gp41, gpl20 has been considered the protein of choice for vaccine development. It is located on the surface of the virus,1"3-7 possesses the domain by which HIV binds to CD4,89 appears to be essential for cell to cell transmission of HIV, ' °- " and elicits antibodies which neutralize HIV-1 infectivity in
Because immunodeficiency
of their location on the surface of the
vitro.12'13
Departments of 'Medicinal and Analytical Chemistry, and 2Molecular Biology, Genentech Inc., 460 Pt San Bruno Blvd, South San Francisco, CA 94080. 357
LUCAS ET AL. For these reasons, a method was devised to produce useful quantities of recombinant gpl20 (rgpl20) from the IIIB isolate of HI V-1 in a continuous Chinese hamster ovary (CHO) cell line.12 The purified protein, which is glycosylated and reacts with CD4 like native gpl20,14 was injected into animals of several species912'15 to examine its immunogenicity. A radioimmunoprecipitation assay (RIP) was developed to monitor the formation and determine the titer of antibodies to rgpl20. This assay provided a simple, quantitative and cost-effective alternative to the commercially available Western blots and allowed us to monitor specifically the immune response to gpl20. In this report, the RIP was evaluated for its precision and specificity, tracer stability, serum effects, and precipitation systems. The assay results were compared with viral neutralization results, as well as to results obtained with a commercial immunoblot assay, as well as with results obtained with the immunoblotting of recombinant gpl20.
MATERIALS AND METHODS
Preparation of antigen Recombinant gpl20 (rgpl20) was produced in a continuous Chinese hamster ovary cell line as described previously,12 and consisted of the N-terminal 25 amino acids from the mature form of the herpes simplex virus 1 glycoprotein D (GD) fused to residue 31 of the mature form of gpl20 from the HIV-1 IIIB isolate.12 The protein was purified by immunoaffinity chromatography using a monoclonal antibody prepared against rgpl20,15 and was approximately 95% pure as judged by silver staining of sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE).1516
Polyacrylamide gel electrophoresis Slab gel electrophoresis was performed in 8-12% gradient gels under dissociating, but nonreducing conditions (0.1% SDS) in 0.05 M Tris-HCl (pH 8.4) according to the method of Laemmli.16 Approximately 10 p.g protein were electrophoresed in each lane and a current of 22 mA per gel was applied. Gels were stained by the silver staining method of Morrissey.17
Preparation of tracers Purified preparations of rgpl20 were radiolabeled with 125I by a lactoperoxidase method similar to that described by Thorell and Johansson.18 Briefly, 100 p.g of rgpl20 diluted in 100 pJ of phosphate-buffered saline (PBS) pH 7.4, was iodinated for 15 min at ambient temperature with 1 mCi (10 p.1) of Na125I (Amersham, Arlington Heights, IL), 7 p,g (20 pJ) of lactoperoxidase (Sigma Chem. Co., St. Louis, MO), and 20 p.1 of 1 raM H202 (Kodak, Rochester, NY). Next, 20 p,l of 1 mM ß-mercaptoethanol (BioRad, Richmond, CA) were added for 2 min to stop the reaction, followed by 20 p.1 of 0.5 M potassium iodide (Sigma) and 50 p.1 of tracer diluent (PBS containing 0.5% bovine serum albumin (BSA) and 0.01 % thimerosal). Radiolabeled rgpl20 was separated from free 125I by filtration over Sephadex G-100 (Pharmacia, Piscataway, NJ) column equilibrated with tracer diluent. Fractions of 0.5 ml were collected and measured for radioactivity in an Apex automatic gamma counter (Micromedic Systems Inc., Horsham, PA). The radioactive protein peak fractions were pooled and precipitability with 10% trichloroacetic acid (TCA) was determined. The tracer was stored for two months at +4°C. Autoradiograms of rgpl20 tracers were obtained by electrophoresing 1000 cpm (60-90 pg) by the SDS-PAGE method of Laemmli16 and exposing the resulting gel to a Kodak X-Omat XAR-2 X ray film for approximately 2 days.
Preparation of antibodies Antisera to the recombinant surface antigen of the hepatitis B virus (rHBs Ag), to herpes simplex virus 2, to Chinese hamster ovary proteins (CHOPS), and to rgpl20 were produced in rabbits or guinea pigs following
358
RADIOIMMUNO ASS AY FOR ANTIBODIES TO
gpl20
standard immunization procedures. '9 The affinity of the rabbit anti rgp 120-positive control used in the assay was determined by Scatchard analysis to be 2 x 109 1/M.
