IgA testing for diagnosis of retroviral infections in the Caribbean

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IgA Testing for Diagnosis of Retroviral

Infections in the Caribbean1

I? R. RIDEL,~ J. C. ARTus, & A. CAFRON~

In Guadeloupe, a Caribbean island with a high prevalence of HTLV-Z infected subjects, the percentage of false positive results for HIV IgG is high, requiring additional time and expense for confirmatory tests. This article describes a simple way of overcoming this problem. First, all IgG and IgG immune complexes are removed by protein G treatment, and then IgA enzyme-linked immunosorbent assay (ELISA) and the Western blot test are performed with minor modification of commercially available kits.

0

ur experience in diagnosing human immunodeficiency virus (HIV) infection of man by means of the IgG

enzyme-linked immunosorbent assay

(ELISA) indicates that about 5% of the sera tested yield false positive results. Moreover, IgG Western blot diagnosis

commonly yields one or more non-

specific bands, the classification of which is difficult-requiring further costly and time-consuming laboratory procedures.

One circumstance apparently aggra- vating this false positives problem on the Caribbean island of Guadeloupe is infection of test subjects with another retro- virus, the human T-cell leukemia virus

*This article will also be published in Spanish in the

Bolefin de la Oficina Sanitaria Panamericana. vol. 112. 1992. This work was supported by funding fro6 the Institut Pasteur of Paris. Correspondence should be addressed to Dr. l? R. Ridel, Institut Pasteur de la Guadeloupe, PO. Box 484, Morne Joivere, P.O. Box 484, 97165, Pointe a Pitre Cedex, F.W.I.

ZInsiitut Pasteur de la Guadeloupe, Pointe-Hitre Cedex, F. W.I.

%stitut Pasteur de Lille, Centre de Biologie et d’humunologie Parasitaire, 8 rue Camille Guerin, 59019 Lille CEDEX, France.

(HTLV), which is highly prevalent on Guadeloupe and various other Caribbean islands. In this regard, we found that 35% to 45% of a group of subjects testing positively for HIV-l/2 by IgG ELISA and negatively for HIV-l/2 by IgG West- ern blot tested positively for HTLV-I infection by both IgG ELISA and IgG West- ern blot.

The study reported here was performed to determine whether we could easily perform a more specific ELISA HIV test directed at IgA rather than IgG antibodies, as described elsewhere (I), under the circumstances prevailing in Gua- deloupe. In contrast to IgG antibodies, neither IgA nor IgM antibodies cross the placenta, and so both are suitable for congenital AIDS diagnosis (2). However, previous studies have found IgA HIV antibodies to persist longer than their IgM counterparts, and so we chose to seek a solution to the nonspecific results problem by testing for IgA antibodies. By way of preparation, we used protein G to remove IgG from the test sera (2, 3). Then, in order to save time and expense, we performed ELISA and Western blot procedures with modified versions of commercially available kits.

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MATERIALS AND METHODS

Patients

The serum samples tested were obtained from 10 Guadeloupe residents who were follow-up patients at the island’s Pasteur Institute. All 10 study subjects had yielded positive ELISA results for HIV-l/2 IgG (Rapid’ ELAVIA Mixt Di- agnostic Pasteur, Marnes la Coquette, France), and the sera of all but two had exhibited one or more nonspecific bands when tested for HIV-l/2 IgG by Western blot (New Lav-Blot I or II Diagnostic Pasteur). These two latter patients (whose cases are designated 9 and 10) were coinfected with HIV-l and HTLV-I.

None of the 10 study sera yielded positive results when tested for HIV-2 (Pepti Lav or New Lav-Blot II, Diagnostic Pasteur). However, all of them yielded positive ELISA results for HTLV-I IgG (Abbott, Paris, France) and showed specific HTLV-I IgG bands (P19, 21, and 24) when tested by Western blot (DuPont de Nemours, Paris, France). These positive ELISA and Western blot results (before removal of IgG) are indicated in Table 2.

Blood Samples

Blood for the investigation was obtained from the 10 study subjects by veni- puncture, and the resulting sera were stored at -20°C until used in the procedures described below.