Radioimmunoprecipitation procedure The RIP method for antibody detection described in this report is essentially a modification of the method developed by Farr20 to study interactions of antibody with antigen and described previously by Chen et al.21 Serum samples (100 pJ, prediluted 1:50) were assayed as a threefold dilution series in assay diluent (PBS, 0.5% BSA, 0.01% thimerosal) in duplicate, incubated overnight at +4°C with a fixed amount of [l25I]rgpl20 tracer (2 ng, 20,000 cpm in 100 pJ of assay diluent). Immune complexes were then precipitated by adding 50 pJ of 1% bovine gamma globulins (Pel Freez, Rogers, AR) in PBS containing 0.01% thimerosal and 1 ml of 12.5% polyethylene glycol (PEG) 8000 (J.T. Baker, Phillipsburg, NJ) to each tube for 30 min at ambient temperature (the final concentration of PEG was 10%). In some experiments, immune complexes were precipitated by adding 50-100 pJ of specific antispecies antiserum. After centrifugation at 2000 x g for 20 min in a Sorvall RC-3B centrifuge, supernatants were decanted and pellets were counted for 1 min each for 125I activity. To keep background levels to a minimum, pellets were washed with assay diluent prior to counting in the gamma counter. The mean nonspecific binding (NSB) was determined by averaging the counts per minute (cpm) of four NSB tubes which contained assay buffer in place of sample. For all the other tubes, the percent B/T (B binding cpm of the sample; T total counts mean cpm of 2 tubes containing 100 pJ tracer) was calculated. Sera were first screened at 1:100 final dilution and determined to be antibody positive if the counts in the pellets amounted to at least twice the amount of counts given by an equivalent dilution of a species-specific negative control serum. Subsequently, titers of positive sera were determined as the log10 of the reciprocal of the dilution equal to 2 X NSB. Titers falling between dilution points were interpolated from a point to point curve-fitting program. In an attempt to simplify the format of the assay, the suitability of calibrating the binding titer relationship, rather than directly titering samples to endpoint, was examined. For this purpose, experimental data were normalized and reduced with a logit-log procedure against a standard curve prepared with representative binding data from 6 serial dilutions of the rabbit anti-rgpl20-positi ve serum. =
=
=
=
Immunoblotting procedures The presence of antibodies to HIV-1 proteins was monitored with commercially available reagents (BioRad Laboratories) following the manufacturer's recommendations. Titration of anti-gpl20 antibodies by immu-
noblotting was performed on 25 p-g of rgp 120 resolved by SDS-PAGE as described above and transferred to nitrocellulose (BioRad Laboratories) according to the method of Towbin et al.22 Electroelution from the gel to nitrocellulose was conducted for 35 min at 1 A. After blocking excess protein binding sites with blocking buffer (50 mM Tris, 5 mM EDTA, 150 mM NaCl, 0.05% Triton X-100, and 0.25% gelatin), the nitrocellulose sheet was cut into 2-mm-wide strips. Individual strips were incubated for 2 h at room temperature with threefold serial dilutions (~3 ml/strip) of the test sera in negative control serum prediluted 1:50 in blocking buffer. Initial dilutions were 1:50 for patient samples and negative controls, or 1:10,000 for the rabbit anti-rgpl20 hyperimmune serum. After rinsing the strips three times with PBS containing 0.05% Tween 20 (Sigma Chem. Co.), HRP-conjugated anti-human IgG (CalTag, South San Francisco, CA) diluted 1:5000 or antirabbit IgG (Tago, Burlingame, CA) diluted 1:3000 in blocking buffer was added for 1 h at room temperature. Nitrocellulose strips were washed again three times with PBS-Tween, and exposed to substrate solution as described by Adams.23 The reaction was stopped with tap water. Animal and human studies Animal sera examined in the RIP were obtained from immunization studies conducted in either guinea pigs, or chimpanzees.15 Additional sera were obtained from mice, goats, or rabbits immunized with 120 for the purpose of generating antibodies. Also, human HIV-seropositive sera were obtained from the rgp of laboratory Dr. J. Groopman.
baboons,
359
LUCAS ET AL.
RESULTS Characterization
of the 125I-rgpl20
tracer
As described in Materials and Methods, the RIP assay is based on the binding of antibodies in test sera to rgpl20 iodinated by lacroperoxidase. It was therefore important to characterize the tracer for use in the assay. Reproducibility of the lactoperoxidase method of radiolabeling rgpl20 was demonstrated by the consistent specific activity of tracers obtained from different lots of rgpl20, ranging from 6 to 8 p.Ci/p.g (720-960 mCi/p,M), for an average labeling of 1 molecule of 125I for every 2-3 molecules of protein (data not shown). The tracer chromatographed as a single radioactive protein peak by filtration over a Sephadex G-100 column (Pharmacia, Piscataway, NJ) equilibrated in PBS and migrated as a single band at 120 kD as detected by autoradiography of SDS polyacrylamide gels (data not shown). This tracer was used for binding studies with a recombinant, soluble form of the CD4 receptor of T-helper cells14 and binding of rgpl20 to CD4 was not altered by the radiolabeling.9 Iodinated rgp 120 bound to anti-gpl20 antibodies, showing a typical binding curve with 92-25% binding between 2 x 102 and 2 x 104 dilutions of a rabbit hyperimmune serum to gp 120 (data not shown). Nonspecific binding by control sera (normal rabbit serum or rabbit antibody to herpes simplex virus type 2) was eliminated at a dilution of approximately 1:500. Subsequent screening of other rabbit negative sera showed variable amount of nonspecific binding, many sera displaying much lower background (data not shown). To verify that iodination did not alter the conformation of rgp 120, competitive
120
100
o
CÛ
m
Unlabeled Protein
(|ig/ml)
'
Displacement of [ 25I]-rgp 120 by unlabeled rgp 120 (•), or the GD protein of herpes simplex type I (P. Berman, Genentech) (O). In this experiment, the rabbit anti-rgpl20 hyperimmune serum at 1:4500 dilution was incubated overnight at +4°C with varying concentrations (0.1-100 pg/ml) of the two proteins. Immune complexes were FIG. 1.
precipitated with goat antirabbit IgG and pellets were counted for radioactivity in an automatic gamma counter. Sixty percent of input counts were precipitated at this dilution of rabbit antiserum. Each data point represents the mean of two
determinations.
360
RADIOIMMUNOASSAY FOR ANTIBODIES TO
gpl20
Table 1. Interassay Precision of the RIP Assay for Detection of Antibodies to rgp 120a
Tracer lot# 1
2
3
4
5
Tracer age assay
at
Rabbit
days)
32 35 41 16 24 25 30 38 45 51 59 1 3 9 14 18 21 1 5 6 15 19 1
Control serum titer0 Baboon
Goat
4.1
4.0 4.1 4.2 4.2 4.1 4.0 4.2
4.4 4.4
4.2 4.2
4.3
4.3
4.2
4.4
4.2
4.4
4.2 4.3
4.4 4.6
4.3 0.11 2.6 7
4.4 0.12
4.5 4.5 4.1 4.2 4.2 4.4 4.3 4.3 4.4 4.2 4.1 4.2 0.16 3.8 19
X a
CV(%) n
2.8 7
aTo titer antibodies to rgp 120, sera were prediluted 1:50 in phosphate-buffered saline containing 0.5% BSA and 0.01% thimerosal, and assayed as a three fold dilution series in this buffer by overnight incubation at+4°C with a fixed amount (20,000 cpm, 2 ng)of [l25I] rgp 120 tracer. Immune complexes were precipitated by adding 50 p\ of 1 % boving gammaglobulins and 1 ml of 12.5% polyethylene glycol for 30 min at ambient temperature. Pellets were measured for in an automatic gamma counter. radioactivity b Titers were determined as the logio of the reciprocal of the dilution equal to 2 X the mean nonspecific binding (NSB, determined with tubes containing assay buffer instead of serum).
binding with unlabeled rgpl20 was examined. The displacement of the tracer by unlabeled rgpl20 is shown in Figure 1 (circles). Displacement curves with increasing ratios of unlabeled to radiolabeled rgp 120 were indistinguishable from the curve shown in Figure 1 and indicated that the radiolabeling did not modify the behavior of the protein in the assay. Precision
of the RIP assay
The precision of the assay was determined by examining the interassay coefficient of variation of titers obtained for positive control sera from three species. Data in Table 1 were accumulated over a period of two months, using five different preparations of tracer representing three lots of purified rgp 120, from 19 assays for the rabbit positive control serum and 7 assays for the baboon and goat control sera. Data from this table show that the standard deviation from a mean of 4.3 was less than 0.2 and that the coefficients of variation were 3.8, 2.6, and 2.8% for the three control sera tested (Table 1). This indicates a high level of reproducibility for the assay and confirms the reproducibility of the lactoperoxidase method of radiolabeling 361
LUCAS ET AL.