IgG Removal

IgG antibodies, including those present in autoantibodies or circulating immune complexes (CIC), tend to interfere with assays for IgA antibodies, reducing the latters’ sensitivity and specificity. Therefore, before testing the 10 study subjects’ sera for IgA, we treated those

sera with protein G (3) to remove all sub- classes of IgG.

The test sera were treated for 90 minutes with protein G (Sepharose-Phar- macia, Uppsala, Sweden) at 37°C using a slightly modified version of the methodology described in Weiblen et al (1). Briefly, 25~1 of sera were incubated with 100,ul of a 50% suspension of protein G agarose in 0.02M phosphate-buffered sa- line at pH 7.6 and 37°C for 90 minutes under constant agitation. The sera were then diluted 125 (for Western blot testing) or 1:50 (for the ELISA) using the diluent reagent provided with each kit.4 The extent to which IgG had been removed was assessed in terms of the turbidity produced by an anti-human IgG serum (Behring Diagnostic, Rueil- Malmaison, France).

ELISA Procedures

The IgG ELISA testing for HIV-l/2 and HTLV-I was performed according to the

manufacturers’ recommendations (see

footnote 4).

In conducting the IgA ELISA testing for HIV-l/2 and HTLV-I, certain modifica- tions were made to the recommendations. These were as follows: (1) The con- jugate was a 1:500 dilution of a F(ab’), phosphatase-conjugated anti-human IgA (Sigma, St. Louis, USA). (2) The substrate buffer was 1M diethanolamine (Sigma) containing 0.5M MgCl, (Sigma) and 0.02% NaN, (Sigma), pH 9.8 with HCl. (3) The chromogenic agent was P-nitrophenyl phosphate employed at lmg per ml of substrate buffer. (4) After 45 minutes of incubation at 37’C, absor- bance was determined using a 405nm filter with a 62Onm reference filter.

4The makers of the kits used were as follows: ELISA HIV-l/2, Pasteur Diagnostic; ELISA HTLV-I, Ab- bott; Western blot Hw, Pasteur Diagnostic; West- ern blot HTLV-I, DuPont de Nemours.

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Calculations and interpretation of the ELISA results were done using the procedure described in the kit booklet. All IgG and IgA results exceeding the calculated cutoff point by 10% or more were considered positive.

Western Blot Procedure

Western blot testing for HIV-l/2 IgG and HTLV-I IgG was done according to the manufacturers’ recommendations (see footnote 4).

IgA immunoblots were performed

with protein G depleted sera diluted 1:25 in the washing solution/diluent supplied with the kit. F(ab’), phosphatase-labeled anti-human IgA (Sigma) was substituted for that included in the kit. The substrate buffer was 1OOmM NaCI, 5mM MgCl,, 1OOrnM TRIS Cl, pH 9.5; and the chromogenic agent was 66~1 of a 5% (W/V) solution of nitro blue tetrazolium (Sigma) and 33~1 of a 5% solution of 5-bromo-4- chloro-3-indolyl phosphate (Sigma) in 10 ml of substrate buffer. The test sera were incubated for two hours at room temperature on a rocker platform, and the alkaline phosphatase-conjugated anti-

human IgA was incubated for one hour at room temperature.

RESULTS

Protein G Treatment

As indicated by the data in Table 1, the protein G treatment effectively reduced the IgG concentration in the test sera by 99.9%, from 12.0-21.7 mglml to 0.007-0.017 mglml.

ELISA

Before treatment with protein G, all the ELISA tests for HIV-l/2 IgG and HTLV-I IgG were positive, yielding values at least 10% above the calculated cutoff levels. As indicated in Table 1, these values ranged from 12% to 63% above the cutoff for HIV-l/2 IgG and from 15% to 54% above the cutoff for HTLV-I IgG .

After IgG depletion with protein G, none of the test sera were ELISA-positive for HIV-l/2 or HTLV-I IgG, all the values obtained being well below the cutoff levels. Specifically, the values obtained

Table 1. Detection by ELISA of IgG, HIV-l/2 IgC, HTLV-I IgG, HIV-l/2 IgA, and HTLV-I IgA antibodies in sera from the 10 study subjects before and after removal of IgG through protein G treatment. The detected levels of IgG are given in mg/ml, while those of the virus-specific antibodies are expressed in terms of a percentage above or below the cutoff value established for the test.

Increase or reduction in the cutoff percentage

Case IgG (mg/ml) I& HIV IgG HTLV

No. Before After Before After Before After IgA HIV IgA HTLV 1 21.7 0.009 l k35% - 70% f27% -52% -38% +24% 2 12.4 0.010 f22% -51% f54% -51 % -50% +27% 3 NDa 0.008 +33% -51 % +40% -61% -53% +39% 4 19.8 0.014 f30% -54% +las -55% -46% + 12 % 5 12.3 0.005 + 14% -65% +1 6% -58% -33% +16%

6 ND 0.014 ND ND +23% -65% -45% +l 6%

7 18.4 0.017 +12% -50% + 15 % -50% -27% + 1 3 % 8 ND 0.010 +24% -56% + 2 3 % -55% -48% +20% 9 12.0 0.007 +63% -71 % +32% -27% +44% +32% 10 15.5 0.014 i-55% -53% +17% -38% +27% + 15 % aND = Not determined.

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for HIV-l/2 IgG were 50% to 71% below the cutoff level and those for HTLV-I IgG were 27% to 65% below the cutoff level- most of the values being close to the titers obtained with negative control sera (provided with the kit or by uninfected subjects).

Regarding IgA, all the test sera except those of cases 9 and 10 yielded negative ELISA results for HIV-l/2 IgA, while all 10 sera tested positively for HTLV-I IgA (see Table 1). In most cases, the percentage by which the HTLV-I IgA value exceeded the cutoff was slightly below the percentage by which the HTLV-I IgG value exceeded the cutoff before protein G treatment (see columns 6 and 9 in Table l), but the differences observed in this small study sample were not statistically significant.

In contrast to the other eight sera, those from the two coinfected patients (cases 9 and 10) yielded ELISA results for HIV-l/2 IgA that were respectively 44% and 27% above the cutoff level-thereby indicating that specific anti-HIV-l/2 IgA was present in those patients.

Western Blot

We observed that treatment with pro-

tein G completely suppressed any

HIV-l/2 or HTLV-I IgG Western blot reactivity, thereby demonstrating the treatment’s effectiveness.

Also, as indicated in Table 2, the HIV IgG bands observed in the eight false positive sera (cases 1 to 8) completely disappeared, indicating that the IgG reactivity observed was probably nonspecific.

As in the case of IgA testing for HIV-l/2 by ELISA, sera from the two coinfected patients tested positively for HIV-l/2 IgA by Western blot. Some of the bands ini- tially observed were not detected by IgA testing, but the principal bands (GPllO, GP160, and P24/25) remained. In contrast to the HIV IgA results, all 10 sera tested

positively for HTLV-I, showing most or all of the specific bands previously observed.

DISCUSSION

AND CONCLUSIONS

Our findings clearly indicate that the presence of nonspecific HIV-l/2 IgG produced the observed false positive HIV-l/2 IgG ELISA results.

Since our test subjects were all infected with HTLV-I, and since HIV and HTLV are known to exhibit cross-reactivity with one another, it seems likely that some of the false HIV-l/2 IgG ELISA results observed were due to antibodies directed against HTLV-I antigen. This is hard to prove, because depletion of specific HTLV-I antibodies would require a large supply of HTIV-I antigens and because, as reported elsewhere, circulating immune complexes as well as many types of autoantibodies can yield nonspecific results (4, 5).

However, when all the IgG was removed with the streptococcal protein G that binds all classes of IgG (3) (including circulating immune complexes), this en- abled us to test the sera for IgA. And when the sera were tested by ELISA for IgA, all the false positive HIV-l/2 results disappeared-indicating that this procedure is more specific than testing for IgG. It should be noted, however, that this IgA methodology is useful only after removal of IgG. (When we first tested the study sera for IgA before protein G treat-

ment, we found that 45% yielded

positive ELISA results for HIV-l/2 or HTLV-I IgA .)