rgpl20. Good tracer reproducibility was also shown, since no significant difference in titer results obtained with different tracer lots or with the use of tracers over a period of two months.
was
Specificity of the RIP assay Specificity was examined by assaying a rabbit antiserum to herpes simplex virus type 2 and indicated no reactivity with rgp 120 at dilutions greater than 1:10. Normal rabbit serum gave somewhat higher background. In addition, an antiserum to proteins originating from the CHO cell line used to produce rgpl20, and two antisera to rHBsAg did not precipitate rgp 120 tracer, when tested at 1:100 dilution (data not shown). A radioimmunoassay formatted with a 1:4500 dilution of the rabbit positive control (allowing the precipitation of 60% of the total counts) assayed against several dilutions of the GD molecule of the herpes simplex type 1 virus resulted in no displacement of the rgp 120 tracer (Fig. 1), also indicating good specificity of the anti-rgpl20 antiserum. cross
Efficiency of PEG precipitation The assay was first optimized with rabbit antiserum and 50 pi of 1 % bovine gammaglobulin added as a carrier for precipitation. The efficiency of precipitating immune complexes, using PEG-8000 at several concentrations, was then assessed with sera from four animal species and compared with precipitations with specific antispecies second antibody. Results in Table 2 showed that for all species, PEG-8000 at 9-10% allowed precipitation of 69-86% of the precipitable counts (corrected for NSB). This compared favorably with 81-91% precipitation using specific second antibody, considering that the use of a PEG precipitation system allowed the handling of sera from several animal species with a single, common, precipitating agent. Since sera are first screened by comparison to a species-specific negative control serum to distinguish positive from negative sera, the effect of serum in the assay was examined by testing negative control sera at several dilutions and comparing the resulting background level to NSB obtained with assay buffer. Table 3 shows that the effect varied with the animal species tested. Background obtained with serum from three animal species (guinea pig, goat, and chimpanzee) was comparable to that obtained with assay buffer (15% B/T or less), whereas rabbit serum had to be diluted at least 1:200 and human serum 1:100. In general, for most animal species, the assay was amenable to a PEG precipitation system provided the sera were diluted 1:100. We also compared endpoint titers obtained when sera were diluted in assay diluent with endpoint titers obtained when the sera were diluted in a constant level of species-specific negative serum (to maintain constant serum concentration throughout the dilution curve). Endpoint titers derived from the two dilution methods were identical (data not shown), thereby validating the derivation of titers from dilution curves prepared in assay diluent. Table 2. Radioimmunoprecipitation Assay for Antibodies to rgp 120: Efficiency of Polyethylene Glycol (PEG) for Precipitating Immune Complexes in Sera from Several Animal Species'1
Final concentration
of PEG-8000 (%)b
-
Animal species Rabbit Guinea
pig Chimpanzee
Baboon
7
9
10
11
12
75 88 66 87
76 86 66 86
72 NDC 69 ND
61 73 ND 81
59 ND
46 ND
Second antibody
precipitation0 81 ND 86 91
Results are presented as percent of counts percipitated, corrected for nonspecific binding (NSB). bIn this experiment, sera from the different animal species were tested at a dilution allowing maximal binding of [125I] rgp 120(1:50 for the primate sera to 1:500 for the rabbit hyperimmune serum). They were incubated overnight a
with a fixed quantity of [l25]rgp 120 (20,000 cpm, 2 ng). Immune complexes were precipitated by adding 50 /il of 1% bovine gamma globulins and 1 ml of varying concentrations of PEG for 30 min at ambient temperature, or 100 /il of specific antispecies antiserum. at +4°C
CND: not determined.