Regarding the Western blot test results, we never observed HIV-specific bands in eight of the 10 study sera. How- ever, our results clearly showed that use of protein G treatment and IgA ELISA testing permitted quick and cost-efficient screening of patients while avoiding any need for additional testing. Moreover,

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Table 2. Data from Western blot testing of the 10 study sera, showing the HIV-l/2 and HTLV-I IgG antibody bands seen before protein G treatment and IgG and IgA bands seen after treatment.

Western blot, HIV-l/2 Western blot, HTLV-I

Case &s, w, kA kfG kG kA,

No. before after after before after after

1 P24l25

2 P24l2.s

3 P24f2.5

NVB= NVB

NVB NVB

NVB NV&

4 P24125

5 P.55

NVB NVB

NV& NVB

6 P55 NVB NVB

7 P18,55 NVB NVB

8 P18,52 NVB NVB

9

10

GP160 NVB CP160 GPllO NVB CPllO P68 NVB NV&

P55 NVB P55

P52 NVB NVB

P40 NVB P40

P24125 NVB P24/25

P18 NVB PI8

CP160 NVB GP160 GPllO NVB GPllO

P68 NVB NVB

P55 NVB P55

P52 NVB NVB

CP41 NVB GP41

P40 NVB P40

P24/25 NVB P24125

P19,21,24,28, GP46 P19,21,24,

28,42, 53 P19,21,24,

26,42, 53, GP46

P19,21,24, 26, 32, GP46

P19, 21,24,26, 28, 32,42, 53, GP46 P19,21,24,

40x, 53, G P46 P19,21,24,28,

32,42, 53, GP46 P19,21,24,

32,40x, 53, GP46 P19,21,24, 28,

32,42, 53, GP46

P19, 21,24, 28,40x, 53, GP46

NVB

NV&

NVB

P19,21,24,40, GP46 P19,21,24,

28,53 P19,21,24,

26,42,53, GP46 P19,21,24, 26,

GP46 P19, 21,24,

26, 32,42, GP46 P19, 21, 24, 53,

GP46 P19, 21,24,28,

32, 42, 53, GP46

P19,21,24,4Ox, 53, GP46

P19,21,24, 26,28, 53, GP46

P19,21,24, GP41,40,18

P18 NVB P18

aNVB = No visible band(s).

the IgA Western blot showed clear bands specific for HTLV-I in all 10 sera and also showed clear bands specific for HIV-l in the two coinfected sera-suggesting that IgA is at least as useful if not more useful than IgG for confirming retroviral infections.

In general, the presence of IgM and IgA antibodies indicates a recently ac-

quired infection, though IgA HIV antibodies appear to persist longer than their IgM counterparts. The IgA class of immunoglobulins, which like the IgM class of immunoglobulins does not cross the placenta, is more useful than IgM for diagnosing intrauterine infection-including acquired intrauterine toxoplasmosis, cytomegalovirus infections, and AIDS (2,

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6, 7). Our experience to date suggests that HIV-l IgA may appear somewhat before HIV-l IgG. Specifically, we found that a patient yielding positive Poly- merase Chain Reaction test results for the presence of HIV-l Gag and Pol antigens had a negative HIV-l/2 IgG ELISA but a positive HIV-l/2 IgA ELISA (l? R. Ridel, R. Goursaud, M. Chalcou, and J. C. Artus, personal communication).

Modification of commercially available kits appears to provide a simple cost-efficient means of testing for HIV IgA. In the Caribbean, where a high prevalence of HTLV-I infection is reported (up to 13% of the population in some countries), the percentage of false positive HIV IgG ELISA results is relatively high. Our results clearly indicate that switching from the HIV IgG ELISA to the more specific HIV IgA ELISA would save time and money, and would also permit more gen- eralized testing for HTLV antibodies. Nevertheless, it also appears advisable that the recommended methodology be further tested elsewhere in the Caribbean in order to confirm the initial findings reported here.

Acknowledgments.

The authors would like to thank Mirette Chalcou and Marie- France Gamblin for technical and secre- tarial assistance, and R@is Goursaud for

providing serum samples used in the

study.

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