362
RADIOIMMUNOASSAY FOR ANTIBODIES TO
gpl20
Table 3. Immunoprecipitation Assay for Detection of Antibodies to rgp 120: Background levels of Negative Sera from Different Animal Species
Animal species* Rabbit
pool
(PelFreez)
Rabbit (X of 12) Individual sera) Guinea pig (1 individual serum)
Goat (X of 4 assays; 1 individual serum) Chimpanzee (X of 6 bleeds from 1 animal) Human pool
Dilution of the sera
%Binding by the buffer (NSB)h
%Binding by the negative sera0
1:25 1:50 1:75 1:100 1:225 1:675 1:2025 1:50
9.3 11.3 9.3 7.9 9.3 9.3 9.3 11.4
21.0 35.6 29.6 19.8 10.1 9.6 9.6 33.6 ± 3.2
1:25 1:75 1:225 1:25
9.3 9.3 9.3 13.0
10.2 8.7 9.0 13.6 ± 1.1
1:50 1:100 1:25 1:75 1:225
11.4 10.8 15.0 15.0 15.0
13.4 10.0+1.0 24.3 18.2 15.3
aIn this experiment, sera from the different animal species were tested as a series of dilutions with the tracer, to determine which dilution reduced the background to a level equivalent to that obtained with buffer alone. After overnight incubation of the serum dilutions with the tracer at +4°C, immune complexes were with 50 pi of 1% bovine gamma globulins and 1 ml of 12.5% polythylene glycol. preciptated b Results are presented as percent of counts precipitated.
Calibration
of the RIP assay
We examined the possibility of calibrating the assay in order to further simplify it and determine antibody titers in test sera from a standard curve prepared with dilutions of a positive serum. For this, standards were prepared with dilutions of the rabbit anti-rgpl20 antiserum and percent of counts precipitated were plotted against the titer values for each dilution (titers were determined previously by the classical endpoint method). Titers of sera from several immunized animal species were determined from this rabbit calibration curve (Fig. 2) and compared with titers obtained experimentally by dilution to endpoint. Since the rabbit antiserum used for calibration was a hyperimmune serum with very high titer, it had to be diluted considerably so that its binding level matched that of the test animal sera. Provided the calibration curve was prepared at lower binding levels (Fig. 2), calibration-derived titers were consistent across several dilutions and correlated well with titers obtained by the endpoint method. The data in Table 4 show good agreement of titers obtained either with PEG or a specific second antibody precipitation system. The maximum disagreement between calibration-derived and endpoint titers ranged from 0.1 titer units for the majority of samples to 0.5 for one high-titered sample (baboon third bleed, Table 4). This approach provided equivalent results using only 1 to 3 eightfold dilutions of samples rather than the 6 or more threefold dilutions typically required for the endpoint method. Table 4 also shows that titers determined with a second antibody precipitation were somewhat higher than titers determined with PEG precipitation, mostly due to the differences in background with the two precipitation systems (the 2 x NSB criterion used for endpoint determination results in lower titers if the background is higher, as was the case with the PEG precipitation).
363
LUCAS ET AL.
Correlation with immunoblot results A panel of 20 HIV-positive human sera titered with the RIP assay were analyzed by immunoblot assay, using commercially available reagents. Figure 3 shows that antibodies to rgp 120 reacted with both native viral gpl60 and gpl20 by immunoblot assay. All samples negative in the RIP were also negative by immunoblot, even when exhibiting high background in this assay (sample # 2). Similarly, RIP positive samples were positive by immunoblot. Sera with higher RIP titers to rgp 120 generally had stronger reactivity in the immunoblots, although this increased reactivity was reflected more with the pl8, p24, p32, p41-43, p51, and p65 bands than with the gp 120-gp 160 bands. Generally, reactivity was stronger with gp 160 than with gp 120. To further investigate the correlation between the RIP assay and immunoblotting, recombinant gpl20 was electrophoresed by SDS-PAGE and transferred to nitrocellulose (see Materials and Methods). Two HIV-positive human sera titered in the RIP (# 18 and 7) and the rabbit positive control were also titered by blot assay on nitrocellulose-bound rgp 120. The results in Figure 4 show good correlation between titers obtained
in the two methods, and demonstrate that the RIP assay is at least as sensitive as the immunoblot assay. Human serum titers of 4.4 and 3.9 by blot assay correlated with RIP titers of 4.9 and 3.9, respectively. The rabbit positive control (RIP titer 4.1) was titered to 5.1 by visual examination of the freshly developed, wet immunoblot (in this method, endpoints are subjectively distinguished above background rather than over 2 x NSB as in the RIP). Relatively high background obtained with rabbit serum in the RIP (Table 3) may explain the lower titer achieved with this method for the rabbit positive control, as compared with immunoblot titer.
CD
2.0
2.2
2.4
2.6
2.8
3.0
3.2
Titer FIG. 2. Calibration curve for antibodies to rgp 120 in the RIP assay. A calibration curve was prepared by incubating 6 dilutions of a rabbit anti-rgp 120 hyperimmune serum with [ ' 25I]rgp 120 and plotting percent of counts precipitated against the titer values for each dilution (titers were previously determined by the classical end point method). After overnight incubation at +4CC, immune complexes were precipitated with 50 pi of 1% bovine gamma globulins and 1 ml of 12.5%
polyethylene glycol (PEG).
364
RADIOIMMUNOASSAY FOR ANTIBODIES TO
gpl20
Table 4. RIP Assay for Detection of Antibodies to rgp 120: Comparison of Endpoint to Calibration-Derived Titers
2nd antibody precip. °
PEG precipitation"
Dilution
factor
Sample Baboon 1st bleed
Titer by calibration0
100 300 900 X
Baboon 2nd bleed
300 900 2700 8100 X 900 2700
Baboon 3rd bleed
8100 24300 X
Chimpanzee
1
100 300 900 2700 X
Chimpanzee
2
100 300 X
Goat
100 300 900 2700 X
Guinea
pig
1350 4050 12150 X
Endpoint titer4
3.1 3.2 3.2 3.1 3.8 4.0 4.1 4.1 4.0 4.3 4.4 4.4 4.4 4.4 3.6 3.7 3.7 3.6 3.6 2.4 2.4 2.4 3.4 3.6 3.5 3.4 3.5 4.6
2.9
3.7
3.9
Titer by calibration0
Endpoint titer4
2.6 3.5 3.4 3.1 4.8 4.4 4.4 4.3 4.5 5.1 4.9 4.8 4.6 4.8
4.8
4.2 4.1 3.9 3.8 4.0
4.2
3.4
4.4
3.6 2.5
3.1
4.4 4.4 4.5
4.4
In this assay, dilutions of sera were incubated overnight at +4°C with [ l25I] rgp 120 tracer. Inmmune complexes precipitated with 50 p\ of 1% bovine gamma globulins and 1 ml of 12.5% polyethylene glycol. bIn this assay, immune complexes were precipitated by adding 50-100 pi of undiluted antispecies antiserum. c For calibration, a curve was prepared by incubating 5 dilutions of rabbit anti-rgp 120 hyperimmune serum with the [ 125I] rgp 120 tracer. Percent of counts precipitated were plotted against the titer values for each dilution. Animal sera were tested at several dilutions with [ ' 25I] rgp 120 and titers for each dilution were extrapolated from the rabbit calibration curve subsequently corrected for dilution. d For endpoint titration, animal sera were assayed as a threefold dilution series and titers were defined as the log i o of the reciprocal of the dilution equal to 2 X the mean nonspecific binding (NSB. determined with tubes containing assay buffer instead of serum). a
were
Correlation with neutralization results
Finally, we examined sera from two immunization studies conducted with rgp 120 in chimpanzees15 or baboons. Neutralization of viral infectivity by the sera was measured by in vitro inhibition of HIV-1-specific reverse transcriptase.1215'24 or by measuring the reduction in HIV-1 antigen production by HIV-1-infected MT2 cells as a result of incubating the viral inoculum with serum prior to infection. A positive correlation was found between titers determined in the RIP assay and the ability of the sera to neutralize where 86-88% of 365
1 2 3
4 5
6 7
8
9 10 11 12 13 14 15 16 17 18 19 20
kD 160 120 -65 —55 Il 41-43
-32 -24 -18
RIP Titers: 0 0 0 0
0 3.8'—3.9—'4.0^4.3—' 4.4 4.5 L 4.6 J 4.95.2 5.3
FIG. 3. Immunoblotting of HIV proteins with several human sera. A commercial kit (BioRad, Richmond, CA) was used and the experiment was performed following the manufacturer's recommendations. Molecular weight markers are shown to the right, serum identification numbers at the top of each immunoblot and titers obtained in the RIP assay at the bottom of each blot. These titers are expressed as the log10 of the reciprocal of the last dilution equal to or greater than 2 x the mean nonspecific binding (NSB).
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2
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